Download THE IDENTIFICATION AND CHARACTERISATION OF THE

THE IDENTIFICATION AND CHARACTERISATION OF THE
BIOSYNTHETIC PATHWAY INVOLVED IN THE PRODUCTION OF
2, 5-DIPHENYLOXAZOLE BY Streptomyces polyantibioticus
I.K. Kemp and P.R. Meyers
Department of Molecular and Cell Biology
University of Cape Town, Private Bag X3, Rondebosch, Cape Town, South Africa, 7701.
E-mail: [email protected]; [email protected]
RESULTS AND DISCUSSION
INTRODUCTION
The actinomycete, Streptomyces polyantibioticus SPRT (Le Roes-Hill & Meyers, 2009), was
isolated from soil collected from the banks of the Umgeni River, KwaZulu-Natal Province, South
Africa, as part of an antibiotic-screening programme. It exhibited antibiosis against M.
tuberculosis H37RvT (the causative agent of tuberculosis), which prompted interest in its antibiotic
production. An antibacterial compound produced by S. polyantibioticus SPRT was isolated and its
structure was determined by X-ray crystallography and nuclear magnetic resonance (NMR) to be
2, 5-diphenyloxazole (DPO). Giddens et al. (2005) reported that DPO showed activity against
non-replicating persistent cells of M. tuberculosis, which are difficult to eradicate using traditional
anti-TB drugs. The authors suggested that simple oxazole derivatives such as DPO may
therefore be feasible options in the search for new antitubercular agents. DPO is currently only
known to be synthesised chemically (Adrova et al., 1956), therefore its discovery from a biological
origin is of great interest. DPO is unusual in that it is a 2,5-disubstituted oxazole, whereas most
other disubstituted oxazoles from biological sources are 2,4-substituted.
1. Isolation of 2, 5-diphenyloxazole from S. polyantibioticus SPRT
The antibacterial compound, 2, 5-diphenyloxazole was isolated from the fermentation broth and
mycelial mass of S. polyantibioticus SPRT. The activity of the compound was detected through
bioautography, showing an Rf of 0.8 with M. aurum A+ as the test organism. Chemicallysynthesized 2, 5-diphenyloxazole was used as a positive control for the detection of the bioactivity
and its Rf value correlated with the biologically-produced compound. Reverse-phase HPLC was
employed to corroborate the results obtained from the bioautography and the retention times of
the biologically-produced DPO and chemically-synthesized DPO were identical. The results of the
bioautography and reverse-phase HPLC confirmed the production of DPO by S. polyantibioticus
SPRT and its activity against M. aurum A+.
2. Isolation of NRPS gene cluster
2.1 Adenylation domain amplification
Based on the structure of DPO (Figure 1), a biosynthetic scheme for the synthesis of this
molecule has been proposed (Figure 2), whereby a non-ribosomal peptide synthetase (NRPS)
condenses a molecule of benzoic acid with 3-hydroxyphenylalanine. The dipeptide is converted
to a diphenyloxazole derivative by heterocyclisation and a final decarboxylation step leads to
DPO. NRPSs are large multifunctional enzymes that are able to synthesize functionally diverse
peptides. They all share a modular structural organization in which each particular module has a
specific function in recognition, activation or modification of a substrate residue in the final peptide
product. A common mechanism is shared by all NRPSs in the biosynthesis of non-ribosomal
peptides, whereby the modules interact in an ordered fashion to catalyse the formation of
successive peptide bonds (Challis et al., 2000; Marahiel et al., 1997).
NRPS adenylation domains were amplified from S. polyantibioticus SPRT by primers specific for
adenylation domain conserved motifs (A3F/A7R) (Ayuso-Sacido & Genilloud, 2005). The
amplified products were subsequently cloned into the pGemT Easy vector, transformed into
competent E. coli DH5α cells and screened to confirm the presence of inserts using colony PCR.
