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Chapter 1 Natural Products 1.1 Introduction The term ‘natural product’ means molecules of life1 but in general it refers to distinct low molecular weight organic compounds produced by living organisms such as bacteria, fungi, lichens, marine invertebrates, plants, insects, mammals etc2 which are recognized for their pharmacological and biological activities. Natural products are also known as secondary metabolites as they are not crucial for basic life processes like growth and reproduction but assist the host organisms in their survival, facilitate interaction and communication, help in adaption to varied environments3 and they may have evolved to provide defence mechanism against pathogens and predators.4 They possess diverse and complex chemical structures which are distinctive of the species or producing organism and are formed at specific stage of the morphological development of the host organism,5 for example during sporulation or pigment production. Secondary metabolites are synthesized from simple intermediate products from primary metabolism, the main precursors are acetyl Coenzyme A, shikimic acid, mevalonic acids and amino acids.6 Their ability to bind biological targets and stimulate bioactive effects has attracted the attention of pharmaceutical industries, natural-product chemists and biologists to explore different natural habitats, terrestrial, aquatic and the microbial world for the presence of natural products and for many years they have been a wealthy source of potential drugs.7 Since primitive times many different plant species have been documented to be used as medicines for human ailments and later many natural product compounds purified from plants prove to be among initial lead drugs. For example, acetyl salicyclic acid 1 (aspirin) is based upon the natural product salicin 2, isolated form the bark of the willow tree Salix alba L.7, 8 The pain killer codeine was synthesized from the alkaloid morphine7 3 obtained from the plant Papaver somniferum L.; digitoxin 11 from Digitalis purpurea L. is used as a cardiotonic to ease congestive heart failure,8 and the antimalarial drug quinine 4 has been used for years to treat fever, malaria and mouth and throat diseases. There are a number of plant derived anti-tumor drugs, for example paclitaxel 9 and baccatin 10 from Taxus brevifolia9 and numerous other anti-tumor 1 compounds are in clinical trials. Various plants’ secondary metabolites display inhibitory activity against microbes for example phenols, quinone 6, coumarin 5, catechin 7, terpenoids and essential oils possess antimicrobial effects, flavones 8 inhibit multiple viruses including HIV and catechin 7 is also used as an anti-coagulant.10 The discovery of the antibiotics penicillin11 161 (see section 3.3) and cephalosporin12 16 from the antibacterial fungi Penicillium notatum and Cephalosporium acremonium respectively, led to the exploration of various microorganisms in search of assorted bioactive metabolites. Fungi from the phyla Basidiomycota and Ascomycota delivered many pharmacological active compounds, 75% were antimicrobial and many others verified to possess antiviral, cytotoxic, antineoplastic, cardiovascular, anti-inflammatory, antitumor and immune-stimulating metabolites.13 The bacterial group of Actinomycetes is also rich with novel metabolites, mainly antibacterial, antiviral, antifungal and antitumor activities.14 The antibiotics vancomycin 15 from Amycolatopsis orientalis7,15 and erythromycin 13 from Saccharopolyspora erythraea are active against a wide range of bacteria and are used in treatment of various infections.7 Doxorubicin 14 isolated from the Streptomyces peucetius is used in the treatment leukaemia, bone sarcomas and lung and thyroid cancer.7,15 A number of immune-suppressive agents such as cyclosporin 17 from the 2 fungus Tolyplocladium inflatum and fujimycin 12 from the bacterium Streptomyces tsukubaensis have enhanced the field of organ transplants.2 Marine natural products also provide a rich source of biodiversity with about 15 bioactive compounds approved from Food and Drug Administration (FDA) and many in clinical trials.14,16 Hence, a number of surveys conducted on drug sources for the treatment of diseases like cancer and infections revealed that 60% of approved drugs are of natural origin.17 These include natural products directly used as drugs as well as semi-synthetic drugs derived from or modelled on natural products.18,19 There are major revolutions in natural product research and drug design. Large libraries of natural product analogues and structural mimics are synthesized through combinatorial chemistry18 and are examined by ‘High Throughput Screening Technology’ against wide range of important biological targets.19 With present-day genomics, cost-effective DNA sequencing of microorganisms helps in annotation of secondary metabolites gene clusters in their respective genomes. This has motivated researchers to express the silent secondary metabolite gene clusters whose chemical 3 products are unidentified by applying different genetic engineering techniques (see section 3.3). Biosynthetic pathways of a large number of novel metabolites have been elucidated from their particular biosynthetic gene clusters by different molecular biology techniques,5 such as gene knock out, RNAi silencing, cloning, homologous recombination and heterologous gene expression in surrogate host. These studies have broadened the knowledge of biosynthetic genes and their encoded enzymes and proteins. These discoveries pave the way for proteins to be used as biocatalysts20 for in vivo synthesis of complex natural products and resolve increasing demand for new drug products. The main classes of natural products are terpenoids, alkaloids, nonribosomal peptides and polyketides. They are briefly introduced in the following sections. 1.2 Terpenoids Terpenoids are made from a five carbon unit (isoprene), which exists in two active structural forms: dimethylallyl diphosphate 18; and isopentenyl diphosphate 19.21 These simple precursors make diverse structures and about 25,000 structures22 of terpenes are reported. The name terpenes was named after the volatile oil of pine tree, turpentine which is composed, among others, of the terpene compound α-pinene 20.1 Terpenoids are identified by their strong aroma, chiefly in essential oils. They have a wide range of functions: they protect plants against attack of pests and some are used as insecticides;22 they serve as a means of communication among organisms; they function as attractants for insects for pollination; and many play a part as mating pheromones and reproductive hormones.21 Many terpenes have a number of bioactivities, for example terpenes from the genus Rubia are reported to possess antitumor, antiinflammatory, antimicrobial, anti-malarial and antidiabetic effects.23 Examples of terpenoids include camphor 21, limonene 22 and gibberelllin GA4 23. 4 1.3 Alkaloids Alkaloids, like terpenoids, are a large and diverse class of compounds, with more than 12,000 examples known at present.24 They contain a basic amine group in their structure and are derived biosynthetically from amino acids. Examples are morphine 3 and the antimalarial drug quinine 4 (see section 1.1). 1.4 Nonribosomal peptides Microorganisms, particularly fungi and bacteria, produce an assorted group of peptide secondary metabolites called nonribosomal peptides (NRP) formed by multifunctional enzymes known as nonribosomal peptide synthetases (NRPS), independent of ribosomal pathway.25 NRP are formed from a wide range of substrates (amino acids) which include both D and L proteiogenic and non-proteiogenic amino acids which explains the numerous complex structures present in this class of natural products.26 The NRP serve as antibiotics, immunosuppressants, cytostatins and siderophores.25 Nonribosomal peptide synthetases (NRPS) are made up of set of modules, each module consists of basic set of catalytic or enzymatic units called domains. Modules are distinct sections in nonribosomal peptides and each module incorporates single amino acids to the growing peptide chain.28 There are three core domains in each module; the adenylation domain (A), the peptidyl carrier protein (PCP) and the condensation domain (C).29 The last module normally contains a termination domain which may be a thiolesterase (TE) or a reductase (R). The adenylation domain selects and activates an amino acid by adenyaltion with ATP, the PCP serves as a transporter of the activated substrate between catalytic domains by binding the substrate to a 4’phosphopantetheine cofactor with a thioester bond. The condensation (C) domain is responsible for peptide bond formation between the amino group of one amino acid 5 and the acyl group of amino acid of the adjacent module. At the end of last module, the peptide chain is usually terminated and released by the thiolesterase domain. Many NRPS may contain additional tailoring domains mainly the epimerization domain, Nmethylation and glycosylation which may further modify the structure of the NRP.30 Examples of NRP include vancomycin 15 and cyclosporin 17 (see section 1.1). 1.5 Fatty acid biosynthesis Before describing the next class of natural products, the polyketides, it is necessary to first discuss the biosynthesis of fatty acids. The biosynthesis of fatty acids has many similar features to polyketides’ pathway and it has always remained a model system to study the latter. Fatty acids are primary metabolites present in all living cells. Structurally they are carboxylic acids with a long saturated chain. They are important sources of fuel for an organism and when metabolized they liberate large quantities of ATP. They are essential components of phospholipids and play a basic structural role in assembly of the cell membrane.31 Fatty acids are synthesized from simple building blocks particularly the two carbon containing acetate in the form of acetyl Coenzyme A (CoA) and malonyl CoA. The former acts as a starter unit and the latter as an extender unit.32 Fatty acid synthesis involves a number of key enzymes in each step. In a general fatty acid pathway an acetyl group from the starter acetyl CoA 24 is transferred by an acyl transferase enzyme (AT) to the thiol group of the 4’-phosphopantetheine arm of acyl carrier protein (ACP). This is followed by the acetyl being transferred to another enzyme β-ketoacyl synthase (KS) at the thiol of the active site cysteine. Meanwhile the extender malonyl CoA 25 is attached to the ACP and condensation between acetyl thiolester 27 and malonyl thiolester 26 is catalysed by the KS in a decarboxylative Claisen condensation with liberation of CO2 and a β-keto thiol ester 28 bound to an ACP is formed.33 ACP serves as a ‘handle’ to carry the growing acyl chain throughout the whole process of fatty acid synthesis (Scheme 1.1). The β-keto thiol ester 28 is then reduced to a secondary alcohol 30 by a βketoacyl reductase (KR) followed by dehydration by a dehydratase enzyme (DH) to from an unsaturated thiolester 31 and finally an enoyl reductase (ER) performs a final reduction to form fully saturated thiolester 32.34 The fully saturated thiolester 32 can 6 enter another cycle of fatty acid chain extension with addition of a second malonyl thiolester by ACP and this process continues until the fatty acid reaches its specific length, for example a sixteen carbon chain in case of palmitic acid and an eighteen carbon chain for stearic acid. The last cycle of the fatty acid is terminated by a thiolesterase enzyme (TE) which liberates the free fatty acid 33 by a hydrolytic release and a long chain carboxylic acid is formed. Thus fatty acid synthases are large multifunctional proteins containing KS, ACP, AT, KR, DH, ER and TE, activities. Scheme 1.1 Series of steps in fatty acid and polyketide biosynthesis. 1.6 Polyketides Polyketides form a large group of secondary metabolites comprising of structurally complex compounds, produced by plants, bacteria, fungi, lichens and marine organisms.35 They have been extensively studied by natural product researchers because of their fascinating biosynthetic pathways and wide range of important biological and pharmaceutical properties. Polyketides have contributed about 50 approved drugs in the pharma industry.36 Some important bioactive compounds of this 7 class include the antibiotics erythromycin 13, rifamycin 38,37 monensin 42,38 doxycycline 43, 39 the antihelminthics avermectin 39,40 the antitumor compounds geldanamycin 4041 and macbecin 41,42 some antifungal polyenes amphotericin 4543 and primaricin 44, the immunosuppressant tacrolimus (FK506) 46,44 the cholesterol lowering agent lovastatin 4745 and many others. Some polyketides are also used as insecticides like spinosad 49,46 while some are toxins, for example aflatoxins 48.47 The basic concept of polyketides biosynthetic origin was presented in 1907 by John Norman Collie who was a professor at University College London and also a mountaineer.48 He proposed that ‘Keten’ groups (CH2=C=O) in the form of aldehydes and ketones can condense and polymerise by simple reactions with liberation of CO2 to form a range of complex compounds, many of them are present in plants. Hence they were named polyketenes and subsequently ‘polyketides’.49 8 During the 1940s, when labelled acetate incorporation was established in fatty acids,50 Robinson supported the polyketide theory presented by Collie.51 Arthur J. Birch worked on the biosynthetic origin of many aromatic polyketides. He showed experimentally the incorporation of radio labelled 14 C-acetic acids in alternating labelling pattern in 6-methyl salicylic acid 51 in Penicillium ariseofuluum (Scheme 1.2). He endorsed that β-keto chain are formed from condensation of acetate units which folds to form aromatic polyketides.49 Scheme 1.2: Biosynthetic steps of 6-MSA 51 showing incorporation of 14C acetate units. Similar studies also demonstrated that methyl groups in fungal polyketides are incorporated from L-methionine.52 When techniques like Mass spectrometry (MS) and Nuclear Magnetic Resonance (NMR) were introduced, the biosynthetic investigations on polyketides were accelerated. Many precursor compounds, mainly acetates were used in the form of singly or doubly labelled 13C or 2H isotopes and fed to the cultures of polyketides producing organisms. As 13 C or 2H enriched compounds are easily studied by NMR, they helped to understand the acetate incorporation patterns and mode of cyclization in many polyketides.53,54 9 Polyketides are formed by a similar biosynthetic pathway to that of fatty acids. They are formed by repetitive decarboxylative condensation of simple acetate units in the form of acetyl CoA 24 and malonyl CoA 25 (in some cases propionate and butyrate units) in a head to tail manner. In polyketide pathway, the first β-keto thiolester 28 is formed by a decarboxylative Claisen condensation of acetyl 27 and malonyl thioloester 26 by the same catalytic units KS, AT and ACP (Scheme 1.1). In fungi, the polyketide chain can also be methylated, receiving a methyl group from S-adenosyl methionine (SAM) to form α-methyl-β-keto thiolester 29 by a unique CMeT domain not active in the FAS pathway. The β keto chain can be further processed by the KR, DH and ER domains.34 The cycle again continues until PKS chain reaches it specific length and the thiolester is hydrolysed from the peptide and released by a termination domain. In fatty acid biosynthesis, the β-keto chain is uniformly reduced from a β-keto group to an alcohol 30, then forms an α, β- double bond in 31 by elimination of water and at the end is fully reduced to a methylene as observed in 32 (scheme 1.1). In the polyketide pathway the reduction process is more controlled, selective and variable. There may not be any reduction in the poly β-keto chain of PKS chain (Scheme 1.1, 35) and is common in aromatic polyketides. The β-keto chain may only be reduced once to remain as a hydroxyl group, or dehydrated to remain as a enoyl group (scheme 1.1, 36), or may be fully reduced to methylene groups to form 34 (Scheme 1.1).33 The polyketide chain may also hold a pendant methyl or ethyl group by incorporation of methyl malonyl or ethyl malonyl units as observed in 37 (Scheme 1.1). Polyketide biosynthesis also sets different stereocentres during reduction of the β-keto chain (Scheme 1.1, 36 and 37). Other factors which determine the structure of the polyketides are choice of the starter units, number of chain extensions and pattern of cyclization.31,55 The choice of all these structural functionalities are governed by the above described enzymes (or domains) and these are collectively called Polyketide synthases (PKS). David Hopwood used the term ‘programming’34 for the way PKS directs different variables or structural features in the synthesis of the diverse polyketides structures.56 These directions are ultimately encoded with in the sequences of PKS gene clusters. Lots of efforts have been served by researchers to understand the core of programming of polyketide synthases and have achieved a considerable success. Advanced genetics and molecular biology tools like sequencing, cloning, gene expression, and enzymes purification have helped to understand the nature and structure 10 of PKS and how they catalyse different reactions or steps in biosynthesis. Many biosynthetic pathways of polyketide metabolites and their respective gene clusters are known.5 Hopwood and co-workers have achieved some pioneering work in PKS genetics and construction of cloning vectors.56 His group discovered the first PKS genes for the antibiotic actinorhordin.53 Different degenerate primers were developed from the initial PKS known genes and were used to screen genome libraries of many bacteria and fungi and pave way to discovery of numerous new PKS genes on the basis of similarities in gene sequences. After allocation of gene clusters many PKS enzymes and proteins were purified and a number of crystal structures were solved, which also gave awareness of different catalytic sites in enzymes. The functions of many enzymes are proved with cell free extracts and in vitro studies. The quest for understanding the Polyketide synthases and their programming behaviour is still in progress and many gene clusters and organisms need to be explored for their exclusive biosynthetic and bioactive properties.57 1.7 Types of Polyketide Synthases PKS enzymes are divided into three main groups according to the protein assembly and arrangement of domains with in the polypeptide. The three main classes are type I, type II and type III PKS.53 1.7.1 Type I PKS In type I PKS, all the multifunctional domains required for polyketide chain elongation and β-keto group processing are located on a single large polypeptide. Type I PKS is further categorized into three types. They are modular PKS, trans-AT modular PKS and iterative PKS.58 1.7.1(a) Modular PKS In modular PKS, the different catalytic units are arranged in the form of sets of domains, called modules. The domains present in each module performs a single chain extension and β-keto processing and then passed it on to the next module for another carbon chain addition and β-keto group processing. Each domain is used once during the cycle and the linear order of the modules and their respective domains can define the structure of the polyketide chain. Modular PKS are common in bacteria.59 11 The best studied example of modular PKS is erythromycin A 13,60 the gene cluster of which was studied in late 1980s.61 It is produced by the Gram positive bacteria Saccharopolyspora erythraea. Erythromycin A 13 biosynthesis consists of an intermediate compound 6-deoxyerythronolide B 52 (6-DEB), which is a 14-membered macrolactone ring and a putative polyketide synthase product.53,62 The polyketide synthase responsible for the biosynthesis of 6-DEB 52 was named as deoxyerythronolide B synthase (DEBS) encoded by three genes eryAI, eryAII and eryAIII, each 10 kb in length (scheme 1.3).59 Scheme 1.3 Biosynthesis of erythromycin by modular Type I PKS. The erythromycin PKS (EryPKS) was among the first PKS to be sequenced among the complex polyketides and formed a model for studying modular PKS. The three genes encode three proteins DEBS1, DEBS2 and DEBS 3. Each DEBS protein holds two modules and each module contains the three basic domains KS-AT-ACP with 12 different combinations of KR, DH and ER depending upon the extent of β-group processing. The last module ends with the thiolesterase (TE) domain. In the first cycle the AT domain of the first module in DEBS1 binds a propionyl CoA and transfers it to the pantotheine arm of the adjacent ACP and then to the next KS domain. The AT then binds the extender unit, a methyl malonyl CoA to the ACP. This results in formation of a diketide by the combination of the starter and the extender unit by the KS, followed by keto-reduction by KR to form a β-hydroxy. The ACP in module 1 then transfers the diketide to the module 2 where it is joined by a second extender unit, followed by keto reduction by KR in module 2. The triketide is passed to the module 3 with condensation with a third extender unit. The β-keto group in module 3 remains unreduced because of a non-functional KR in module 3 as apparent in C-9 in 52, in the fourth cycle the β-keto group undergoes subsequent reduction, dehydration and enoyl reduction to form a methylene functionality at C-7 because of presence of KR, DH and ER in module 4. Similar keto group condensation and reduction continues in cycle 5 and 6 depending on the domains present in the respective modules. In the last, a 15 carbon PKS chain is released from the ACP of DEBS 3 by the action of the last TE domain in module 6 and forms a macrolide 52 structure by the combination of C-1 to carboxylate of C-13 hydroxyl. The post PKS steps includes attachment of a 6-deoxy sugars D-desosamine at position 5, a L-cladinose at position 3 and P450 mediated hydroxylations at C-6 and C12 to from erythromycin A 13.59 Trans-AT PKS There are a number of modular type I PKS gene clusters reported, which lack the regular cis-AT, a domain found in association with ACP and KS and other domains in a standard module.63 In trans-AT modular PKS, the prescribed function of acyl transferase is accomplished by stand-alone AT domain(s) which act in trans with all the other modules and serves the same function of supplying acyl building blocks to all the respective modules in the PKS. There are a number of polyketide metabolites biosynthesized by a trans-AT modular PKS pathway for example leinamycin 55, 64 pseudomonic acid A 54 65 and lankacidin C 53. 65 Scheme 1.4 shows the gene cluster of leinamycin (LNM) produced by Streptomyces atroolivaceus S-140. 64 The gene cluster consists of six PKS, one NRPS module encoded by the genes lnmI and lnmJ, among these all the six PKS modules lack the key AT domain. The AT function is provided by 13 LnmG which loads the malonyl CoA units to all the PKS modules to biosynthesize the compound leinamycin 55 (scheme 1.4). Scheme 1.4 Biosynthesis of leinamycin 55 by trans-modular PKS. 1.7.1(b) Iterative Type IPKS Type I iterative PKSs (IPKS) are made up of a single set of multifunctional domains found in a large polypeptide. The sole set of domains carry out all cycles of carbon chain extension and the respective β-keto chain processing, and many domains are used repeatedly, hence the name iterative. IPKS have long been the attention of biosynthetic investigations because the single set of enzymes can act differently in every cycle of chain extension and processing, portraying a high level of complex programming. IPKS are found most commonly in fungi. IPKSs are divided into three classes according to the extent of β-keto group reduction and on the presence of KR, DH and ER domains in the protein architecture. They are non-reducing (NR-PKS), 14 partially reducing (PR-PKS) and highly reducing (HR-PKS). This classification was first presented by Simpson.34 Non-reducing IPKS As the name indicates, non-reduced polyketides are formed from the condensation of intact and non-reduced poly β-keto chain which cyclise forming aromatic compounds with a mono or multiple ring structures. Some common nonreduced polyketides are orsellinic acid 56, emodin 57 and norsolonic acid 58.57 Orsellinic acid 56, a tetraketide, was among the early discovered fungal PKS from Penicillium madriti in 1968.34,66 A typical NR-PKS is composed of an N-terminal loading component, a chain extension component and a C-terminal processing component. The loading component is made of a starter unit-acyl transferase (SAT). It can load acyl CoA and in many cases complex FAS or PKS elements as starter units. For example norsolorinic acid 58 incorporates a hexanoate unit,34 dehydrocurvularin 59 accepts a HR-tetraketide as starter unit,67 zearalenone 60 uses a HR-hexaketide as a starter unit.68 The chain extension component consists of KS, followed by AT. The AT is specifically a malonyl loading domain. Following the AT, is the product template domain (PT). It is believed to be involved in chain length control34 and in some reports it also takes part in PKS chain cyclization.57 PT is followed by ACP. Some NR-PKS may end with ACP but many possess a distinct C-terminal processing component. The C-terminal components may end in a Claisen cylcase-thiolesterase (CLC/TE) or may further consist of methyl transferase (CMeT), 69 additional ACPs or thiolester reductase (R) domains. The CLC/TE is involved in chain length decision, chain release and cyclisation via intramolecular Claisen condensation.57,34 An example of NR-PKS with an active CMeT domain is 3-methylorcinaldehyde 61 synthase where a methyl group is provided by SAM (S-adenosyl methionine) (Scheme 1.5).69 The PKS structure of 3methyl orcinaldehyde synthase (MOS) consists of N-terminal (NT) domain responsible for selecting the starter unit, followed by KS, AT, PT, an ACP, C-MeT domain and a Cterminus NADPH dependent thiolester reductase (R) domain. 15 Fig 1.1 Examples of non-reduced polyketides, the complex starter units are highlighted in green in norsolorinic acid 58, dehydrocurvularin 59 and zearalenone 60. Scheme 1.5 Non-reduced PKS gene cluster of 3-methylorcinaldehyde 61. Partially reducing IPKS The protein architecture of PR-PKS consists of KS, AT, DH, a unique core domain, followed by KR domain and at the end an ACP domain.34 PR-PKS compounds are formed by successive condensation of acetyl starter and malonyl extender units, forming poly β-keto chain, while β-keto processing does not necessarily occur in every cycle; therefore they are termed as partially reduced PKS. The core domain is believed to maintain integration and functional stability between the domains. A well-studied PR-PKS is 6-methylsalicylic acid (6-MSA) 51. 