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.     

Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase

The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions.     

Inter-domain movements in polyketide synthases: a molecular dynamics study

Insights into the structure and dynamics of modular polyketide synthases (PKS) are essential for understanding the mechanistic details of the biosynthesis of a large number of pharmaceutically important secondary metabolites. The crystal structures of the KS-AT di-domain from erythromycin synthase have revealed the relative orientation of various catalytic domains in a minimal PKS module. However, the relatively large distance between catalytic centers of KS and AT domains in the static structure has posed certain intriguing questions regarding mechanistic details of substrate transfer during polyketide biosynthesis. In order to investigate the role of inter-domain movements in substrate channeling, we have carried out a series of explicit solvent MD simulations for time periods ranging from 10 to 15 ns on the KS-AT di-domain and its sub-fragments. Analyses of these MD trajectories have revealed that both the catalytic domains and the structured inter-domain linker region remain close to their starting structures. Inter-domain movements at KS-linker and linker-AT interfaces occur around hinge regions which connect the structured linker region to the catalytic domains. The KS-linker interface was found to be more flexible compared to the linker-AT interface. However, inter-domain movements observed during the timescale of our simulations do not significantly reduce the distance between catalytic centers of KS and AT domains for facilitating substrate channeling. Based on these studies and prediction of intrinsic disorder we propose that the intrinsically unstructured linker stretch preceding the ACP domain might be facilitating movement of ACP domains to various catalytic centers.     

Characterization of the biosynthetic gene cluster for the antifungal polyketide soraphen A from Sorangium cellulosum So ce26

A genomic DNA region of over 80 kb that contains the complete biosynthetic gene cluster for the synthesis of the antifungal polyketide metabolite soraphen A was cloned from Sorangium cellulosum So ce26. The nucleotide sequence of the soraphen A gene region, including 67,523 bp was determined. Examination of this sequence led to the identification of two adjacent type I polyketide synthase (PKS) genes that encode the soraphen synthase. One of the soraphen A PKS genes includes three biosynthetic modules and the second contains five additional modules for a total of eight. The predicted substrate specificities of the acyltransferase (AT) domains, as well as the reductive loop domains identified within each module, are consistent with expectations from the structure of soraphen A. Genes were identified in the regions flanking the two soraphen synthase genes that are proposed to have roles in the biosynthesis of soraphen A. Downstream of the soraphen PKS genes is an O-methyltransferase (OMT) gene. Upstream of the soraphen PKS genes there is a gene encoding a reductase and a group of genes that are postulated to have roles in the synthesis of methoxymalonyl-acyl carrier protein (ACP). This unusual extender unit is proposed to be incorporated in two positions of the soraphen polyketide chain. One of the genes in this group contains distinct domains for an AT, an ACP, and an OMT.     

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.     

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.     

Analysis of the enzymatic domains in the modular portion of the coronafacic acid polyketide synthase