After confirmation, the clones were sent for sequencing and the resulting sequences were
subjected to both nucleotide and protein BLAST analysis in order to confirm their identity as
adenylation domains. Thirty-four clones were identified as carrying an insert with homology to
known adenylation domains contained within the NCBI database (Table 1). Thirteen (13) unique
substrate specificities were determined when the protein sequences were inserted individually into
the NRPSpredictor2 program (Röttig et al., 2011; Rausch et al., 2005), which gives an indication
of the most probable specificity of the binding pocket of each domain.
Table 1. Specificity Binding Pocket Code and Amino Acid Specificity
Clone AD-2
Clone AD-16
Clone A-1
Clone A-7
Clone A-8
Clone A-11
Clone A-12
Clone A-16
Clone A-18
Clone A-19
Clone A-22
Clone A-28
Clone A-31
Clone A-40
Clone A-48
Clone A-60
Clone A-69
Clone A-74
Clone A-32
Clone A-51
Clone A-5
Clone A-15
Clone A-58
Clone A-66
Clone A-68
Clone A-70
Clone A-14
Clone A-33
Clone A-36
Specificity Binding Pocket Code Residue and
Position
23 23 23 27 29 30 32 33 33 51
5
6
9
8
9
1
2
0
1
7
D V Q
F
N A H M V
D A
F
F
L G V T
F
D M V Q
F
G L V Y
D
F W N V G M V H
D M V Q
F
G L V Y
D M V Q
F
G L V Y
D
F W N V G M V H
D A
F
F
L G A T
F
D C G T A A A V D
D M V Q
F
G L V Y
D
F W N V G M V H
D M E N L G L
I
N
D A
F
F
L G A T
F
D M V Q
F
G L V Y
D V G
F
V
D A
F
F
L G A A T
D M V Q
F
G L V Y
D A A D V G
F
V D
G
I
Y H L G L
L C
D V W H
F
S
L
I
D
D
F W N V G M V H
D V W H
F
S
L
I
D
D A
F
F
L G V T
F
D C G T A A A V D
D M V Q
F
G L V Y
D M V Q
F
G L V Y
D M V Q
F
G L V H
G
I
Y H L G L
L C
D V W H
F
S
L
I
D
-
Clone A-53
D
M
V
Q
F
G
L
V
Y
Clone A-63
D
V
W
H
F
S
L
I
D
Adenylation
domain Source
Figure 1. The structure of 2,5-diphenyloxazole (DPO).
Figure 2 Proposed reaction scheme for the synthesis of DPO in S. polyantibioticus SPRT. (A) Benzoic Acid, (B)
β-Hydroxyphenylalanine, (C) Benzoyl-β-Hydroxyphenylalanine, (D) 4-Carboxy 2,5-Diphenyloxazole, (E) DPO.
Reactions are shown by red arrows or lines, 1- peptide bond formation, 2- Cyclization, 3- Decarboxylation. An
NRPS is proposed to catalyse the condensation of (A) and (B), as well as the proposed heterocyclization of (C)
to form (D) (Stegmann, 2011).
It seems likely that DPO is synthesized non-ribosomally by S. polyantibioticus SPRT due to the
condensation between benzoic acid and β-hydroxyphenylalanine and the heterocyclization across
the peptide bond to form 4-carboxy 2,5-diphenyloxazole. The NRPS responsible for the
biosynthesis of DPO is proposed to have Aryl carrier protein (ArCP), Cyclization, Adenylation,
Oxidation, Peptidyl carrier protein (PCP) and Thioesterase domains (Figure 3).
Figure 3 Proposed module arrangement for a DPO producing NRPS. Benzoate and β-hydroxyphenylalanine
bind to the serine residue belonging to the 4‘-phosphopantetheine (4‘-PP) arm of their respective carrier protein
domains to form an activated thioester derivative. Benzoate binds to the 4‘-PP of ArCP forming a benzoyl
intermediate (A) and β-hydroxyphenylalanine binds to the 4‘-PP of PCP forming a β-hydroxyphenylalanyl
intermediate (B). Domains: ArCP - aryl carrier protein, Cy – cyclization domain, A – adenylation domain, Ox –
oxidation domain, PCP - peptidyl carrier protein, TE – thioesterase domain (Stegmann, 2011).
