Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases

Modular polyketide synthases (PKSs) are large multi-enzymatic, multi-domain megasynthases, which are involved in the biosynthesis of a class of pharmaceutically important natural products, namely polyketides. These enzymes harbor a set of repetitive active sites termed modules and the domains present in each module dictate the chemical moiety that would add to a growing polyketide chain. This modular logic of biosynthesis has been exploited with reasonable success to produce several novel compounds by genetic manipulation. However, for harnessing their vast potential of combinatorial biosynthesis, it is essential to develop knowledge based in silico approaches for correlating the sequence and domain organization of PKSs to their polyketide products. In this work, we have carried out extensive sequence analysis of experimentally characterized PKS clusters to develop an automated computational protocol for unambiguous identification of various PKS domains in a polypeptide sequence. A structure based approach has been used to identify the putative active site residues of acyltransferase (AT) domains, which control the specificities for various starter and extender units during polyketide biosynthesis. On the basis of the analysis of the active site residues and molecular modelling of substrates in the active site of representative AT domains, we have identified a crucial residue that is likely to play a major role in discriminating between malonate and methylmalonate during selection of extender groups by this domain. Structural modelling has also explained the experimentally observed chiral preference of AT domain in substrate selection. This computational protocol has been used to predict the domain organization and substrate specificity for PKS clusters from various microbial genomes. The results of our analysis as well as the computational tools for prediction of domain organization and substrate specificity have been organized in the form of a searchable computerized database (PKSDB). PKSDB would serve as a valuable tool for identification of polyketide products biosynthesized by uncharacterized PKS clusters. This database can also provide guidelines for rational design of experiments to engineer novel polyketides.     

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.     

Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations.

Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced. Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains. Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4. This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.     

The acyltransferase homologue from the initiation module of the R1128 polyketide synthase is an acyl-ACP thioesterase that edits acetyl primer units

Type II polyketide synthases (PKSs) synthesize polyfunctional aromatic polyketides through iterative condensations of malonyl extender units. The biosynthesis of most aromatic polyketides is initiated through an acetate unit derived from decarboxylation of malonyl-acyl carrier protein (ACP). Modification of this primer unit represents a powerful method of generating novel polyketides. We have demonstrated that recombination of the initiation module from the R1128 PKS with heterologous elongation modules afforded regioselectively modified polyketides containing alternative primer units. With the exception of the role of the acyltransferase homologue ZhuC, the catalytic cycle of the initiation module has been well explored. ZhuC, along with the ketosynthase III homologue ZhuH and the ACP(p) ZhuG, is essential for the in vivo biosynthesis of aromatic polyketides derived from non-acetate primer units. Here we have studied the role of ZhuC using PKS proteins reconstituted in vitro. We show that the tetracenomycin (tcm) minimal PKS can be directly primed with non-acetate acyl groups. In the presence of approximately 10 microM hexanoyl-ZhuG or approximately 100 microM hexanoyl-CoA, the tcm minimal PKS synthesized hexanoyl-primed analogues of octaketides SEK4 and SEK4b, as well as acetate-primed decaketides SEK15 and SEK15b at comparable levels. Addition of ZhuC abolished synthesis of the acetate-primed decaketides, resulting in exclusive synthesis of the hexanoyl-primed octaketides. In the absence of alternative acyl donors, ZhuC severely retarded the activity of the tcm minimal PKS. The editing capabilities of ZhuC were directly revealed by demonstrating that ZhuC has 100 times greater specificity for acetyl- and propionyl-ACP as compared to hexanoyl- and octanoyl-ACP. Thus, by purging the acetate primer units that otherwise dominate polyketide chain initiation, ZhuC (and presumably its homologues in other PKSs such as the doxorubicin and frenolicin PKSs) allows alternative primer units to be utilized by the elongation module in vivo. The abilities of other alkylacyl primer units to prime the tcm minimal PKS were also investigated in this report.     

Engineered biosynthesis of a novel amidated polyketide, using the malonamyl-specific initiation module from the oxytetracycline polyketide synthase

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.     

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.     

