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STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF POLYKETIDE SYNTHASE
GENE CLUSTERS FOUND IN NEWLY SEQUENCED BACTERIAL GENOME
Kitti Csepregi1, Andrea Valasek1, Ágota Pénzes1,2, Zsuzsanna Tóth1, Éva Írisz Kiss1, Ildikó Kerepesi1,
Judit Hunyadkürti1,3, Balázs Horváth3, István Nagy3 and Csaba Fekete1
Department of General and Environmental Microbiology, University of Pécs, Hungary1; PannonPharma Pharmaceutical Ltd., Pécsvárad, Hungary2;
Bay Zoltán Nonprofit Ltd., Szeged, Hungary3
Introduction
Even though remarkable advances were made in medical research and treatments
over the last decade, due to emerging and re-emerging pathogens and increase
of
multi-resistant
strains,
infectious
diseases
remain
among
the
primary
causes of death worldwide. In respect of the clinical needs, isolation of new microbial natural
products (e.g. antimicrobials) and screening for potential producing microorganisms are
being facilitated by several new strategies. Polyketides are large and structurally diverse
group of natural products biosynthesized by multifunctional enzymes called polyketide
synthases (PKSs). To provide new insights into the molecular basis of secondary metabolites
biosynthesis, Saccharomonospora azurea strain SZMC 14600 was discovered. In the
present study we give a detailed overview of structural and functional characterisation of the
identified PKS gene cluster. Complementary to the structural genomics approach, to get
insight into the relationship of PKS cluster stucture and function, digital transcriptome profiling
(RNA-seq) has also been performed.
Some of the complex polyketides are synthesized by bacterial type I modular PKS. PKS is a
large multifunctional, multimodular protein, in which each module responsible for a well
defined function. The basic domains are presented in each module such as acyltransferase
(AT), keto synthase (KS) and acyl carrier protein (ACP). In addition, the modules might be
contain other chain-modification domains e.g. β-ketoreductase (KR), dehydratase (DH) and
enoyl reductase (ER) domains. The growing polyketide chain is passed from module to
module until the completed chain is released from the terminal module by a specific chainreleasing enzyme, thioesterase (TE). The PKS gene cluster contains genes are not closely
related to the basic steps of polyketides, however they involved in processes of multiple postmodifications.
Gene
Size
product aa
ID Protein
% homologue
Gene
product
Putative
homologue function
Size
aa
ID
%
Protein
homologue
Putative
homologue function
SapkI
187
89
S. cyanea
Transcriptional regulator
SapkM1 501
46
Streptomyces sp.
Hypothetical protein
SapkJ
199
89
S. viridis
Dihydrofolate reductase
SapkN1 318
40
S. violaceusniger
Hypothetical protein
SapkK
103
56
S. cyanea
Hypothetical protein
SapkA
S. zinciresistens
Acyl transferase
SapkL
132
59
S.glauca
Hypothetical protein
SapkO1 763
A. mediterranei
Hypothetical protein
SapkM
148
89
S. cyanea
Hypothetical protein
SapkB
1998 60
S. violaceusniger
Beta-ketoacyl synthase
SapkN
153
79
S. cyanea
Acetyltransferase
SapkC
3702 58
S. violaceusniger
SapkO
237
75
S. cyanea
Hydrolase , acyltransferase
SapkD
5860 55
SapkP
100
85
S. cyanea
SapkE
642
SapkQ
129
83
S. cyanea
SapkF
6354 55
S. ambofaciens
Acyl transferase
SapkR
254
87
S. cyanea
Transcriptional regulator
Glyoxalase/bleomycin
resistance protein
Regulatory protein TetR
SapkG
1659 53
A. orientalis
SapkS
386
81
S. cyanea
Hypothetical protein
SapkH
1828 52
S. violaceusniger
SapkT
144
68
S. erythraea
SapkP1
375
58
S. roseum
SapkU
535
66
A. mediterranei
SapkQ1 67
45
S. glauca
SapkV
774
53
S. spinosa
Cytidine deaminase
Acetyl/propionyl-CoA
carboxylase
Penicillin amidase
Acyl transferase
Modular polyketide
synthase
Agmatinase
SapkR1 331
66
S. griseoflavus
SapkZ
393
55
S. scabiei
Hypothetical protein
SapkI1
112
34
S. marina
Hypothetical protein
SapkS1
269
62
S. coelicolor
SapkJ1 262
69
Streptomyces sp. ABC transporter
SapkT1
398
53
Streptomyces sp
SapkK1 517
44
SapkU1 212
70
Streptomyces sp
SapkL1 428
48
Streptomyces sp. Hypothetical protein
Cytochrome p450-like
S. erythraea
enzyme
Hypothetical protein
ABC transporter ATPbinding protein
ABC-transporter
transmembrane protein
Two-component
histidine kinase
Response regulator
SapkV1
45
T. curvata
ATPase-like protein
5141 52
52
64
982
Beta-ketoacyl synthase
Modular polyketide
S. avermitilis
synthase
S. paurometabolica Acyl transferase
Table 1. Deduced functions of the sapk genecluster. ID, indicate percentage identity (%) to the homologues using BlastP; aa: amino acid; PKS subunits
are highlighted by grey, and blue colored lines indicate those peptide encoding genes which were significantly upragulated in the transcriptome analysis.