[NOTE: As illustrated above, the Ox domain would not be required, as the phenylalanine side chain has already
been hydroxylated. The proposed Ox domain has been suggested for hydroxylation of phenylalanine if the
NRPS binds Phe rather than 3-OH Phe]
The overall aim of this project is to elucidate the biosynthetic pathway involved in DPO synthesis,
which consists of the identification and characterisation of the genes involved in the production of
DPO, thereby confirming whether it is in fact synthesized by an NRPS. This work will lay the
foundations for future combinatorial biosynthetic studies to develop a range of oxazole derivatives
that can be used to test for enhanced antitubercular activity and therefore could become
candidates for development as novel drugs to treat drug resistant tuberculosis.
RESEARCH STRATEGY
SPRT
The identification of an adenylation domain within the genome of S. polyantibioticus
with a
binding pocket substrate specificity for phenylalanine or β-hydroxyphenylalanine would most likely
indicate an NRPS responsible for the biosynthesis of DPO. PCR amplification, using suitable
degenerate primers, would allow for the detection of these adenylation domains within S.
polyantibioticus SPRT and their amino-acid binding specificities could be determined by
comparison with the binding-pocket specificities of adenylation domains for which the amino-acid
substrates are known. Larger fragments of the gene cluster may then be detected by Southern
hybridisation and further sequencing would reveal the remainder of the genes constituting the
DPO gene cluster. This would be one of only a handful of oxazole biosynthetic gene clusters
characterised from actinomycetes (Onaka et al., 2005: Zhao et al., 2006; Pulsawat et al., 2007).
The identification of cyclization domains within the genome of S. polyantibioticus SPRT may be
analysed in a similar fashion to the adenylation domains and their presence would indicate
heterocyclization across the peptide bonds they form in the biosynthesis of non-ribosomal peptide
products. PCR primers specific for oxazole and thiazole producing cyclization domains may be
used to amplify such domains in the genome.
In addition to searching for NRPS genes, the presence of an orthologue of the encP gene, coding
for phenylalanine ammonia-lyase (PAL), a key enzyme in the generation of benzoic acid for the
synthesis of enterocin in ‗Streptomyces maritimus‗ strain DSM 41777T, within the genome of S.
polyantibioticus SPRT would be of great interest. This is not only because of the rarity of benzoic
acid biosynthesis in bacterial systems, but also because S. polyantibioticus SPRT is proposed to
utilize benzoic acid in the synthesis of 2,5-diphenyloxazole (DPO). The encP orthologue could be
amplified from the genome of S. polyantibioticus SPRT using suitable PCR primers.
If benzoic acid is not synthesized via a PAL-mediated pathway in S. polyantibioticus SPRT, it could
be synthesized via a novel variation on the phenylacetate pathway for the degradation of
phenylalanine. Besides the aerobic process catalysed by PAL in ‗S. maritimus‘ strain DSM
41777T, other aerobic and anaerobic pathways for the production of benzoyl-CoA and derivatives
do exist, such as in the β-subclass proteobacterium Azoarcus evansii, whereby phenylacetic acid
(PA) is degraded via an anaerobic mechanism to benzoyl-CoA (Gescher et al., 2005). It has been
reported that phenylacetate-coenzyme A ligase (PA-CoA ligase) catalyses the initial reaction in
this pathway which involves the activation of PA to PA-CoA. The gene coding for PA-CoA ligase
(paaK) has also been identified in Streptomyces spp. (Pometto III and Crawford, 1985) and since
PA-CoA ligase or an isoenzyme could be involved in either aerobic or anaerobic metabolism,
searching for a similar gene to paaK in S. polyantibioticus SPRT could indicate its ability to
synthesise benzoyl-CoA aerobically from PA (Stegmann, 2011). However, since the chorismate
pathway is a common pathway for generating molecules with benzene rings in bacteria (and other
organisms), S. polyantibioticus SPRT could perhaps also use a novel variant of one of the
aromatic biosynthetic pathways to generate benzoic acid. Amino acid catabolism has been linked
to antibiotic synthesis in the production of macrolides in Streptomyces ambofaciens and
Steptomyces fradiae and therefore it is possible that an aromatic amino acid degradation pathway
could be involved in the production of a benzoic acid intermediate for DPO biosynthesis (Tang et
al., 1994).
