Evolution of metabolic diversity: Insights from microbial polyketide synthases

Polyketides are a family of complex natural products that are built from simple carboxylic acid building blocks. In microorganisms, the majority of these secondary metabolites are produced by exceptionally large, multifunctional proteins termed polyketide synthases (PKSs). Each unit of a type I PKS assembly line resembles a mammalian type fatty acid synthase (FAS), although certain domains are optionally missing. The evolutionary analysis of microbial PKS has revealed a long joint evolution process of PKSs and FASs. The phylogenomic analysis of modular type I PKSs as the most widespread PKS type in bacteria showed a large impact of gene duplications and gene losses on the evolution of type I PKS in different bacterial groups. The majority of type I PKSs in actinobacteria and cyanobacteria may have evolved from a common ancestor, whereas in proteobacteria most type I PKSs were acquired from other bacterial groups. The modularization of type I PKSs almost unexceptionally started with multiple duplications of a single ancestor module. The repeating modules represent ideal platforms for recombination events that can lead to corresponding changes in the actual chemistry of the products. The analysis of these “natural reprogramming” events of PKSs may assist in the development of concepts for the biocombinatorial design of bioactive compounds.     

Cloning and characterization of a bacterial iterative type I polyketide synthase gene encoding the 6-methylsalicyclic acid synthase

Unusual polyketide synthases (PKSs), that are structurally type I but act in an iterative manner for aromatic polyketide biosynthesis, are a new family found in bacteria. Here we report the cloning of the iterative type I PKS gene chlB1 from the chlorothricin (CHL) producer Streptomyces antibioticus DSM 40725 by a rapid PCR approach, and characterization of the function of the gene product as a 6-methylsalicyclic acid synthase (6-MSAS). Sequence analysis of various iterative type I PKSs suggests that the resulting aromatic or aliphatic structure of the products might be intrinsically determined by a catalytic feature of the paired KR-DH domains in the control of the double bond geometry. The finding of ChlB1 as a 6-MSAS not only enriches the current knowledge of aromatic polyketide biosynthesis in bacteria, but will also contribute to the generation of novel polyketide analogs via combinatorial biosynthesis with engineered PKSs.     

Probing the modularity of megasynthases by rational engineering of a fatty acid synthase Type I

Modularity is a fundamental property of megasynthases such as polyketide synthases (PKSs). In this study, we exploit the close resemblance between PKSs and animal fatty acid synthase (FAS) to re‐engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by sequence and structural information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS‐like modules as well as bimodular constructs. The novel re‐engineered modules resemble all four common types of PKSs and demonstrate that this approach can be a powerful tool to deliver products with higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS‐based biosynthetic pathways for custom compound design.     

Structural insights into dehydratase substrate selection for the borrelidin and fluvirucin polyketide synthases

Engineered polyketide synthases (PKSs) are promising synthetic biology platforms for the production of chemicals with diverse applications. The dehydratase (DH) domain within modular type I PKSs generates an α,β-unsaturated bond in nascent polyketide intermediates through a dehydration reaction. Several crystal structures of DH domains have been solved, providing important structural insights into substrate selection and dehydration. Here, we present two DH domain structures from two chemically diverse PKSs. The first DH domain, isolated from the third module in the borrelidin PKS, is specific towards a trans-cyclopentane-carboxylate-containing polyketide substrate. The second DH domain, isolated from the first module in the fluvirucin B₁ PKS, accepts an amide-containing polyketide intermediate. Sequence-structure analysis of these domains, in addition to previously published DH structures, display many significant similarities and key differences pertaining to substrate selection. The two major differences between BorA DH M3, FluA DH M1 and other DH domains are found in regions of unmodeled residues or residues containing high B-factors. These two regions are located between α3–β11 and β7–α2. From the catalytic Asp located in α3 to a conserved Pro in β11, the residues between them form part of the bottom of the substrate-binding cavity responsible for binding to acyl-ACP intermediates.