70 The 6-MSA was originally obtained from Penicillium patulum biosynthesized by 6-methylsalicylic synthase (MSAS). During biosynthesis of 51, one acetate 24 and 3 malonyl extender units 25 condense in subsequent extensions to form a tetraketide 62. The KR domain functions after the second extension in the presence of NADPH forming an alcohol group. After the third 16 cycle, the PKS chain undergoes cyclisation and dehydration to form 6-MSA 51 (Scheme 1.6).53 Scheme 1.6 Biosynthetic pathway of 6-MSA 51. Highly Reducing IPKS A typical HR PKS consists of KS, AT, DH followed by a C-MeT domain in most cases. The next domain is the ER, but many HR-PKS may not contain a functional ER. The ER is followed by a KR and the PKS most normally terminates with an ACP domain. With the presence of all three β-keto processing domains used iteratively, HR PKS synthesize structures with high level of complexity and advanced programming.34 For example lovastatin 47, is a HR polyketide produced by Aspergillus terreus. It is biosynthesized by two PKS proteins, LovB and LovF.71,105 lovB encodes lovastatin nonaketide synthase (LNKS) and lovF encodes lovastatin diketide synthase (LDKS). LNKS with the assistance of LovC, a trans acting ER (the ER domain in LNKS is nonfunctional) synthesize a nonaketide PKS intermediate compound Monacolin J 63. LDKS then produces a methylated diketide 64, which is loaded on to Monacolin J 63 at C-10 hydroxy by a specialized acyltransferase encoded by a gene lovD to form 47 (Scheme 1.7). Squalestatin S172 65 is also made by two PKS chains, a main hexaketide and a tetraketide sidechain. Other examples of HR PKS include the longest polyketides Fumonisin B1 68 produced by Gibberella fujikuroi 73 and T-toxin 74 69 produced by 17 maize pathogen Cochliobolus heterostrophus. Solanopyrone A 66 and alternaric acid 67 produced by the plant pathogen Alternaria solani are also produced by HR PKS. 34 Scheme 1.7 Lovastatin biosynthetic pathway. Hybrid IPKS-NRPS Hybrid highly reduced polyketide synthases fused with nonribosomal peptide synthetase (HRPKS-NRPS) are an important class of synthetases often found in fungi. The protein architecture consists of the HRPKS domains (KS, AT, DH, MT, inactive ER, KR, ACP) and nonribosomal peptide catalytic units (C, A, T and terminal R domain) forming a megasynthase.75 PKS-NRPS have been extensively studied in recent years because of their intriguing iterative programming code and important biological 18 activities demonstrated by the hybrid compounds.57,34 All PKS-NRPS discovered up till now possess non-functional ER sequence and if ER activity is required, it is provided by a distinct trans-acting ER encoding gene homologous to LovC. This property makes them closely related to the lovastatin 47 gene cluster and presents a common origin.76 The HRPKS synthesizes a polyketide chain from an acetyl starter unit and subsequent malonyl extender units with methyl groups delivered from SAM by a methyltransferase. The adenylation domain of the NRPS selects and activates an amino acid and transfers it to the thiolation domain. The condensation domain binds the amino acid and the polyketide chain by an amide bond. The hybrid polyketide and peptide chain is released from the megasynthase by the terminal R domain by either of two release mechanisms.75 It can either be released in the form of an aldehyde forming pyrrolinone 70 by a Knoevenagel condensation (Scheme 1.8, A) reported in pseurotin A 78, isoflavipucine 79 and chaetoglobosin A 80 biosynthesis or as a tetramic acid (pyrrolidone 71) by direct Dieckmann cyclisation (Scheme 1.8, B) detected during tenellin 87, desmethylbassianin (DMB) 88, equisetin 81 and cyclopiazonic acid 85 biosynthesis.75,57 The hybrid polyketide-peptide compound is further modified by tailoring enzymes encoded by genes clustered near the megasynthase. A B Scheme 1.8 A, Release mechanism by a Knoevenagel condensation by reductase domain; B, release mechanism by Dieckmann cyclisation. The first PKS-NRPS gene cluster was identified for fusarin C 77 from Fusarium moniliforme and Fusarium venenatum in an attempt to search for C-methyltransferase domains.77 The fusA gene encodes the synthesis of a tetramethylated heptaketide 72 fused by an amide bond to L-homoserine 73 by the C domain to form a covalently 19 bound intermediate 74 and is probably released by an R domain to form the aldehyde 75.78 Further Knoevenagel condensation forms the putative prefusarin 76. Subsequent modifying steps of carboxylation, epoxidation and hydroxylation forms Fusarin C 77 (Scheme 1.9).75 Other examples of hybrid PKS-NRPS includes aspyridone A 84, xyrrolin 86, militarinone C 82 and pramanicin 83.75 Scheme 1.9 Fusarin C biosynthesis. Figure 1.2 Examples of hybrid PKS-NRPS compounds, the NRPS part is highlighted in red. 20 1.7.2 Type II PKS In contrast to type I PKS, the enzymatic activities for the β- keto chain elongation and processing in type II PKSs are present in separate polypeptides, and each domain is used iteratively.53,58 A well-studied model of type II PKS is actinorhodin 89 biosynthesis (Scheme 1.10). Scheme 1.10 Actinorhordin pathway showing Type II PKS system. 1.7.3 Type III PKS Type III PKSs (Scheme 1.11) were originally identified in plants but recently have also been isolated from several bacteria. In contrast to type I and type II polyketide biosynthesis, the β-keto chain is elongated and processed at a single multifunctional active site in type III PKSs.53 It does not require an acyl carrier protein and distinctively accepts acyl coenzyme A building units, for example in chalcone 90 biosynthetic pathway. Scheme 1.11 Chalcone synthase biosynthesis. 21 1.8 AIMS The main objective of our research is to contribute in understanding the programming code veiled in the iterative protein structure of the hybrid PKS-NRPS pathways. We aimed to study the PKS-NRPS systems of three compounds; tenellin 87, desmethylbassianin 88 and aspyridone A 84. Tenellin 87 and desmethylbassianin 88 are hybrid polyketide-peptide metabolites produced by two different strains of enthomopathogenic fungi B. bassiana. The tenellin 87 pathway has been elaborately studied in the Bristol Polyketide Group with different genetic and chemical analysis and it helped understand the basic biosynthetic pathway of tenellin 87. We aimed to continue the biosynthetic studies by carrying out further heterologous expression of tenellin 87 genes or its components in a heterologous host. These include expression of PKS-NRPS encoding gene in A. oryzae without the tailoring genes and expression of tenellin polyketide synthase alone in A. oryzae without its NRPS counterpart. The objective was to determine the product as well as the intricate role of each component of the megasynthase in tenellin 87 biosynthesis. Desmethyl bassianin 88 (DMB) and tenellin 87 gene clusters have 90% sequence identity but DMB 88 differs in structure from tenellin 87 in having an additional carbon chain extension and a methyl group less than tenellin 87. We next intended to study the co-expression of tenellin 87 genes together with DMB 88 tailoring genes in different combinations in A. oryzae. This was designed to see whether the two similar yet different biosynthetic genes are compatible to work together in one expression system, to obtain new engineered natural products and determine the programming role of the particular genes. The function of trans-acting enoyl reductase enzyme in tenellin 87 is encoded by a discrete gene, tenC. The RNAi silencing of tenC has been successfully achieved before in the native fungus using a strong constitutive promoter.79 We planned to perform the RNAi silencing of tenC, again, this time using an inducible promoter and grow the silenced transformants in different growth conditions. We desired to investigate whether we can control the level of gene silencing and obtain new compounds reflecting varying degree of silencing. 22 The last objective was to study aspyridone 84 biosynthesis. Aspyridone A 84, a PKS-NRPS compound is produced from a silent gene cluster in Aspergillus nidulans and its pathway has been proposed but not proved experimentally.80 We aimed to investigate aspyridone pathway using an effective heterologous expression system in A. oryzae and analyse the transformants. The objective was to determine the order of different biosynthetic steps, role of each gene in the pathway and discover potential of the megasynthase in synthesizing new bioactive natural products. The detail description of the aims has been separately described in each chapter with the respective analytical methods. 23 Chapter 2 Elucidation of new compounds from different genetic studies in Beauveria bassiana 2.1 Introduction Beauveria bassiana belongs to a group of entomopathogenic fungi. These fungi are parasitic to insects and kill or disable them completely. They invade the insects initially by their microscopic spores called conidia.81 These spores attach to the insect cuticle and the conidia swells by secretion of lytic enzymes which helps it to breach and penetrate the outer layer of cuticle. This is followed by development of morphological structures such as appressorium on the cuticle, infection pegs and penetrant hyphae in the epicuticle and procuticle helping the conidia hyphae to reach the body cavity of the insect (hoemocoel). The fungal hyphae continue to proliferate causing damage to the host tissue and nutrient exhaustion eventually leaves the insect body dead or destroyed.82 Figure 2.1: Insect infected by fungus Beauveria bassiana. 83 Beauveria bassiana (Balsamo) Vuillemin has a long taxonomic history.84,85 Agostino Bassi (1835) first described this fungus as the causal agent of ‘mark disease’ also known as white muscardine disease in France.86 This disease caused destruction of silkworm larvae in Southern Europe during the 18th and 19th centuries resulting in huge economic losses to the silk industry. Bassi discovered that microbes can act as contagious pathogens of animals and this formed the basic fundamentals of the ‘germ theory of disease’.87 The first taxonomic recognition of the muscardino fungus was proposed by Balsamo-Crivelli. He recognized Bassi’s discovery by naming this fungus 24 Botrytis bassiana. Beauverie stated that the fungus should belong to an undescribed genus and Vuillmen established in 1912 the genus as Beauveria in his honour and Botrytis bassiana Bals.Criv as the type species.86,88 Beauveria is distributed worldwide. They are soil borne hygomycetes (having naked spores)89 and are pathogenic towards several different orders of insects including Lepidoptera, Coleoptera, Hemiptera, Hymenoptera and Orthoptera.85 They are easy to culture. There are no toxic metabolites from beauveria reported to enter the food chain or accumulating in the environment.90 These features make B. bassiana a model system for studying entomogenetics and effective biological control of pests. B. bassiana produces several secondary metabolites of varied structures but the contribution of these metabolites to pathogenesis is mostly unknown.91 Some metabolites reported from B. bassiana include bassianin92 91, beauvericin93 93, bassianolide91 92, beauverolide A94 94, oosporein95 95 and tenellin96 87. Among these, bassianolide 92 has been identified in dead silkworm larvae infected by B. bassiana.91 Beauvericin 93 is known to have mycotoxic properties.93 A high molecular weight protein toxin Bassiacridin is stated to be isolated from B. bassiana strain obtained from a locust.97 The Bristol polyketide group have identified that tenellin 87 is not involved in pathogenicity.98 The present chapter involves work on tenellin 87 and its biosynthesis. 25 Tenellin 87 is a prominent secondary metabolite of B. bassiana and its structure was elucidated by Wat and co-workers, together with another similar compound bassianin 91.96 Both compounds are known by their distinctive yellow colour apparent during fermentation and in organic extracts. Tenellin 87 possesses a 5- substitiuted 2 pyridone ring with an acylated moiety at C-3.99 Tenellin 87 has been the focus of many biosynthetic studies over a period of many years. The most distinctive reason has been that it is formed from a combination of an amino acid and a polyketide chain.99 The key work to determine the precursors of tenellin 87 was reported by McInnes and co-workers.100 They used [1, 2-13C2]-acetate 96, L-[methyl-13C] methionine 97, (±)-[1-13C] phenylalanine and (±)-[2-13C] phenylalanine 98. The results analysed by 13 C-NMR indicated that C-2, C-3 and C-7 to C-14 of tenellin 87 were alternately enriched with doubly labelled acetate 96 and both methyl groups at C-15 and C-16 showed enhanced peaks for labelled 13 C-methionine 97. The carboxy carbon C-1 of phenylalanine 98 forms C-4 of tenellin 87 and C-2 of phenyl alanine 98 becomes C-6 of tenellin 87. There is an intramolecular rearrangement of phenylalanine which causes migration of the carboxy carbon adjacent to the aromatic ring forming C-4 of 87 and the alpha carbon of phenylalanine separates to form C-6 of tenellin 87 (Scheme 2.1). They confirmed that tenellin 87 is formed by condensation of methylated polyketide chain having five acetates with an entity comprising all carbons from phenylalanine.100 Scheme 2.1: Incorporation of labelled acetate, methionine and phenylalanine in tenellin 87. 26 Another similar study by Leete and coworkers101 supported the intramolecular rearrangement of phenylalanine in tenellin 87. They fed phenylalanine labelled at two carbon positions [1, 3-13C2]. They indicated in 13 C-NMR spectrum additional satellite peaks adjacent to singlet peaks of C-4 and C-5 due to spin spin coupling confirming intramolecular rearrangement of the phenylalanine side chain in tenellin 87. They argued that had there been ‘intermolecular’ movement of carboxyl group, the 13C-NMR would only show singlets peaks for both C-5 and C-4. Further isotope feeding on tenellin 87 was carried out by Wright et al.102 They also used singly and doubly labelled sodium acetate, labelled methionine and three different labelled forms DL-[carboxy-13C], DL-[α-13C] and L-[15N] phenylalanine. Their results supported previous feeding studies by McInnes.100 Incorporation of N-labelled phenylalanine established that it is fused to the polyketide chain with no loss of nitrogen. In addition they also showed severely reduced incorporation of radioactive L[U-14C] tyrosine suggesting that tyrosine is not a direct precursor of tenellin 87. Wright and coworkers proposed a route for tetramic acid formation. First, condensation of phenylalanine 99 with the ten carbon polyketide chain 100 and then hydroxylation of the aromatic ring by an oxygenase enzyme give quinomethine 101. This would then undergo rearrangement of the tetramic acid to form the pyridone of tenellin 87 (Scheme 2.2). 27 Scheme 2.2: Proposed tenellin 87 biosynthesis by Wright et al.102 Another biosynthetic proposal suggested by Cox and O’Hagan was that phenylalanine 99 rearranges early and then condenses with a polyketide 100 to give the six membered pyridone directly (Scheme 2.3).103 They synthesized and fed DL-[3-13C] and [3-14C]-3-amino-2-phenylpropionic acids 102 to the cultures of B. bassiana. However, the 13 C NMR analysis of this experiment did not show that 3-amino-2- phenylpropionic acid 102 is a genuine intermediate in tyrosine 87 biosynthesis. Scheme 2.3: Proposed biosynthesis by Cox and O’Hagan.103 28 On the basis of the Wright et al.102 hypothesis, Moore et al.99 synthesized acyl tetramic acid in two isotopically labelled forms [4-13C] in 103 and [phenyl-2H5] in 104 and carried out feeding experiments with B. bassiana fermentations. They found that these compounds were not incorporated into tenellin 87 and there was no proof for its presence in B. bassiana extracts. They observed a single minor metabolite 105, the purified sample of metabolite on 1H NMR showed it to be para-substituted aromatic moiety without N-hydroxylation. They concluded that para-hydroxylated acyl tetramic acid emerged as a late intermediate and a precursor or a reduced metabolite in tenellin biosynthetic pathway (Scheme 2.4). They also carried out isotopic feeding of tyrosine 13 DL-[3- C] and [1-13C] phenylalanine and indicated that both tyrosine and phenylalanine are efficiently incorporated into tenellin 87. By this result, in contrast to the hypothesis of Wright et al.,102 Moore and coworkers proposed that phenolic hydroxylation in tenellin 87 is introduced by tyrosine and not at a later stage modification of the aryl ring. Scheme 2.4: Labelled acyl tetramic acid feeding in tenellin 87 by Moore et al.99 In recent years with advancement in molecular biology and discovery of new tools to manipulate DNA, investigations of biosynthetic routes of natural compounds from their specific gene clusters have been very remarkable. Lately the Bristol Polyketide Group have carried out quite a number of successful genetic studies on polyketides genes of tenellin 87 from B. bassiana 110.25. They discovered specific 29 genes responsible for different stages and intermediate steps in tenellin biosynthesis. All these stages reflect cryptic programming of PKS genes. Eley et al. identified a hybrid PKS-NRPS gene cluster from the genomic DNA of B. bassiana and showed by gene knock out experiments that this cluster is involved in tenellin 87 biosynthesis.98 Analysis of this cluster revealed four open reading frames (ORF) (Figure 2.2). BLAST analysis revealed that ORF1 and 2 were homologous to cytochrome P450 enzymes. ORF3, a putative Zn dependent oxidoreductase,104 showed high homology to enoyl-reductase (ER) enzymes. ORF4 consists of an approximately 12-kb biosynthetic gene that encodes β-ketoacyl synthase (KS), acyl transferase (AT), dehydratase (DH), CMeT (methyltransferase), β-ketoacyl-reductase (KR) and acyl carrier protein (ACP) domains typical of a fungal iterative type I PKS, followed by condensation (C), adenylation (A), thiolation (T) and putative thiol (R ester reduction) domains, characteristic of an NRPS module. A directed gene knockout (KO) experiment confirmed ORF4 to be involved in tenellin production, and ORF4 was therefore renamed tenS (tenellin synthetase). In this work they also proved that B. bassiana tenS KO and WT strains are equally pathogenic towards insect larvae suggesting that tenellin 87 is not involved in insect pathogenesis of B. bassiana.98 Figure 2.2: Tenellin gene cluster identified by Eley et al.98 They proposed a pathway for the early stages of tenellin 87 biosynthesis from its respective PKS and NRPS enzymes (Scheme 2.5). The double methylated pentaketide 106 bound to ACP is synthesised by TENS PKS and the A domain of TENS NRPS first activates tyrosine 107 by adenylation and transfer to the thiol group of the T domain. The polyketide and the amino acid are fused by the C domain to form N-β-ketoacyl amino thiolester 108. The reduction domain R carries out reduction of the thiol ester 108 with the help of NADPH and release in the form of a peptide aldehyde 109. The aldehyde can cyclise to form pre-tenellin 110, which was assumed to be a precursor of tenellin 87 (Scheme 2.5). 30 Scheme 2.5: Tenellin 87 pathway proposed by Eley et al.98 Halo et al. expressed the tenS gene encoding tenellin synthetase (TENS), in Aspergillus oryzae M-2-3.104 It led to the production of three new compounds, identified as acyl tetramic acids, prototenellin A 111, prototenellin B 112 and prototenellin C 113. Protenellin C 113 was not fully characterized due to the low concentration of the purified compound (Table 2.1). These compounds didn’t have the methylation pattern of tenellin 87 and the polyketide chain length in prototenellin B 112 was shorter than tenellin 87. In addition there were double bonds between C-11 and C-12 of prototenellin A 111 and between C-9 and C-10 of prototenellin B 112 whereas this bond is always saturated in 87. This shows that the enoyl reductase domain present within the TENS protein is defective and fails to carry out reduction in the first cycle of the polyketide chain formation. These results depict that enzymes in tenS gene when expressed on its own lose the ‘fidelity’ in the programming of the polyketide side chain of the compound. Halo et al. also carried out another important experiment which was coexpression of tenS with the gene encoding the enoyl reductase enzyme (ORF3) which led to the production of single acyl tetramic acid compound, pretenellin A 114 (Table 2.1). This compound was concluded to be a genuine precursor to 87, possessing the 31 same methylation and chain length. Pretenellin A 114 has a saturated bond in the first keto chain extension similar to tenellin 87 proving that the enoyl reduction is carried out by ORF3 and not by ER in the TENS protein. This result showed that in the presence of the ORF3, the tenellin gene cluster undergoes its normal pattern of bond formation and methylation of the polyketide unit leading to correctly programmed compound structure. Similar stand-alone enoyl-reductase encoding genes like lovC and apdC in PKS gene clusters are reported from lovastatin 47105 and aspyridone A80 84 respectively, where ER domain present in the PKS synthase is inoperative. The ORF3 gene in tenellin synthase is known as tenC. The first precursor compound pretenellin A 114 produced from coexpression of TENS and TENC was different than pre-tenellin 110 hypothesized by Eley et al.98 in being hydroxylated at C-4 (Table 2.1). Halo et al. described that the C-terminal R domain of the NRPS after releasing thiolester 108, does not undergo a reductive reaction at this step but must catalyse a Dieckmann cyclisation of the N-β-ketoacyl amino thiolester 108 directly to form the tetramic acid, pretenellin A 114 (Scheme 2.6). This concept was further supported by similar result reported by Sims and Schmidt.106 They carried out in vitro experiments, reacting purified proteins from R domains of equisetin synthetase (EqiS) with synthetic substrate analogues. They obtained equisetin tetramic acids and did not observe any reduced or aldehyde intermediates thus giving evidence for Dieckmann cyclisation activity of R domains of EqiS. Tang et al. also produced in vitro a 3-acyltetramic acid preaspyridone A 224 by incubating Aspergillus nidulans PKS-NRPS encoding gene apdA with its enoyl reducatse encoding gene apdC in the absence of any oxidative enzymes, proving that its R domain also actually catalyses a Dieckmann cyclisation.107 Scheme 2.6: Release and formation of pretenellin A 114 by Dieckmann cyclase domain of TENS. 32 In another study Halo et al. revealed late stage oxidations during the biosynthesis of the 2-pyridone tenellin 87 in B. bassiana by a combination of gene knockout, gene silencing by antisense RNA and gene coexpression studies.108 They concluded that the putative cytochrome P450 oxidase encoded by tenA catalyzes the oxidative ring expansion required to convert the tetramic acid of pretenellin A 114 (Table 2.2) to the pyridone of tenellin 87. Gene knockout and silencing of tenB produced pretenellin B 115, which confirmed that tenB catalyzes N- hydroxylation of 115 (Table 2.2) to form 87. The tenB gene encodes a rare kind of cytochrome p450 which N-hydroxylates only 2-pyridones and not tetramic acids. Another experiment confirmed the N-hydroxylation function of tenB when (tenA + tenB + tenS) were coexpressed in A. oryzae producing pretenellin B 115. The above studies on the tenellin gene cluster revealed the highly programmed nature of the PKS-NRPS proteins. After successful studies on the tenellin 87 gene cluster, Heneghan et al.109 carried out screening of several other species of Beauveria bassiana to search for compounds similar to tenellin 87. Their aim was to compare new gene clusters and their sequences of proteins to that of tenellin 87. After analysing various B. bassiana species they found and characterized the similar compound Desmethyl bassianin 88 (DMB) from B. bassiana strain 992. Desmethylbassianin (DMB) 88 differs from tenellin 87 in having a single methylation in its side chain and it also has an additional chain extention. Heneghan et al. identified the DMB gene cluster by Southern Blot by using tenS probe (Figure 2.3). The biosynthetic gene cluster of DMB 88 had 90% sequence identity to the tenellin 87 gene cluster.109 It has the same four open reading frames dmbA, dmbB, dmbC and dmbS (which is the PKS-NRPS). Figure 2.3: DMB gene cluster identified by Heneghan et al.109 33 To evaluate and confirm that dmbS is responsible for DMB 88 production,109 they carried out a double silencing and knockout strategy to disrupt the dmbS gene and the results showed total loss of production of any DMB 88 or related compound. Similar results to tenellin 87 were obtained when dmbS was expressed in A. oryzae (M-2-3) producing protoDMB-B 116 and protoDMB-C 117 which cannot be regarded as precursor of DMB 88. When dmbS was co-transformed with dmbC, it gave the preDMB A 118 (Table 2.3) giving the same methylation and chain length pattern as DMB 88. It confirmed that in the presence of dmbC, dmbS undergo correct reduction in the polyketide chain of the DMB compounds, show high conformity in programming and the yield of compounds is also increased. In both tenellin 87 and the DMB 88 gene clusters there is more than 90% similarity between the tenellin and DMB PKS proteins but still both these compounds were different in their PKS chain length and methylation pattern. After successful expression studies in both individual gene clusters, it was possible to create hybrid tenellin and dmb genes expression in Aspergillus oryzae to know which proteins is responsible for difference in programming in closely related gene clusters. Heneghan et al. carried out some co-expression experiments. These showed that DMBC and TENC are interchangeable; the programme of the PKS is influenced by tenS and dmbS. This led to the idea of ‘fidelity’- the extent to which the PKS makes ‘mistakes’. When dmbS and dmbC were co-transformed in A. oryzae, it gave preDMB A 118, while cotransformation of dmbS with tenC again gave preDMB A 118 (Table 2.4). This indicated that tenC and dmbC had identical effects in assisting correct programmed compounds production. Coexpression of tenS with dmbC gave pretenellin A 114 which is identical when tenS is expressed with tenC. These results showed that the PKS controls the programming of polyketide chain in the presence of either of the trans-acting ER proteins.109 In the absence of the trans acting ER encoded by tenC or dmbC the PKS displays low fidelity. But when the trans-ER is present the PKS displays high fidelity and high productivity. In another experiment Heneghan and coworkers created a hybrid gene consisting of tenSPKS with dmbNRPS to know the effect of NRPS in programming.109 This swap produced prototenellin A 111, prototenellin B 112 and prototenellin C 113 (Table 2.4). This means that the NRPS does not have an effect in PKS programming but it only 34 functions to connect the amino acid to the polyketide chain and plays role as off-loading mechanism for the PKS.109 This swap produced the same compounds when tenSPKS was expressed in A. oryzae (M-2-3).104 The productive heterologous gene expression, silencing and gene knockout studies in DMB 88 and tenellin 87 PKS-NRPS genome resolved the characteristic function of their respective genes. The oxidative enzymes encoded by tenA, tenB and dmbA, dmbB carry out important ring expansion and N-hydroxylation steps. The enoyl reductase enzymes tenC and dmbC were observed to play crucial part in production of correctly programmed precursor compounds pretenellinA 114 and preDMB 118 (Scheme 2.7). Knocking out the hybrid PKS-NRPS genes dmbS and tenS completely eliminated the production of tenellin and DMB in their respective fungi. Scheme 2.7: Individual domains in TENS and DMBS proteins producing precursor compounds. Recently Fisch and colleagues revealed the catalytic role undergone by individual domains in DMBS and TENS proteins. Their work also solved the important queries about the methylation and chain length difference in tenellin 87 and DMB 88 structure.110 They took tenS as a host gene and exchanged its constituent domains with domains from dmbS one at a time in each experiment and expressed it in A. oryzae with 35 tenC. They started initially by replacing the KS-AT domain of tenS by dmbS and then the DH in a second experiment until the entire tenS PKS gene was swapped over with domains from dmbS PKS. Scheme 2.8: Key metabolites desmethyl pretenellin A 119, preDMB A 118 and prebassianin 120 produced in different domain swaps between TENS and DMBS proving catalytic role of CMeT and KR domains in programming. No change in programming was detected by including KS-AT-DH domains from DMBS and the clones still produced pretenellin A 114. Variations in programming 36 were observed when CMeT and KR domains were included in TENS. (KS-AT-DHCMeT) domains from DMBS produced a new compound desmethyl pretenellin A 119 possessing pentaketide side chain length of pretenellin A 114 but a single methylation in the PKS chain (Scheme 2.8). This indicated that the CMeT from DMBS brings about single methylation pattern of DMB 88. In another swap by adding KR in the TENS host together with DMBS (KS-AT-DH-CMeT-ER) the clone produced the hexaketide predmbA 118, proving that KR is the chain length determining enzyme. Another swap supporting this result was observed when only KR from DMBS was located in TENS. This clone produced a compound prebassianin 120 possessing hexaketide chain length of predmbA 118 but with double methylation in PKS side chain (Scheme 2.8). This study revealed that KR and CMeT domains exhibit the major part in selecting number of methylation and chain elongation in iterative HR-PKS NRPS.110 The potential and diversity of secondary metabolites production from B. bassiana was further examined by treating this fungus with epigenetic modifying chemicals.111 B. bassiana strain 110.25 fungus was grown in the presence of genetic modifiers, 5-azacytidine (5AC) 121 and suberoyl bis-hydroxamic acid (SBHA) 122 (Scheme 2.9). Epigenetic modifiers are small chemicals that bring about modification of gene activation and expression without modifying its nucleotide sequence. Histones are the main protein component of chromatin providing a framework for the DNA around which it winds and forms a structure. In addition chromatin also exhibits an important role in gene regulation. It affects gene expression by removing acetyl groups from its N-acetyl lysine amino acid with the help of deacetylase enzymes. This deacetylation makes the DNA wrap around histones more firmly leading to compressed chromatin which makes the genes inactive. Some chemicals can act as histone deacetylase inhibitors such as suberoyl bis-hydroxamic acid (SBHA) 122. These inhibitors block this action of deacetylase enzymes which increase lysine acetylation leading to activation of silenced genes. The expression of genes in cells is also dependent on DNA methylation. This involves addition of methyl group to cytosine or adenine of DNA nucleotide. This methylation inactivates the expression of certain genes. 