-
Amino Acid
Specificity
Pro
Ile, Leu, Val
Gly, Ala, Val
Ser, Thr, Dht
Gly, Ala, Val
Gly, Ala, Val
Ser, Thr, Dht
Ile, Leu, Val
Phe
Gly, Ala, Val
Ser, Thr, Dht
Orn, Lys, Arg
Ile, Leu, Val
Gly, Ala, Val
NO HIT
Val, Leu, Ile
Gly, Ala, Val
Glu, Gln, Asp, Asn
Dhpg, hpg
Ser, Thr
Ser, Thr, Dht
Ser, Thr
Val, Leu, Ile
Phe
Gly, Ala, Val
Gly, Ala, Val
Gly, Ala, Val
Dhpg, hpg
Ser, Thr
Gly, Ala, Val
Ser, Thr
The sequences of clones A-18 and A-66 were identical and determined by the NRPSpredictor 2 to
be specific for phenylalanine. Protein BLAST analysis of the amino acid sequence of both clones
showed a high similarity to an amino acid adenylation protein in Granuliella mallensis MP5ACIX8
(YP_00507340.1) with an amino acid similarity of 57%, an amino acid adenylation protein in
Streptomyces griseus XYLEBKG1 (YP_08236938.1) with an amino acid similarity of 60% and a
NRPS in Streptomyces netropsis (BAH_68437.1) with an amino acid similarity of 54%. All of the
sequences showed similar substrate specificities to clones A-18 and A-66. The A-18/A-66
adenylation domain may be used for the recognition and activation of phenylalanine as a starting
molecule in the biosynthesis of DPO. However, it is possible that clones A-18 and A-66 have
substrate specificities for amino acids other than phenylalanine and this is due to the fact that the
NRPSpredictor 2 predicts aromatic substrates less reliably due to the observed promiscuity of the
A-domains utilizing these substrates (Rausch et al., 2005). It is necessary to obtain further
sequence information surrounding the phenylalanine-specific adenylation domain, utilizing
Southern hybridisation, in order to confirm its substrate specificity and reveal the remainder of the
genes comprising the DPO gene cluster. Each of the open reading frames can then be subjected
to BLAST analysis so that the sequenced genes can be annotated and a likely function assigned
to each one. Gene knock-out experiments, using the method of homologous recombination, can
then be performed on selected genes from the DPO cluster in order to elucidate the complete
biosynthetic pathway involved in DPO biosynthesis. Briefly, each chosen gene would be
disrupted by insertional inactivation, cloned into a suicide vector individually and transformed into
S. polyantibioticus strain SPRT. The vector would be allowed to recombine and integrate into the
chromosome of S. polyantibioticus strain SPRT creating a mutant lacking the functional target
gene. Once this has been achieved, loss of the ability to synthesise DPO would indicate that the
target gene is involved in the proposed DPO biochemical pathway.