     

A new modular polyketide synthase in the erythromycin producer Saccharopolyspora erythraea

A previously unidentified set of genes encoding a modular polyketide synthase (PKS) has been sequenced in Saccharopolyspora erythraea, producer of the antibiotic erythromycin. This new PKS gene cluster (pke) contains four adjacent large open reading frames (ORFs) encoding eight extension modules, flanked by a number of other ORFs which can be plausibly assigned roles in polyketide biosynthesis. Disruption of the pke PKS genes gave S. erythraea mutant JC2::pSBKS6, whose growth characteristics and pattern of secondary metabolite production did not apparently differ from the parent strain under any of the growth conditions tested. However, the pke PKS loading module and individual pke acyltransferase domains were shown to be active when used in engineered hybrid PKSs, making it highly likely that under appropriate conditions these biosynthetic genes are indeed expressed and active, and synthesize a novel polyketide product.     

Novel intermolecular iterative mechanism for biosynthesis of mycoketide catalyzed by a bimodular polyketide synthase

In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-beta-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel "modularly iterative" mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.     

Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up < 1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.     

PKSI-AT结构域及在组合生物合成研究中的应用

I型聚酮合酶（PKSI）的模块型分子结构组织方式非常适合于组合生物合成研究.结构域和模块通过二级组织方式构成了PKSI的催化单元,其它结构多肽则作为“支架”.在“支架”上对结构域和模块两个水平进行突变、替换、插入、缺失等基因操作形成重组PKS,可以理性设计并获得复杂多样的新活性或高活性的聚酮化合物.利用PKSI进行组合生物合成以期获得新聚酮化合物的研究迄今已有约25年,但是目前仍不能够对PKS进行完美的理性设计,快速合成目标活性的新聚酮化合物.PKS中的酰基转移酶结构域的研究在PKS的组合生物合成研究中一直发挥着重要作用.本文结合本课题组的研究基础,对AT结构域的结构、功能及在组合生物合成研究中的最新研究成果作以分析总结.     

Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC 1067

Fungal type I polyketide (PK) compounds are highly valuable for medical treatment and extremely diverse in structure, partly because of the enzymatic activities of reducing domains in polyketide synthases (PKSs). We have cloned several PKS genes from the fungus Xylaria sp. BCC 1067, which produces two polyketides: depudecin (reduced PK) and 19,20-epoxycytochalasin Q (PK-nonribosomal peptide (NRP) hybrid). Two new degenerate primer sets, KA-series and XKS, were designed to amplify reducing PKS and PKS-NRP synthetase hybrid genes, respectively. Five putative PKS genes were amplified in Xylaria using KA-series primers and two more with the XKS primers. All seven are predicted to encode proteins homologous to highly reduced (HR)-type PKSs. Previously designed primers in LC-, KS-, and MT-series identified four additional PKS gene fragments. Selected PKS fragments were used as probes to identify PKS genes from the genomic library of this fungus. Full-length sequences for five PKS genes were obtained: pks12, pks3, pksKA1, pksMT, and pksX1. They are structurally diverse with 1-9 putative introns and products ranging from 2162 to 3654 amino acids in length. The finding of 11 distinct PKS genes solely by means of PCR cloning supports that PKS genes are highly diverse in fungi. It also indicates that our KA-series primers can serve as powerful tools to reveal the genetic potential of fungi in production of multiple types of HR PKs, which the conventional compound screening could underestimate.     

The biosynthetic gene cluster for the anticancer drug bleomycin from Streptomyces verticillus ATCC15003 as a model for hybrid peptide–polyketide natural product biosynthesis

The hybrid peptide–polyketide backbone of bleomycin (BLM) is assembled by the BLM megasynthetase that consists of both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules. BlmIX/BlmVIII/BlmVII constitute a natural hybrid NRPS/PKS/NRPS system, serving as a model for both hybrid NRPS/PKS and PKS/NRPS systems. Sequence analysis and functional comparison of domains and modules of BlmIX/BlmVIII/BlmVII with those of nonhybrid NRPS and PKS systems suggest that (1) the same catalytic sites appear to be conserved in both hybrid NRPS–PKS and nonhybrid NRPS or PKS systems, with the exception of the KS domains in the hybrid NRPS/PKS systems that are unique; (2) specific interpolypeptide linkers may play a critical role in intermodular communication to facilitate transfer of the growing intermediates between the interacting NRPS and/or PKS modules; and (3) posttranslational modification of the BLM megasynthetase has been accomplished by a single PPTase with a broad substrate specificity toward the apo forms of both acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). Journal of Industrial Microbiology & Biotechnology (2001) 27, 378–385. Received 08 June 2001/ Accepted in revised form 18 July 2001     