5-azacytidine (5AC) 121 is a chemical analogue of cytidine which is a nucleoside present in DNA and RNA. 5AC 37 can bring about changes in the cell genome and its behaviour by removing methyl groups from the DNA thus activating the silenced genes. There a number of studies in which epigenetic modifiers stimulated silent gene clusters in fungi producing new compounds and adding variety in the library of natural products.112 Scheme 2.9: New compounds produced by B. bassiana WT after growing with epigenetic chemicals 5AC and SBHA. Yakasai et al. reported that B. bassiana showed three times higher production of tenellin related compounds when B. basssian WT cultures were grown in the presence of 5AC 121 and SBHA 122.111 They also produced new compounds 3’,4’-antipyridomacrolidin-A 123, 3’,4’-syn-prepyridomacrolidin-A 124, 3’,4’-syn- prepyridomacrolidin-B 125, and reprogrammed compounds prototenellin A 111 and protenellin E 126 possessing different methylation pattern than pretenellin A 114 (Scheme 2.9). In the same study two different clones tenA-silenced strain and tenB-silenced strain were grown in the presence of epigenetic chemicals. The tenA-silenced strain 38 produced new compounds 12-hydroxy pretenellin A 127 and 128 (both syn and anti diastereomer), 14-hydroxy pretenellin A 129 (Scheme 2.10) and reprogrammed compound protenellin E 126 but not any pyridone compounds. This showed that silenced genes of tenA were not switched on by these chemicals. Scheme 2.10 New compounds produced by tenA aRNA transformant after growing with epigenetic chemicals 5AC and SBHA. tenB silenced clones in the presence of 5AC 121 and SBHA 122 produced a variety of different new compounds (10, 11-Z ) pyridovericin 130, (10, 11-Z )-syn-13hydroxy pretenellin B 131, (10, 11-Z )-anti-13-hydroxy pretenellin B 132, prototenellin F 133, anti-13, 15-dihydroxy pretenellin B 134, syn-13-hydroxy pretenellin B 135 and anti-13 hydroxy pretenellin B 136. In this, epigenetic modifiers overcome the silencing factor of tenB producing tenellin 87 which is an N-hydroxylated compound. These chemicals demonstrated the rich tendency of PKS-NRPS genes for crafting new compounds (Scheme 2.11). 39 Scheme 2.11: New compounds produced by tenB aRNA strain after growing with epigenetic chemicals 5AC and SBHA. 2.2 Aims and Objective of the Chapter Tenellin 87 is an example of a secondary metabolite in entomopathogenic fungus Beauveria bassiana, the biosynthesis of which is composed of highly programmed steps directed by an organized enzyme complex. This enzyme complex is encoded by a hybrid gene cluster of iterative polyketide synthases-non ribosomal peptide synthetases (PKS-NRPS). The modern tools of biotechnology help to manipulate and study genes in fungi. These techniques include different gene expression in host fungi and silencing or knock-out of target genes in native organisms. The results of these techniques are examined with a number of the latest laboratory instruments particularly Liquid Chromatography-Mass Spectrometry. New chemical compounds are detected and their structures are elucidated. Different molecular genetic techniques combined with chemistry apparatuses help to explore the relation between compounds and their 40 corresponding proteins and genes. These help plot the biosynthetic routes of natural product compounds. The various genetic studies on tenellin 87 and DMB 88 PKS-NRPS gene clusters discussed in section 2.1 encouraged further transformation experiments in A. oryzae. The objective of this Chapter is to analyse various strains prepared from heterologous expression of tenellin 87 PKS genes and hybrid co-expression of tenellinDMB genes in A. oryzae. The hybrid tenellin and DMB genes co-expression was carried out in quest to know the difference in programming of both tenellin and DMB gene clusters. We also intended to analyse tenC-aRNA silenced clones of B. bassiana carried out using amyB promoters. All these strains were grown under standard fermentation conditions and their organic extracts were examined using LC-MS which displays UV diode array and mass spectrometry data of the compounds in the crude extract. Any interesting compounds detected in these extracts were purified using preparative HPLC and the isolated pure compounds were subjected to various structure techniques including IR, NMR, HRMS and X-ray diffraction. 2.3.0 Results 2.3.1 Heterelogous Expression of tenS in A. oryzae M-2-3 The effective tenS gene knock out experiment in B. bassiana reported by Eley et al.98 resulted in loss of production of tenellin 87. This generated interest to explore the function of TENS PKS-NRPS protein. Halo et al. carried out heterologous expression of tenS in A. oryzae M-2-3 using the pTAex3 expression system.104 Three new compounds were produced from this experiment which were prototenellin A 111 (C21H23NO4, m/z 353), prototenellin B 112 (C18H19NO4, m/z 313) and prototenellin C 113 (C21H25NO6, m/z 387). Prototenellin C 113 was not fully characterized previously, due to lack of material and instability during purification.104 The experiment was repeated to characterize prototenellin C 113. Spores of A. oryzae pTAex3-tenS were first grown on plates (DPY media) for 7-10 days. A spore solution was made using sterile deionized water and 1 ml of spore solution was added in 41 100 ml liquid culture in 500 ml Erlenmeyer flasks (10 × 100 ml) for 7 days with 200 rpm, at 25 °C. After completion of fermentation the cultures were homogenized and extracted with ethyl acetate (section 4.11). The ethyl acetate extracts were dried and evaporated to yield a crude extract of 70 mg, which was dissolved in HPLC methanol (10 mg/ml). This extract was analyzed by LCMS (Method 1, section 4.4). Analytical LCMS showed peaks of prototenellin C at Rt 29.4 mins (Figure 2.4). ES+ and ESshowed masses of 388 and 386 respectively indicating a mass of 387 corresponding to prototenellin C 113 (Figure 2.5). This peak was purified by mass-directed preparative LCMS. 26 Injections were made using a 20 minute program (Method 1, section 4.5). The purified fractions of prototenellin C were collected and dried under nitrogen gas into pale yellow solid (2.4 mg/L). Figure 2.4: Diodearray chromatogram of tenS expression in A. oryzae producing prototenellin C. 42 Figure 2.5: ES-, ES+ and UV spectrum of prototenellin C 113. 2.3.1(a) Characterization of Prototenellin C 113 The pure prototenellin C 113 was dissolved in deutrated methanol and 1D and 2D NMR experiments were carried out using a 500 MHz spectrometer. The 1H NMR showed two prominent sets of doublets (Figure 2.6) which are characteristic of the parasubstituted hydroxy phenol found in tenellin and related compounds. In the 1H-1H COSY spectrum, the two diastereotopic protons H-16a and H-16b showed correlation with H-5 which is a typical pattern of a tetramic acid (Figure 2.7). This was further confirmed in the HMBC spectrum where the benzylic protons at H-16 showed two and three bonds correlation to the phenolic protons and also to the H-5 proton of the tetramic acid ring (Figure 2.7). Three methyl signals corresponding to H-13, H-14 and H-15 were present in the alkane region. The HMBC correlation of H-15 with C-7 and quaternary carbon C-6 confirmed methyl H-15 to be attached to C-7 indicating that the polyketide chain of prototenellin C possess the same methylation pattern as prototenellin A 111. HMBC signals at H-10 showed the occurrence of two hydroxyl groups at H-11 and H-12. The signals at H-10 and H-14 are two overlapping doublets and singlets respectively, providing evidence that the compound is a mixture of two diastereomers. Collective NMR data confirmed prototenellin C structure to be 113. The molecular formula and molecular mass was confirmed by HRMS to be C21H25NO6. 43 Fig 2.6: 1H NMR spectrum of prototenellin C 113 in methanol-d4. Figure 2.7: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in prototenellin C 113. 2.3.2 Heterelogous Expression of tenSPKS-dmbNRPS in A. oryzae M-2-3 Prototenellin C 113 was also produced from another strain tenSPKS-dmbNRPS cloned in A. oryzae (This transformation was done by Katherine Williams in the School of Biological Sciences). A. oryzae clones were grown in liquid culture (10 × 100 ml) for 7 days, 200 rpm, at 25 °C. After fermentation the cultures were homogenized and extracted with ethyl acetate (section 4.11). The ethyl acetate extracts were dried, evaporated (yield was 137 mg) and dissolved in HPLC methanol (to a final 44 concentration of 10 mg/ml). This extract was analyzed by LCMS. The LCMS analysis showed peak of prototenellin C. The ES+ and ES- showed obvious peaks of 386 and 388 respectively corresponding to the 387 mass of prototenellin C. This peak was purified by mass- directed preparative LCMS. The purified fraction of prototenellin C were combined and dried under nitrogen gas to give a pale solid mass of 9.6 mg. 1 H NMR was carried out from this purified fraction on a 500 MHz spectrometer. The 1H NMR showed the same proton signals as observed from the Prototenellin C 113 obtained from tenS expression clone (Figure 2.8). Figure 2.8: Overlay of 1H NMR of prototenellin C 113 from A, A. oryzae pTAex3-tenS expression clone; B, tenSPKSdmbNRPS clone. 2.3.3 Analysis of A. oryzae dmbS–tenC Expression Clone Different hybrid combinations of DMB 88 genes and tenellin 87 genes were expressed in A. oryzae (This transformation was done by Mary N. Heneghan in the School of Biological Sciences, University of Bristol) in order to investigate their mode of programming in terms of chain length and methylation. Many different transformants were produced. This section is an explanation of analysis of the LCMS of different transformants and isolation of new compounds. 45 An A. oryzae dmbS–tenC transformant was grown in CMP liquid culture (10 flasks × 100 mL) at 25 °C at 200 rpm. The fermentation cultures were homogenized and extracted as described in section 4.11. A crude extract of 111 mg was obtained and was made into a solution of 10 mg/ml in HPLC methanol. This extract was analyzed by analytical LCMS. A prominent peak at Rt 19 mins was observed (Figure 2.9). Mass analysis indicated ES+ and ES- of 368 and 366 respectively (Figure 2.10). This peak was purified with 25 preparative runs on LCMS (Method 1, section 4.5). The pure compound was collected in a number of different tubes. They were all collected and dried under nitrogen gas in the form of bright yellow powder (23 mg). Figure 2.9: Diode array chromatogram of A, dmbS-tenC expression in A. oryzae; B, Aspergillus oryzae Wild type. Figure 2.10: ES+, ES- and UV spectrum of the 19min peak. 46 2.3.3(a) Characterization of preDMB A 118 The high resolution mass spectrum (HRMS) of this fraction gave a molecular formula of C22H26NO4 (observed 368.1851; calculated 368.1856 for M[H]+). The pure compound was dissolved in deuterated methanol and 1D and 2D NMR experiments were done on the purified fraction. The 1H NMR showed two separate doublets in the aromatic region for H-19/23 and H-20/22 and diasteorotopic protons H-17a and H-17b characteristic of benzylic protons of a tetramic acid (Figure 2.11). The diastereotopic protons showed COSY connection with methine proton H-5 and HMBC correlation to the C-19/23 of the phenol ring which confirms the tetramic acid structure (Figure 2.12). There were two methyl signals, one for terminal H-15 and second for H-16. The HMBC and COSY correlations confirmed that the pendant methyl at position 16 is attached to C-13 (Figure 2.12). Six olefinic protons H-7, H-8, H-9, H-10, H-11 and H-12 in the alkene region of the spectrum confirmed three double bonds typical for the DMB 88 polyketide chain. In the COSY spectrum the olefinic protons showed couplings of H-7 to H-8, H-9 to H-10 and H-11 to H-12. The alkene proton H-7 displayed a two bond correlation to the quaternary carbon C-6 in HMBC. All 1D and 2D NMR data confirmed the structure to be 118 which was named preDMB A. 47 Figure 2.11: Spectrum of 1H NMR of preDMB A 118 run in methanol-d4. Figure 2.12: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in preDMB A 118. 2.3.4 Heterologous expression of dmbS-dmbC in A. oryzae The clone dmbS + dmbC gene (This transformation was done by Mary N. Heneghan in the School of Biological sciences, University of Bristol) was analyzed having both dmbS synthases and dmb enoyl reductase encoding genes cloned in A. oryzae. It was grown in liquid culture (10 flasks × 100 ml) at 25 °C at 200 rpm. After completion of fermentation the cultures were homogenized and extracted with ethyl acetate (section 4.11). The ethyl acetate extracts were dried, evaporated (yield was 363 mg) and dissolved in HPLC methanol (10 mg/ml). The LCMS analysis showed peak of 48 predmbA at Rt 19 mins. The ES+ and ES- showed masses 368 and 366 respectively which corresponds a mass of 367 of preDMB A 118. The peak was purified by massdirected preparative LCMS. This purification was accomplished by 18 injections on 20 minutes program with Method 1 described in section 4.5. The tubes containing purified fractions were all collected and dried. The dried preDMB A was bright yellow with yield of 17.6 mg. The 1H NMR showed the same proton signals as the preDMB A obtained from dmbS+ tenC clone as illustrated in Figure 2.13. Figure 2.13: Overlay of 1H NMR of preDMB A 118 from A, dmbS-dmbC clone; B, dmbS-tenC expression in A. oryzae. 2.3.5 Heterologous expression of tenSPKS – dmbC in A. oryzae In the course of investigating the biosynthetic potential of the tenellin 87 polyketide synthase-nonribosomal synthatase encoding genes, we now know that TENS in the absence of tailoring enzymes is capable of producing tetramic acids prototenellin A 111, protenellin B 112 and protenellin C 113 possessing different methylation and reduction pattern than tenellin 87. When tenC is expressed with tenS, the ‘correct’ precursor pretenellin A 114 is produced. These results encouraged us to further expand 49 the gene expression experiments using the tenellin gene cluster. We now aimed to express the HRPKS of TENS without its counterpart NRPS in A. oryzae. This expression also included the enoyl reductase encoding gene dmbC from DMB cluster. As the NRPS module affords a peptide or an amino acid, in its absence we expected that the ACP bound pentaketide 106 synthesized by TENSPKS can either get hydrolysed and released in the form of an open chain pentaketide 137 (Scheme 2.12) or it can enter another chain extension cycle by addition of a malonyl CoA 25 by the KS forming hexaketide 138. The hexaketide 138 can offload from the TENS by cyclisation to form a pyrone 139 (Scheme 2.12). Scheme 2.12: Possible compounds from expression of tenSPKS+ dmbC in A. oryzae. Similar pyrone structures are also reported from other polyketide synthase enzymes from other fungi. Tang et al. investigated the programming role of HRPKSNRPS encoding gene apdA involved in aspyridone A 84 biosynthesis in A. nidulans.107 He manipulated the megasynthase ApdA in different in vitro assays and reported production of a number of α-pyrones. The in vitro assay of ApdA without the enoyl reductase ApdC produced pentaketide and hexaketide pyrone 142 and 139 respectively (Scheme 2.13, A). The NRPS module of ApdA is very specific to recognize only fully reduced tetraketide synthesized from the ApdA PKS. This might be the reason that unsaturated tetraketide in the absence of ApdC enters further chain extension cycles and methylations and get offloaded by the ACP in the form of pyrones without being processed by the NRPS module. Another tetraketide 140 and pentaketide 141 pyrones 50 without any methylations is observed in in vitro assay where methyltransferase domain is removed from ApdA. The in vitro assay of ApdA-PKS with ApdC independent of the NRPS module also produced a pyrone 143 with saturated linear chain similar to aspyridone A 87. Kennedy et al. reported two pyrones 144 and 145 when they over expressed lovastatin polyketide synthase LNKS in Aspergillus nidulans without the enoyl reductase LovC (Scheme 2.13, B).71, 105 A B Scheme 2.13: A, α-pyrones reported from different in vitro assays with PKS-NRPS megasynthase ApdA of aspyridone 84 by Tang et al.107; B, pyrones reported from LNKS, LovB from lovastatin gene cluster. 2.3.6 Analysis of tensPKS–dmbC transformants A total of 11 transformants were analysed (this transformation was carried out by Dr. Walid Bakeer). They were grown in the same fermentation conditions for 7 days and extracted with ethyl acetate (see section 4.11). The crude extracts were all in range of 5 – 10 mg per 100 ml of liquid media. Each crude extract was made 10 mg/ml in HPLC methanol and analysed by LCMS. In seven transformants we observed two prominent peaks at 7.5 and 8.1 minutes (Figure 2.14) in a 15 minute program (Method 51 3, section 4.4). These were not observed in the A. oryzae wild type (M-2-3) strain. The mass spectra of these two peaks were similar to the open chain polyketide 137, we expected from this experiment (Scheme 2.12). The peak at 7.5 minutes showed mass of 213 in ES+ and 211 in ES- and was named compound A (Figure 2.15). The peak at 8.1 minutes gave a mass of 211 in ES+ and 209 in ES- and was named compound B (Figure 2.16). Neither compound showed a strong uv absorption. Figure 2.14: A, LCMS chromatogram of tenSPKS-dmbC producing two new compounds at 7.5 mintues and 8.1 minutes; B, Wild type A. oryzae (M-2-3). We thought there might be new polyketide compounds and thus planned to purify these two peaks. These compounds were produced in low titre from each 100 ml liquid culture. We chose one transformant producing this compound and grew large scale fermentation of three litres liquid medium. The total crude extract was 239 mg. A 50 mg/ml sample was made in HPLC methanol for purification. About 24 runs of a 20 min program as stated in Method 2 (section 4.5) were carried out. The purified compound A with mass (MH+) 213 was 9.5 mg and the second compound B with mass (MH+) 211 was 9 mg. 52 Figure 2.15: A, ES- of compound A; B, ES+ of compound A; C, wavelength. Figure 2.16: A, ES- of compound B; B, ES+ of compound B; C, wavelength. 2.3.6(a) Identification of Compound A Compound A had a chemical formula of C11H17O4 (observed 213.1130; calculated 213.1121 for M[H]+) on High Resolution Mass Spectrometry. The 9.5 mg of pure compound was dissolved in 0.65 ml of deuterated chloroform. The structure of the 53 compound was elucidated using 1D and 2D 1H and 13C NMR spectroscopic analysis. A number of 1D and 2D NMR experiments were carried out on 500 MHz NMR Spectroscopy. The structure of the compound A 146 is given in Figure 2.17. The carbon spectra revealed the presence of eleven carbons, which included two carbonyl groups at δC 176.4 (C-11) and δC 170.1 (C-9), four methylene groups in the alkane region of the spectra and one terminal methyl carbon at δC 14.4 (C-1) (Figure 2.18). The 1H-13C HSQC gave eight protonated carbons. The 1H NMR showed two distinct doublets in the alkene region at δH 5.95 and δH 6.54 which were assigned to two geminal methylene protons of H-10. One of the H-10 methylene protons shows 1H-13C HMBC correlation to an alkene carbon at δC 136.1 which was assigned C-8. Both geminal methylene protons (H-10) show HMBC connections to carbonyl carbon at δC 170.1 (C-9) and sp3 carbon at δC 45.2 (C-7). The triplet at δH 3.65 was assigned to methine proton (H-7) (Figure 2.17) which show a 1H-13C HMBC connection to terminal carbonyl of a carboxylic acid at δC 176.4 (C-11), carbonyl carbon at δC 170.1 (C-9) and an alkene carbon at δC 136.1 (C-8) (Figure 2.19). Figure 2.17: 1H NMR of compound A run in chloroform-d. 54 Figure 2.18: 13C NMR of compound A run in chloroform-d. The methine proton at δH 3.65 (H-7) show 1H-1H COSY connections to two geminal methylene protons at δH 2.01 and 2.55 (H-6). Both geminal methylene protons of H-6 show 1H-1H COSY and 1H-13C HMBC connections to a methine proton at δH 4.43 (C-5) linked to an oxygen atom (Figure 2.19). The chemical shifts of δC 79.6 for C5 support its link to an oxygen atom. The HMBC correlations of this compound provided valid evidence for a pyran structure attached to a carboxylic acid at C-7. The methine protons at δH 4.43 (H-5) and methylene protons at δH 2.01 and 2.55 (H-6) show 1 H-1H COSY connections to two other geminal protons at δH 1.65 and 1.81 (H-4). Two multiplets at δH 1.37 and δH 1.46 were assigned to two geminal methylene protons H-3 which display 1H-1H COSY link to one of the methylene at δH 1.8 (H-4). The broad signal at δH 1.35-1.39 was assigned to methylene protons H-2 showing connections to δC 27.8 (C-3) in HMBC spectrum and to terminal methyl at δH 0.92 (H-1). Both HMBC and COSY correlations show an aliphatic chain (C1-C4) attached to the pyran at methine proton H-5. 55 Figure 2.19: Important 1H-1H COSY and 1H-13C HMBC connections in compound A 146. Figure 2.20: Arrangement of protons in the pyran ring of 146 around the stereo centre at C-7 and C-5. In the pyran ring of compound 146, the spatial orientation of the protons at the two stereocentres C-5 and C-7 was established with the help of calculating the coupling constant of H-5 and H-7 with their adjacent methylene protons at δH 2.01 (H-6a) and δH 2.55 (H-6b). The coupling constant of 6 Hz between H-5 and H-6b indicated that H6b is equatorial. The J value of 10 Hz between H-5 and H-6a was 10 Hz, placing H-6a in axial position. The coupling constant between the two geminal methylene protons H6a (2.00) and H-6b (2.55) was 12 Hz, which confirmed the conformation of H-6a to be axial and H-6b as equatorial. The methine proton H-7 displayed J value of 12 Hz between H-6a and 9 Hz with H-6b. These values depicts that H-7 orientation is axial. The predicted conformation for the pyran of compound 146 from J values is given in Figure 2.20 showing H-6a, H-7 and H-5 in axial direction and H-6b to be aligned in equatorial position. Further confirmation of these spatial arrangements of atoms came from 1D NOE, where both H-7 and H-5 show NOE correlations to each other which can occur when both protons are in axial position (Figure 2.21 and 2.22). 56 Figure 2.21: In 1D-NOE, on irradiating H-5, signals of H-7 is observed in close proximity. Figure 2.22: Irradiation of H-7 in 1D NOE, signals of H-5 are observed. 2.3.6(b) Identification of compound B The structure of compound B 147 was interpreted with the help of HRMS and NMR analysis and by comparison to compound A 146. The chemical formula given by HRMS was C11H14O4 (observed 233.0799; calculated 233.0784 for [M]Na+). The 9 mg of the pure compound was dissolved in 0.65 ml of deuterated chloroform. The ESI gave 57 idea that this compound may have the same structure with two protons less than compound A 146. It was further confirmed by different NMR experiments. The 13C NMR displayed the presence of 11 carbons including two carbonyls at δC 169.5 (C-9) and 171.7 (C-11), a methine carbon at δC 80.8 (C-5) attached to an oxygen atom, a methyl group at δC 14.0 (C-1), three methylene groups and four olefinic carbons at 125.0 (C-7), δC 128.5 (C-8), δC 133.6 (C-10) and δC 153.5 (C-6) (Figure 2.23). Figure 2.23: 13C NMR of compound B 147 run in chloroform-d. The 2D HSQC showed 7 protonated carbons. In the 1H NMR, the broad doublet signal downfield at δH 7.96 was assigned to the methine (H-6) (Figure 2.24) attached to the olefinic carbon at δC 153.5 (C-6) in the 1H- 13C HSQC. In 1H-13C HMBC spectra, H6 shows linkage to methine carbon at δC 80.8 (C-5) and to the olefinic carbon (C-7) at δC 125.0. This confirmed the presence of a double bond between C-6 and C-7, which is the difference in structure between compound 146 and 147. H-6 also shows HMBC correlations with the olefin carbons, C-8 and C-11 (Figure 2.25). The two broad signals at δH 6.79 and δH 7.19 were identified as geminal methylene protons H-10 showing correlations to the methine protons H-5 and H-6 in 1H- 1H COSY and 1H- 13 C HMBC connections to the olefin carbon at δC 125.0 (C-7) and to the carbonyl group at δC 169.5 (C-9) (Figure 2.25). 58 Figure 2.24: 1H NMR of compound B run in chloroform-d. The triplet signal at δH 4.99 was assigned to the methine H-5, displaying HMBC correlations with the methylene groups at δC 27.3 (C-3) and δC 33.1 (C-4) and to olefin carbons at δC 125.0 (C-7) and δC 153.5 (C-6). The two multiplet signals at δH 1.71 and δH 1.79 were assigned to geminal methylene protons (H-4) linked to C-2 and C-3 at δC 22.6 δC 27.3 respectively in HMBC. The terminal methyl signal at δH 0.92 (H-1) showed HMBC connections to the methylene groups at δC 22.6 (C-2) and δC 27.3 (C-3). The 1H-13C HMBC and 1H- 1H COSY shows an aliphatic chain comprising of C-1 to C-4 attached to the methine proton H-5. From NMR analysis, this compound was elucidated to be a carboxylic acid pyran ring attached to an aliphatic chain and possess a double bond between C-6/C-7. Figure 2.25: Important 1H-1H COSY and 1H-13C HMBC connections in compound B 147. 59 2.3.6(c) Discussion The TENS-PKS synthesizes a polyketide chain from condensation of five acetate units in four cycles, carrying out methylation and reduction in the first cycle and another methylation in second cycle producing an open chain pentaketide 106. The structures of compounds 146 and 147 were different than the compounds 137 and 139, we proposed to be produced from this transformation. We concluded that they are not products of TENS-PKS pathway. Both compounds 146 and 147 have not been reported before. We searched for similar compounds in the literature and came across a number of similar compounds (Figure 2.26). Horhant et al. isolated three paraconic acids, protolichesterinic acid 148, lichesterinic acid 149 and roccellaric acid 150 from lichen, Cetraria islandica (L.) Ach.113 Dahiya and Tewari characterized three plant growth factors from the fungus Alternaria brassica, one of them was 3-carboxy-2methylene-4-pentenyl-4-butenolide 153. They reported that 153 reduces plant growth by causing chlorosis.114 The same structure 153 was reported by Park et al.115 They named it methylenolactocin 153 (αmethylene-γ-lactone) and obtained it from a penecillium sp.24-4 (FERM P-9437). They stated that methylenolactocin 153 possess antimicrobial activity against Gram positive bacteria. Huneck and Höfle isolated and characterized δ-lactone structures acaranoic acid 151 and acarenoic acid 152 from the lichen Acarospora chlorophana.116 Figure 2.26: Similar compounds to A 146 and B 147, reported in literature. 60 Seshime et al. over expressed a type III PKS gene csyB in A. oryzae (M-2-3) under the effect α-amylase promoter and produced a novel metabolite csypyrone B1 154 which is a 3-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl) proponoic acid (Scheme 2.14).117 In a [1, 2- 13 C2] feeding, csypyrone B1 154 showed incorporation of five acetate units which confirmed it to be a product of a PKS pathway. They presented a proposed biosynthetic pathway for csypyrone B1 154 in which a succinyl CoA condenses with three malonyl CoA and by pyrone ring cyclization form csypyrone B1 154. Scheme 2.14: Proposed biosynthetic pathway for csypyrone B1 154. It seemed highly unlikely that compound A 146 and compound B 147 could be product of the tenellin PKS. We thus re-examined wild type A. oryzae. Although peaks for 146 and 147 could not be observed by uv, their characteristic masses could be detected at the correct retention times using a more sensitive 60 minutes programme with Method 5 described in section 4.4 (Figure 2.27). We proposed a hypothetical pathway for the biosynthesis of compound 146 and 147 (Scheme 2.15). Figure 2.27: Section of Single ion monitoring chromatogram of compound A in ES+ and ES- mode observed at 29.1 minutes in A. oryzae tenSPKS-dmbC and Wild type A. oryzae. 61 Scheme 2.15: Proposed biosynthetic pathway for compound A 146 and compound B 147. 2.3.7 tenC RNAi Silencing in B. bassiana with amyB promoter Messenger RNA is an important type of ribonucleic acid (RNA) and it possesses an important role in gene expression. During gene expression, the DNA molecule transcribes the genetic information required for a protein synthesis to a single strand of messenger RNA. This is carried out by RNA polymerase enzymes in a process called transcription. The messenger RNA travels outside the nucleus carrying the genetic code, which is then translated into proteins with the support of ribosomes and transfer RNA by an organised cell process called translation. Figure 2.28: Process of gene expression from DNA to proteins. 