2.2 Southern hybridisation using the Phe adenylation domain probe
A Southern hybridisation experiment was performed using a collection of single restriction
endonuclease digestions (PstI, NotI and SacII) of S. polyantibioticus SPRT genomic DNA,
resulting in the detection of numerous bands of varying size containing the target adenylation
domain. Due to the fact that 13 unique adenylation domains were identified from PCR
amplification, cloning and sequencing, it is proposed that the multiple hybridisation bands indicate
the presence of other adenylation domains in the genome. Genomic DNA can be digested using
the same restriction endonucleases and DNA of the appropriate size can be purified and cloned.
The sequencing of these clones should yield sequencing data of DNA flanking the adenylation
domains such as condensation, cyclization or thioesterase domains. We may also be able to
identify novel adenylation domains within S. polyantibioticus SPRT in this way (Stegmann, 2011).
2.3 Cyclization domain amplification
Degenerate cyclization-domain PCR primers were designed based on sequences from
characterised Streptomyces and Streptoalloteichus thiazole and oxazole producers . PCR was
performed on genomic DNA isolated from S. polyantibioticus SPRT using the various sets of
cyclization domain primers (CyF/CyR, CyF/SmobR, VVFTS_F/A7R, QTPQV_F/A7R), but no
amplification was observed. The lack of amplification of a cyclization domain fragment may be
due to the fact that the target sequence is not present in the genome of S. polyantibioticus SPRT,
but a more likely scenario is that the primers do not bind to the target sequences in S.
polyantibioticus SPRT. A lack of primer binding is plausible given the low degree of homology
observed in the multiple sequence alignment of cyclization domains from the various thiazole and
oxazole producers. Moreover, a cyclization domain is absolutely required for the formation of the
heterocyclic ring and oxazoline intermediate involved in DPO biosynthesis and thus the
sequencing of regions flanking the adenylation domain fragments detected by Southern
hybridisation may be the most effective strategy for finding the DPO NRPS, as the cyclization
domain is always adjacent to (and on the N-terminal side of) the adenylation domain.
3. Isolation of genes involved in benzoic acid biosynthesis
3.1 Amplification of PAL/HAL
It has been shown that the presence of the PAL-encoding gene encP is absolutely required for
benzoyl-CoA formation in ‗S. maritimus‘ and therefore encP is a prime candidate to screen for
when searching for benzoyl-CoA biosynthetic potential (Xiang & Moore, 2003). PCR amplification
was performed using the designed encP primers on genomic DNA extracted from S.
polyantibioticus SPRT and ‗S. maritimus‘. Amplification was observed for ‗S. maritimus‘ genomic
DNA resulting in a clear, single band of about 0.7 kb. There was no similar band observed for
encP amplification from S. polyantibioticus SPRT.
The PAL amino acid sequence from ‗S. maritimus‘ was used in a multiple sequence alignment
together with the HAL amino acid sequences from 11 different Streptomyces strains in order to
design new degenerate primers for the amplification of a PAL/HAL from S. polyantibioticus SPRT
(PalHal_F/PalHal_R). The HAL sequences from various Streptomyces strains share a high
degree of homology with each other and to the PAL sequence of ‗S. maritimus‘. A 350 bp
fragment was amplified from S. polyantibioticus SPRT genomic DNA, which was sequenced and
identified as a HAL after analysis using BLASTX. It was therefore concluded that the existence of
a PAL in the genome of S. polyantibioticus SPRT is unlikely and the synthesis of benzoic acid for
the incorporation into DPO could be produced in a novel manner, perhaps via the phenylalanine
degradation pathway mentioned earlier.
3.2 Southern hybridisation using EncP probe
Southern hybridisation was performed using various pairwise restriction endonuclease digestions
of S. polyantibioticus SPRT genomic DNA along with a pairwise restriction endonuclease digestion
of ‗S. maritimus‘ genomic DNA. There was no hybridisation observed for any of the S.
polyantibioticus SPRT genomic digests, while a weak hybridisation band was observed for the ‗S.
maritimus‘ positive control. This result agrees with that for the PCR amplification experiment and
suggests that there is no encP or similar gene in S. polyantibioticus SPRT (Stegmann, 2011).