三烷基取代芳香聚酮gombapyrones生物合成基因簇的确认及其生物合成推导

【目的】本研究旨在确认链霉菌Streptomyces rubellomurinus ATCC 31215来源芳香聚酮化合物(gombapyrones, GOMs)的生物合成基因簇(biosynthetic gene cluster, BGC),并对其生物合成途径进行推导。【方法】对链霉菌S. rubellomurinus ATCC 31215进行大规模发酵及提取分离,得到GOM-B和GOM-D;以三烷基取代芳香聚酮生物合成途径保守存在的P450单氧化酶的蛋白序列作为探针,在GOMs产生菌S. rubellomurinus基因组中进行BLAST搜索获得潜在的GOMs生物合成基因簇(gom BGC);通过对gom BGC中的聚酮合成酶(polyketide synthase, PKS)结构基因进行同框缺失突变,对突变株发酵产物进行高效液相色谱-质谱(highperformanceliquidchromatography-massspectrometry,HPLC-MS)分析以确认gomBGC与GOMs的产生相关;基于生物信息学分析,推导GOM-B的生物合成途径。【结果】从S. rubell...     

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C₁₈ chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides.     

Initiation of polyene macrolide biosynthesis: interplay between polyketide synthase domains and modules as revealed via domain swapping, mutagenesis, and heterologous complementation

Polyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT(0) in the nystatin PKS loading module NysA with the propionate-specific AT(1) from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT(0) domain by site-directed mutagenesis were accomplished. However, none of the NysA mutants constructed was able to initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids were functional, providing for different levels of nystatin biosynthesis. An interplay between certain residues in AT(0) and an active site residue in the ketosynthase (KS)-like domain of NysA in initiation of nystatin biosynthesis was revealed. Some hybrids between the NysA and RimA loading modules carrying the NysA AT(0) domain were able to prime rimocidin PKS with both acetate and butyrate units upon complementation of a rimA-deficient mutant of the rimocidin/CE-108 producer Streptomyces diastaticus. Expression of the PimS0 loading module from the pimaricin producer in the same host, however, resulted in production of CE-108 only. Taken together, these data indicate relaxed substrate specificity of NysA AT(0) domain, which is counteracted by a strict specificity of the first extender module KS domain in the nystatin PKS of Streptomyces noursei.     

Cloning and characterization of a type III polyketide synthase from Aspergillus niger

Type III polyketide synthases (PKSs) are the condensing enzymes that catalyze the formation of a myriad of aromatic polyketides in plant, bacteria, and fungi. Here we report the cloning and characterization of a putative type III PKS from Aspergillusniger, AnPKS. This enzyme catalyzes the synthesis of alkyl pyrones from C2 to C18 starter CoA thioesters with malonyl-CoA as an extender CoA through decaboxylative condensation and cyclization. It displays broad substrate specificity toward fatty acyl-CoA starters to yield triketide and tetraketide pyrones, with benzoyl-CoA as the most preferred starter. The optimal temperature and pH of AnPKS are 50°C and 8, respectively. Under optimal conditions, the enzyme shows the highest catalytic efficiency (k(cat)/K(m)) of 7.4×10(5)s(-1)M(-1) toward benzoyl-CoA. Homology modeling and site-directed mutagenesis were used to probe the molecular basis of its substrate specificity. This study should open doors for further engineering of AnPKS as a biocatalyst for synthesis of value-added polyketides.     

6-Deoxyerythronolide B analogue production in Escherichia coli through metabolic pathway engineering

The erythromycin precursor polyketide 6-deoxyerythronolide B (6-dEB) is produced from one propionyl-CoA starter unit and six (2S)-methylmalonyl-CoA extender units. In vitro studies have previously demonstrated that the loading module of 6-deoxyerythronolide B synthase (DEBS) exhibits relaxed substrate specificity and is able to accept butyryl-CoA, leading to the production of polyketides with butyrate starter units. We have shown that we can produce butyryl-CoA at levels of up to 50% of the total CoA pool in Escherichia coli cells that overexpress the acetoacetyl-CoA:acetyl-CoA transferase, AtoAD (EC 2.8.3.8), in media supplemented with butyrate. The DEBS polyketide synthase (PKS) used butyryl-CoA and methylmalonyl-CoA supplied in vivo by the AtoAD and methylmalonyl-CoA mutase pathways, respectively, to produce 15-methyl-6-dEB. Priming DEBS with endogenous butyryl-CoA affords an alternative and more direct route to 15-Me-6-dEB than that provided by the chemobiosynthesis method [Jacobsen, J. R., et al. (1997) Science 277, 367-369], which relies on priming a mutant DEBS with an exogenously fed diketide thioester. The approach described here demonstrates the utility of metabolic engineering in E. coli to introduce precursor pathways for the production of novel polyketides.     