62 Transcription is a process which can be interrupted by RNA interference (RNAi). RNAi is an effective tool in eukaryotes often used in recent advances in molecular genetic techniques.118 It is used to diminish the function of a specific gene by destroying the messenger RNA translation stage of the target gene; this can reduce, or completely prevent, protein production. In this technique a single stranded RNA comprising of reverse sequence or complementary sequence to that of target gene is introduced into the cell. This single strand binds to the native single strand RNA to form double stranded RNA. The double stranded RNA is considered as abnormal condition by the cell and is then degraded by enzymes called dicer (Figure 2.29). The number of native RNAs expressed into proteins becomes less and hence eliminates the gene product. This reverse sequence dependant genetic tool is also referred to as ‘gene silencing’. RNA silencing does not diminish the target gene but it causes reduction of the function of the gene so it is also named as a ‘gene knock down’ technique.119 This method helps to understand the product and function of the target gene and aids in the investigation of the biosynthetic pathways of natural products and secondary metabolites from their gene clusters. A lot of efforts are underway to apply this gene silencing technique in curing many diseases by destroying function of genes which are causal factor of viral or tumour diseases in humans. The concept of RNA interference system came into limelight among researchers when in a number of cases of introducing homologous RNA for high gene expression actually diminished the outcome of the native gene unexpectedly.120 In 1990, Napoli and Jorgensen in their attempt to investigate the enzyme responsible for colouration of petunia petals, brought forward the hypothesis of RNA silencing for the first time.121 They overexpressed chalcone synthase in the native plant, which is the putative enzyme for colouration in violet petunias. Instead of obtaining deep violet colour, the petals of flowers produced were white. This led to the conclusion that the original gene of the flower was suppressed by the transgene introduced. This suppressing factor was later discovered to be interfering RNA in the cell which diminishes the expression of the genes. 63 Figure 2.29: RNA silencing pathway. In the course of transcription of genes, ‘promoters’ play a vital role in initiating conversion of the structural gene to proteins. Promoters are regulatory regions consisting of DNA sequences and are located upstream of the gene they transcribe. The promoter provides a binding site for RNA polymerase (the enzyme responsible for generation of mRNA) and for transcriptional factors (proteins that initiate RNA 64 polymerase). The transcription factors are responsible for activation or suppression of transcription. Figure 2.30: Promoters are responsible for initiating gene expression. Similarly in designing gene silencing procedures, promoters are a key feature which initiates the gene silencing pathway. There are different kinds of promoters used in gene silencing depending on the goal of an experiment. Most commonly utilized are constitutive and inducible promoters. Constitutive promoters always direct expression of genes and are independent of environmental or endogenous factors in cells. The activity of inducible promoters are dependent on external stimuli such as light, temperature and different media sources like alcohol, different nutrients including carbon or many herbicides and antibiotics. The use of inducible promoters makes it possible to control the level and time of expression of target genes. Fuji reported that the starch inducible α-amylase promoter (PamyB) is an effective promoter for heterologous expression systems of Iterative fungal polyketide synthases in Aspergillus oryzae host.122 Halo et al. successfully expressed hybrid polyketide synthase – nonribosomal peptide synthatase (PKS-NRPS) encoded by tenS in A. oryzae using PamyB.104 PamyB is induced (switched on) by starch and repressed (switched off) by glucose. The activity of genes driven by PamyB can thus be controlled by the addition of starch or glucose to growth media. A good example of a strong constitutive promoter is A. nidulans PgpdA (glyceraldehyde-3-phosphate dehydrogenase promoter). Halo et al. obtained productive results by using PgpdA to carry out RNA silencing (iRNA) for knocking down the function of two oxidase enzymes encoded by tenA and tenB in B. bassiana.108 65 The enoyl reductase encoding gene tenC is known to be vital for correct programming of tenellin 87 production.104 Yakasai and colleagues carried out RNAi silencing of tenC in B. bassiana with the constitutive PgpdA promoter.111 The B. bassiana tenC RNAi transformant not only produced WT compounds tenellin 87, pretenellin A 114 and prototenellin D 155 but also reprogrammed compounds obtained in tenS expression in A. oryzae, prototenellin A 111, prototenellin B 112. This silencing experiment produced a new reprogrammed compound prototenellin E 156. The presence of WT compounds in the culture show small varied level of TenC proteins present in the transformant. Figure 2.31: B. bassiana WT and reprogrammed compounds produced from RNAi tenC transformants by gpdA promoter.111 The aim of the present project is to examine tenC RNAi transformants produced under the effect of the inducible promoter amyB in B. bassiana. Here we design an experiment to know whether the ‘concentration’ of TenC affects programming and new or reprogrammed compounds are produced or not. The idea was to use an ‘inducible’ promoter ‘amyB’ the expression of which is dependent on the carbon source used in liquid media. The expression of amyB is stimulated in the presence of maltose or starch and is repressed by glucose. 66 The construction of the PamyB/tenC silencing vector and its transformation into B. bassiana was done by Dr. Walid Bakeer. My role was to grow the six selected silenced clones in different carbon sources and analyse the produced compounds. The silenced clones (A, B, C, D, E and F) were grown along with wild type B. bassiana using three carbon sources, mannitol, maltose and glucose. 2.3.7(a) Growth of B. bassiana transformants in TPM (mannitol) The spores of the six silenced clones (A, B, C, D, E and F) and wild type (WT) B. bassiana were each grown in standard tenellin production medium (TPM) (see section 4.8). In TPM, mannitol is used as the carbon source. WT A B C D E F Figure 2.32: tenC silencing clones and WT B. bassiana grown in standard TPM media. The cultures were grown in 100 ml of TPM in 500 ml Erlenmeyer flasks. They were incubated at 25 °C in shakers at 150 rpm. After ten days the cultures were filtered. All cultures had varying degrees of yellow colour, which is an indication of tenellin compounds. The mycelia in each flask were extracted in acetone (200 ml). The acetone extract was concentrated under vacuum to a brown aqueous extract. It was further diluted with deionized water (200 ml) and then extracted into ethyl acetate (200 ml). The ethyl acetate layer was separated and dried with MgSO4. The extracts were concentrated in vacuum to give a brown solid. All crude extracts of silenced clones were in range of 5-8 mg and WT was 10 mg. A solution of 10 mg/ml for all extracts was made with HPLC grade methanol and analysed with LCMS with Method 3 (section 4.4). The chromatograms of all six silenced clones and wild type are given in figure 2.34 and 2.35. 67 The wild type B. bassiana produces four major compounds which are 15hydroxy tenellin 157 at 9.3 minutes, prototenellin D 155 at 10.0 minutes, pretenellin A 114 at 10.7 minutes and tenellin 87 at 11 minutes (Figure 2.33). The silenced clones A, B, C, E and F still produce prototenellin D 155, pretenellin A 114 and tenellin 87. Clone D produced only compound tenellin 87 and 157 (Figure 2.34 and 2.35). These chromatograms showed that all the silenced clones still produce more or less the wild type compounds. We did not observe any newly programmed or reprogrammed compounds. This suggests that under these conditions tenC is not silenced and the amyB promoter is inactive. Figure 2.33: Diode array chromatogram of WT B. bassiana grown in TPM (mannitol) media showing production of 15hydroxytenellin 157 at 9.3 minutes, prototenellin D 155 at 10.0 minutes, pretenellin A 114 at 10.7 minutes and tenellin 87 at 11.0 minutes. 68 Figure 2.34: Diode array chromatograms of WT B. bassiana, Clone A, Clone B, Clone C grown in mannitol. Figure 2.35: Diode array chromatograms of WT B. bassiana, Clone D, Clone E and Clone F grown in mannitol. 2.3.7(b) Growth of B. bassiana transformants in maltose media The next experiment was to grow the six silenced clones A, B, C, D, E, F and wild type B. bassiana in TPM medium but using maltose (30 g/L) as carbon source instead of mannitol. As amyB promoter is turned on when grown with maltose medium, we expected strong silencing of tenC. 69 WT A B C D E Fig 2.36: tenC silencing clones and WT B. bassiana grown in maltose. The six clones and WT B. bassiana were grown in the same conditions for ten days at 25 °C at 150 rpm. All cultures were pale white colour, but produced mycelia. The cultures were then extracted in the same way first with acetone, and then concentrated and later diluted with deionized water and in the last extracted with ethyl acetate. All dried extracts weighed from 28-34 mg from 100 ml liquid medium. The concentrated extracts (10 mg/ml) in HPLC methanol were analysed in LCMS. The chromatograms of silenced clones with WT B. bassiana are given in Figure 2.37 and 2.38. Figure 2.37: Diode array chromatograms of WT B. bassiana, Clone A, Clone B, Clone C grown in maltose. 70 Figure 2.38: Diode array chromatograms of WT B. bassiana and tenC silencing Clone D, Clone E and Clone F grown in maltose. In maltose media the B. bassiana WT and tenC silenced clones did not produce tenellin related compounds strongly. Only wild type produced small peaks of 15hydroxy tenellin 157 and prototenellin D 155. Tenellin 87 at 11 minutes was not observed, the ESI only showed mass MH+ 354 at 11.08 minutes which may be pretenellin B 115 but the uv was not strong or convincing. All the silenced clones failed to produce compounds, even the wild type compounds were not detected. So, this indicates that although amyB promoter is believed to be activated when there is maltose, if mannitol is not used as the basic carbon source B. bassiana fails to produce any compounds. 2.3.7(c) Growth of B. bassiana transformants in glucose media After growing B. bassiana clones in maltose, the next experiment was to grow them in TPM medium using glucose as carbon source (20 g/L). As the amyB promoter is turned off when grown with glucose medium, we expected to observe an absence of or weak silencing of tenC. 71 WT A B C D E F Fig 2.39: tenC silencing clones and WT B. bassiana grown in glucose. The six clones and WT B. bassiana were grown in the same conditions for ten days at 25 °C at 150 rpm. They all showed very faint colour which show poor production of tenellin compounds. The cultures were extracted first with acetone, then concentrated and diluted with deionized water and in the last extracted with ethyl acetate. The dried extracts weighed 9-10 mg from 100 ml liquid media. The concentrated extracts were made 10 mg/ml in HPLC methanol and analysed in LCMS. The chromatograms of silenced clones with WT B. bassiana is given in Figures 2.40 and 2.41. In glucose medium the WT and all six silenced clones failed to produce any tenellin related compounds. Figure 2.40: Diode array chromatograms of WT B. bassiana and tenC silencing Clone A, Clone B and Clone C grown in glucose. 72 Figure 2.41: Diode array chromatograms of WT B. bassiana and tenC silencing Clone D, Clone E and Clone F grown in glucose. 2.3.7(d) Growth of transformants in mannitol in combination with 1% maltose In the previous three experiments we observed that B. bassiana produces tenellin 87 and related compounds only when mannitol is used in TPM medium. In maltose and glucose even the WT could not produce. Here we used mannitol with 1% maltose to see if maltose makes silencing in the presence of mannitol. The WT B. bassiana and silenced clones were grown in the same conditions as explained in section 4.10. The crude extracts were made 10 mg/ml in HPLC methanol and analysed by LCMS. The diode arrays of all extracts are given in Figure 2.42 and 2.43. In all chromatograms wild type compounds, 15-hydroxy tenellin 157, prototenellin D 155, pretenellin A 114 and tenellin 87 were produced. 73 Figure 2.42: Diode array chromatograms of WT B. bassiana and tenC silencing Clone A, Clone B and Clone C grown in mannitol with 1% maltose. Figure 2.43: Diode array chromatograms of WT B. bassiana and tenC silencing Clone D, Clone E and Clone F grown in mannitol with 1% maltose. 2.3.7(e) Growth of transformants in mannitol in combination with 1% glucose The WT and silenced clones of B. bassiana were grown on mannitol with 1% glucose to see the effect of glucose on production of compounds. The WT B. bassiana and silenced clones were grown in the same conditions as explained in section 4.10. The 74 crude extracts were made up to 10 mg/ml in HPLC methanol and analysed by LCMS. The diode arrays of all extracts are given in figure 2.44 and 2.45. Wild type compounds 157, 155, 114 and 87 are observed in silenced clone A, B and D. Clones C, E and F also produced more and less WT compounds. Figure 2.44: Diode array chromatograms of WT B. bassiana and tenC silencing Clone A, Clone B and Clone C grown in mannitol with 1% glucose. Figure 2.45: Diode array chromatograms of WT B. bassiana and Clone D, E and F grown in mannitol with 1% glucose. 75 2.3.7(f) Growth of transformants in mannitol with combination of 5% maltose In this experiment the WT and silenced clones of B. bassiana were grown in mannitol with increased percentage of maltose, 5%. The crude extracts were made 10 mg/ml in HPLC methanol and analysed by LCMS. The chromatograms of all extracts are given in Figure 2.46 and 2.47. WT B. bassiana produces only compound protenellin D 155 and tenellin 87 when percentage of maltose is increased. The silenced produce WT compounds 157, 155 and 87 in different concentrations. Figure 2.46: Diode array chromatograms of WT B. bassiana and Clone A, B and C grown in mannitol with 5 % maltose. Figure 2.47: Diode array chromatograms of WT B. bassiana and Clone D, E and F grown in mannitol with 5 % maltose. 76 2.3.7(g) Growth of transformants in mannitol with combination of 5% glucose In last experiment the WT and silenced clones of B. bassiana were grown on mannitol with increased percentage of glucose 5%. The crude extracts were made 10 mg/ml in HPLC methanol and analysed by LCMS. The chromatograms of all extracts are given in figure 2.48 and 2.49. Wild type compounds prototenellin D 155 and tenellin 87 are produced in some clones. Figure 2.48: Diode array chromatograms of WT B. bassiana and Clone A, B and C grown in mannitol with 5 % glucose. Figure 2.49: Diode array chromatograms of WT B. bassiana and Clone D, E and F grown in mannitol with 5 % glucose. 77 These experiments showed that silencing a gene with a promoter whose function is dependent on the carbon source was not successful in this case. B. bassiana will always require mannitol in TPM media for production of its primary and secondary metabolites. We suggest that if we want tenC silencing in varying degrees in B. bassiana, we may use another kind of promoter, the expression of which is not dependent on carbon source for example, alcohol dehydrogenase PalcDH which is stimulated by glycerol or lactose in the media. 2.4 Conclusions Heterelogous expression of tenellin genes alone and co-expression with DMB genes was effectively accomplished in A. oryzae (M-2-3) and their chemical products were isolated. The structure of prototenellin C 113 was elucidated and fully characterized. Prototenellin C along with prototenellin A 111 and prototenellin B 112 were produced from two clones; A. oryzae tenSPKS-NRPS and A. oryzae tenSPKS-dmbNRPS. The TENS PKS-NRPS in absence of enoyl reductase TenC produce re-programmed or unusual tetramic acids. The pattern of reduction, methylation and in case of 112, even chain length was deviated than tenellin 87. This shows that TENS PKS controls the programming of the polyketide chain while NRPS proves to possess a broader substrate specificity to accept altered PKS chain from the TENS in case of 111, 112 and 113. The NRPS deliberately serves its role to select and combine tyrosine with the PKS chain and provide offloading mechanism for the chemical product. The production of 111, 112 and 113 from A. oryzae tenSPKS-dmbNRPS verifies successful development of a Hybrid PKS-NRPS system, with proteins obtained from two different fungal strains working together admirably. This paves way for further manipulation of PKS-NRPS systems and exploring their enzymes for obtaining desired compounds. The yield of prototenellin C 113 from A. oryzae tenSPKS-dmbNRPS was more (9.6 mg/L) than 113 obtained from A. oryzae tenSPKS-NRPS (2.4 mg/L), which further adds to the efficacy of the hybrid TENS-PKS: DMB-NRPS system. PreDMB A 118 was produced from A.oryzae dmbS –dmbC and A.oryzae dmbStenC clones. PreDMB A 118 has similar polyketide chain pattern as DMB 88. This 78 heterologous expression shows that like tenellin pathway, the DMB PKS-NRPS in the presence of enoyl reductase DMBC, produced the correctly programmed precursor compound preDMB A 118. The expression of DMBS with TENC again produced preDMB A 118. This shows that the enoyl reductase does not play any role in programming of the polyketide chain but the presence of enoyl reductase, either DMBC or TENC, DMBS ‘does not’ loses the fidelity of programming and compounds with correct methylation and chain length are formed. Heterologous expression tenSPKS-dmbC in A. oryzae without the NRPS produced two new compounds 146 and 147, but they were not the products of tenellin pathway. We ascertained that they are native wild type A. oryzae compounds. This demonstrates that expression of TENS PKS without NRPS is a challenging experiment and requires a more efficient expression system. The silencing of tenC with amyB promoter in B. bassiana was carried out with the purpose to achieve different level of concentration of the enoyl reductase and obtain new compounds. This experiment was unsuccessful as substituting the carbon source (mannitol) with maltose and glucose to obtain induction and repression of silencing, severely abrogated the production of tenellin or related compounds. We suggested that alternative inducible promoters can be considered in future, the induction of which is not dependant on carbon source. 79 Chapter 3 Investigation of the Role of Genes from the Aspyridone Pathway using Heterologous Expression and Structural Elucidation of new Compounds 3.1 Introduction Aspergillus is a large genus of moulds which acquired its name because of its distinctive spore bearing structure similar to an aspergillum (a holy water sprinkler). All aspergilli have characteristic morphology consisting of a foot cell, elongated hyphae called a conidiophore and a round vesicle bearing the asexual spores, the conidiospores (Figure 3.1). In many species, the colour of the spores serves as identification, for example Aspergillus niger produces black spores, Aspergillus ochraceus have yellow or brown spores and the colour of spores from A. nidulans, A. fumigatus and A. flavus are green.123 Because of their asexual airborne spores and their ability to grow with minimal nutrients, aspergilli are widespread in all ecosystems.124 They mostly occur in terrestrial habitats, soil and mostly on plant and animal debris. They are saprophytes. After they get in contact to their food they first breakdown complex ingredients by secreting enzymes and acids and then absorb the nutrients. The aspergilli play an important part in decay and decomposition of organic matter driving carbon and other important minerals back into the environment by natural recycling.125 They also provide a means for supply of nutrients and food for a large number of other soil dwelling microorganisms. Their ability of bio deterioration and degradation is a major problem in spoiling foods, textiles, paper and even historic paintings.126 There are about 250 species in the genus Aspergillus. Many of the species aid in fermentation and are important in the food industry. Since the early 20th century Aspergillus niger has been used in the production of citric and gluconic acids and has also been used in the pharmaceutical industry.127 Aspergillus terreus is used in the production of synthetic polymer.128 Aspergillus oryzae is used in the production of rice vinegars, soy sauce, alcohol beverages,130 kojic acid used in a range of Japanese food 80 and also in making koji and synthesis of flavour enhancers.131 Many aspergillus species are important in providing commercial enzymes.132 Aspergillus species can be easily grown in the laboratory on simple organic media and have been studied extensively by molecular biologists and in studies for developing biotechnology tools. A. oryzae has been an effective host for the heterologous expression of different biosynthetic gene clusters of many secondary metabolites133 and A. niger is used as a host for construction of heterologous proteins.134 Figure 3.1: A, microscopic view of Aspergillus spores; B, A. oryzae; C, A. terreus. Most Aspergillus species produce a large number of secondary metabolites and many of them are important natural product drugs. Lovastatin 47, a cholesterol lowering drug was isolated from A. terreus,135 cholecystokinin antagonist asperlicin 157 from A. alliaceus,136 ion channel ligands137 and anti-fungal compounds 158 are reported from aspergillus species.138 Besides many beneficial species of Aspergillus, there are many fungi which are causal agents of human and animal diseases. They produce a number of mycotoxins139 mainly aflatoxin 48, patulin 159 and ochratoxin 160 which deteriorate stored seeds and also cause loss of poultry and loss of domestic animals.140 The air borne spores may cause respiratory tract disease like asthma, hay fever and cause a number of allergies.141 Aspergillosis caused mainly by A. fumigatus may become fatal in immunosuppressed individuals.142 81 3.2 Aspergillus nidulans Aspergillus nidulans (also known as Emericella nidulans) propagates both by asexual spores called conidia and sexual spores called ascospores. Conidia are produced on specialized structures called coniodiophores and ascospores are grown inside round sexual fruiting bodies called cleistothecia.143 A. nidulans has been used as a model organism for over fifty years, on which various research areas of mycology, eukaryotic cell biology and genetics were initiated. For example, the parasexual cycle was first discovered and studied in A. nidulans by Pontecorvo and was used as a means to produce strain of fungus with desired genetic traits before the advent of modern biotechnology techniques.144 Ronald Morris analysed the genetics of mitosis by studying this organism.145 A. nidulans has also been used as a model to study genetic metabolic diseases, genetic recombination,146 explain intron splicing, chromatin, DNA repair and regulatory pathways.123 It has a defined sexual cycle which is used as a guide to investigate reproductive mechanism in other fungi where the sexual phase is not clearly defined. Figure 3.2: Aspergillus nidulans strain 2.2 grown on plate. 82 The genome of A. nidulans has been sequenced.147 It shows that the organism is distantly related to A. oryzae and A. fumigatus. The genome sequence indicates the presence of various secondary metabolite gene clusters, of which 27 are polyketide synthases, 14 are nonribosomal peptide synthetases, 6 fatty acid synthases, one sesquiterpene cyclase and two dimethylallyl tryptophan synthase genes.148 3.3 Aspergillus nidulans metabolites The role of secondary metabolites in the life cycle of a fungus is considered mostly ambiguous but they often exhibit important bioactivities; most importantly antitumor, antibacterial, antifungal and other vital pharmaceutical properties.5 Aspergillus nidulans is a producer of a range of biologically active metabolites, some of which are toxic. But the numbers of secondary metabolite genes predicted from sequencing of A. nidulans genome are more than the reported metabolites from this fungus. There are a number of elements which govern the formation of these metabolites. These include environmental factors for example temperature, light, pH and more important are the availability of different nutrients, nitrogen and carbon sources.143 Synthesis of some metabolites is also related to fungal vegetative growth and morphology. Recently it has been reported that G-proteins regulate growth of asexual spores as well as mycotoxin production.149 A main reason for the number of compounds discovered being less than predicted from the genome sequencing is that many genes encoding the biosynthesis of metabolites are silent under normal fermentation conditions and they require signals or stimulants to be activated and expressed in the form of natural products. Many efforts over a long period of time have been made to isolate metabolites from A. nidulans and identify genes involved in the biosynthetic pathway for the characterized metabolites. Mostly this has been achieved by targeted gene deletion assisted by sequence comparison of the genes with those from the known library of metabolites.150 The genes involved in the biosynthesis of a particular metabolite are usually clustered; this helped the natural product chemists to recognize boundaries for 83 genes encoding all essential enzymes and catalysing each step in the biosynthesis of the natural product. The data provided by the genome sequence of A. nidulans encouraged researchers to devise methods to activate silent gene clusters by different genetic engineering using modern molecular tools and advanced genomics. Considerable success has been achieved in this regard with the discovery and over expression of global regulators of secondary metabolism genes such as laeA151 and replacing pathway specific transcription regulators with inducing promoters.152 Moreover, manipulating chromatin modifying proteins and epigenetic modifiers has facilitated the up-regulation of previously silent clusters of many bioactive compounds and has unveiled hidden biosynthetic potential present in this fungus.153 A review of recognized metabolites of A. nidulans is explained below. The well-known β-lactam antibiotic compound penicillin 161 is produced from A. nidulans. It is produced from three amino-acids: L-α-aminoadipic acid, L-cysteine and L-valine. The biosynthesis of penicillin 161 is encoded by three genes namely, acvA, ipnA and aat.154 Understanding the genetic and molecular regulation of penicillin 161 biosynthesis in A. nidulans can help devise ways to increase production of this antibiotic. Sterigmatocystin 164 is a carcinogen polyketide mycotoxin, produced by about 20 species of Aspergillus including Aspergillus nidulans.155, 156, 157 There is a great concern of contamination caused by mycotoxins in food and feed products resulting in health issues and huge economic loss. It also causes high level of genotoxicity in liver samples. It was necessary to study the biosynthesis of sterigmatocystin 164 on enzymatic level and control them by molecular studies. A lot of research had been made to investigate the metabolic pathway of strigmatocystin 164 and determine its gene cluster. Brown et al. identified a 60 kb cluster in A. nidulans comprising of 25 genes reported to conduct all the steps essential for sterigmatocystin 164 biosynthesis.150 In this cluster a NADPH dependent reductase gene stcU158 and a P450 monooxygenase 84 gene stcS converts the intermediate compound versicolorin A 162 to sterigmstocystin 164159 and stcP encoding a methyltransferase carry out methylation of the intermediate demethylsterigmatocystin 163 to form sterigmatocystin 164.160 Scherland and Hertweck identified four unique prenylated quinolone alkaloids, aspoquinolones A-D 165, 166, 167 and 168 from Aspergillus nidulans (HKI 0410) by growing the fungus in rice medium.161 They predicted that like other alkaloids, these quinolones might have produced from its precursor compound anthranilic acid. The presence of anthranilate synthase (AS) like genes in A. nidulans sequence supports this assumption. The conidia spore pigmentation in A. nidulans was proposed to be encoded by a polyketide synthase gene wA. Heterologous expression of this PKS in A. oryzae produced a yellow coloured, novel heptaketide naphthopyrone compound known as YWA1 169. This compound was regarded as the intermediate in pigmentation of mature green spores.162, 163 Fernandez et al. isolated shamixanthone 170, emiricellin 171, dehydroaustinol 172 and austinol 173 from Aspergillus nidulans. They reported that an essential 4’phosphopantetheinyl transferase (PPTase) encoding gene cfwA is required for the production of secondary metabolites in A. nidulans particularly polyketides and NRPS 85 compounds. Xanthones are phenolic compounds and exhibit important biological activities including antimicrobial, antioxidant, cytotoxic and neuropharmacological activities.164 In recent years after genome sequencing of A. nidulans revealed that there are many more secondary metabolite clusters present as compared to the isolated metabolites form this fungus, a lot of efforts has been served to uncover factors responsible for the repression of metabolites expression. Bok et al. detected a gene called cclA, similar to an orthologous gene in S. cerevisae which is used in chromatin mediated gene silencing by DNA modifications involving methylation of lysine of Histones. The cclA deletion mutants in A. nidulans gave production of six aromatic compounds not observed before in A. nidulans. They were monodictyphenone 174, emodin 57 and emodin analogs 175, 176, 177 and 178 and two anti-osteoporosis polyketides F-9775A 179 and F-9775B 180.153 Sanchez et al. reported two more xanthone compounds from A. nidulans variecoxanthone A 181 and epishamixanthone 182. The gene cluster of xanthose comprise of a cluster of ten genes including a PKS gene mdpG which is also involved in monodictophenone 174 biosynthesis. They stated that 174 and emodin 57 are precursors 86 of prenyl xanthones. They also identified three prenyl transferase genes necessary for encoding prenyl transferase part of the xanthone structures.165 Schroek et al. reported the production of the aromatic tetraketide orsellinic acid 56, a lichen metabolite lecanoric acid 183 and the two antiosteoporosis polyketides F9775A 179 and F-9775B 180 by growing A. nidulans in conjunction with a collection of soil dwelling bacteria actinomycetes.166 They revealed that a NRPKS encoding gene orsA (AN7909) is required for the biosynthesis of orsellinic acid 56, lecanoric acid 183, 179 and 180. Sanchez et al. confirmed orsA to be involved in orsellinic acid production and in AN7909 deletion mutant isolated two bioactive aromatic compounds in A. nidulans, gerfelin 184 and diorcinol 185.167 Nielsen et al. grew A. nidulans on eight different media and observed not only a number of known metabolites but also arugosin A 186, arugosin H 187, antibiotic compounds violaceol I 188 and violaceol II 189 not known before from this fungus.168 They obtained 32 PKS deletion mutants and linked violaceol I 188 to the orsA gene and biosynthesis of arugosin 186 to the monodictophenone 174 gene mdpG. 87 Szewczyk et al. reported a new metabolite, asperthecin 192 by deleting a gene SumO in Aspergillus nidulans which encodes a regulatory protein.169 They also identified the gene cluster for this compound by a following a number of gene deletion strategies. The gene cluster consists of an NR-PKS gene aptA, a gene aptB which encodes a hydrolase and a monooxygenase gene, aptC. They proposed that the aromatic structure 190 of asperthecin 192 is synthesized from one acetyl-CoA and seven malonyl-CoA assembled by a NR-PKS, encoded by aptA. The PKS chain 190 is hydrolysed by AptB into 191 and AptC carries out later oxidation to form asperthecin 192 (Scheme 3.1). Scheme 3.1: Proposed biosynthetic pathway of asperthecin 192. Bok and Keller identified a nuclear methyltransferase protein LaeA which globally regulates transcription of secondary metabolites in A. nidulans.151 In the laeA over expressed mutants, Bok and collegues (during genetic profiling of the mutants) recognized an antitumor compound terrequinone 198 not reported before in A. nidulans.170 They related a five open reading frame gene cluster to the biosynthesis of terrequinone 198 named tdi. The cluster consists of a mono-modular NRPS encoding gene tdiA encoding a protein comprising an adenylation domain, a thiolation domain and a thioesterase domain but a condensation domain typical of NRPS was lacking. 