There may well be a novel PAL-like enzymatic process in S. polyantibioticus SPRT involved in the
synthesis of benzoic acid, but which has a very dissimilar nucleotide sequence to the ‗S.
maritimus‘ encP gene. This would explain the inability to amplify/detect a similar size fragment
from S. polyantibioticus SPRT genomic DNA.
3.3 Amplification of paaK
Despite S. polyantibioticus SPRT appearing not to possess a PAL gene similar to encP, it may
produce benzoyl-CoA via another mechanism such as the phenylacetate pathway for the
degradation of phenylalanine. The initial step of the PA pathway is catalyzed by a PA-CoA ligase
and degenerate primers (Paak_AveF/Paak_AveR) were used to amplify a 700 bp fragment of the
homologue of the gene, paaK, encoding this enzyme in S. polyantibioticus SPRT. The presence of
this gene in S. polyantibioticus SPRT suggests that the phenyalacetate pathway does exist and
thus could indicate the ability of the organism to produce benzoyl-CoA. It is possible that the
genes involved in benzoic acid synthesis would be clustered together with the NRPS involved in
DPO biosynthesis and therefore sequencing further upstream and downstream of the amplified
paaK fragment may help to prove our hypothesis.
3.4 Southern hybridisation using paaK probe
Southern hybridisation using the paaK probe resulted in the detection of single bands in each lane
of single restriction enzyme digested genomic DNA. These bands were excised and purified from
genomic DNA and cloned into the plasmid vector pSK. After identification of the clones carrying
the desired insert, using the method of colony PCR with the paaK primer set, they will be
sequenced. This should result in the elucidation of the genes situated both upstream and
downstream of the amplified paaK fragment.
MATERIALS AND METHODS
Isolation of 2, 5-diphenyloxazole from S. polyantibioticus SPRT and confirmation of antibacterial
activity
DPO was isolated from the fermentation broth and mycelial mass of S. polyantibioticus SPRT
according to the method described by Le Roes (2005). Bioautography with Mycobacterium aurum
strain A+ was performed according to the protocol described by Betina (1973).
PCR amplification and sequencing
All PCR amplifications were performed using genomic DNA isolated according to the method
described by Everest and Meyers (2008). Primers were obtained from published work or
designed based on multiple sequence alignments of amino acid and nucleotide sequences
obtained from GenBank. Cycling conditions used for the A3F/A7R, CyF/CyR, CyF/SmobR, EncPF/EncP-R, VVFTS/A7R and QTPQV/A7R primer sets were as follows: initial denaturation at 95 oC
for 5 minutes, followed by 35 cycles of denaturation at 95 oC for 30 seconds; annealing and
elongation at 72 oC for 4 minutes, with a final elongation at 72 oC for 10 minutes. The annealing
temperature and duration for each primer set varied and can be found in Table 2. Cycling
conditions for the PalHal_F/PalHal_R primers were the same as above, except that denaturation
was for 15 seconds and elongation was for 10 seconds, with a final elongation at 72 oC for 5
minutes. Cycling conditions for the Paak_AveF/Paak_AveR primers were also the same, except
that denaturation was for 30 seconds and elongation was for 60 seconds, with a final elongation at
72 oC for 5 minutes. PCR reactions consisted of: 200-1500 ng of DNA, 2 U SuperThem Taq
polymerase (JMR Holdings, USA), 0.5- 1.5 μM of each primer, 0.8 mM of each dNTP, 2-4 mM
MgCl2 and 3% - 8% (v/v) glycerol in a total volume of 50μl.
Table 2. Oligonucleotide primers used in this study.