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant pathogen Pseudomonas syringae. The CFA polyketide synthase (PKS) consists of two open reading frames (ORFs) that encode type I multifunctional proteins and several ORFs that encode monofunctional proteins. Sequence comparisons of the modular portions of the CFA PKS with other prokaryotic, modular PKSs elucidated the boundaries of the domains that are involved in the individual stages of polyketide assembly. The two β-ketoacyl:acyl carrier protein synthase (KS) domains in the modular portion of the CFA PKS exhibit a high degree of similarity to each other (53%), but are even more similar to the KS domains of DEBS, RAPS, and RIF. Cfa6 possesses two acyltransferases- AT0, which is associated with a loading domain, and AT1, which uses ethylmalonyl-CoA (eMCoA) as a substrate for chain extension. Cfa7 contains an AT that uses malonyl-CoA as a substrate for chain extension. The Cfa6 AT0 shows 35 and 32% similarity to the DEBS1 and NidA1 AT0s, respectively, and 32 and 36% similarity to the Cfa6 and Cfa7 AT1s. Sequence motifs have previously been identified that correlate with AT substrates. The motifs in Cfa6 AT1 were found to correlate reasonably well with those predicted for methylmalonyl-CoA (mMCoA) ATs. The motifs in the AT of Cfa7 correlated more poorly with those predicted for MCoA ATs. Three ACP domains occur in the modular proteins of the COR PKS. The loading domain-associated ACP0 showed 38% similarity to the loading domain ACP0s of DEBS1 and NidA1 and 32–36% similarity to the two module-associated ACPs of the COR PKS. It exhibited a higher degree of similarity to the module-associated ACPs of RAPS. The two module-associated ACPs show 39% similarity to each other, but appear more closely related to module-associated ACP domains in RAPS and RIFS. Furthermore, the DH and KR domains of Cfa6 and Cfa7 show greater similarity to DH and KR domains in RAPS and RIFS than to each other. The CFA PKS includes a thioesterase domain (TE I) that resides at the C-terminus of Cfa7 and a second thioesterase, which exists as a separate ORF (Cfa9, a TE II). Analysis of a Cfa7 thioesterase mutant demonstrated that the TE domain is required for the production of CFA. The co-existence of TE domains within modular PKSs along with physically separated, monofunctional TEs (TE IIs) has been reported for a number of modular polyketide and non-ribosomal peptide synthases (NRPS). An analysis of the two types of thioesterases using Clustal X yielded a dendrogram showing that TE IIs from PKSs and NRPSs are more closely related to each other than to domain TEs from either PKSs or NRPSs. Furthermore, the dendrogram indicates that both types of TE IIs are more closely related to TE domains associated with PKSs than to TE domains in NRPSs. Finally, the overall % G+C content and the % G+C content at the third codon for all of the PKS genes in the COR cluster suggest that these genes may have been recruited from a gram-positive bacterium.     

Determination of the extent of phosphopantetheinylation of polyketide synthases expressed in Escherichia coli and Saccharomyces cerevisiae

A sensitive fluorescent assay was developed to measure the extent of phosphopantetheinylation of polyketide synthase (PKS) acyl carrier protein (ACP) domains in polyketide production strains. The in vitro assay measures PKS fluorescence after transfer of fluorescently labeled phosphopantetheine from coenzyme A to PKS ACP domains in crude protein extracts. The assay was used to determine the extent of phosphopantetheinylation of ACP domains of the erythromycin precursor polyketide synthase, 6-deoxyerythronolide B synthase (DEBS), expressed in a heterologous Escherichia coli polyketide production strain. The data showed that greater than 99.9% of DEBS is phosphopantetheinylated. The assay was also used to interrogate the extent of phosphopantetheinylation of the lovastatin nonaketide synthase (LNKS) heterologously expressed in Saccharomyces cerevisiae. The data showed that LNKS was efficiently phosphopantetheinylated in S. cerevisiae and that lack of production of the lovastatin precursor polyketide was not due to insufficient phosphopantetheinylation of the expressed synthase.     

Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.     

Starter unit specificity directs genome mining of polyketide synthase pathways in fungi

Search of the protein database with the aflatoxin pathway polyketide synthase (PKS) revealed putative PKSs in the pathogenic fungi Coccidioides immitis and Coccidioides posadasii that could require partnerships with a pair of fatty acid synthase (FAS) subunits for the biosynthesis of fatty acid-polyketide hybrid metabolites. A starter unit:acyl-carrier protein transacylase (SAT) domain was discovered in the nonreducing PKS. This domain is thought to accept the fatty acid product from the FAS to initiate polyketide synthesis. We expressed the C. immitis SAT domain in Escherichia coli and showed that this domain, unlike that from the aflatoxin pathway PKS, transferred octanoyl-CoA four times faster than hexanoyl-CoA. The SAT domain also formed a covalent octanoyl intermediate and transferred this group to a free-standing ACP domain. Our results suggest that C. immitis/posadasii, both human fungal pathogens, contain a FAS/PKS cluster with functional similarity to the aflatoxin cluster found in Aspergillus species. Dissection of the PKS and determination of in vitro SAT domain specificity provides a tool to uncover the growing number of similar sequenced pathways in fungi, and to guide elucidation of the fatty acid-polyketide hybrid metabolites that they produce.     