88 Bouhired et al. proposed that the gene tdiD, which encodes a putative aminotransferase is responsible for the deamination of L-tryptophan 193 to indole pyruvic acid 194, tdiA encodes a protein which then adenylates the pyruvic acid 194 and dimerises it to a quinone structure 195. A presumed prenyl transferase encoded by tdiB catalyses a first prenylation to form 196 and then a reductase (tdiC) accomplishes hydroquinone reduction 197 before a second prenylation (tdiE) to form terriquinone 198.171 Scheme 3.2: Proposed pathway of terriquinone A. Emericellamide, an antibiotic known previously from marine emericella species, is formed by the fusion of a polyketide and a nonribosomal peptide. Chiang et al. identified emericellamide A 207 and its analogues in gene deletion studies of series of NRPS sequences during genome mining experiments in A. nidulans. They also deduced that emericellamides are synthesized from a gene cluster comprising of four contigious open reading frames. The HR-PKS EasB forms a carboxylic acid polyketide chain 199 and is converted to CoA thiolester 200 by EasD (CoA ligase) and loaded on to the acyltransferase (AT) of EasC, 201 (Scheme 3.3). The polyketide is then loaded to the EasA which is an NRPS consisting of five modules. Each module of the EasA delivers 89 an amino peptide group to the growing chain, glycine and valine being the amino acid provided in the first two cycles of the NRPS subsequently forming 202, 203, 204 and 205. At the end of the biosynthetic cycle the linear chain 206 is released, cyclised and assembles to form emericellamide A 207 and its analogues C 208, D 209, E 210 and F 211.17 Scheme 3.3: Proposed biosynthetic pathway of emericellamides. Dohren reviewed the non-ribosomal peptide synthetase encoding genes in A. nidulans and listed 27 NRPS and NRPS related genes. He reported a number of peptide 90 and aminoacid metabolites reported from A. nidulans including echinocandin 212, emericellamide 207, triacetylfusigen 214, fusarinine 215, terriquinone 198, emerin 213 and aspyridone 84.173 Wang et al. recognized a silent gene cluster in Aspergillus nidulans which consist of two adjacent PKS genes, one a NR-PKS (afoE) and other a HR-PKS (afoG) in the same cluster.152 They triggered the cluster by replacing a putative transcription activator gene (afoA) with the inducible alcohol dehydrogenase promoter alcA. This led to the production of a new metabolite asperfuranone 219 with subsequent gene deletion experiments they identified five genes involved in its biosynthesis (Scheme 3.4). They proposed a biosynthetic pathway for asperfuranone 219 which shows that the HRPKS that (afoG) synthesizes a 3, 5-dimethyloctadienone moiety 216 from acetyl-CoA, three malonyl-CoA and two S-adenosyl methionine (SAM). The 3, 5-dimethyloctadienone 216 is loaded on to the next NR-PKS (afoE) by the SAT domain. The NR-PKS extends 216 by condensing with four malonyl CoA and a SAM to form the first intermediate 217. A gene (afoD) encoding hydroxylase enzyme carries out hydroxylation at C-3 to 91 form 218. The afoF, encoding an FAD dependant oxygenase hydroxylates C-7 and afoC encoding a hydrolase is involved in furan ring formation. A gene AN1030.3 encoding an oxidoreducatse catalyzes the last reduction step forming asperfuranone 219 (Scheme 3.4).174 Scheme 3.4: Proposed biosynthetic pathway of asperfuranone. 3.4 Aspyridone pathway in A. nidulans Hertweck and co-workers reported an 11.9 kilobase (kb), putative hybrid polyketide synthase- nonribosomal peptide synthetase encoding gene in A. nidulans which was named apdA.80 ApdA is homologous to TenS discussed in chapter 2. They described the PKS-NRPS to be comprised of a number of domains, which are ketosynthase (KS), acyltransferase (AT), ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), C-methyltransferase (C-MeT), acylcarrier protein (ACP), adenylation domain (A), condensation domain (C), peptidyl carrier protein (PCP) and a reducase domain (R). The apdA gene is clustered with a number of putative oxidoreductase encoding genes; two of the genes encode cytochrome P450 monooxygenases and are 92 named as apdB and apdE and one is an FAD-dependent monooxygenases called apdD. The PKS-NRPS is bordered downstream by a putative exporter gene apdF, an activator gene apdR and an acyl-CoA dehydrogenase encoding gene apdG. The gene also has an additional trans acting ER domain, apdC which is homologous to tenC. Hertweck et al. believed this cluster to be silent as A. nidulans extracts grown in different laboratory mediums do not contain any PKS-NRPS metabolites.80 They perceived that the sequence of the activator gene apdR was similar to a transcription factor found in Aspergillus fumigatus. Over-expression of apdR under the influence of the inducible alcohol dehydrogenase promoter alcAp was achieved by homologous recombination in A. nidulans. The mutant A. nidulans strain produced two new pyridone compounds Aspyridone A 84 and Aspyridone B 226. Scheme 3.5: Proposed biosynthesis pathway of aspyridone by Bergmann et al.80 93 Hertweck and coworkers proposed that the PKS synthesizes a tetraketide 220 from three malonyl CoA, an acetyl CoA and two SAM (S-adenosyl-methionine). The enoyl reductase enzyme in ApdA was believed to be inactive and this function is catalysed by the stand alone reductase protein ApdC acting in trans. This characteristic is similar to the gene clusters of lovastatin, tenellin and desmethyl bassianin. 104,109,105 The tetraketide 220 fuses with the tyrosine 107 catalysed by the condensation domain to form a hybrid polyketide-peptide 221. Based on knowledge at the time, it was assumed that a reductive release was catalysed by the reductase domain to form an aldehyde intermediate 222 which after Knoevenagel closure forms pyrrolinone 223. The cytochrome P450 encoded by apdB was proposed to perform the first oxidation to form the tetramic acid 224 and the second oxidase encoding gene apdE makes the hydroxylation of the tetramic acid forming an intermediate 225. The oxidative ring expansion from tetramic acid to 2-pyridone is also proposed to be catalysed by the P450 enzymes to form aspyridone A 84. The last step of phenol hydroxylation of aspyridone to form aspyridone B 226 is performed by the FAD dependent monooxygenase apdD (scheme 3.5). Tang et al. confirmed the biosynthesis of the aspyridone pathway by reconstructing the function of apdA and apdC in in vitro studies and S. cerevisiae was used as the expression host. They reported the production of preaspyridone A 224 by incubating purified apdA and apdC in the presence of co-factors and building blocks (Figure 3.3, A). The production of 4-hydroxy preaspyridone proves that the hybrid Ltyrosine-tetraketide thiolester 221 after its release undergo ring closing by a Dieckman cyclisation catalysed by the last C-terminal domain of the NRPS (Figure 3.3, B) and disproves the presence of a reductase domain and consequent aldehyde intermediate 222 (Scheme 3.5). They highlighted the flexibility of the adenylation domain towards different aromatic amino-acids by showing incorporation of L-tryptohan, L-4fluorophenylalanine and L-phenylalanine by apdA to form 227, 228 and 229.107 94 A B C Figure 3.3: A, in vivo synthesis of preaspyridone A 224; B, Dieckmann cyclisation in preaspyridone A 224; C, incorporation of different amino acid analogues by ApdA. 3.5 Objectives of the Chapter Heterologous expression of biosynthetic genes in a foreign host, particularly fungi, has been an important biotechnology tool to investigate the various steps in biosynthetic pathways of novel natural products. It allows the determination of the role of each gene in the gene cluster and is of particular use in the case of silent gene clusters.175 Among different fungi, Aspergillus oryzae is considered as an effective heterologous host, mainly because of its established use in producing large amount of proteins.176 The Bristol Polyketide Group successfully studied the activities of a number of HRPKS-NRPS gene clusters in detail by heterologous expression of the specific gene clusters in A. oryzae, for example fusarin C 77,77 squalestatin 65,72 tenellin 87,104 desmethyl bassianin 88.109 These heterologous expressions experiments resolved the methylation and chain length factors and cryptic programming between two similar yet different compounds tenellin 87 and desmethyl bassianin 88.110 This encouraged us to revisit and study the iterative PKS-NRPS gene cluster of aspyridone 84. We planned to 95 study heterologous expression of aspyridone biosynthetic genes in A. oryzae by using a recently reported multiple gene expression plasmid system for transformation. A. oryzae 176 We also aimed to investigate and determine the function of each tailoring enzyme, particularly the role of the different oxidative-enzyme-encoding genes in the aspyridone gene cluster by expressing the genes in different groups in A. oryzae. These experiments would also produce intermediate compounds and the structure elucidation of these will aid in understanding the order of biosynthetic steps in the aspyridone pathway. The A. oryzae transformants obtained will be analysed. The new metabolites will be isolated by preparative mass directed LCMS and their structures will be elucidated and characterized. The stereochemistry of any crystallised compound will be studied under X-ray crystallography. The chemical structure of a compound in a particular gene expression will help to deduce the function and biosynthetic potential of the genes in the cluster. 3.6 Heterologous Expression system used in fungal transformation The number of secondary metabolite gene clusters annotated in A. nidulans genome is more than the natural products known from the fungus. One of the reasons is the mass metabolic background in the native fungus due to which many gene clusters fail to express their products. A suitable way to determine the product of a secondary metabolite encoding gene cluster is to express it in an appropriate host by fungal transformation.178 Heterologous expression also helps in engineering fungal genes and provides an alternative method for high production of novel metabolites which are formed in low titre in their native host fungus. The first successful DNA-mediated fungal transformation was reported in 1979.179,180 Since then, fungal transformation protocols are still evolving and have improved through years but also have their limitations. Fungal PKS genes encode large megasynthases and appropriate hosts with an effective expression system are required for their expression. Quite a number of organisms have been used for fungal PKS transformation181 for example yeast, bacteria, plants and also fungi like A. nidulans and A. oryzae. For many PKS genes A. oryzae has 96 been a favourable host. This is because it belongs from the same fungal genus and due to similar cellular mechanism the expressed genes function properly.122 A. oryzae has the ability to translate mRNA from eukaryotes, carry out post-translational modifications, produce large amounts of enzymes and secretes secondary metabolites in the medium in high amount. It is among those fungal species which are generally regarded as safe (GRAS) and they are easily fermented under simple laboratory conditions. The genomic analysis as well as analysing the LCMS data of A. oryzae transformants is easy to study. A. oryzae can also splice introns present in the PKS genes.182 The heterologous expression plasmid used in fungal transformations usually consists of the target genes themselves, suitable promoters and appropriate terminator sequences and gene for the selection markers. A number of selection markers are used in transformation, such as nutritional markers argB, amdS, pyrG or niaA used in auxotrophic condition or different antibiotic resistance markers against hygromycin B, phleomycin or benomyl.183 In our fungal transformations we will use the arginine auxotrophic A. oryzae strain (M-2-3) as the host organism which is unable to grow in the absence of arginine – i.e. on minimal media. The plasmid contains the argB gene of A. nidulans. Thus transformed cells will be able to grow on minimal medium, whereas the un-transformed cells will be unable to survive. In a standard fungal transformation procedure the required genomic DNA is extracted from the subject fungus, amplified and cloned in the respective plasmid used for the heterologous expression. The spores of the host fungus are subjected to enzymatic treatment to break down the cell wall and liberate protoplasts. The protoplasts are incubated with transforming DNA in a medium containing CaCl2 and other additives and then grown on medium containing selective nutrients allowing only heterologous transformants to grow (Figure 3.4).175 97 Figure 3.4: Steps in fungal transformation. The PKS-NRPS encoding genes, like other fungal secondary metabolite genes, exist in clusters and a simple precursor compound is synthesized by encoding of a megasynthase enzyme accompanied by tailoring enzymes. This provoked biologists to devise a multiple gene expression vector which can express four genes in a single transformation experiment. This has been recently accomplished in the Lazarus group in the School of Biological Sciences, University of Bristol.176 The vector comprises of three constitutive promoters, PgpdA from A. nidulans, Padh (alcohol dehydrogenase) and Peno (enolase) to express maximum of three tailoring genes from a gene cluster. For expression of megasynthases like PKS-NRPS it consists of inducible PamyB (promoter of taka-amylase coding gene in A. oryzae) and TamyB, the amyB terminator. The vector 98 contains of the argB gene as a selectable marker for expression in A. oryzae auxotroph (M-2-3). This vector is termed as pTAYAGSargPage. We planned to use the pTAYAGSargPage vector to transform the silent gene cluster from the A. nidulans aspyridone pathway. The strong constitutive promoters provided in this vector will allow the PKS-NRPS and the tailoring genes to trigger and express into proteins. Figure 3.5: Multiple gene expression vector pTAYAGSargPage used in fungal transformation. 3.7.0 Results 3.7.1 Heterologous expression of apdA and apdC in A. oryzae (M-2-3) From in vitro studies by Tang et al.107 and the aspyridone pathway proposed by Bergmann et al.,80 we knew that the megasynthase HRPKS-NRPS encoded by apdA and the enoyl reductase apdC synthesize preaspyridone A 224, the first compound in the aspyridone pathway. This step is similar to the biosynthesis of pretenelllin A 114104 and preDMB A 118109 in B. bassiana. So, to confirm this hypothesis in vivo we carried out heterologous expression of apdA and apdC in A. oryzae. The cloning and fungal transformation was done by Dr. Khomaizon Pahirulzaman from the School of Biological Sciences, University of Bristol. The pTAYAGSargPage vector was used to combine the iterative HRPKS-NRPS apdA and apdC genes to form the vector pTAYAargAC. Transformation of this vector into A. oryzae produced a number of transformants (denoted A. oryzae apdAC) and the 99 incorporation and expression of the genes apdA and apdC was confirmed by qRT-PCR. The best producing transformants were then selected for chemical analysis and identification and purification of novel compounds by LCMS and NMR. Wild-type A. oryzae M-2-3 was grown in parallel in all experiments as a control. Figure 3.6: Expression vector pTAYAargAC containing apdA and apdC. A B Figure 3.7: A, A. oryzae mycelia in liquid media in a flask; B, A. oryzae apdAC expression clone on CDA plate. The A. oryzae transformant was grown first on Czapek Dox agar (CDA) (Figure 3.7, B) and later on DPY solid media for maximum sporulation. The transformant was grown on plates for 7-10 days. The mature spores were scratched with a sterile loop and spores were collected in deionized water. 1 ml of the spore solution was added to 100 100 ml of CMP liquid media (see section 4.9) in a 250 ml baffled Erlenmeyer flask. The cultures were grown at 28 °C in a shaker at a speed of 200-250 rpm for 7 days. The A. oryzae transformants mycelia grow in a form of small balls (Figure 3.7, A). After 7 days the cultures were removed from the shaker, and the entire fermentation mixture (mycelia with the liquid) was acidified (pH= 3) and homogenized with ethyl acetate (section 4.11). The organic extract was separated, then concentrated under vacuum on a rotary evaporator and then the residue was defatted. The mass of the dried crude extract was 40 mg (from 100 ml fermentation). A solution of 10 mg/ml of the crude extract was made with HPLC grade methanol and 20 µl was injected and analysed by a Waters 2795HT HPLC system. It measures wavelength between 200 and 400 nm with a Waters 998 diode array detector and provides an electrospray (ES) mass spectrum with Waters ZQ spectrometer sensing masses between the ranges of 150 to 600 m/z units. The LCMS chromatogram of A. oryzae apdAC displayed two new peaks, one minor peak at 13.2 minutes and the second major peak at 14.0 minutes which were not present in the A. oryzae wild type used as a control (Figure 3.8). Both the peaks showed m/z 332 [M]H+ and a λmax of 279 nm which is the same as described for preaspyridone 224107 by Tang et al. although they reported that only a single compound was produced in their experiments. The ESI chromatogram showed two peaks of identical masses indicating that they are probably isomers. To confirm the production of preaspyridone 224 and determine the two isomers we purified each compound for NMR structural determination. The purified minor (0.61 mg) and major (60.7 mg) components were dissolved in deuterated chloroform (CDCl3) and 1D, 2D 1H NMR and 13 C NMR spectroscopic studies were carried out. Both compounds were identified as preaspyridone A 224 and the chemical shifts of the NMR spectra matched with those reported by Tang et al.107 The 1D 1H and 13C chemical shifts of both minor and major components in CDCl3 were same with only negligible differences of 0.01ppm. But NMR spectra run in dimethyl sulfoxide-d6 discovered key differences between the structures of the minor and major compounds. Reinjection of the pure compounds showed that they did not interconvert. 101 14.0 ZW-II-92H A 1.5e+2 AU 1.25e+2 3.45 1.0e+2 7.5e+1 2.65 5.0e+1 6.23 13.2 2.5e+1 0.0 -0.00 2.00 4.00 6.00 8.00 10.00 12.00 28.77 26.93 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 ZW-II-92M 3.22 B 6.0e+1 3.45 5.0e+1 Wildtype A. oryzae AU 4.0e+1 5.80 3.0e+1 2.0e+1 4.25 1.0e+1 27.43 2.60 0.0 Figure 3.8: A, Diode array chromatogram of A. oryzae apdAC expression clone showing two new peaks at 13.2 and 14.0 minutes; B, Diode array chromatogram of Wild type A. oryzae used as a control. A 100 B 330 [M]H ZW-II-92H 246 (13.218) 100 ZW-II-92H 793 (13.200) 222 330 [M]H- ZW-II-92H 262 (14.078) 279 apdA,C-repeat ZW-II-92H 841 (14.000) 223 8.0e-2 2.4 2.2 7.0e-2 2.0 280 1.8 6.0e-2 1.6 AU AU 5.0e-2 1.0 % % 1.4 1.2 4.0e-2 3.0e-2 8.0e-1 6.0e-1 2.0e-2 331 300 305 313 316 321 325 332 2.0e-1 1.0e-2 341 346 352 331 362363 366 371376 332 [M]H+ 0 297302 308309 315 317 320 325 ZW-II-92H 263 (14.105) 100 332 0.0 337 344 348 355 363 366 368 332 [M]H+ % 100 357 333 341 336 342 % 0 4.0e-1 333 358 365 370 373 334 Figure 3.9: A, ES+, ES- and UV spectrum of minor isomer at 13.2 minutes; B, major isomer at 14.0 minutes observed in A. oryzae apdAC expression clone 3.7.1(a) Identification of minor isomer of preaspyridone A 224 The minor compound eluting at 13.2 minutes was purified in the form of pale crystalline solid. HRESIMS gave a molecular formula of C19H26NO4 (observed 332.1852; calculated 332.1856 for M[H]+). 102 30.00 The 13C NMR indicated the presence of 19 carbons which included two carbonyl groups at δC 175.4 (C-2) and δC 191.9 (C-6), an enol group at δC 194.4 (C-4), a quaternary carbon at δC 99.9 (C-3), a methine carbon at δC 62.3 (C-5), two sp3 carbons at δC 33.4 (C-7) and δC 31.5 (C-9), two methylene groups at δC 39.6 (C-8) and δC 28.7 (C-10) and three methyl carbons at δC 16.7 (C-13), δC 18.9 (C-12) and δC 10.8 (C-11). Figure 3.10: 1H NMR of minor component of preaspyridone A 224 run in DMSO-d6. The 1D 1H NMR contains two distinct doublets in the aromatic region at δ H 6.88 (2H, H-16, H-20) and δH 6.59 (2H, H-17, H-19) (Figure 3.10) which is characteristic of a para- substituted phenol ring and is further corroborated by 1H-13C HMBC correlation of both the aromatic protons with the C-OH at δc 155.7 (C-18) (Figure 3.12). The two singlets at δH 9.17 and δH 8.93 were assigned to the para- substituted hydroxyl group at H-18 and to H-1 attached to a nitrogen atom respectively by HMBC correlations. The multiplet peaks at δH 2.82 were assigned to the diastereotopic protons (H-14a, H-14b) linked to the methine proton at δH 4.08 (H-5) in 1H-1H COSY (Figure 3.12) and to a quaternary carbon at δC 125.3 (C-15), aromatic carbon at δC 130.6 (C-16/20) and 103 carbonyl group at δC 194.4 (C-4) in 1H-13C HMBC and confirms benzylic protons linked to the pyrrolidine ring at C-5 (Figure 3.11). Figure 3.11: Segment of 1H-13C HMBC NMR spectrum of minor isomer of preaspyridone A 224 showing correlations of benzylic protons H-14 with C-5, C-16, C-15 and C-4. The diastereotopic protons (H-14a, H-14b) and the methine proton H-5 are three different nuclei (A, B, X) coupled to each other and have separate chemical shifts. Each of these proton signals are doublets of doublets but the signals of H-14a and H-14b have merged into each other creating distorted peaks typical pattern of an ABX system and the signals at H-5 have combined to a broad peak (Figure 3.10). The quartet at δH 3.53 was assigned to H-7 linked to the carbonyl carbon at δC 191.9 (C-6), the methyl carbon at δC 17.1 (C-13) and methylene group at δC 39.6 (C-8) in HMBC. The broad multiplet between δH 1.24-1.27 was assigned to the methylene protons H-8 and to the methine proton H-9. The multiplet at δH 1.09 and δH 1.26 corresponds to the geminal protons of H-10. The doublet at δH 1.03 was assigned to the methyl group at C-13 exhibiting a HMBC connection to C-6, C-7 and C-8. The terminal multiplet at δH 0.80-0.82 was consigned to the last methyl H-11 and methyl group H-12 showing HMBC connections to C-10, C-9 and C-8. 104 Figure 3.12: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in preaspyridone minor isomer 224. Crystals of the minor component 224 were formed in chloroform and were analyzed on a Microstar X-ray instrument. The crystal data (ccdc code 941137) gave the relative stereochemistry of the three chiral centres in minor preaspyridone as (5R, 7S, and 9R). The methyl groups at C-7 and C-9 possess anti-configuration in the crystal data. Bergmann et al. reported syn configuration for both methyls in aspyridone A 84 2, 4-dimethylhexanoyl side chain.80 Figure 3.13: Crystal structure of the minor isomer of preaspyridone A 224. 3.7.1(b) Identification of major isomer of preaspyridone A 230 The major compound eluting at 14.0 minutes was purified in the form of pale brown solid. HRESIMS gave a molecular formula of C19H25NO4Na (observed 354.1677; calculated 354.1676). The 13C NMR revealed the presence of 19 Carbons (Figure 3.15) having similar chemical shifts as the 13 C NMR spectra of minor preaspyridone 224. The 1H NMR of the major component was similar to minor preaspyridone A 224 in having similar aromatic doublets at δH 6.59 (H-17, H-19), δH 6.90 (H-16, H-20) and the singlet at δH 9.17 (H-18) which is distinctive of para- substituted phenol in preaspyridone A. The 1H NMR showed difference in the signals of three protons H-14, H-8 and H-13 (Figure 3.16). The splitting pattern of the diastereotopic protons H-14a and H-14b in major 105 preaspyridone 230 was different because both protons appeared at the same chemical shift δH 2.82 as a doublet coupled to methine proton H-5 with J constant of 4.5 Hz, unlike in minor preaspyridone 224 where both H-14 protons appeared at separate ppm (Figure 3.10). The methylene group at H-8 in major preaspyridone 230 appeared as geminal protons resonating at two separate chemical shifts δH 1.29 and δH 1.38. The chemical shift of the methyl H-13 was also different as it produced a doublet at δH 0.95 (Figure 3.16) as compared to δH 1.03 in the minor isomer. From the above variations in the 1H NMR and the crystal structure of the minor preaspyridone 224, we deduced that both the minor and major compound observed in the apdAC expression clone, are diastereomers of preaspyridone A being epimeric at C-5 (Figure 3.13). The 1H- 13 C HMBC showed similar correlations as observed in minor preaspyridone 224 (Figure 3.14). Figure 3.14: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in preaspyridone major isomer 230. Figure 3.15: 13C NMR of major isomer of preaspyridone A 230 run in DMSO-d6. 106 Figure 3.16: 1H NMR of major isomer of preaspyridone A 230 run in DMSO-d6. 3.7.1(c) Discussion on the biosynthesis of preaspyridone A 224 and 230 Heterologous expression of apdA and apdC in A. oryzae (M-2-3) produced two diastereomers of preaspyridone A, 224 and 230, which confirms the in vitro reconstitution of apdA and apdC function by Tang and colleagues.107 There are a number of 3-acyl tetramic acids reported from analogous iterative HRPKS-NRPS gene clusters from other fungi. For example, fusarin C 77, pramanicin 83, militarinone C 82, equisetin 81, pseurotin A 78, chaetoglobosin A 80, 2-oxo-cyclopiazonic acid 85 (Figure 3.17), pretenellin A 114 and predesmethylbassianin A 118 (see chapter 1). The biosynthetic pathway of preaspyridones is more similar to pretenellin A 114. The minor 224 and major 230 preaspyridone A are epimers at methine proton H5. This illustrates that the adenylation domain of the NRPS is able to select D-tyrosine 231 during biosynthesis of minor preaspyridone 224 and L-tyrosine 232 to form the major preaspyridone 230 (Scheme 3.5). This further confirms the in vitro studies by Tang and coworkers where they reported the flexibility of the adenylation domain of apdA to incorporate different aromatic amino acids.107 We also proposed that minor preaspyridone 224 might have formed by reduction of 267. 