Characterization of two novel non-reducing polyketide synthase genes from the lichen-forming fungus Hypogymnia physodes

Lichens are known to produce a variety of secondary metabolites including polyketides, which have valuable biological activities. Some polyketides are produced solely by lichens. The biosynthesis of these compounds is primarily governed by iterative type I polyketide synthases. Hypogymnia physodes synthesize polyketides such as physodic, physodalic and hydroxyphysodic acid and atranorin, which are non-reducing polyketides. Two novel non-reducing polyketide synthase (PKS) genes were isolated from a fosmid genomic library of a mycobiont of H. physodes using a 409bp fragment corresponding to part of the reductase (R) domain as a probe. H. physodes PKS1 (Hyopks1) and PKS2 (Hypopks2) contain keto synthase (KS), acyl transferase (AT), acyl carrier protein (ACP), methyl transferase (ME) and R domains. Classification based on phylogeny analysis using the translated KS and AT domains demonstrated that Hypopks1 and Hypopks2 are members of the fungal non-reducing PKSs clade III. This is the first report of non-reducing PKSs containing the R domain-mediated release mechanisms in lichens, which are also rare fungal type I PKS in non-lichenized filamentous fungi.     

Genome mining reveals the evolutionary origin and biosynthetic potential of basidiomycete polyketide synthases

Numerous polyketides are known from bacteria, plants, and fungi. However, only a few have been isolated from basidiomycetes. Large scale genome sequencing projects now help anticipate the capacity of basidiomycetes to synthesize polyketides. In this study, we identified and annotated 111 type I and three type III polyketide synthase (PKS) genes from 35 sequenced basidiomycete genomes. Phylogenetic analysis of PKS genes suggests that all main types of fungal iterative PKS had already evolved before the Ascomycota and Basidiomycota diverged. A comparison of genomic and metabolomic data shows that the number of polyketide genes exceeds the number of known polyketide structures by far. Exploiting these results to design degenerate PCR primers, we amplified and cloned the complete sequence of armB, a PKS gene from the melleolide producer Armillaria mellea. We expect this study will serve as a guide for future genomic mining projects to discover structurally diverse mushroom-derived polyketides.     

Site-specific mutagenesis and domain substitutions in the loading module of the nystatin polyketide synthase,and their effects on nystatin biosynthesis in Streptomyces noursei

The loading module for the nystatin polyketide synthase (PKS) in Streptomyces noursei is represented by the NysA protein composed of a ketosynthase (KS(S)), acyltransferase, dehydratase, and an acyl carrier protein. The absolute requirement of this protein for initiation of nystatin biosynthesis was demonstrated by the in-frame deletion of the nysA gene in S. noursei. The role of the NysA KS(S) domain, however, remained unclear, since no data on the significance of the "active site" serine (Ser-170) residue in the loading modules of type I PKSs were available. Site-specific mutagenesis of Ser-170 both in the wild-type NysA and in the hybrid loading module containing malonyl-specific acyltransferase domain from the extender module had no effect on nystatin biosynthesis. A second mutation (S413N) of the NysA KS(S) domain was discovered that completely abolished the ability of the hybrids to restore nystatin biosynthesis, presumably by affecting the ability of the resulting proteins to catalyze the required substrate decarboxylation. In contrast, NysA and its Ser-170 mutants bearing the same S413N mutation were able to restore nystatin production to significant levels, probably by using acetyl-CoA as a starter unit. Together, these data suggest that the KS(S) domain of NysA differs from the KS(Q) domains found in the loading modules of several PKS type I systems in that the active site residue is not significant for its activity.     

Cloning, sequencing and characterization of the biosynthetic gene cluster of sanglifehrin A, a potent cyclophilin inhibitor

Sanglifehrin A (SFA), a potent cyclophilin inhibitor produced by Streptomyces flaveolus DSM 9954, bears a unique [5.5] spirolactam moiety conjugated with a 22-membered, highly functionalized macrolide through a linear carbon chain. SFA displays a diverse range of biological activities and offers significant therapeutic potential. However, the structural complexity of SFA poses a tremendous challenge for new analogue development via chemical synthesis. Based on a rational prediction of its biosynthetic origin, herein we report the cloning, sequencing and characterization of the gene cluster responsible for SFA biosynthesis. Analysis of the 92 776 bp contiguous DNA region reveals a mixed polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) pathway which includes a variety of unique features for unusual PKS and NRPS building block formation. Our findings suggest that SFA biosynthesis requires a crotonyl-CoA reductase/carboxylase (CCR) for generation of the putative unusual PKS starter unit (2R)-2-ethylmalonamyl-CoA, an iterative type I PKS for the putative atypical extender unit (2S)-2-(2-oxo-butyl)malonyl-CoA and a phenylalanine hydroxylase for the NRPS extender unit (2S)-m-tyrosine. A spontaneous ketalization of significant note, may trigger spirolactam formation in a stereo-selective manner. This study provides a framework for the application of combinatorial biosynthesis methods in order to expand the structural diversity of SFA.     