Ketosynthases in the initiation and elongation modules of aromatic polyketide synthases have orthogonal acyl carrier protein specificity

Many bacterial aromatic polyketides are synthesized by type II polyketide synthases (PKSs) which minimally consist of a ketosynthase-chain length factor (KS-CLF) heterodimer, an acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT). This minimal PKS initiates polyketide biosynthesis by decarboxylation of malonyl-ACP, which is catalyzed by the KS-CLF complex and leads to incorporation of an acetate starter unit. In non-acetate-primed PKSs, such as the frenolicin (fren) PKS and the R1128 PKS, decarboxylative priming is suppressed in favor of chain initiation with alternative acyl groups. Elucidation of these unusual priming pathways could lead to the engineered biosynthesis of polyketides containing novel starter units. Unique to some non-acetate-primed PKSs is a second catalytic module comprised of a dedicated homodimeric KS, an additional ACP, and a MAT. This initiation module is responsible for starter-unit selection and catalysis of the first chain elongation step. To elucidate the protein-protein recognition features of this dissociated multimodular PKS system, we expressed and purified two priming and two elongation KSs, a set of six ACPs from diverse sources, and a MAT. In the presence of the MAT, each ACP was labeled with malonyl-CoA rapidly. In the presence of a KS-CLF and MAT, all ACPs from minimal PKSs supported polyketide synthesis at comparable rates (k(cat) between 0.17 and 0.37 min(-1)), whereas PKS activity was attenuated by at least 50-fold in the presence of an ACP from an initiation module. In contrast, the opposite specificity pattern was observed with priming KSs: while ACPs from initiation modules were good substrates, ACPs from minimal PKSs were significantly poorer substrates. Our results show that KS-CLF and KSIII recognize orthogonal sets of ACPs, and the additional ACP is indispensable for the incorporation of non-acetate primer units. Sequence alignments of the two classes of ACPs identified a tyrosine residue that is unique to priming ACPs. Site-directed mutagenesis of this amino acid in the initiation and elongation module ACPs of the R1128 PKS confirmed the importance of this residue in modulating interactions between KSs and ACPs. Our study provides new biochemical insights into unusual chain initiation mechanisms of bacterial aromatic PKSs.     

A new family of type III polyketide synthases in Mycobacterium tuberculosis

The Mycobacterium tuberculosis genome has revealed a remarkable array of polyketide synthases (PKSs); however, no polyketide product has been isolated thus far. Most of the PKS genes have been implicated in the biosynthesis of complex lipids. We report here the characterization of two novel type III PKSs from M. tuberculosis that are involved in the biosynthesis of long-chain alpha-pyrones. Measurement of steady-state kinetic parameters demonstrated that the catalytic efficiency of PKS18 protein was severalfold higher for long-chain acyl-coenzyme A substrates as compared with the small-chain precursors. The specificity of PKS18 and PKS11 proteins toward long-chain aliphatic acyl-coenzyme A (C12 to C20) substrates is unprecedented in the chalcone synthase (CHS) family of condensing enzymes. Based on comparative modeling studies, we propose that these proteins might have evolved by fusing the catalytic machinery of CHS and beta-ketoacyl synthases, the two evolutionarily related members with conserved thiolase fold. The mechanistic and structural importance of several active site residues, as predicted by our structural model, was investigated by performing site-directed mutagenesis. The functional identification of diverse catalytic activity in mycobacterial type III PKSs provide a fascinating example of metabolite divergence in CHS-like proteins.     

Hybrid peptide-polyketide natural products: biosynthesis and prospects toward engineering novel molecules

The structural and catalytic similarities between modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) inspired us to search for hybrid NRPS-PKS systems. By examining the biochemical and genetic data known to date for the biosynthesis of hybrid peptide-polyketide natural products, we show (1) that the same catalytic sites are conserved between the hybrid NRPS-PKS and normal NRPS or PKS systems, although the ketoacyl synthase domain in NRPS/PKS hybrids is unique, and (2) that specific interpolypeptide linkers exist at both the C- and N-termini of the NRPS and PKS proteins, which presumably play a critical role in facilitating the transfer of the growing peptide or polyketide intermediate between NRPS and PKS modules in hybrid NRPS-PKS systems. These findings provide new insights for intermodular communications in hybrid NRPS-PKS systems and should now be taken into consideration in engineering hybrid peptide-polyketide biosynthetic pathways for making novel "unnatural" natural products.     