107 Scheme 3.5: Incorporation of amino acids in isomers of preaspyridone A 224 and 230. The structures of the diastereomers of preaspyridone A 224 and 230 also reflects the exclusive potential of the enoyl reductase domain (ApdC) of aspyridone gene cluster, particularly setting the opposite R and S stereo centres at the pendant methyls in the 2, 4-dimethyl hexanoyl side-chain of preaspyridone A 224 and 230. In the initial cycle of the polyketide chain biosynthesis the AT and KS domains form a diketide, followed by methyl group transfer by CMeT domain and ketoreduction by the KR. The dehydratase (DH) further reduces the diketide to an enol group. The enoyl reductase is defective in the ApdA protein and the reduction is carried out by the stand alone ER protein encoded by apdC. The ER reduces the enoyl group to a saturated bond and at the same time sets the stereochemistry of the methyl chain, and in pre aspyridone A the first methyl group is arranged in R configuration 233. The same steps repeat in the second cycle by the iterative domains and the second methyl group is settled in S configuration 234 by the ER domain (Scheme 3.6). In the last cycle a β-keto tetraketide 220 is formed. 108 Scheme 3.6: Proposed biosynthetic steps of polyketide chain in pre aspyridone A 224 and 230. A similar stereoselective programming is observed by KR domain in (6’S, 10’S)-7, 8’-dehydrozearalenol (DHZ 236) biosynthesis, which is an intermediate of hypothemycin 237. The HRPKS Hpm8 synthesizes a hexaketide 235 where KR catalyzes opposite stereo reduction at C-6’ and C-10’. The hexaketide 235 is transferred to a NRPKS which combines with three malonyl coenzyme A to form DHZ 236 and later steps eventually form hypothemycin 237 (Scheme 3.7).184 Scheme 3.7: Stereoselective reduction during biosynthesis of hypothemycin 237. 109 Figure 3.17: 3-acyl tetramic acids reported from hybrid PKS-NRPS pathway, amino acids are highlighted in red. 3.7.2 Heterologous expression of apdACE in A. oryzae (M-2-3) After successful expression of apdA and apdC, pTAYAGSargPage was modified to develop another plasmid pTAYAargACE, containing apdA, apdC and apdE, which was then transformed into A. oryzae M-2-3. The apdE gene encodes a cytochrome P450 monooxygenase and it shares 48% protein identity to the ring expandase P450 enzyme TenA present in B. bassiana 110.2.108 This expression was aimed to delineate the role of P450 enzymes in aspyridone A 84 pathway. Selected transformants were screened for production of new metabolites by LCMS. The A. oryzae ACE expression clone was grown on DPY plates and mature spores were inoculated in CMP liquid media for 7 days at 28 °C (see section 4.10). The A. oryzae culture was homogenised, acidified and extracted with ethyl acetate. The organic extract was concentrated under vacuum and defatted. The dried extract (112.6 mg from 100 ml of culture) was made to a solution of 10 mg/ml in HPLC grade methanol and analysed by LCMS. 110 14.5 ZW-II-92K-50-70ch3cn A 14.9 8.8 3.0e+2 2.0e+2 3.28 1.0e+2 3.72 2.58 0.0 -0.00 ZW-II-92M 6.0e+1 2.00 4.00 24.8 6.4 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 3.22 B Wildtype A. oryzae 3.45 5.0e+1 4.0e+1 5.80 3.0e+1 2.0e+1 1.0e+1 4.25 2.60 27.43 0.0 Figure 3.18: A, Diode array chromatogram of A. oryzae apdACE expression clone showing four new peaks at 8.8, 14.5, 14.9 and 24.8 minutes; B, Diode arrat chromatogram ofvWild type A. oryzae used as a control. The diode array chromatogram of A. oryzae apdACE expression clone indicated four new peaks which were not present in the A. oryzae wild type (WT) extract. The first peak at 8.8 minutes possessed a λmax (222, 281) which is similar to uv absorption of the 3-acyl tetramic acid preaspyridone A 224. The ESI spectrum of this peak showed m/z 348 [M]H+ which is 16 mass units more than that of preaspyridone A 224. The second peak at 14.5 minutes showed mass ion of 238 [M]H+ and a longer λmax (229, 325 nm). The peak eluting at 14.9 minutes showed a uv absorption (λmax 246, 344) and ESI (m/z 330 [M]H+), spectra corresponding to aspyridone A 84. The last peak at 24.8 minutes had a mass ion of m/z 316 [M]H+, which is 16 mass units less than preaspyridone A 224 (m/z 332 [M]H+) and a λmax 281 nm. The transformant was grown on large scale (100 ml × 10 flasks) and massdirected purification of the above peaks was performed on a Waters LCMS autopurification system (see section 4.5). The dried crude extract (1303 mg/L) was used to prepare a solution of 50 mg/ml and about 200 µl was injected in each preparative run. The gradient use on a 30 duration programme was acetonitrile Method 5 (section 4.5) on a C18 Phenomenex LUNA column. The purified fractions of each compound were collected and dried under nitrogen gas. Structural elucidation was achieved using 1D and 2D NMR spectroscopy and High Resolution Mass Spectrometry. 111 3.7.2(a) Identification of 14-Hydroxypreaspyridone A 238 The compound eluting at 8.8 minutes was obtained in the form of waxy light brown solid (67 mg/L). HRESIMS gave a molecular formula of C19H25NO5 (observed 370.1624; calculated 370.1625 for M[Na] +). The 13 C NMR in methanol-d4 showed many signals coming at similar chemical shifts as preaspyridone A 224. These included two methylene carbons at δC 41.2 (C-8) and δC 30.2 (C-10), a methine carbon at δC 68.5 (C-5), two sp3 carbons at δC 33.5 (C-9) and δC 35.5 (C-7), three methyl groups at δC 17.5 (C-13), δC 19.6 (C-12) and δC 11.4 (C-11), two aromatic carbon at δC 115.5 (C-17, C-19) and δC 129.6 (C-16, C-20) and carbonyl group at δC 195.2 (C-6). A new signal at δC 74.8 indicated carbon attached to a hydroxyl (OH) group. The 1H NMR presented a similar spectrum to preaspyridone A 224 spectra. A doublet at δH 4.99 attached to δC 74.8 (C14) in the 1H-13C HSQC was assigned to H-14 (Figure 3.20). The aromatic doublets at δH 7.11 (H-16, H-20) and δH 6.65 (H-17, H-19) exhibited HMBC correlations to parasubstitited phenol at δC 158.6 (C-18), quaternary carbon at δC 130.3 (C-15) and benzylic carbon δC 74.8 (C-14) (Figure 3.19). The doublet at δH 4.22 was assigned to methine H5 showing a 1H-1H COSY to H-14 and HMBC correlation to benzylic carbon at C-14 which confirmed para-substituted phenol attached at hydroxy-benzyl to a pyrrolidine nucleus at methine H-5 (Figure 3.21). The quartet at δH 3.55 (H-7) was linked to C-8 and C-13 in HMBC. The multiplet spread between δH 1.29-1.40 was assigned to methylene protons (H-8) and δH 1.09-1.33 to methylene groups (C-10) and methine at C-9. The terminal methyl at δH 0.81 (H-11) and methyl at 0.82 (H-12) showed HMBC connection to C-13, C-9 and C-8 confirming a similar 2,4-dimethylhexanoyl side chain possessed by preaspyridone A 224 (Figure 3.19). All correlations confirmed the structure to be 14-hydroxypreaspyridone A 238. Figure 3.19: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in 14-hydroxypreaspyridone 238. 112 Figure 3.20: 1H NMR of 14-hydroxy preaspyridone A 238 run in methanol-d4. Figure 3.21: Segment of 1H-13C HMBC NMR spectrum of 14-hydroxy preaspyridone A 238 showing key correlations of hydroxyl benzyl at C-14 with a methine at H-5 and aromatic proton H-16. 113 Figure 3.22: ES+ and UV spectrum of 14-hydroxy preaspyridone A 238 eluted at 8.8 mins. 3.7.2(b) Identification of 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239 The compound eluting at 14.5 minutes was obtained as pale white solid (63 mg/L). The HRESIMS gave a molecular formula of C13H20NO3 (observed 238.1428; calculated 238.1437 for M[H] +). The 13C NMR in DMSO-d6 indicated the presence of all 13 carbons consisting of two carbonyl groups at δC 177.6 (C-4) and δC 211.9 (C-7), two methylene groups at δC 40.0 (C-9) and δC 29.7 (C-11) and three methyl groups at δC 11.1 (C-12), δC 16.7 (C-14) and δC 18.8 (C-13). The 1H NMR showed a triplet downfield at δH 7.60 and a doublet at δH 5.92 which were assigned to H-6 and H-5 respectively (Figure 3.24), linked to an amide proton at δH 11.49 (H-1) in 1H-1H COSY (Figure 3.23). The aromatic protons H-5 and H-6 showed 1H-13C HMBC correlations to quaternary carbon C-3 (δC 105.9), a hydroxyl group at C-4 (δC 177.6) and carbonyl group at δC 161.8 (C-2) verifying the structure of 2-pyridone (Figure 3.25). The quartet at δH 4.25 was assigned to a methine proton H-8 linked to the carbonyl group at δC 211.9 (C-7), methyl group at C-14 and to a methylene group at C-9 (Figure 3.23). The methyl group at δH 1.02 (H-14) showed HMBC connection to carbonyl at C-7 and methine at C8 which established that it is attached to C-8. The multiplets at δH 1.21 and δH 1.49 were assigned to geminal protons at H-9 and multiplets at δH 1.09 and δH 1.25 were allocated to geminal protons at H-11. A number of COSY and HMBC showed the structure to be a 2, 4-dimethylhexanoyl side chain attached to 2-pyridone at C-3 and was named as 4hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239. 114 Figure 3.23: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2pyridone 239 and crystal structure of 239. Figure 3.24: 1H NMR of 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239 run in DMSO-d6. Figure 3.25: Section of 1H-13C HMBC NMR spectrum of 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239 showing important correlations of pyridone protons H-5 and H-6 with quaternary carbons C-2, C-4 and C-3. 115 Figure 3.26: ES+ and UV spectrum of 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239. 3.7.2(c) Identification of aspyridone A 84 The compound separating at 14.9 minutes (Figure 3.18) was obtained as light brown solid. The HRESIMS gave a molecular formula of C19H24NO4 (observed 238.1692; calculated 330.1699 for M[H]+). From 1D 1H and 13C NMR and 2D NMR the compound was identified as aspyridone A 84.80 The two aromatic doublets at δH 7.26 (H-16, H-20) and δH 6.80 (H-17, H-19) (Figure 3.28) exhibited HMBC correlations to para-substituted phenol carbon at δC 158.3 (C-18) and quaternary carbon at δC 125.2 (C15). The singlet at δH 7.47 was assigned to methine proton H-6 which displayed 1H- 13C HMBC connections to a hydroxyl group at δC 177.6 (C-4) and carbonyl group at C-2 (δC 163.9) (Figure 3.27). The H-6 singlet also showed HMBC connection to quaternary carbon C-15 which confirmed 2-pyridone linked to para-substituted phenol at C-15. The quartet at δH 4.39 was allotted to methine proton H-8 linked to methylene group at δC 41.2 (C-9) and to methyl group at δC 17.6 (C-14). The last methyl at δH 0.87 (H-12) showed correlations to C-10 (δC 33.7) and methylene group at δC 33.7 (C-11). The methyl group at δH 0.90 (H-13) showed connections to C-9, C-10 and C-11 giving 116 evidence that a similar polyketide chain of preaspyridone A 224 has combined intact to 2-pyridone at C-7 (δC 214.4) to give a structure of aspyridone A 84. Crystals of aspyridone A 84 were formed. The methyl groups at C-8 and C-10 possess anti-configuration in the crystal data, similar to preaspyridone A 224 minor isomer and 239. Figure 3.27: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in aspyridone A 84 and crystal structure of aspyridone A 84. Figure 3.28: 1H NMR of aspyridone A 84 run in methanol-d4. 117 Figure 3.29: ES+ and UV spectrum of aspyridone A 84. 3.7.2 (d) Identification of 18-deshydroxypreaspyridone A 240 The final compound from A. oryzae apdACE expression clone separated at 24.8 minutes (Figure 3.18). It was obtained as light brown solid (12 mg/L) and HRESIMS showed molecular formula of C19H25NO3 (observed 338.1735; calculated 338.1726 for M[Na]+). The 13C NMR revealed the presence of 15 carbons and quaternary carbons were observed in the 2D NMR. A significant difference in the 1H NMR of compound 240 was observed due to the presence of distinct multiplets in the aromatic region (Figure 3.31) in contrast to the distinct pair of doublets which are characteristic of the parasubstituted phenol present in the spectra of preaspyridone 224 and aspyridone A 84 (Figure 3.10 and 3.28). The multiplets from δH 7.16-7.22 were assigned to three aromatic protons (H-17/19), (H-18) and (H-16/20) (Figure 3.31). The aromatic protons H-16/20 and H-17/19 displayed HMBC correlations to quaternary carbon at δC 136.9 (C-15) and benzylic carbon at δC 38.3 (C-14) (Figure 3.32). Two set of doublets of doublets at δH 2.98 and δH 3.07 were assigned to two diastereotopic protons at the benzylic position, H-14 and showed connection to δC 130.7 (C-17/19), C-15 and to methine carbon at δC 63.5 (C-5) confirming the structure to be benzene ring attached to pyrrolidine ring at C-5 connected through benzylic carbon, C-14. The multiplet at δH 3.65 was allotted to methine proton H-7 linked to a carbonyl group at δC 196.0 (C-6), methylene group at C-8 (δC 41.3), a methine at δC 33.5 (C-9) and a methyl group at δC 118 17.6 (C-13) (Figure 3.30). The multiplets at δH 1.33 and 1.44 were assigned to geminal protons at H-8 correlated to C-6, C-13, C-12 (δC 19.5), C-10 (δC 30.3), C-9 (δC 33.5) and C-7 in 1H-13C HMBC. The multiplets at δH 1.09 and δH 1.34 were assigned to a second methylene group at H-10 linked to C-11 (δC 11.5), C-12 and C-9 in the HMBC correlations. All the 1D and 2D NMR elucidated the structure to be 18deshydroxypreaspyridone A 240. Figure 3.30: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in 18-deshydroxypreaspyridone A 240. Figure 3.31: 1H NMR of 18-deshydroxypreaspyridone A 240 run in methanol-d4. 119 Figure 3.32: Segment of 1H-13C HMBC NMR of 18-deshydroxypreaspyridone A 240. Figure 3.33: ES+ and UV spectrum of 18-deshydroxypreaspyridone A 240. 3.7.2 (e) Role of apdE in aspyridone pathway Heterologous expression of hrPKS-NRPS ApdA, enoyl reductase ApdC and cytochrome P450 enzyme ApdE in A. oryzae was accomplished using the 120 pTAYargACE expression vector. The transformant produced three new products; 14hydroxy preaspyridone 238, 4-hydroxy-3-(2,4-dimethylhexanoyl)2-pyridone 239, aspyridone A 84 and 18-deshydroxypreaspyridone A 240. This experiment exemplified the role of ApdE in the aspyridone pathway as the new compounds 238, 239 and 240 were not observed in the A. oryzae apdAC expression clone. The apdE gene encodes a monooxygenase enzyme which belongs to a large family of cytochrome P450 oxidases. Cytochrome P450 enzyme in fungi are known to perform important bioconversions and reactions in the biosynthesis of many natural products, for example selective olefin epoxidation and oxidation of methyl groups of natural product compounds. They also execute hydroxylation of complex polyaromatic hydrocarbons, steroids such as progesterone. Some plant pathogenic fungi are reported to encode P450 enzymes.185 Cytochrome P450 contain a heme cofactor and are known as hemo proteins. Scheme 3.8 illustrates the reaction by which Ferrous (II) ions in heme uses molecular oxygen as the oxidant and carries out oxidation of organic substrates by introduction of oxygen into C-H bond. Scheme 3.8: The heme in Cytochrome P450 uses molecular oxygen for oxidation of organic substrates. In aspyridone biosynthesis, ApdE catalyses three important reactions which are: conversion of preaspyridone A 224 and 230 to aspyridone A 84 by oxidative ring expansion; hydroxylation of preaspyridone A to form 238; and oxidative dephenylation of preaspyridone A to from 239. A similar oxidative ring expansion reaction takes place in tenellin 87 biosynthesis in B. bassiana 110.0 by a cytochrome P450 enzyme known as TenA. It drives the formation of pretenellin B 115 from pretenellin A 114 by oxidative ring expansion of 3-acyl tetramic acid to 2-pyridone (see chapter 2). Prototenellin D 155 is another compound similar to 14-hydroxy preaspyridone A 238 in tenellin pathway. It is formed by hydroxylation of pretenellin A by direct rebound 121 mechanism by an unknown oxidase enzyme in B. bassiana as it is only produced in the native organism and not observed during heterologous expression of tenellin 87 genes in A. oryzae. Halo and colleagues have comprehensively studied and presented a hypothesis for the mechanism of oxidative ring expansion and benzylic hydroxylation of tetramic acid in tenellin 87 pathway.104,108 Scheme 3.9: Biosynthesis of pretenellin B 115 and protonellin D 155 in B.bassiana. Scheme 3.10: A, Proposed oxidative mechanisms for biosynthesis of hydroxyl tetramic acid 238; B, Proposed mechanism for oxidative ring expansion during biosynthesis of 2-pyridone 84. 122 We proposed similar biosynthetic routes for compounds 238, 84 and 239 (Scheme 3.10, 3.11). Cytochrome P450 initiates by hydrogen atom abstraction from the benzylic position and forms carbon-centred radical 241. The C-centred radical reacts with an iron bound hydroxyl radical and gets hydroxylated directly by the oxygen rebound mechanism186 to form 14-hydroxy preaspyridone 238 (Scheme 3.10, A). The carbon centred radical 241 can seemingly also follow another route of single electron transfer and form cyclopropyl oxy-radical 242,187 followed by another short lived intermediate 243, which consequently leads to ring expansion and form 2-pyridone compound aspyridone A 84 (Scheme 3.10, B). Scheme 3.11: Proposed oxidative dephenylation for biosynthesis of 239. A third mechanism was proposed for the biosynthesis of 4-hydroxy-3-(2,4dimethylhexanoyl) 2-pyridone 239. A peroxo-iron intermediate 244 can make hydroxylation at the phenyl-bridge head carbon of preaspyridone A 224 which results in formation of assumed hydroxyquinone 245 (Scheme 3.11). Further electron transfer leads to ring expansion of the pyrrolidone ring to 2-pyridone 246 and the hydroquinone separates causing dephenylation. The 2-pyridone rearranges and forms 239 (Scheme 3.11). A number of related natural products are known. For example Torrubiellone A 247 and Torrubiellone B 248 from the spider-pathogenic fungi Torrubiella sp. BCC 2165,188 (+)-N-deoxymilitarinone A 249 and militarinone A 250 from entomopathogenic fungi Paecilomyces farinosus,189 all feature structures which have been hydroxylated at the carbon corresponding to the oxidation target in the proposed mechanism, while jacaglabroside B 251190,191 features a more highly oxidised version of the same structural feature. 123 TenA in the tenellin 87 gene cluster has a similar catalytic activity to ApdE, yet the latter displays a broader chemical diversity as no dephenylated or similar compounds were reported from the tenellin 87 pathway. There are a number of reported structures which are similar to dephenylated compound 239. Piericidins A 252 and B 253 are natural insecticides isolated from Streptomyces mobaraensis.192 They exhibit specific inhibitory action for electron transport system in mitochondria. Sapinopyridione 254 and 255 were purified from a fungal pathogen of conifers, Sphaeropsis sapinea.193 Atpenins 256, 257, 258 are antifungal metabolites of molds Penicillium sp.FO-125. They are known to inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II.194 The atpenins also possess pendant methyls arranged in anti-configuration. 124 3.7.3 Heterologous Expression of apdABC in A. oryzae The apdB gene encodes a second cytochrome P450 monooxygenase in the aspyridone cluster.80 The pTAYAargABC plasmid was constructed to reveal the role of apdB. Selected transformants were grown to be analysed for production of new metabolites. The transformants were grown on DPY medium on plates and spores were afterwards inoculated in CMP (section 4.9.b) growth media for seven days according to methods dercribed in section 4.10. The cultures of A. oryzae apdABC transformants were extracted in the usual way and the crude extract was defatted with wet methanol and hexane and then evaporated (80.5 mg from 100 ml). A 14.0 apdA,B,C ZW-II-92D 1.5e+2 3.32 1.0e+2 5.0e+1 2.67 0.0 -0.00 ZW-II-92M 6.0e+1 5.0e+1 4.52 2.00 4.00 13.2 6.10 6.00 8.00 10.00 12.00 29.25 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 3.22 B Wildtype A. oryzae 3.45 4.0e+1 5.80 3.0e+1 2.0e+1 1.0e+1 4.25 2.60 27.43 0.0 Time Figure 3.34: A, Diode array chromatogram of A. oryzae apdABC expression clone showing peaks of preasyridone A minor isomer 224 at 13.2 and major preaspyridone A 230 at 14.0 minutes; B, Wild type A. oryzae used as a control. A solution of 10 mg/ml in HPLC grade MeOH of the crude extract was injected for analysis on LCMS. The diode array chromatogram showed two peaks at 13.2 and 14.0 minutes. They were identified as the minor and major isomers of preaspyridone A 224 and 230 which were characterised from the apdAC expression clone. We observed an increase in the titres of both compounds in the apdABC expression clone to be 2.2 mg/L and 141.0 mg/L for the minor and major components of preaspyridone A, respectively. This was double the amount of preaspyridone A in comparison to apdAC expression clone where the yields of minor and major component were 0.6 mg/L and 60 125 mg/L, respectively. This expression experiment showed that the ApdB cytochrome P450 cannot chemically act on the tetramic acid preaspyridone A, but it may act later in the pathway. 3.7.4 Heterologous Expression of apdACEB in A. oryzae The A. oryzae apdABC expression clone did not produce any new compounds, compared to A. oryzae apdAC. We next planned to express the apdB gene in the presence of the megasynthase ApdA, enoyl reductase ApdC and monooxygenase ApdE in A. oryzae. The pTAYAGSargPage vector has a capacity to express maximum of four genes owing to the presence of four promoters.176 Thus pTAYAGSargPage was modified to construct a plasmid pTAYAargASP inserting all four genes apdA, apdB, apdC and apdE (Figure 3.35). The expressions of all the genes were confirmed by qRTPCR. Figure 3.35: (left) pTAYAargASP expression plasmid, (right) A. oryzae apdACEB expression clone on Czapek Dox Agar. The A. oryzae clones were initially grown on selection media and later on DPY media (section 4.7.b) for maximum production of spores. The one week old spores were inoculated in liquid media (CMP) at 28 °C at 200 rpm for 7 days (section 4.10). The fermentations were extracted in the usual way and the organic layer was concentrated under vacuum and then defatted with wet methanol and hexane. The resulting crude 126 extract was dried to a thick mass weighing 30 mg/100ml. 10 mg/ml of the crude extract was made with HPLC methanol and 20 µl was injected in LCMS. A 14.2 13.5 3.43 4.18 2.25 2.00 3.00 B 4.00 5.00 6.00 7.00 8.00 9.00 20.0 20.3 16.5 9.1 10.00 11.00 12.00 13.00 14.00 3.22 15.00 16.00 17.00 18.00 19.00 20.00 21.00 20.00 21.00 Wildtype A. oryzae 3.45 5.8 4.25 2.60 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 Figure 3.36: A, Diode array chromatogram of A. oryzae apdACEB expression clone showing production of 14hydroxypreaspyridone A 238, minor 224 and major isomer of preaspyridone A 230, aspyridone A 84 and three new compounds at 16.5, 20.0 and 20.3 minutes; B, A. oryzae Wild type. The LCMS chromatogram displayed a number of peaks which were not present in the diode array chromatogram of the Wild type (WT) A. oryzae. The wavelength and mass spectrum of first peak at 9.2 min. was identified as 14-hydroxy preaspyridone 238, the second peak at 13.6 min. was minor isomer of preaspyridone A 224, third peak at 14.2 min. was major isomer of preaspyridone A 230 and the compound eluting at 14.5 min was aspyridone A 84 (Figure 3.36). These compounds were later confirmed by 1H NMR. Three new peaks were detected at 16.5, 20.0 and 20.3 min. (Figure 3.36). The mass spectrum for the compound eluting at 16.5 min. was m/z 254 [M]H+, 16 mass units more than the dephenylated aspyridone 239 and observed λmax of 278, 335 nm. The peak at 20.0 minutes showed a uv absorption 228, 330 nm and ESI spectrum presented m/z 268 [M]H+. The last peak at 20.3 min. showed a higher uv absorption (294, 376 nm) and m/z 330 [M]H+. For purification of all the peaks 1 litre (100 ml x 10 flasks) of the cultures of the transformant was grown according standard fermentation conditions (section 4.10). The 127 Time crude extract obtained after extraction and concentration was 1.13 g per liter. A concentration of 50 mg/ml was made with HPLC grade methanol. 200 µl of the solution was injected in Waters LCMS mass directed purification in each preparative run. The purification was achieved with an acetonitrile and water solvent system with Method 3 (section 4.5) in a 30 min. duration programme. The purified fraction of each peak was collected and dried under nitrogen. The last peak at 20.3 min. was not collected due to lower titres (it was later purified in another experiment explained in section 3.7.5). The structures of pure compounds were each studied with the help of 1D and 2D NMR spectrometer and high resolution mass spectrum. 3.7.4 (a) Identification of 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2- pyridone 259 The compound eluting at 16.5 min. was obtained as light brown solid 6.2 mg/L. The HRESIMS provided the molecular formula to be C13H19NO4 (observed 276.1218; calculated 276.1206 for [M]Na+). The 13C NMR showed the presence of all 13 carbons, with many chemical shifts similar to dephenylated aspyridone 239. The 13 C NMR composed of two carbonyls groups at δC 213.6 (C-7) and δC 160.0 (C-2), a quaternary carbon at δC 107.4 (C-3), a hydroxyl group at C-4 (δC 176.0), two methylene groups at δC 31.0 (C-11) and δC 41.1 (C-9) and three methyl groups at δC 19.3 (C-13), δC 17.3 (C14) and δC 11.7 (C-12). A quartetet at δH 4.31 allocated to H-8 displayed 1H-1H COSY correlation to H-14 (δH 1.11) and to the methylene carbon C-9 in the 2D 1H-13C HMBC. The multiplets at δH 1.33 and δH 1.62 were assigned to geminal protons at H-9 linked to C-7, C-14, C-13, C-11, C-10 and C-8 in the HMBC correlations (Figure 3.37 and 3.39). The methine proton H-10 signals at δH 1.41 and multiplets at δH 1.18 and δH 1.36 were consigned to the two geminal protons at H-11. These correlations verified that the aliphatic chain is the same as in dephenylated preaspyridone 239 and no hydroxylation has occurred on the polyketide chain. The pair of aromatic douplets at δH 5.97 and δH 7.94 were assigned to protons H-5 and H-6 respectively, of the 2-pyridone (Figure 3.38). The distance between the doublets was more than observed in the 1H NMR of dephenylated aspyridone 239 and this was ascribed to the presence of a hydroxyl group attached to the pyridone nitrogen. From the NMR correlations and HRMS the structure was established to be 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 259. 128 Figure 3.37: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in 1, 4-dihydroxy-3-(2, 4dimethylhexanoyl) 2-pyridone 259. Figure 3.38: 1H NMR of 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 259 run in methanol-d4. Figure 3.39: Key 1H-13C HMBC correlations in 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 259. 129 Figure 3.40: ES+ and UV spectrum of 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 259. 3.7.4 (b) Identification of 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2- pyridone 260 The compound 260 separating at 20.0 minutes was purified in the form of light brown solid (2.7 mg/L) and HRESIMS analysis presented a molecular formula of C14H21NO4 (observed 290.1368; calculated 290.1362 for [M]Na+). The 13 C NMR displayed presence of 14 Carbons. Many chemical shift values were similar to dephenylated aspyridone A 239 as it consists of three methyl groups at δC 11.7 (C-12), δC 17.3 (C-14) and δC 19.3 (C-13), two methylene groups at δC 31.0 (C-11) and δC 41.1 (C-9), two methine at δC 42.2 (C-8) and δC 33.5 (C-10), two carbonyls at δC 213.6 (C-7) and δC 159.7 (C-2) and aromatic carbons at δC 100.7 (C-5) and δC 143.0 (C-6). A new carbon signal was observed at δC 65.6 which indicated that it is attached to an oxygen atom. The 1H NMR consists of two sets of doublets at δH 6.02 (H-5) and δH 8.03 (H-6), assigned to aromatic protons of 2-pyridone (Figure 3.43). The 1H NMR presented a singlet at δH 4.01 integrating to three protons which was not observed in dephenylated aspyridone A 239. It was attached to δC 65.6 in 1H-13C HSQC. A characteristic feature which aided in elucidation of the structure was revealed in 1D NOESY when δH 8.03 130 (H-6) showed correlation to δH 4.01, which determined that an O-methyl is attached to nitrogen atom at position 1 in 2-pyridone (Figure 3.41). Figure 3.41: 1D NOESY showing connection of H-6 with methoxy group at N-1. In 1H-13C HMBC spectrum, H-5 presented two bond and three bond correlations with hydroxyl group at C-4 (δC 177.3) and quaternary carbon (δC 108.2) respectively (Figure 3.44). The aromatic proton H-6 displayed connections to carbonyl group at C-2, aromatic carbon C-5 and hydroxyl group at C-4. We didn’t observe any HMBC correlations from H-15 to C-2 or C-6; this might be because four bond correlations are rarely seen. The terminal methyl at δH 0.87 (H-12) was linked to methylene group at δC 31.0 (C-11) and methine at C-10 (δC 33.5) and adjacent methyl signal at δH 0.92 (H-13) diplayed HMBC correlations to C-10, C-11 and C-9 (Figure 3.42). The geminal protons at δH 1.31 and δH 1.61 at H-9 shows connections to carbonyl group at C-7 (δC 213.6), methine carbon at δC 42.2 (C-8), C-10, C-11 and C-13. This confirms a 2, 4dimethylhexanoyl side chain identical to aspyridone A 84. These correlations establish the structure to be 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 260. 