STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF POLYKETIDE SYNTHASE GENE CLUSTERS FOUND IN NEWLY SEQUENCED BACTERIAL GENOME
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.
Kitti Csepregi
1, Andrea Valasek
1, Ágota Pénzes
1,2, Zsuzsanna Tóth
1, Éva Írisz Kiss
1, Ildikó Kerepesi
1, Judit Hunyadkürti
1,3, Balázs Horváth
3, István Nagy
3and Csaba Fekete
1Department of General and Environmental Microbiology, University of Pécs, Hungary
1; PannonPharma Pharmaceutical Ltd., Pécsvárad, Hungary
2; Bay Zoltán Nonprofit Ltd., Szeged, Hungary
3Introduction
Methods
Results
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.
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.
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 chain- releasing 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 post- modifications.
Gene product
Size aa
ID
%
Protein
homologue
Putative
homologue function
SapkI 187 89 S. cyanea Transcriptional regulator SapkJ 199 89 S. viridis Dihydrofolate reductase SapkK 103 56 S. cyanea Hypothetical protein
SapkL 132 59 S.glauca Hypothetical protein SapkM 148 89 S. cyanea Hypothetical protein SapkN 153 79 S. cyanea Acetyltransferase
SapkO 237 75 S. cyanea Hydrolase , acyltransferase SapkP 100 85 S. cyanea Transcriptional regulator SapkQ 129 83 S. cyanea Glyoxalase/bleomycin
resistance protein
SapkR 254 87 S. cyanea Regulatory protein TetR SapkS 386 81 S. cyanea Hypothetical protein SapkT 144 68 S. erythraea Cytidine deaminase SapkU 535 66 A. mediterranei Acetyl/propionyl-CoA
carboxylase
SapkV 774 53 S. spinosa Penicillin amidase SapkZ 393 55 S. scabiei Hypothetical protein SapkI1 112 34 S. marina Hypothetical protein SapkJ1 262 69 Streptomyces sp. ABC transporter SapkK1 517 44 Streptomyces sp. Hypothetical protein SapkL1 428 48 S. erythraea Cytochrome p450-like
enzyme
Gene product
Size aa
ID
%
Protein
homologue
Putative
homologue function
SapkM1 501 46 Streptomyces sp. Hypothetical protein SapkN1 318 40 S. violaceusniger Hypothetical protein SapkA 5141 52 S. zinciresistens Acyl transferase SapkO1 763 52 A. mediterranei Hypothetical protein SapkB 1998 60 S. violaceusniger Beta-ketoacyl synthase SapkC 3702 58 S. violaceusniger Beta-ketoacyl synthase SapkD 5860 55 S. avermitilis Modular polyketide
synthase
SapkE 642 64 S. paurometabolica Acyl transferase SapkF 6354 55 S. ambofaciens Acyl transferase SapkG 1659 53 A. orientalis Acyl transferase SapkH 1828 52 S. violaceusniger Modular polyketide
synthase SapkP1 375 58 S. roseum Agmatinase
SapkQ1 67 45 S. glauca Hypothetical protein SapkR1 331 66 S. griseoflavus ABC transporter ATP-
binding protein SapkS1 269 62 S. coelicolor ABC-transporter
transmembrane protein SapkT1 398 53 Streptomyces sp Two-component
histidine kinase
SapkU1 212 70 Streptomyces sp Response regulator SapkV1 982 45 T. curvata ATPase-like protein
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.
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.
• Among the gene clusters related to certain secunder metabolite synthesis, partial NRPS and NRPS/PKS hybid (~100 kbp) gene clusters were indentified.
• 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.
• 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.
• 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).
183316 bp 70637 bp
sapkA sapkB sapkC sapkD sapkE sapkF sapkG sapkH
ACP KS AT DH KR ACP KS AT KR ACP
SapkA module 1
KS AT DH ER KR ACP KS AT DH ER KR ACP KS AT KR ACP
KS AT KR ACP
module 2 module 3 module 4 module 5 module 6
SapkB SapkC
SapkD
KS AT KR ACP KS AT KR ACP KS AT KR ACP
module 7
KS AT KR ACP
KS
SapkE
module 8 module 9 module 10
AT DH KR ACP KS AT KR ACP KS AT DH ER KR ACP
KS AT KR ACP KS
module 11 module 12 module 13
SapkF
SapkG
AT DH ER KR ACP KS AT KR ACP TE
SapkH
module 14 module 15 module 16
A
B
lmod
C
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
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.