Methods
The genome sequencing was performed by combining cycled ligation sequencing on the
SOLiD 3Plus system with 454 FLX pyrosequencing. The annotated draft genome sequence
has been deposited at DDBJ/EMBL/GenBank under accession number AHBX00000000. PKS
gene cluster was identified and analysed by antiSMASH program tools. Predicted peptids
were used to serach for homology in GeneBank by BlastP. Domain analysis and motif search
were done by SMART, MAPSI, SBSPKS and MEME respectively. Sequences were aligned
by different MSA programs.
Results
• Among the gene clusters related to certain secunder metabolite synthesis, partial NRPS
and NRPS/PKS hybid (~100 kbp) gene clusters were indentified.
A
70637 bp
183316 bp
sapkA
B
module 1
module 3
module 2
module 8
KS AT DH ER KR
module 10
module 9
SapkG
KS AT KR ACP KS
ACP KS AT DH ER KR ACP KS AT KR ACP
SapkF
module 11
KS
module 6
module 5
SapkE
KS AT KR ACP KS AT KR ACP KS AT KR ACP KS AT KR ACP
module 14
SapkC
module 4
SapkD
sapkG sapkH
sapkF
SapkB
ACP KS AT DH KR ACP KS AT KR ACP KS AT KR ACP
module 7
sapkE
sapkD
sapkC
SapkA
lmod
• Furthermore, we have discovered a novel type of modular PKS (I) gene cluster (113 kbp)
on the AHBX00000216 contig named sapk.
• Detailed structural characterisation of the newly described PKS (I) cluster was carried out.
sapkB
module 12
module 13
AT DH KR ACP KS AT KR ACP KS AT DH ER KR ACP
SapkH
module 16
module 15
AT DH ER KR ACP
KS AT KR ACP TE
C
• That cluster includes eight PKS subunits encoding genes sapkA, sapkB, sapkC, sapkD,
sapkE, sapkF, sapkG, sapkH and 29 additional genes listed in the Table 1. and Fig.1. A.
• In silico analysis showed that the full length PKS consists of 16 modules. All modules
responsible for extension process has a minimal set of enzime- domains: AT, KS, and ACP.
Modules 4, 5, 13, and 15 contain ER, modules 1, 4, 5, 11, 13 and 15 contain DH domains.
Special loading module contains only ACP domain (Fig. 1. B).
• Analysis of the active cenrum of AT domains indicate that 5 ATs (modules 1, 2, 3, 15, 16)
are specific for methylmalonyl-CoA and 10 ATs (modules 4-14) for malonyl CoA precursor.
Fig. 1. Organization of sapk gene cluster. A, The top line denotes the PKS carrying chromosomal region of S. azurea. ORFs are shown to scale as
arrows. Gene names are given above the arrows. B, Modules are boxed on top, and abbreviations of the enzymatic domains are indicated in the grey
rectangles. Members of the PKS subunits SapkA, SapkB, SapkC, SapkD, SapkE, SapkE, SapkF, SapkG, SapkH are indicated above the grey open
arrow. Intermodular linkers are marked with black squares, and docking domains with red spaces. C. Prediction of chemical structure of the synthesized
secunder metabolite. Methylmalonyl-CoA specific AT domains are indicated by violet, and malonyl CoA specific AT labeled by black. Lmod denotes
loading module.
sapkA
sapkB
sapkC
sapkD
sapkE
sapkF
sapkG sapkH
• 11 KRs (modules 1-7, 9, 11, 13) have characteristic motifs for B-type and 5 KRs (modules
10, 12, 14, 16) have A-type domains.
• Based on the structural of PKS, chemical structure of the synthesized secunder metabolite
was predicted (Fig.1. C).
• Three inter polypeptide linker sequences, frequently called as docking domain, were
indentified between SapkA-SapkB, SapkD-SapkE and SapkG-SapkH (Fig. 1. B).
• Structural comparison of the entire sapk PKS cluster was performed against the closely
related avermectin encoding gene cluster of Setreptomyces avermitilis (Fig. 2).
• Digital transcriptome profiling (RNA-seq) revealed that, SapkK1 and SapkQ1 hypothetical
protein encoding genes are significantly upregulated in the producer strain in comparison
to non-producer mutans strain (Table.1).
Fig. 2. Structural comparison of the entire sapk PKS cluster against the closely related avermectin encoding gene cluster of Setreptomyces avermitilis
Genes with the same color are putative homologues based on significant Blast hits between them.
Summary and Conclusions
These data clearly indicate that rare Actinomycetes species such as S. azurea is potentially
prolific sources of pharmaceutically important secondary metabolites.
Structural characterization of the newly identified PKS (I) open the possibility to redesign gene
cluster to produce higher amount or novel antibiotics.
This work was supported in part by the grant of SROP-4.2.1.B-10/2/KONV-2010-0002, SROP-4.2.2/B-10/1-2010-0029 Developing the South-Transdanubian Regional University Competitiveness, Baross grant DA07-DA TECH 07-2008-0045, and NKTH Teller program grant OMFB-00441/2007.