131 Figure 3.42: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in 1-methoxy, 4-hydroxy-3-(2, 4dimethylhexanoyl) 2-pyridone 260. Figure 3.43: 1H NMR of 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 260 run in methanol-d4. Figure 3.44: Key 1H-13C HMBC correlations in 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 260. 132 Figure 3.45: ES+ and UV spectrum of 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 260. 3.6.4 (c) Discussion on role of apdB The apdABCE expression clone produced a range of compounds including the two diastereomers 224 and 230 observed when megasynthase ApdA was expressed with enoyl reducatse ApdC and 14-hydroxy preaspyridone A 238, dephenylated aspyridone 239 and aspyridone A 84 which are products of the monooxygenase ApdE. This illustrates that as more genes from a cluster are expressed in a heterologous experiment, we observe novel biosynthetic potential of the specific gene cluster. The production of 1-methoxy dephenylated pyridone 260 and N-hydroxy dephenylated pyridone 259 ascribes a role for the cytochrome P450 enzyme ApdB, that it is involved in N-hydroxylation of 5-dephenylated pyridone 239 to form 259 and 260. It exhibits substrate specificity for dephenyalted pyridines only, because we didn’t observe any N-hydroxylation in tetramic acids 224 and 230 and neither in aspyridone A 84. In tenellin, the cytochrome P450 enzyme TenB also displays N-hydroxylation for only pretenellinB to form tenellin 87 and TenB does not N-hydroxylates tetramic acids pretenellin A 114 (see chapter 2). We assumed that the O-methylation in 260 is carried out by a native enzyme present within A. oryzae as there is no enzyme for putative methyl transferase characteristic in aspyridone cluster.80 There are a number of reported compounds which possess N-methoxy group, for example Cordypyridone C 133 261 and Cordypyridone D 262 from the insect pathogenic fungus Cordyceps nipponica.195 Kumarihamy et al. isolated four new N-methoxy-2-pyridinone compounds from the plant pathogen Septoria pistaciarum, which are 17-hydroxy-N-(O-methyl) septoriamycin A 263, 17-acetoxy-N-(O-methyl) septoriamycin A 264, 13-(S)-hydroxyN-(O-methyl) septoriamycin A 265 and 13-(R)-hydroxy-N-(O-methyl) septoriamycin A 266.196’’ 3.7.5 Heterologous expression of apdACED in A. oryzae ApdD encodes a FAD dependent monooxygenase. We planned to coexpress the apdD gene in the presence of the megasynthase ApdA, enoyl reductase ApdC and monooxygenase ApdE in A. oryzae to illustrate the role of ApdD in aspyridone biosynthesis. The pTAYAargASP plasmid was used to construct pTAYAargACED consisting of apdA, apdE, apdC and apdD and this was transformed into A. oryzae. The transcription levels of all the genes were confirmed by qRTPCR (This transformation was done by Dr. Khomaizon Pahirulzaman). The selected transformant was grown on DPY plates and when mature spores were visible after seven days, a spore solution was made in sterilised deionized water and was inoculated in each 100 ml liquid media kept in 250 ml Erlenmeyer flask. 1 Litre of the media (10 x 100 ml flasks) was grown with constant shaking at 200 rpm at 28 °C. After seven days the fungal cultures were extracted with ethyl acetate (section 4.11). The organic layer was concentrated and defatted with hexane. Drying the crude extract under nitrogen, gave a brown mass (1.128 g / 1000 ml). A 10 mg/ml solution was made with HPLC grade methanol and analysed on LCMS using CH 3CN/H2O analytical programme with a gradient (50-65%) with 30 min. duration (Figure 3.46). 134 A 16.6 17.3 3.55 24.4 4.02 4.00 3.22 10.2 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 B Wildtype A. oryzae 3.45 5.80 4.25 Figure 3.46: A, Diode array chromatogram of apdACED consisting of 14-hydroxy preaspyridone A 238 at 10.2 minutes, dephenylated pyridone 239 at 16.6 minutes, aspyridone A 84 at 17.3 minutes and a new compound at 24.4 minutes (green); B, Diode array chromatogram of wild type A. oryzae. The LCMS chromatogram displayed four noticeable peaks which were not present in the wild type A. oryzae. The peak at 10.2 min. was identified as 14-hydroxy preaspyridone 238 possessing a mass of m/z 348 in [M]H+ and λmax of (221, 283 nm); the second peak at 16.6 min. was recognized as dephenylated pyridone 238 with a λmax of (221, 283 nm) and a m/z of 238 in the ES+. The third peak at 17.3 min. had a uv absorption of (246, 345 nm), characteristic of aspyridone A 84, exhibiting a molecular ion of m/z 330 [M]H+. The last peak at 24.4 min. showed a m/z of 330 as [M]H+ and a λmax of (294, 376 nm). This peak was observed in the apdABCE expression clone but was not purified and characterised previously because of low titre (section 3.7.4). The crude extract (1.13g) was prepared to a concentration of 100 mg/ml and 200 µl was injected in each run in mass directed preparative LCMS. The gradient used was (50-65%) in acetonitrile/water (Method 6, section 4.5). Each of the above peaks were purified, collected and dried under nitrogen. The first three compounds at 10.2, 16.6 and 17.3 min. after purification were analysed by 1D 1H NMR and confirmed to be 14hydroxy preaspyridone 238, dephenylated pyridone 239 and aspyridone A 84. The last peak which was a new compound was studied with 1D 1H and 13 C and 2D NMR experiments. 135 From the above experiment we couldn’t observe any precise role for ApdD. Bergmann et al. guessed that apdD performs hydroxylation of aspyridone A 84 to form aspyridone B 226.80 But we did not observe any peak confirming the production of aspyridone B 226 in the expression clone. However, we observed that the presence of ApdD in the expression clone with ApdE reduced the production of metabolites to 71.5 mg as compared to A. oryzae apdACE where total titres of metabolites were 390.6 mg. (section 3.7.10). 3.7.5(a) Identification of Z-5, 14-anhydropreaspyridone A 267 The compound eluting at 24.4 min. was obtained as a bright yellow solid (4 mg) after repurification. The molecular formula in HRESIMS analysis was given as C19H23NO4 (observed 352.1528; calculated 352.1519 for [M]Na+). The 13 C NMR spectra consisted of three methyl groups at δC 11.6 (C-11), δC 17.5 (C-13), δC 19.5 (C12), two methylene groups at δC 30.6 (C-10) and δC 41.5 (C-8) and a methine group at δC 33.6 (C-9), two aromatic signals at 116.9 (C-17, C-19) and 132.3 (C-16, C-20), a quaternary signal at 126.5 (C-15) and a hydroxyl signal at C-18 (δC 159.4). A quaternary carbon at δC 184.0 observed in 2D HMBC was assigned for C-4 and an alkene carbon at δC 110.4 in 2D HSQC was assigned for C-14. A structural feature of this compound was revealed in the 1H NMR by the presence of a singlet at δH 6.52 (Figure 3.47), joined to δC 110.4 in the 1H-13C HSQC. The signal at δH 6.52 displayed 1H- 13 C HMBC connection to 4-hydroxy of the pyrrolidine nucleus at δC 184.0 (C-4) and to aromatic carbons at C-16/20 (Figure 3.49 and 3.50). From the above correlations the signal at δH 6.52 was assigned to H-14 attached at the benzylidene position of a tetramic acid. The aromatic doublet at δH 7.40 assigned to H-16/H-20 displayed HMBC connections to benzylidene carbon at δC 110.4 (C-14) and hydroxyl group at C-18. The second aromatic doublet at δH 6.83 assigned to H-17 and H-19 show couplings to quaternary carbon C-15 and para hydroxyl group at C-18 (Figure 3.49 and 3.50). 136 Figure 3.47: 1H NMR of Z-5, 14-anhydropreaspyridone A 267 run in methanol-d4. In order to determine the orientation of the olefin between C-5 and C-14, a three bond long range J coupling between H-14 and quaternary carbon C-4 was measured from a 2D selective EXSIDE NMR experiment.191 The EXSIDE NMR is a 2D spectrum and displays cross peak similar to a HMBC spectrum. The cross peak is a split in the Carbon dimensions and this splitting gives the value of J (C-H) constant. Figure 3.48 shows cross peaks between H-14 and C-4, labelled in Hertz. The second number in each peak is the 13C frequency in Hertz and the difference between both peaks (22974.16 Hz -23019.82 Hz) is 45 Hz. A 15-fold J-scaling factor is used which give a value of 3 Hz coupling constant between H-14 and C-4. The value of 3JHC for two possible E/Z isomers of 267 by DFT calculations gave a value of 7.5 and 4.5 Hz respectively, which further supported the EXSIDE experiment and the double bond between C-5 and C-14, was confirmed to be a cis configuration.191 (DFT calculations were performed by Dr. Craig Butts, School of Chemistry, University of Bristol). We failed to determine the geometry of this compound from 1D NOESY and 1D ROESY because of exchangeable protons between N-H and hydroxyl group at C-18. 137 Figure 3.48: Selective EXSIDE NMR spectrum showing cross peaks coupling between H-14 and C-4 to be 3 Hz in 267. The multiplet at δH 3.85, assigned to methine at H-7 showed couplings to pendant methyl group at C-13 and to methylene group at C-8. The geminal protons at δH 1.40 and δH 1.59 were assigned to H-8, linked to methyls at C-13, C-12 and to methylene at C-10 and to methine at C-9. The multiplets at 0.87 and 0.90 were allocated to terminal methyl H-11 and pendant methyl H-12 respectively displaying HMBC correlations to C-9, C-10 and methylene C-8 (Figure 3.49). These connections showed that this compound also possess a similar dimethylated tetraketide chain as observed in preaspyridone 224 and 230. The above correlations decided the structure to be Z-5, 14anhydropreaspyridone A 267. Figure 3.49: 1H-1H COSY (solid lines) and 1H-13C HMBC correlations (arrows) in Z-5, 14-anhydropreaspyridone A 267. 138 Figure 3.50: Key 1H-13C HMBC NMR correlations in Z-5, 14-anhydropreaspyridone A 267. Figure 3.51: ES+ and UV spectrum of Z-5, 14-anhydropreaspyridone A 267. 3.7.6 Coexpression of apdG with apdACEB in A. oryzae The apdG gene, located downstream of megasynthase apdA and exporter gene apdR in the gene cluster, encodes an acyl dehydrogenase. Bergmann et al.80 predicted that it assists in tetramic acid ring closing and reductive release of preaspyridone A 224 from the PKS-NRPS domains (section 3.4). However, the production of preaspyridone 139 A 224 and 230 from A. oryzae apdAC expression clone and from in vitro analysis by Tang et al. (section 3.4), contradicts Bergmann et al. hypothesis. In order to explore the function of ApdG, we planned to co-express apdG with apdA, apdB, apdC and apdE in A. oryzae. The vector pTAYAargASP carried apdABCE and can express only four genes. To express apdG Dr. Khomaizon Pahirulzaman designed another plasmid which contained a different selection marker, the bleomycin resistance gene, ble. The argB gene in the original vector pTAYAGSargPage was replaced by bleomycin resistance gene ble and the insertion of this gene was driven by PtrpC. This plasmid was used to enclose apdG inserted next to PgpdA, this plasmid was named pTAYAGSbleG (Figure 3.52). A. oryzae (M-2-3) was co-expressed with pTAYAargASP (carrying apdABCE) and pTAYAGSbleG (carrying apdG). The minimal media was supplemented with antibiotic for selection of transformants. Figure 3.52: Vector pTAYAGSbleG carrying apdG used in A.oryzae apdABCEG expression clone. The selected transformant was grown on DPY plates for a week, spores were prepared in sterile deionized water and then transferred to liquid media (CMP media, section 4.9.b). One flask (100 ml media in 250 ml Erlenmeyer flask) was grown for 140 seven days at 28 °C with constant shaking at 200 rpm. The mycelia and liquid media were homogenized and acidified before extraction and defatting in the usual way to give crude extract weighing 62.2 mg/100 ml. A 10 mg/ml solution was made and analysed with LCMS on a 30 minutes analytical programme using CH3CN/H2O (50-70% gradient) (Figure 3.53). 14.4 8.9 14.9 A 6.00 21.2 17.0 6.48 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 Wildtype A. oryzae B 8.28 6.00 7.00 8.00 10.02 9.00 10.00 13.27 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 Time 23.00 Figure 3.53: A, Diode array chromatogram of apdABCEG expression clone showing the production of 14-hydroxy preaspyridone 238 at 8.9 minutes, diastereomers of preaspyridone A 224 and 230 at 13.9 and 14.4 minutes, aspyridone A 84 at 14.9 minutes, N-hydroxy dephenylated pyridone 259 at 17 minutes and O methoxy dephenylated pyridone 260 at 21.2 minutes; B, Diode array chromatogram of wild type A. oryzae. The yield of these compounds from 1 litre fungal culture was calculated by quantification experiment (see section 3.6.10). The quantity of 14-hydroxy preaspyridone 238 was 42.6 mg/L, major diasteomer of preaspyridone A 230 was 98 mg/L, aspyridone A 84 was 12.6 mg/L, N-hydroxy dephenylated pyridone 259 was 15.8 mg/L and N-O methoxy dephenylated pyridone 260 was 8.2 mg/L. The above mentioned compounds are the same as observed in the apdABCE expression clone only they are produced in higher titres in apdABCEG expression clone (see section 3.7.4). From the above heterologous expression we couldn’t settle a precise role for apdG only that when it was expressed with other four genes from the cluster it displayed increase in the production of metabolites. 141 3.7.7 Heterologous expression of apdAC and tenA in A. oryzae The tenA gene encodes a cytochrome P450 enzyme during tenellin 87 biosynthesis in B. bassiana 110.25 and the protein encoded by apdE in the aspyridone gene cluster displays 48 % sequence similarity to TenA. TenA catalyses the oxidative ring expansion of the pyrrolidone in pretenellin A 114 to a 2-pyridone compound, pretenellin B 115.108 We planned to express megasynthase apdA and enoyl reductase encoding gene apdC with tenA in A. oryzae to determine if TenA displays broader substrate selectivity and turnover of preaspyridone A 224 and 230 produced by apdAC to aspyridone A 84. Earlier the Cox group achieved effective gene swaps between tenellin 87 and DMB 88 when enoyl reductase dmbC from DMB 88 cluster and PKSNRPS tenS were co-expressed in A. oryzae, they produced a precursor compound of tenellin 87 pathway, pretenellin A 114.109 Dr. Khomaizon Pahirulzaman constructed a vector pTAYAargACtenA carrying PKS-NRPS coding gene apdA, enoyl reductase encoding gene apdC and ring expandase gene tenA and expressed it in A. oryzae. One producing transformant was grown on DPY solid media and later mature spores were inoculated in aspyridone growth media (CMP media) after growing for 7-10 days. The fungal culture was grown in 100 ml liquid media in 250 ml flask at 28 °C on shakers at a speed of 200 rpm for seven days. Mature mycelia were homogenised and extracted with ethyl acetate (see section 4.11). The organic layer was concentrated and defatted. A crude extract of 65.3 mg/100 ml was obtained after drying. The crude extract prepared in HPLC grade methanol (10 mg/ml) was analysed on analytical LCMS programme with CH3CN/H2O having a gradient of 55-65% in 30 minutes (Figure 3.54). 142 14.3 3.38 A 2.62 4.55 2.00 4.00 13.3 6.07 6.00 8.00 10.00 12.00 28.52 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 3.22 Wildtype A. oryzae 3.45 5.80 B 4.25 27.43 2.60 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 Time Figure 3.54: A, Diode array chromatogram of apdACtenA expression clone showing production of minor preaspyridone A diastereomer 224 at 13.3 min. and major preaspyridone A diastereomer 230 at 14.3; B, Diode array chromatogram of wild type A. oryzae. The LCMS chromatogram displayed production of major and minor diasteomers of preaspyridone A 224 and 230 presenting a molecular ion of 332 [M]H+ and a uv spectrum of 222, 278 nm. This experiment indicated that TenA has restricted substrate specificity and didn’t convert preaspyridone A 224 to aspyridone A 84. But the titres of minor component of preaspyridone A 224 (2.2 mg/L) and major preaspyridone A 230 (121 mg/L) in apdACtenA expression clone was higher than produced in apdAC expression (see section 3.7.1). A similar effect in increase in preaspyridone A 224 and 230 was observed in apdABC expression. With presence of ApdB and TenA we didn’t observe any new metabolites but the production of preaspyridone A 224 and 230 was increased. 3.7.8 Heterologous expression of apdACE and tenB in A. oryzae TenB, a cytochrome P450 protein in tenellin 87 gene cluster catalyses the N- hydroxylation of the 2-pyridone in pretenellin B 115 and froms tenellin 87.108 In an A. oryzae transformation we planned to express tenB with apdACE to explore the catalytic activity of TenB and whether it N-hydroxylates the 2-pyridone compound, aspyridone A 143 84. Dr.Khomaizon Pahirulzaman modified the vector pTAYAargASP to form pTAYAargACEtenB carrying genes apdACE and tenB. A number of A. oryzae transformants were obtained and one was selected for screening by LCMS. The transformant was grown on DPY media and after 7-10 days when spores were well grown, spore solution was inoculated in CMP liquid media (1 x 100 ml) in a 250 ml flask for seven days at 28 °C (see section 4.10). The fungus culture was extracted and defatted in the usual way. A solid mass weighing 27.4 mg/100ml was obtained. 10 mg/ml in HPLC methanol was made from the crude extract and analysed by LCMS with CH3CN/H2O with gradient 55-65% in 30 minutes. 15.4 4.42 4.78 A 5.02 2.00 4.00 14.5 9.5 2.78 6.00 3.22 8.00 10.00 12.00 14.00 15.9 16.00 18.00 20.00 22.00 24.00 Wildtype A. oryzae 3.45 5.80 B 4.25 2.60 Figure 3.55: A, Diode array chromatogram of A. oryzae apdACEtenB expression clone displaying production of 14hydroxy preaspyridone A 238, preaspyrdione A 230, dephenylated aspyridone 239 and aspyridone A 84; B, Diode array chromatogram of wild type A. oryzae. The LCMS chromatogram revealed the production of 14-hydroxy preaspyridone A 238 (m/z 348 [M]H+, λmax 223, 281 nm), preaspyridone A 230 (m/z 332 [M]H+, λmax223, 280 nm), dephenylated aspyridone 239 (m/z 238 [M]H+, λmax229, 325 nm ) and aspyridone A 84 (m/z 330 [M]H+, λmax247, 344 nm). One litre (10 x 100 ml) of apdACEtenB clone was grown for purification of compounds. The crude extract achieved after extraction and concentration was 764 mg/L. The extract was made to a 144 concentration of 50 mg/ml and 100 µl was injected in each run of auto purification on mass directed preparative LCMS. The solvents used were acetonitrile/water with a gradient of 50-75% carried out in 30 min (Method 3) programme. The purified fractions of each compound was collected and dried. All above stated compounds were verified with 1H NMR spectroscopy. The yield of 14-hydroxy preaspyridone A 238 was 5.2 mg, preaspyridone A 230 was 11 mg, dephenylated aspyridone 239 was 28 mg and aspyridone A 84 was 2.3 mg. We didn’t observe any new or N-hydroxylated 2-pyridone compounds which confirm that tenB is highly selective and didn’t take any 2-pyridone compounds from aspyridone pathway as its substrate. 3.7.9 [1-13C] L-Tyrosine feeding Earlier aspyridone A 84 and preaspyridone A 224 were reported from hybrid PKS-NRPS gene cluster by Bergmann et al.80 and Tang107 and co-workers. They are synthesized from the fusion of a tetraketide 220 and an amino acid tyrosine 107. With hetererologous expression of apdACE genes and consequential later experiments we achieved novel dephenylated aspyridone compound, 4-hydroxy-3-(2, 4- dimethylhexanoyl) 2-pyridone 239. We carried out [1-13C] L-tyrosine feeding with 5dephenylated pyridone 239 producing clone apdACEtenB, firstly to determine if 5dephenylated pyridone 239 utilize L-tyrosine as its amino acid precursor and secondly to ascertain that dephenylated 2-pyridone 239 is an oxidative breakdown product of aspyridone pathway. 25 mg of [1-13C] L-tyrosine was dissolved in 3 ml of deionized water with 5µl of 1 molar NaOH. 1 ml of this solution was added in three flasks each containing 100 ml of 3 days old fungal culture of A. oryzae apdACEtenB in 250 ml Erlenmeyer flask. This was repeated on 4th and 5th day as well. One 250 ml flask containing 100 ml of media was grown as a control with no [1-13C] feeding. The fungal cultures were grown according to the standard fermentation conditions (see section 4.10). On the seventh day the fungal mycelia was homogenized, acidified and extracted with ethyl acetate. The organic layer was semi concentrated and defatted. After defatting the extract was dried to a brown mass weighing 71.7 mg/300 ml. A 50 mg/ml solution was made with HPLC 145 grade methanol and 50µl was injected in each run of mass directed auto-purfication on LCMS instrument. The gradient used on a CH3CN/H2O solvent was 55-60 % in 30 min. duration. The purified fractions were collected and fully dried under nitrogen gas. The yield of 14-hydroxy preaspyridone 238 was 2.8 mg, preaspyridone A 230 was 6.8 mg, 5-dephenylated pyridone 239 was 9.8 mg and aspyridone A 84 was 1.4 mg. Each of these compounds was studied by 1D 13C NMR spectroscopy and compared parallel with un-labelled pure compounds. A 13 C feeding No feeding B Figure 3.56: A, 13C NMR showing incorporation of [1-13C] L-tyrosine at C-4 in preaspyridone A 230; B, 13C NMR of preaspyridone A 230 without isotope feeding. The products of A. oryzae apdACEtenB expression clone are 14- hydroxy preaspyridone A 238, preaspyridone A 230, dephenylated 2- pyridone 239 and aspyridone A 84. In [1-13C] L-tyrosine feeding preaspyridone A 230 exhibited 30% incorporation of L-tyrosine at C-4 (Figure 3.56) and dephenylated 2-pyridone 239 displayed 13% incorporation of [1-13C] L-tyrosine at C-4 (Figure 3.57). We didn’t observe any incorporation of isotope labelled tyrosine in 14- hydroxy preaspyridone A 238 and aspyridone A 84. The feeding experiment confirmed that dephenylated 146 pyridone 239 derives the same PKS-NRPS biosynthetic pathway as preaspyridone A 230 and aspyridone A 84. It also confirms that the adenylation domain of the NRPS selects the amino acid, L-tyrosine. 13 C feeding A No feeding B Figure 3.57: A, 13C NMR showing incorporation of [1-13C] L-tyrosine at C-4 in dephenylated 2-pyridone 239; B, 13C NMR of dephenylated 2-pyridone 239 without isotope feeding. 3.7.10 Quantification of metabolites The titres of different compounds produced in various heterologous expression clones can vary owing to differences in metabolic and environmental conditions. For this reason, we carried out quantification of all aspyridone metabolites by measuring standard calibration curves. Serial dilutions of pure compounds from 30 µg/ml-1000 µg/ml were made with HPLC grade methanol and diode array chromatograms were obtained on LCMS with acetonitrile/water using gradient 50-70% in 20 min. Integration values of absorption peaks were recorded at the λmax value for each compound. Graphs were plotted between integration values versus different concentration used in serial dilution. This gave a standard calibration curve and linear equation for each compound. 147 100 ml of each A. oryzae transformant in a 250 ml flask was grown and crude extracts were obtained according to the method given in section 4.11. LCMS was run on these samples and peaks were first identified by standard retention time, uv spectra and mass spectra. The yields of each compound in the crude extracts were then calculated from their respective standard linear equation at their respective retention time and λmax value. The values are arranged in Table 3.1. 148 Table 3.1: Quantification of metabolites in mg/L. Transformant 238 224 230 84 260 259 239 267 240 Total yield apdA,C 430 mg 0 0.61 60.70 0 0 0 0 0 trace 61.3 apdA,C,E 1126 mg 183.6 0 0 119.8 0 0 72.2 0 15 390.6 apdA,B,C 805 mg 0 2.2 141.0 0 0 0 0 0 trace 143.2 apdA,C,tenA 653 mg 0 2.2 121.0 0 0 0 0 0 trace 123.2 apdA,C,E,B 300 mg 2.6 5.7 34.6 1.8 2.7 6.2 trace 4 trace 53.6 apdA,C,E,tenB (274mg from 100ml) 2.9 0 5.3 3.2 0 0 6.9 6 0 24.3 apdA,C,E,D 448 mg apd A,B,C,E,G (62.2 mg from 100ml) 8.2 0 0.3 5.1 0 0 49.9 8 0 71.5 42.6 0 98 12.6 8.2 15.8 0 trace trace 177.2 149 3.7.11 Discussion and Conclusions Aspyridone A 84 and aspyridone B 226 were reported to be the exclusive products of a silent hybrid PKS-NRPS gene cluster in A. nidulans.80 It was discovered by the Hertweck group80 and they activated the cluster by overexpressing the transcription regulator apdR. In this Chapter we studied the gene cluster of aspyridone 84 using a heterologous gene expression technique in A. oryzae via a lately devised multi gene expression plasmid pTAYAGSargPage.176 It consists of strong constitutive promoters for each subject gene and resulted in successful transcription of apd genes in the heterologous host. The coexpression of apd genes in A. oryzae in an orderly scheme disclosed advance oxidative modification and programming potential of respective genes leading to a number of new compounds. Heterologous expression of apdAC formed the previously known precursor compound preaspyriodone A 224.107 In our project preaspyridone A was isolated as a major and a minor diastereomer compounds 224 and 230, being epimers at C-5 (section 3.7.1). The formation of crystals of minor preaspyridone A isomer 224 presented antiarrangement of the pendent methyl groups of the alkane chain which is opposite to that reported before.80 We established that aspyridone A 84 is the product of three genes apdAC and apdE. Similar result is reported by Niehaus et al.,197 they studied the biosynthesis of fusarin C in the fungus Fusarium fujikuroi. The fusarin gene cluster consists of nine genes but they proved that only four genes are required for the biosynthesis of fusarin C. So, we concluded that it is difficult to predict what a gene actually does and how many genes are required for the biosynthesis of a single compound. The A. oryzae apdACE expression clone showed distinctive catalytic feature of cytochrome P450 apdE that it performs oxidative ring expansion of preaspyridone A 224 to form aspyridone A 84, benzylic hydroxylation of 224 to form 14-hydroxy preaspyridone A 238 and loses a phenoxide to form the novel compound, 5dephenylated pyridone 239. The apparent incorporation of [1-13C]-L-tyrosine by 5dephenylated pyridone 239 and the precursor compound preaspyridone 230 confirms that tyrosine is the precursor amino acid and biosynthesis of 239 is linked to preaspyridone 230. The structure of 5-dephenylated pyridone 239 and its derivatives 259 and 260 are similar to known fungal metabolites, the Atpenins 256-258. The 150 biosynthetic pathway of Atpenins is not reported and we suggest that they must be products of a similar PKS-NRPS cluster like the aspyridone genes. The polyketide chain in Atpenins and in 84, 224, 239, possess pendant methyls arranged in anti configuration. The methyls are derived form S-adenosyl methionine by the CMeT domain. The ER domain in ApdA is inactive and the stand alone enoyl reductase ApdC acting in trans sets the stereochemistry of the carbon bearing the methyls. The ApdC remarkably sets an opposite stereochemistry of the two methyls during the first and second cycle of the tetraketide producing anti dimethylation pattern. The characterization of 18-deshydroxy preaspyridone A 240 in apdACE and in trace amounts in other transformants (Table 3.1) shows a wider specificity of the adenylation domain to incorporate other amino acids than L-tyrosine. This is also in agreement of the in vitro studies performed by Tang and coworkers.107 The gene apdB encodes a cytochrome P450 enzyme which didn’t accomplish any chemical step when expressed with apdAC alone but was observed to increase the titres of the metabolites. However, when expressed with apdACE in A. oryzae apdACEB expression it was resolved that it catalyses N- hydroxylation of dephenylated 2 pyridone 239 to form a new compound 259. The apdACEB was an exclusive clone to form nine different compounds (Table 3.1) owing to the presence of two cytochrome P450s but the overall yield of this transformant was less (53.6 mg/L) as compared to apdACE (390 mg/L). We did not perceive a role for the FAD dependent mono oxygenase apdD. As we didn’t observe the production of aspyriodone B 226, we suspect it might have been catalysed by a gene outside the cluster which is possible in transcription mediated recombination where simulataneously many genes are turned on. The presence of apdD also lowered the yield (71.5 mg/L) of metabolites in apdACED expression than achieved in apdACE transformant (Table 3.1). The gene apdG didn’t exhibit any catalytic role in apdACEBG expression clone but increased the titres of metabolites produced. This might due to the fact that biosynthetic proteins work in clusters and with more proteins in a heterologous expression there is more protein-protein interaction and more production. But it cannot be concluded decisively without further experimental studies. The expression of tenellin genes with apd genes showed high substrate specificity of tenellin biosynthetic genes as we didn’t observe their particular role in the 151 transformants. The presence of tenA with apdAC increased the titres of the compounds with a similar effect as apdB in apdABC expression clone. Thus heterologous expressions of apd genes in suitable host, A. oryzae produced new compounds and help understand the role of specific genes in aspyridone cluster of A. nidulans. 