Naturally mosaic operons for secondary metabolite biosynthesis: variability and putative horizontal transfer of discrete catalytic domains of the epothilone polyketide synthase locus

A putative instance of horizontal gene transfer (HGT) involving adjacent, discrete beta-ketoacyl synthase (KS), acyl carrier protein (ACP) and nonribosomal peptide synthase (NRPS) domains of the epothilone Type I polyketide biosynthetic gene cluster from the myxobacterium Sorangium cellulosom was identified using molecular phylogenetics and sequence analyses. The specific KS domain of the module EPO B fails to cluster phylogenetically with other epothilone KS sequences present at this locus, in contrast to what is typically observed in many other Type I polyketide synthase (PKS) biosynthetic loci. Furthermore, the GC content of the epoB KS, epoA ACP and NRPS domains differs significantly from the base composition of other epothilone domain sequences. In addition, the putatively transferred epothilone loci are located near previously identified transposon-like sequences. Lastly, comparison with other KS loci revealed another possible case of horizontal transfer of secondary metabolite genes in the genus Pseudomonas. This study emphasizes the use of several lines of concordant evidence (phylogenetics, base composition, transposon sequences) to infer the evolutionary history of particular gene and enzyme sequences, and the results support the idea that genes coding for adaptive traits, e.g. defensive natural products, may be prone to transposition between divergent prokaryotic taxa and genomes.     

Targeted gene replacements in a Streptomyces polyketide synthase gene cluster: role for the acyl carrier protein

A methodology was developed to construct any desired chromosomal mutation in the gene cluster that encodes the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2). A positive selection marker (resistance gene) is first introduced by double crossing-over into the chromosomal site of interest by use of an unstable delivery plasmid. This marker is subsequently replaced by the desired mutant allele via a second high-frequency double recombination event. The technology has been used to: (i) explore the significance of translational coupling between two adjacent PKS genes; (ii) prove that the acyl carrier protein (ACP) encoded by a gene in the cluster is necessary for the function of the actinorhodin PKS; (iii) provide genetic evidence supporting the hypothesis that serine 42 is the site of phosphopantetheinylation in the ACP of the actinorhodin PKS; and (iv) demonstrate that this ACP can be replaced by a Saccharopolyspora fatty acid synthase ACP to generate an active hybrid PKS.     

Chain initiation on type I modular polyketide synthases revealed by limited proteolysis and ion-trap mass spectrometry

Limited proteolysis in combination with liquid chromatography-ion trap mass spectrometry (LC-MS) was used to analyze engineered or natural proteins derived from a type I modular polyketide synthase (PKS), the 6-deoxyerythronolide B synthase (DEBS), and comprising either the first two extension modules linked to the chain-terminating thioesterase (TE) (DEBS1-TE); or the last two extension modules (DEBS3) or the first extension module linked to TE (diketide synthase, DKS). Functional domains were released by controlled proteolysis, and the exact boundaries of released domains were obtained through mass spectrometry and N-terminal sequencing analysis. The acyltransferase-acyl carrier protein required for chain initiation (AT(L)-ACP(L)), was released as a didomain from both DEBS1-TE and DKS, as well as the off-loading TE as a didomain with the adjacent ACP. Mass spectrometry was used successfully to monitor in detail both the release of individual domains, and the patterns of acylation of both intact and digested DKS when either propionyl-CoA or n-butyryl-CoA were used as initiation substrates. In particular, both loading domains and the ketosynthase domain of the first extension module (KS1) were directly observed to be simultaneously primed. The widely available and simple MS methodology used here offers a convenient approach to the proteolytic mapping of PKS multienzymes and to the direct monitoring of enzyme-bound intermediates.     

Expression and characterization of polyketide synthase module involved in the late step of cephabacin biosynthesis from Lysobacter lactamgenus

The cephabacins produced by Lysobacter lactamgenus are beta-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, beta-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of His6-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-LAla- L-3H-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/ PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with 14C-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.