Primer
Primer sequence (5ʹ→3ʹ)
Reference
Annealing temperature and time
A3F
GCSTACSYSATSTACACSTCSGG
Ayuso-Sacido & Genilloud, 2005
64 oC for 90 seconds
A7R
SASGTCVCCSGTSCGGTAS
Ayuso-Sacido & Genilloud, 2005
64 oC for 90 seconds
CyF
AGCCITTCYCSCTSACSSMBSTSCAG
Stegmann, 2011
54 oC to 68 oC for 90 seconds
CyR
GICSAGSWISSWSGTGAASACSAC
Stegmann, 2011
54 oC to 68 oC for 90 seconds
Smob-CyR
AGGCAGGTCGGAGGTGAAGACGAC
Stegmann, 2011
54 oC to 68 oC for 90 seconds
EncP-F
GACTCGCACCTGGCGGTCAAC
Stegmann, 2011
60 oC for 30 seconds
EncP-R
GTAGTCGGTGATGGTCTCGTC
Stegmann, 2011
60 oC for 30 seconds
VVFTS_F
TSGTSTTCACSWSSIHSYTS
This study.
54 oC to 68 oC for 60 seconds
QTPQV_F
TSWSSCAGACSCGSCAGG
This study.
54 oC to 68 oC for 60 seconds
PalHal_F
GGSCTSGCSCTSMTCAACGGCAC
This study.
56 oC for 45 seconds
PalHal_R
GSRCASCGBABSGARTASGCGTCC
This study.
56 oC for 45 seconds
Paak_AveF
CCBTCSTACMTGCTSACSCTSCTSGACG
This study.
60 oC for 45 seconds
Paak_AveR
GSAGSASGATCTCCTCGARCTGSSTGG
This study.
60 oC for 45 seconds
Cloning and sequencing
All cloning and sequencing procedures were performed as described by Stegmann (2011).
Southern hybridisation
For all Southern hybridisations, total genomic DNA of S. polyantibioticus SPRT was digested with
the following single restriction endonucleases: PstI, NotI and SacII, except for the Southern
hybridisation using the encP probe, in which DNA of S. polyantibioticus SPRT was digested with
pairs of restriction endonucleases: SphI & StuI, PvuII & SphI, AvrII & SphI and AvrII & StuI, while
‗S. maritimus‘ genomic DNA was digested with SphI & StuI. Reaction volumes were 50 μl and
contained approximately 40 μg of DNA, 1.5 U of each restriction endonuclease and the
appropriate restriction buffer. Digestions were performed overnight at 37oC.
The primers specific for amplification of encP from ‗S. maritimus‘, paaK from S. polyantibioticus
SPRT and the NRPS adenylation domain fragment from S. polyantibioticus SPRT were used to
create probes by PCR using the PCR DIG Probe Synthesis Kit (Roche).
The Southern blot hybridisation protocol was performed according to the method outlined by
Stegmann (2011).
CONCLUSION
Unfortunately, progress on this project has been slow and this may be related to our hypothesis
on how DPO is synthesized. NRPSs are commonly involved in the biosynthetic strategy for the
production of heterocyclic molecules like oxazoles, methyl oxazoles and thiazoles in bacteria,
which is why the current hypothesis seems most likely to be correct. We have managed to identify
an adenylation domain that has been predicted to have a binding pocket specificity for
phenylalanine. This is promising in our attempts to elucidate the pathway involved in DPO
biosynthesis, but the method of benzoic acid synthesis in S. polyantibioticus SPRT is still
uncertain.
The sequencing of the S. polyantibioticus SPRT genome is currently underway and once this
enormous amount of sequence data becomes available and the open reading frames have been
detected, the task of identifying the DPO genes should become easier. Once we have identified
the DPO biosynthetic gene cluster, the predicted functions of selected DPO biosynthetic genes
will be determined by gene disruption experiments. A method for transforming S. polyantibioticus
SPRT is being optimised.
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Acknowledgements
Kyle Kemp is the recipient of an Innovation
Doctoral Scholarship from the National
Research Foundation of South Africa
(NRF). This research is supported by
grants provided to Paul Meyers by the
Medical Research Council (MRC) and the
University Research Committee (UCT).