Active-site residue,domain and module swaps in modular polyketide synthases

Sequence comparisons of multiple acyltransferase (AT) domains from modular polyketide synthases (PKSs) have highlighted a correlation between a short sequence motif and the nature of the extender unit selected. When this motif was specifically altered in the bimodular model PKS DEBS1-TE of Saccharopolyspora erythraea, the products included triketide lactones in which acetate extension units had been incorporated instead of propionate units at the predicted positions. We also describe a cassette system for convenient construction of hybrid modular PKSs based on the tylosin PKS in Streptomyces fradiae and demonstrate its use in domain and module swaps.     

The use of PCR for detecting genes that encode type I polyketide synthases in genomes of actinomycetes

PCR screening of type I polyketidesynthase genes (PKS) was conducted in genomes of actinomycetes, producers of antibiotics. Some DNA fragments from the Streptomyces globisporus 1912 strain, a producer of a novel angucycline antibiotic landomycin E, were amplified. These fragments shared appreciable homology with type I PKS controlling the biosynthesis of polyene antibiotics (pymaricin and nistatin). The cloned regions were used to inactivate putative type I PKS genes in S. globisporus 1912. Strains with inactivated genes of PKS module do not differ from the original strain in the spectrum of synthesized polyketides. Apparently, these are silent genes, which require specific induction for their expression. The method of PCR screening can be used in a large-scale search for producers of new antibiotics.     

Evolutionary affiliations within the superfamily of ketosynthases reflect complex pathway associations

Abstract Type I polyketide synthases are known to produce a wide range of medically and industrially important polyketides. The ketosynthase (KS) domain is required for the condensation of an extender unit onto the growing polyketide chain during polyketide biosynthesis. KSs represent a superfamily of complex biosynthetic pathway-associated enzymes found in prokaryotes, fungi, and plants. Although themselves functionally conserved, KSs are involved in the production of a structurally diverse range of metabolites. Degenerate oligonucleotide primers, designed for the amplification of KS domains, amplified KS domains from a range of organisms including cyanobacterial and dinoflagellates. KS domains detected in dinoflagellate cultures appear to have been amplified from the less than 3-μm filtrate of the nonaxenic culture. Phylogenetic analysis of sequences obtained during this study enabled the specific identification of KS domains of hybrid or mixed polyketide synthase/peptide synthetase complexes, required for the condensation of an extender unit onto an amino acid starter unit. The primer sets described in this study were also used for the detection of novel KS domains directly from environmental samples. The ability to predict function based on primary molecular structure will be critical for future discovery and rational engineering of polyketides.     

Structural and biochemical studies of the hedamycin type II polyketide ketoreductase (HedKR): molecular basis of stereo- and regiospecificities

Bacterial aromatic polyketides that include many antibiotic and antitumor therapeutics are biosynthesized by the type II polyketide synthase (PKS), which consists of 5-10 stand-alone enzymatic domains. Hedamycin, an antitumor antibiotic polyketide, is uniquely primed with a hexadienyl group generated by a type I PKS followed by coupling to a downstream type II PKS to biosynthesize a 24-carbon polyketide, whose C9 position is reduced by hedamycin type II ketoreductase (hedKR). HedKR is homologous to the actinorhodin KR (actKR), for which we have conducted extensive structural studies previously. How hedKR can accommodate a longer polyketide substrate than the actKR, and the molecular basis of its regio- and stereospecificities, is not well understood. Here we present a detailed study of hedKR that sheds light on its specificity. Sequence alignment of KRs predicts that hedKR is less active than actKR, with significant differences in substrate/inhibitor recognition. In vitro and in vivo assays of hedKR confirmed this hypothesis. The hedKR crystal structure further provides the molecular basis for the observed differences between hedKR and actKR in the recognition of substrates and inhibitors. Instead of the 94-PGG-96 motif observed in actKR, hedKR has the 92-NGG-94 motif, leading to S-dominant stereospecificity, whose molecular basis can be explained by the crystal structure. Together with mutations, assay results, docking simulations, and the hedKR crystal structure, a model for the observed regio- and stereospecificities is presented herein that elucidates how different type II KRs recognize substrates with different chain lengths, yet precisely reduce only the C9-carbonyl group. The molecular features of hedKR important for regio- and stereospecificities can potentially be applied to biosynthesize new polyketides via protein engineering that rationally controls polyketide ketoreduction.