Synthetic biology of polyketide synthases

Complex reduced polyketides represent the largest class of natural products that have applications in medicine, agriculture, and animal health. This structurally diverse class of compounds shares a common methodology of biosynthesis employing modular enzyme systems called polyketide synthases (PKSs). The modules are composed of enzymatic domains that share sequence and functional similarity across all known PKSs. We have used the nomenclature of synthetic biology to classify the enzymatic domains and modules as parts and devices, respectively, and have generated detailed lists of both. In addition, we describe the chassis (hosts) that are used to assemble, express, and engineer the parts and devices to produce polyketides. We describe a recently developed software tool to design PKS system and provide an example of its use. Finally, we provide perspectives of what needs to be accomplished to fully realize the potential that synthetic biology approaches bring to this class of molecules.     

Characterization of the enzymatic domains in the modular polyketide synthase involved in rifamycin B biosynthesis by Amycolatopsis mediterranei

Five clustered polyketide synthase (PKS) genes, rifA–rifE, involved in rifamycin (Rf) biosynthesis in Amycolatopsis mediterranei S699 have been cloned and sequenced (August, P.R. et al., 1998. Chem. Biol. 5, 69–79). The five multifunctional polypeptides constitute a type I modular PKS that contains ten modules, each responsible for a specific round of polyketide chain elongation. Sequence comparisons of the Rf PKS proteins with other prokaryotic modular PKSs elucidated the regions that have an important role in enzyme activity and specificity. The β-ketoacyl:acyl carrier protein synthase (KS) domains show the highest degree of similarity between themselves (86–90%) and to other PKSs (78–85%) among all the constituent domains. Both malonyl-coenzyme A (MCoA) and methylmalonyl-coenzyme A (mMCoA) are substrates for chain elongation steps carried out by the Rf PKS. Since acyltransferase (AT) domains of modular PKSs can distinguish between these two substrates, comparison of the sequence of all ten AT domains of the Rf PKS with those found in the erythromycin (Er) (Donadio, S. and Katz, L., 1992. Gene 111, 51–60) and rapamycin (Rp) (Haydock, S. et al., 1995. FEBS Lett. 374, 246–248) PKSs revealed that the AT domains in module 2 of RifA and module 9 of RifE are specific for MCoA, whereas the other eight modules specify mMCoA. Dehydration of the β-hydroxyacylthioester intermediates should occur during the reactions catalysed by module 4 of RifB and modules 9 and 10 of RifE, yet only the active site region of module 4 conforms closely to the dehydratase (DH) motifs in the Er and Rp PKSs. The DH domains of modules 9 and 10 diverge significantly from the consensus sequence defined by the Er and Rp PKSs, except for the active site His residues. Deletions in the DH active sites of module 1 in RifA and module 5 in RifB and in the N- and C-terminal regions of module 8 of RifD should inactivate these domains, and module 2 of RifA lacks a DH domain, all of which are consistent with the proposed biosynthesis of Rf. In contrast, module 6 of RifB and module 7 of RifC appear to contain intact DH domains even though DH activity is not apparently required in these modules. Module 2 of RifA lacks a β-ketoacyl:acyl carrier protein reductase (KR) domain and the one in module 3 has an apparently inactive NADPH binding motif, similar to one found in the Er PKS, while the other eight KR domains of the Rf PKS should be functional. These observations are consistent with biosynthetic predictions. All the acyl carrier protein (ACP) domains, while clearly functional, nevertheless have active site signature sequences distinctive from those of the Er and Rp PKSs. Module 2 of RifA has only the core domains (KS, AT and ACP). The starter unit ligase (SUL) and ACP domains present in the N-terminus of RifA direct the selection and loading of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA), onto the PKS. AHBA is made by the products of several other genes in the Rf cluster through a variant of the shikimate pathway (August, P.R. et al., inter alia). RifF, produced by the gene immediately downstream of rifE, is thought to catalyse the intramolecular cyclization of the PKS product, thereby forming the ansamacrolide precursor of Rf B.