152 Chapter 4 Experimental 4.1 General Chemicals and Equipment All chemicals and reagents used were of analytical grade and obtained from Sigma Aldrich, Fischer, Fluka analytical and BDH laboratories. All solvents used in HPLC and purification were HPLC grade. Deionized water was used in all experiments. Weighing balances were of Sartorius AX224. Small centrifuge for obtaining clear sample were from AG Hanburry 22331. Sterilizations were carried out using Astell Autoclave at 121 ºC for 15 minutes. Optical rotations were measured with an ADP 220 polarimeter at 589 nm. Melting points were determined using Electrothermal apparatus. IR data were obtained using a Perkin–Elmer FTIR instrument. 4.2 Mass Spectrometry Electrospray ionization (ESI) mass spectra were recorded on a VG Quattro-Mass spectrometer or Bruker microtof mass spectrometer. 4.3 Nuclear Magnetic resonance Spectroscopy NMR experiments were conducted on Varian VNMRS-500 spectrometer, 1H NMR at 500 MHz and 13 C NMR at 125 MHz. Chemical shifts were recorded in parts per million (ppm) and coupling constant (J) in Hz. 4.4 Analytical LCMS All crude extracts were prepared to a concentration of 10 mg/ml in HPLC grade methanol, centrifuged for 60 seconds and supernatant was placed in LCMS vials. 20 µl of the extracts were injected and analysed on a Waters 2795HT HPLC system. Detection was achieved by uv between 200 and 400 nm using a Waters 998 diode array detector, and by simultaneous electrospray (ES) mass spectrometry using a Waters ZQ spectrometer detecting between 150 and 600 m/z units. Chromatography (flow rate 1 ml/min) was achieved using a Phenomenex LUNA column (5 μ, C18, 100 Å, 4.6 × 250 mm) equipped with a Phenomenex Security Guard precolumn (Luna C5 300 Å) or Chromatography was achieved using a Phenomenex Kinetex column (2.6 µ, C18, 100 153 Å, 4.6 x 100 mm) equipped with a Phenomenex Security Guard precolumn (Luna C5 300 Å). Solvents used were: A, HPLC grade H2O containing 0.05 % formic acid; B, HPLC grade MeOH containing 0.045 % formic acid; and C, HPLC grade CH3CN containing 0.045 % formic acid. The following gradients were used: Method 1. Luna/MeOH: 0 min, 25% B; 5 min, 25% B; 51 min, 95% B; 53 min, 95% B; 55 min, 25% B; 59 min, 25% B; 60 min, 25% B. Method 2. Luna/MeOH: 0 min, 25% B; 13 min, 95% B; 15min, 95% B; 17 min, 25% B; 20 min, 25% B. Method 3. Kinetex/MeOH: 0 min, 10% B; 10 min, 90% B; 12 min, 90% B; 13 min, 10% B; 15 min, 10% B. Method 4. Kinetex/CH3CN: 0 min, 10% C; 10 min, 90% C; 12 min, 90% C; 13 min, 10% C; 15 min, 10% C. Method 5. Luna/CH3CN: 0 min, 5% C; 5 min, 5% C; 45 min, 75% C; 46 min, 95% C; 50 min, 95% C; 55 min, 5% C; 60 min, 5% C. 4.5 Preparative HPLC Purification of compounds was generally achieved using a Waters mass-directed autopurification system comprising of a Waters 2767 autosampler, Waters 2545 pump system, a Phenomenex LUNA column (5µ, C18, 100 Å, 10 × 250 mm) equipped with a Phenomenex Security Guard precolumn (Luna C5 300 Å) eluted at 4 ml/min. Solvents used were: A, HPLC grade H2O + 0.05% formic acid; solvent B, HPLC grade MeOH + 0.045% formic acid; solvent C, HPLC grade CH3CN + 0.045% formic acid. The postcolumn flow was split (100: 1) and the minority flow was made up with solvent A to 1 ml/min for simultaneous analysis by diode array detector (Waters 2998), evaporative light scattering (Waters 2424) and ESI mass spectrometry in positive and negative modes (Waters Quatro Micro). Method 1: 0 min, 25% B; 13 min, 95% B; 15 min, 95% B; 17 min, 25% B; 20 min, 25% B. Method 2: 0 min, 40% C; 15 min, 80% C; 15.50 min, 95% C; 16.50 min, 95% C; 17 min, 40% C; 20 min, 40% C. Method 3: 0 min, 50% C; 22 min, 75% C; 24 min, 95% C; 26 min, 95% C; 27 min, 50% C; 30 min, 50% C. 154 Method 4: 0 min, 55% C; 22 min, 60% C; 24 min, 95% C; 26 min, 95% C; 27 min, 55% C; 30 min, 55% C. Method 5: 0 min, 55% C; 22 min, 75% C; 24 min, 95% C; 26 min, 95% C; 27 min, 55% C; 30 min, 55% C. Method 6: 0 min, 50% C; 22 min, 65% C; 24 min, 95% C; 26 min, 95% C; 27 min, 50% C; 30 min, 50% C. 4.6 X-ray Crystallography X-ray diffraction data were analysed on a Bruker Microstar rotating anode diffractometer using Cu-Kα radiation (λ = 1.54178 Å). Data collections were performed using a CCD area detector from a single crystal mounted on a glass fibre. Absorption corrections were based on equivalent reflections using TWINABS or SADABS. The structures were solved using direct methods using SHELXS and refined against all Fo2 data with hydrogen atoms on carbon and oxygen atoms riding in calculated positions using SHELXL. 4.7 Solid media for growth of fungal spores The Aspergillus oryzae transformants and were first grown on Czapek Dox Agar (minimal media) and for further production of spores, they were inoculated and grown on DPY solid media for 7-10 days at 25 °C. Beauveria bassiana WT and RNAi strains of B. bassiana were grown on Potato Dextrose Agar (PDA) for 10 days at 25 °C. 4.7(a) Czapek Dox Agar 50 grams of Czapek Dox agar was dissolved on 1 litre of deionized water and sterilized in autoclave. 4.7(b) DPY media The spores of A. oryzae transformants were grown on plates in DPY media (dextrin-peptone-yeast extract). It is made by the following ingredients 2% (w/v) dextrin, 1% (w/v) polypeptone, 0.5% (w/v) yeast extract, 0.5% (w/v) potassium dihydrogen phosphate, 0.05% (w/v) magnesium sulphate and 2.5% (w/v) agar. 155 4.7(c) PDA media B. bassiana was grown on potato dextrose agar for growth of spores. 39 gram of PDA was dissolved in 1 litre of deionized water and sterilized by autoclave. 4.8 Preparation of Tenellin production Media The liquid media used for growing B. bassiana spores was Tenellin production media (TPM). The ingredients include D-mannnitol (50 g), KNO3 (5 g), KH2PO4 (1 g), MgSO4·7H2O (0.5 g), NaCl (0.1 g), CaCl2 (0.2 g), FeSO4·7H2O (20 mg) and mineral ion solution (10 ml, ZnSO4·7H2O (880 mg), CuSO4·5H2O (40 mg), MnSO4·4H2O (7.5 mg), boric acid (6 mg), and (NH4)6Mo7O24·4H2O (4 mg) made up to 1 L in deionized water) made up to 1 L in deionized water. This medium solution was divided into 100 ml each in 500 ml Erlenmeyer flask, covered with foam bung covered with aluminium foil and sterilized by autoclaving. 4.9 Czapek-Dox minimal medium (CD) A. oryzae tenPKS-dmbC strain was grown in CD minimal media. It was prepared by adding 10 g peptone, 20 g glucose, and 30 g sucrose, 50 ml of solution A (40 g NaNO3, 40 g KCl, 10 g MgSO4.7H2O, 0.2 g FeSO4.7H2O in 1 litre deionized water) and 50 ml of solution B (20 g K2HPO4 in 1 litre deionized water) in 1 litre deionized water. 100 ml of the media was divided in 500 ml conical Erlenmeyer flasks, covered with bung form and alumium foil and autoclaved. The A. oryzae spores were inoculated in flasks and were incubated for 4 days at 28 °C and shaken at 200 rpm. This was followed by changing the media (under sterile condition) with induction medium for production of secondary metabolites. The inducing medium was made by dissolving 20 g starch, 10 g peptone, 50 ml solution A and 50 ml solution B in 1 litre deionized water and incubated under the same previous conditions over 5-7 days. 4.9(a) Czapek-Maltose-Polypeptone (CMP) medium A This medium was prepared by adding 20 g maltose, 10 g polypeptone, 50 ml solution A and 50 ml solution B in 900 ml deionized water. The medium was divided into 100 ml each in 500 ml Erlenmeyer flask, covered with foam bung and aluminium foil and then sterilized by autoclaving. 156 4.9(b) Czapek-Maltose-Polypeptone (CMP) medium B The liquid medium for A. oryzae transformants studied in aspyridone biosynthesis (chapter 3) was made by adding 30 g sucrose, 20 g maltose, 10 g polypetone, 50 ml solution B and 50 ml solution C (60 g NaNO3, 10 g KCl, 10 g MgSO4.7H2O and 0.2 g FeSO4.7H2O in 1 litre deionized water) in 900 ml deionized water. The medium (100 ml) was divided in 500 ml Erlenmeyer flasks as described previously. 4.10 Culturing and inoculation of fungal spores in liquid media 10 mL of deionized water was added on plates having 10 days old growing spores of the fungus. The spores were made to pass into the deionized water by careful scratching the surface with sterilized loop. The spore suspension (1 ml) from the plate was added in each 100 mL liquid medium contained in 500 ml Erlenmeyer flask. The A. oryzae spores were allowed to grow in the liquid culture for 7 days on shakers at 200 rpm at 25 °C- 28 °C and B. bassiana spores were grown on shakers for at least 10 days at 25 °C at 150-200 rpm. 4.11 Extraction of A. oryzae transformants Cells and media (1 L) were homogenized using a hand-held electric blender and then acidified to pH 4.0 using 37% aqueous HCl. An equal volume of ethyl acetate was added and stirred for 10 min. The resulting mixture was vacuum filtered through Whatman no. 1 filter paper. The filtrate was transferred into a separating funnel and shaken vigorously. The mixture was allowed to stand to separate the layers. The organic layer was washed once with concentrated brine solution and then with deionized water. The organic phase was dried (MgSO4), filtered and evaporated to dryness. The crude extract was dissolved in 10 % aqueous methanol and defatted by extraction with hexane. The methanolic layer was evaporated to dryness and then made into a solution of 10 mg/ml in HPLC grade methanol and analysed by LCMS. For purification of compounds the crude extract was made into a solution of 50 mg/ml in HPLC grade methanol and 200 µl aliquots were injected in each run of mass-directed HPLC preparative purification. 157 4.12 Extraction of B. bassiana transformants The 10 days old cultures of B. bassiana were vacuum filtered and the mycelia was collected and kept in equal volume of acetone overnight. The acetone layer was separated from the mycelia by filtration with Whatman filter paper and acetone was concentrated by vacuum to form a brown aqueous extract. It was further diluted with deionized water and then extracted with ethyl acetate (2 × 500 ml). The ethyl acetate layer was separated from the water layer by a separating funnel. MgSO4 was added to absorb any trapped water droplets. The ethyl acetate was filtered and concentrated under vacuum to obtain a semi solid crude extract. The extract was dissolved in 10 % aqueous methanol and defatted by extraction with hexane. The methanolic layer was obtained in separated glass vial and dried. The crude extract was prepared to a concentration of 10 mg/ml in HPLC grade methanol and analysed by LCMS. 4.13 Purification of metabolites from A. oryzae pTAex3-tenS and A. oryzae tenSPKS-dmbNRPS The 1 litre culture of A. oryzae pTAex3-tenS was extracted by the procedure explained above and a crude extract of 70 mg was achieved. The extract was dissolved in HPLC grade methanol to a concentration of 50 mg/ml and used for mass-directed HPLC preparative purification. 100 – 200 μl of the crude solution was injected during successive rounds of a 20 minute HPLC program (Method 1). Fractions corresponding to prototenellin C 113 were collected and evaporated to yield 2.4 mg of pure compound. Similar protocols were used in purifying the prototenellin C 113 from 137 mg of a crude extract of A. oryzae tenSPKS-dmbNRPS obtained from 1 L fermentation and produced 9.6 mg of the pure compound. 4.13(a) Characterization of prototenellin C 113109 Prototenellin C 113, pale yellow solid, 1H NMR (CD3OD, 500 MHz) δ = 1.15 (d, J = 6.4 Hz, 3H, H-13), 1.29 (s, 3H, H-14), 1.93 (s, 3H, H-15), 2.85 (m,1H, H-16a), 2.99 (m, 1H, H-16b), 3.62 (m, 1H, H-12), 3.94 (m, 1H, H-5), 6.15 (d, 1H, J = 15 Hz, H-10), 6.68 158 (d, J = 8.2 Hz, 2H, H-19, H-21), 6.71 (m, 1H, H-9), 6.98 (m, 1H, H-8), 7.03 (d, J = 8.2 Hz, 2H, H-18, H-22); 13 C NMR (CD3OD, 125 MHz), δ = 11.5 (C-15), 16.3 (C-13), 22.9 (C-14), 36.3 (C-16), 61.2 (C-5), 73.3 (C-12), 75.3 (C-11), 114.4 (C-19, C-21), 124.2 (C-9), 126.3 (C-17), 130.5 (C-18, C-22), 131.7 (C-7), 138.9 (C-8), 145.5 (C-10), 155.0 (C-20), 163.1 (C-2), 188.3 (C-6), 194.0 (C-4). The 13 C signal for C-3 was not observed. HRMS calculated for C21H25NO6Na: 410.1580; found 410.1578 [M]Na+. Purification of metabolites from A. oryzae dmbS –tenC and A. oryzae dmbS- 4.14 dmbC A 1 litre fermentation media was grown for A. oryzae dmbS –tenC at 25 °C (10 flasks × 100 mL) for 7 days on shakers at 200 rpm. The fungal cultures were extracted with protocol explained in section 4.14, which gave a dark brown crude extract of 111 mg. This was made to a solution of 50 mg/ml with HPLC grade methanol for purification of compounds on mass- directed preparative HPLC. 100µl - 200µl of crude extract was injected in each successful preparative run. Pure fractions having bright yellow colour of predmB A were collected after 25 preparative runs and dried to evaporation. 23 mg of pure compound was achieved. 363 mg of crude extract was obtained from a 1 litre culture of A. oryzae dmbS-dmbC and 17.6 mg of pure predmb A was produced by following a similar protocol as explained above. 4.14(a) Characterization of predmbA 118 PreDMB A 118, brownish yellow solid; IR (neat): νmax 3277, 2961, 2922, 2876, 1648, 1594, 1514 cm-1; 1H NMR (CD3OD, 500 MHz) δ = 0.92 (t, J = 7.5 Hz, 3H, H15), 1.09 (d, J = 7.5 Hz, 3H, H-16), 1.41 (m, 2H, H-14), 2.20 (m, 1H , H-13), 2.90 (brd, 1H, H-17a), 3.00 (brd, 1H, H-17b), 4.08 (brs, 1H, H-5), 5.98 (dd, J = 7.8, 15.2 Hz, 1H, H-12), 6.27 (dd, J = 10.8, 15.2 Hz, 1H, H-11), 6.43 (dd, J = 11.9, 14.5 Hz, 1H, H9), 6.70 (d, J = 8.1 Hz, 2H, H-20, H-22), 6.83 (dd, J = 14.5, 10.8 Hz, 1H, H-10), 7.00 (d, J = 8.1 Hz, 2H, H-19, H-23), 7.21 (d, J = 15.4 Hz, 1H, H-7), 7.53 (dd, J = 11.9, 15.4, 159 Hz, 1H, H-8), 13C NMR (CD3OD, 125 MHz) δ = 10.7 (C-15), 18.7 (C-16), 29.2 (C-14), 36.2 (C-17), 38.8 (C-13), 62.8 (C-5), 114.7 (C-20, C-22), 119.7 (C-7), 126.3 (C-18), 128.7 (C-11), 128.8 (C-9), 130.4 (C-19, C-23), 144.3 (C-10), 145.2 (C-8), 147.8 (C-12), 155.9 (C-21), 173.3 (C-6), 173.7 (C-2), 195.9 (C-4). The 13 C signal for C-3 was not observed. HRMS calculated for C22H26NO4: 368.1856; found 368.1851 [M]H+. 4.15 Purication of metabolites from A. oryzae tensPKS –dmbC 3 litres of A. oryzae tenSPKS-dmbC strain (30 flasks × 100 ml) were grown at 28 °C at 200 rpm. After extraction of fungal cultures with ethyl acetate (see section 4.14), a crude extract of 239 mg was obtained. HPLC grade methanol was added to make a 50 mg/ml solution for mass-directed purification of compounds on preparative HPLC. The crude extract was subjected to 24 successful preperative runs with Method 2 and two pure metabolites were obtained. The yield of compound 146 was 9.5 mg and compound 147 was 9 mg. 4.15(a) Characterization of Compound A 146 Light brown viscous oil, [α]22D -16.9 (c = 0.23, MeOH); IR (neat): νmax 2932, 2872, 2342, 1761, 1631, 1355, 1190 1084 cm-1; 1H NMR (CDCl3, 500 MHz) δ = 0.92 (t, J = 7 Hz, 3H, H-1), 1.36 (m, 2H, H-2), 1.37 (m, 1H, H-3a), 1.45 (m, 1H, H-3b), 1.65 (m, 1H, H-4a), 1.81 (m, 1H, H-4b), 2.01 (ddd, J = 12, 10.5, 12 Hz 1H, H-6a), 2.55 (ddd, J = 9, 6, 12 Hz, 1H, H-6b), 3.65 (t, J = 9, 12 Hz, 1H, H-7), 4.43 (m, 1H, H-5), 5.95 (b, 1H, H10a), 6.54 (b, 1H, H-10b), 13C NMR (CDCl3, 125 MHz) δ = 14.4 (C-1), 22.9 (C-2), 27.8 (C-3), 35.5 (C-4), 36.1 (C-6), 45.2 (C-7), 79.6 (C-5), 131.8 (C-10), 136.1 (C-8), 170.1(C-9), 176.4 (C-11). HRESIMS calculated for C11H17O4: 213.1121; observed 213.1130 [M]H+. 160 4.15(b) Characterization of compound B 147 light color viscous oil, IR (neat): νmax 2962, 2930, 2873, 1736, 1582, 1454, 1045, 878 cm-1; 1H NMR (CDCl3, 500 MHz) δ = 0.92 (t, J = 7 Hz, 3H, H-1), 1.38 (m, 2H, H-2), 1.46 (m, 2H, H-3), 1.71 (m, 1H, H-4a), 1.79 (m, 1H, H-4b), 4.99 (t, J = 6.2 Hz, 1H, H5), 6.79 (b, 1H, H-10a), 7.19 (b, 1H, H-10b), 7.96 (b, 1H, H-6); 13C NMR (CDCl3, 125 MHz) δ = 14.0 (C-1), 22.6 (C-2), 27.3 (C-3), 33.1 (C-4), 80.8 (C-5), 125.0 (C-7), 128.5 (C-8), 133.6 (C-10), 153.5 (C-6), 169.5 (C-9), 171.7 (C-11). HRESIMS calculated for C11H14O4Na: 233.0784; observed 233.0799 [M]Na+. 4.16 Purication of metabolites from A. oryzae apdACE A brown crude extract of 1303 mg was formed from 1 litre culture of A. oryzae apdACE strain by growing the fungus according to standard conditions and extraction with ethyl acetate. The crude extract was made to a solution of 50 mg/ml with HPLC grade methanol for purification of metabolites on the preparative HPLC by following the gradient given in Method 5. Pure fractions of corresponding metabolites were collected and dried to evaporation. Characterisation of metabolites from A. oryzae apdACE 4.16(a) 14- Hydroxy preaspyridone A 238 Brown viscous oil, [α]22D -158.8 (c = 1.29, MeOH); IR (neat): νmax 3317, 3020, 2964, 1651, 1214 cm-1; 1H NMR (CD3OD, 500 MHz) δ = 0.81 (m, 3H, H-11), 0.82 (m, 3H, H-12), 0.95 (d, J = 6.5 Hz, 3H, H-13), 1.09 (m, 1H, H-10a), 1.23 (m, 1H, H-9), 1.29 (m, 1H, H-8a), 1.33 (m, 1H, H-10b), 1.40 (m, 1H, H-8b), 3.55 (q, J = 7 Hz, 1H, H-7), 4.22 (d, J = 3.5 Hz, 1H, H-5), 4.99 (d, J = 4 Hz, 1H, H-14), 6.65 (d, J = 8.5 Hz, 2H, H-17, H-19), 7.11 (d, J = 8.5 Hz, 2H, H-16, H-20) ; 13C NMR (CD3OD, 125 MHz) δ = 11.4 161 (C-11), 17.5 (C-13), 19.6 (C-12), 30.2 (C-10), 33.5 (C-9), 35.5 (C-7), 41.2 (C-8), 68.5 (C-5), 74.8 (C-14), 115.5 (C-17, C-19), 129.6 (C-16, C-20), 130.3 (C-15), 158.6 (C18), 195.2 (C-6). The 13 C signals for C-2, C-3 and C-4 were not observed. HRESIMS calculated for C19H25NO5Na: 370.1624; observed 370.1624 [M]Na+. 4.16(b) 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239 Light brown solid, mp 120 °C; [α]22D -24.3 (c = 0.82, MeOH); IR (neat): νmax 3408, 3298, 2923, 2287, 1734, 1601, 1462, 1227 cm-1; 1H NMR (DMSO, 500 MHz) δ = 0.79 (m, 3H, H-12), 0.81 (m, 3H, H-13), 1.02 (d, J = 7 Hz, 3H, H-14), 1.09 (m, 1H, H-11a), 1.21(m, 1H, H-9a), 1.25 (m, 1H, H-11b), 1.33 (m, 1H, H-10), 1.49 (m, 1H, H-9b), 4.25 (q, J = 7 Hz, 1H, H-8), 5.92 (d, J = 7.5 Hz, 1H, H-5), 7.60 (t, J = 6.5, 7 Hz, 1H, H-6), 11.49 (s, 1H, H-1); 13C NMR (DMSO, 125 MHz) δ = 11.1 (C-12), 16.7 (C-14), 18.8 (C13), 29.7 (C-11), 31.7 (C-10), 39.9 (C-8), 40.0 (C-9), 99.1 (C-5), 105.9 (C-3), 142.8 (C6), 161.8 (C-2), 177.6 (C-4), 211.9 (C-7). HRESIMS calculated for C13H20NO3: 238.1437; observed 238.1428 [M]H+. Crystals were formed by slow evaporation in methanol. Crystals size/mm3 = 0.29 × 0.25 × 0.06, formula C13H19NO3, (M =237.29): monoclinic, space group P21 (no. 4), a = 9.0939(16) Å, b = 30.695(5) Å, c = 9.1390(16) Å, β = 94.405(5)°, V = 2543.5(8) Å3, Z = 8, T = 100(2) K, μ/mm-1 = 0.713, Dcalc = 1.239 g/mm3, range for data collection = 9.7 to 133.62°, reflections collected 60175, independent reflections 8598 (Rint = 0.0426), final R1 was 0.0294 and wR2 was 0.0759, Largest diff. peak/hole/e Å-3 = 0.12/-0.19, flack parameter 0.03(8), ccdc code 941139. 162 Aspyridone A 8480 4.16(c) Light brown solid, mp 178 °C; [α]22D -8.2 (c = 0.48, MeOH); IR (neat): νmax 2961, 2928, 1648, 1610, 1516, 1458, 1378, 1217, 1175, 992, 835, 588 cm-1; 1H NMR (CD3OD, 500 MHz) δ = 0.87 (t, J = 7.5 Hz, 3H, H-12), 0.90 (d, J = 6.5 Hz, 3H, H-13), 1.13 (d, J = 6.5 Hz, 3H, H-14), 1.16 (m, 1H, H-11a), 1.33 (m, 1H, H-9a), 1.34 (m, 1H, H-11b), 1.41 (m, 1H, H-10), 1.64 (m, 1H, H-9b), 4.39 (q, J = 6.8 Hz, 1H, H-8), 6.80 (d, J = 8.5 Hz , 1H, H-17, H-19), 7.26 (d, J = 8.5 Hz, 1H, H-16, H-20), 7.47 (s, 1H, H-6); 13 C NMR (CD3OD, 125 MHz) δ = 11.7 (C-12), 17.6 (C-14), 19.4 (C-13), 31.0 (C-11), 33.7 (C -10), 41.2 (C-9), 41.9 (C-8), 107.0 (C-3), 116.0 (C-5), 116.15 (C-17, C-19), 125.2 (C-15), 131.5 (C-16, C-20) 140.6 (C-6), 158.3 (C-18), 163.9 (C-2), 177.6 (C-4), 214.4 (C-7). HRESIMS calculated for C19H24NO4: 330.1699; observed 330.1692 [M]Na+. The specific rotation and melting point of aspyridone A 84 are not reported before in literature. Crystals were formed by slow evaporation in methanol and diethyl ether. Brown crystals, formula C19H23NO4, M =329.38, monoclinic, space group P21, a = 7.3372(7) Å, b = 22.647(3) Å, c = 20.618(2) Å, β = 90.112(6)°, V = 3426.0(6) Å3, Z = 8, T = 100(2) K, μ/ mm-1 = 0.727, Dcalc = 1.277 g/mm3, range for data collection = 3.9 to 132.38°, reflections collected 56007, independent reflections 5995 (Rint = 0.0792), final R1 was 0.0452 and wR2 was 0.1139, Largest diff. peak/hole/e Å-3 = 0.28/-0.29, flack parameter 0(10), ccdc code 941138. 4.16 (d) 18-deshydroxypreaspyridone A 240 Brown viscous oil, [α]22D -142.3 (c = 0.15, MeOH); IR (neat): νmax 3019, 2962, 2875, 1709,1656, 1600, 1214 cm-1; 1H NMR (CD3OD, 500 MHz) δ = 0.83 (m, 3H, H-11), 163 0.84 (m, 3H, H-12), 1.02 (d, J = 7 Hz, 3H, H-13), 1.09 (m, 1H, H-10a), 1.27 (m, 1H, H9), 1.34 (m, 1H, H-10b), 1.33 (m, 1H, H-8a), 1.44 (m, 1H, H-8b), 2.98 (dd, J = 5.5, 14 Hz, 1H, H-14a), 3.07 (dd, J = 4, 14 Hz, 1H, H-14b), 3.65 (q, J = 6.7 Hz, 1H, H-7), 4.11 (t, J = 4.5 Hz, 1H, H-5), 7.16 (m, 2H, H-17, H-19), 7.17 (m, 1H, H-18), 7.22 (m, 2H, H16, H-20); 13 C NMR (CD3OD, 125 MHz) δ = 11.5 (C-11), 17.6 (C-13), 19.5 (C-12), 30.3 (C-10), 33.5 (C-9), 36.0 (C-7), 38.3 (C-14), 41.3 (C-8), 63.5 (C-5), 128.1 (C-18), 129.5 (C-16, C-20), 130.7 (C-17, C-19), 136.9 (C-15), 196.0 (C-6), 197.7 (C-4). The 13 C signals for C-2 and C-3 were not observed. HRESIMS calculated for C19H25NO3Na: 338.1726; observed 338.1735 [M]Na+. 4.17 Purification of metabolites from A. oryzae apdACEB 1 litre culture (100 ml x 10 flasks) of the fungal strain A. oryzae apdACEB was grown in CMP medium B at 28 °C for 7 days at 200 rpm. The culture was then extracted with ethyl acetate according to standard method given in section 4.11. A crude extract of 1.13 g was obtained after vacuum concentration of the ethyl acetate layer and 50 mg/ml solution was made with HPLC grade methanol. For purification of metabolites 200 µl of the crude extract was injected in each successive run on the preparative HPLC following the gradient in Method 3. The purified fractions of respective metabolites were collected and dried. Characterization of new metabolites from A. oryzae apdACEB 4.17(a) Preaspyridone A (minor) 224 Pale white crystalline solid, mp 146-148 °C; [α]22D 98.4 (c = 0.51, CHCl3); IR (neat): νmax 3262, 2962, 1693, 1650, 1589 cm-1; 1H NMR (DMSO, 500 MHz) δ = 0.80 (m, 3H, H-12), 0.82 (m, 3H, H-11), 1.03 (d, J = 6 Hz, 3H, H-13), 1.09 (m, 1H, H-10a), 1.24 (m, 1H, H-9), 1.26 (m, 1H, H-10b), 1.27 (m, 2H, H-8), 2.82 (td, J = 4.5, 15.5 Hz, 2H, H14), 3.53 (q, J = 6.8 Hz, 1H, H-7), 4.08 (brs, 1H, H-5), 6.59 (d, J = 8 Hz, 2H, H-17, H164 19), 6.88 (d, J = 8.5 Hz, 2H, H-16, H-20), 8.93 (s, 1H, H-1), 9.17 (brs, 1H, H-18); 13C NMR (125 MHz, DMSO) δ = 10.8 (C-11), 16.7 (C-13), 18.9 (C-12), 28.7 (C-10), 31.5 (C-9), 33.4 (C-7), 35.4 (C-14), 39.6 (C-8), 62.3 (C-5), 99.9 (C-3), 114.7 (C-17, C-19), 125.3 (C-15), 130.6 (C-16, C-20), 155.7 (C-18), 175.4 (C-2), 191.9 (C-6), 194.4 (C-4). HRESIMS calculated for C19H26NO4: 332.1856; observed 332.1852 [M]H+. Crystals were formed by slow evaporation in methanol. Formula C19H25NO4, M =331.40, monoclinic, space group P21, a = 8.3357(13) Å, b = 11.294(2) Å, c = 9.9646(17) Å, α = 90.00°, β = 103.551(8)°, V = 912.0(3) Å3, Z = 2, T = 100(2) K, m/ mm-1 = 0.683, Dcalc = 1.207 g/mm3, range for data collection = 9.12 to 132.5°, reflections collected 10439, independent reflections 3021( Rint = 0.0533), final R1 was 0.0467 and wR2 was 0.1226, Largest diff. peak/hole/e Å-3 = 0.28/-0.29, flack parameter 0.2 (2), ccdc code 941137. 4.17(b) Preaspyridone A (major diastereomer) 230107 Brown viscous oil, [α]22D -166.3 (c = 0.95, CHCl3) ; IR (neat): νmax 3020, 2964, 1653, 1602, 1214 cm-1. 1H NMR (500 MHz, DMSO) δ = 0.80 (m, 3H, H-12), 0.79 (m, 3H, H11), 0.95 (d, J = 7 Hz, 3H, H-13), 1.07 (m, 1H, H-10a), 1.28 (m, 1H, H-9), 1.29 (m, 1H, H-10b), 1.29 (m, 1H, H-8a), 1.38 (m, 1H, H-8b), 2.82 (d, J = 4.5 Hz, 2H, H-14), 3.51 (q, J = 6.6 Hz, 1H, H-7), 4.08 (brs, 1H, H-5), 6.59 (d, J = 8.5 Hz, 2H, H-17, H-19), 6.90 (d, J = 8 Hz, 2H, H-16, H-20), 8.92 (s, 1H, H-1), 9.17 (brs, 1H, H-18); 13C NMR (125 MHz, DMSO), 10.9 (C-11), 17.1 (C-13), 18.9 (C-12), 28.6 (C-10), 31.7 (C-9), 33.5 (C-7), 35.7 (C-14), 39.1 (C-8), 62.4 (C-5), 99.8 (C-3), 114.7 (C-17, C-19), 125.4 (C-15), 130.6 (C-16, C-20), 155.9 (C-18), 175.6 (C-2), 191.9 (C-6), 194.3 (C-4). HRESIMS calculated for C19H26NO4Na: 354.1676; observed 354.1677 [M]Na+. 165 4.17 (c) 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 259 Brown viscous oil, [α]22D -14.3 (c = 0.20, MeOH) ; IR (neat): νmax 3104, 2928, 1731, 1635, 1611, 1453, 1200, 751 cm-1; 1H NMR (CD3OD, 500 MHz) δ = 0.87 (t, J = 7.2 Hz, 3H, H-12), 0.92 (d, J = 7 Hz, 3H, H-13), 1.11 (d, J = 6.5 Hz, 3H, H-14), 1.18 (m, 1H, H-11a), 1.33 (m, 1H, H-9a), 1.36 (m, 1H, H-11b), 1.41 (m, 1H, H-10), 1.62 (m, 1H, H9b), 4.31 (q, J = 7 Hz, 1H, H-8), 5.97 (d, J = 8 Hz , 1H, H-5), 7.94 (d, J = 7.5 Hz, 1H, H-6); 13C NMR (CD3OD, 125 MHz) δ = 11.7 (C-12), 17.3 (C-14), 19.3 (C-13), 31.0 (C11), 33.5 (C -10), 41.1 (C-9), 42.1 (C-8), 98.9 (C-5), 107.4 (C-3), 142.3 (C-6),160.2 (C2), 176.0 (C-4), 213.6 (C-7). HRESIMS calculated for C13H19NO4Na: 276.1206; observed 276.1218 [M]Na+. 4.17(d) 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 260 Brown viscous oil, [α]22D -32.5 (c = 0.12, MeOH) ; IR (neat): νmax 2961, 2930, 1661, 1611, 1465, 1388, 975 cm-1; 1H NMR (DMSO, 500 MHz) δ = 0.80 (t, J = 7.2 Hz, 3H, H-12), 0.84 (d, J = 6.5 Hz, 3H, H-13), 1.03 (d, J = 7 Hz, 3H, H-14), 1.13 (m, 1H, H11a), 1.25 (m, 1H, H-9a), 1.25 (m, 1H, H-11b), 1.37 (m, 1H, H-10), 1.49 (m, 1H, H-9b), 3.93 (s, 3H, H-15), 4.15 (q, J = 7 Hz, 1H, H-8), 5.99 (d, J = 7.5 Hz , 1H, H-5), 8.24 (d, J = 7.5 Hz, 1H, H-6); 13C NMR (DMSO, 125 MHz) δ = 11.1 (C-12), 16.5 (C-14), 18.6 (C-13), 29.4 (C-11), 31.8 (C-10), 39.6 (C-9), 39.9 (C-8), 64.4 (C-15), 98.4 (C-5), 146.2 (C-6), 109.9 (C-3), 159.9 (C-2), 177.3 (C-4), 214.5 (C-7). HRESIMS calculated for C14H21NO4Na: 290.1362; observed 290.1368 [M]Na+. 4.18 Purification of metabolites from A. oryzae apdACED The A. oryzae apdACED was grown in 1 litre CMP medium B (10 flasks ×100 ml) at 28°C for 7 days with constant shaking at 200 rpm. The fungal cultures were then 166 extracted with ethyl acetate following the protocol explainedin section xx. A dried crude extract of 1.128 g was obtained which was made to a concentration of 50 mg/ml with HPLC grade methanol for purification of metabolites on preparative HPLC with gradient given in Method 6. The pure fractions of metabolites were collected and dried. Characterization of new metabolites from A. oryzae apdACED 4.18(a) Z-5, 14-anhydropreaspyridone A 267 Bright yellow solid, [α]22D 15.1° (c = 0.13, MeOH); IR (neat): νmax 3667, 3190, 2961, 2876, 2366, 1691, 1584 cm-1; 1 H NMR (DMSO, 500 MHz) δ = 0.79 (m, 3H, H-11), 0.82 (m, 3H, H-12), 1.05 (d, J = 6.5 Hz, 3H, H-13), 1.08 (m, 1H, H-10a), 1.32 (m, 1H, H-9), 1.32 (m, 1H, H-10b), 1.38 (m, 1H, H-8a), 1.47 (m, 1H, H-8b), 3.75 (q, J = 6.6 Hz, 1H, H-7), 6.37 (s, 1H, H-14), 6.78 (d, J = 8.5 Hz, 2H, H-17, H-19), 7.46 (d, J = 8.5 Hz, 2H, H-16, H-20); 13C NMR (DMSO, 125 MHz) δ = 10.9 (C-11), 16.8 (C-13), 18.8 (C12), 28.7 (C-10), 31.5 (C-9), 40.4 (C-8), 110.4 (C-14), 115.6 (C-17, C-19), 124.8 (C15), 131.5 (C-16, C-20), 158.8 (C-18), 184.0 in CD3OD (C-4). The 13C signals for C-2, C-3, C-4, C-5, C-6 and C-7 were not observed. HRESIMS calculated for C19H23NO4Na: 352.1519; observed 352.1528 [M]Na+. 167 Chapter 5 Summary and Future Perspective Iterative HRPKS-NRPS produce a wide array of diverse, bioactive and biosynthetically intriguing compounds. The programming rules embedded in the PKSNRPS enzymes have been under investigation for many years and still provide undiscovered horizons for researchers. In this research we isolated and characterized new compounds produced from different genetically modified transformants (particularly from heterologous expression and gene silencing). The structures of the isolated compounds gave clues to the biosynthetic potential of the PKS-NRPS enzymes and also the tailoring enzymes present in the PKS-NRPS clusters. We characterized two new putative A. oryzae wild type compounds 146 and 147 from A. oryzae tenSPKS-dmbC but couldn’t determine the chemical product of TENSPKS acting without its NRPS moiety. The tenC silencing in B. bassiana by the carbon inducible promoter PamyB failed to deliver any outcome because B. bassiana didn’t produce tenellin compounds when we changed the carbon source in TPM media from mannitol to maltose and glucose. The investigation of the aspyridone pathway of A. nidulans and heterologous expression of apd genes in A. oryzae gave insight into the unique catalytic capabilities of the cytochrome P450 present in the apd cluster and the biosynthetic potential of the PKS-NRPS. The cytochrome P450 ApdE has oxidative ring expanding and unusual dephenylation activity. ApdB catalyses N-hydroxylation in dephenylated 2-pyridones. The production of 18-deshydroxy preaspyridone 240 displays wider amino acid selectivity besides tyrosine. The compounds 239, 259, 260 and 267 show more diverse chemical activities for apd PKS-NRPS enzymes as compared to tenellin and desmethyl bassianin PKS-NRPS gene clusters. 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