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.     

A mechanism based protein crosslinker for acyl carrier protein dehydratases

Recent advances in the structural study of fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic enzymes have illuminated our understanding of modular enzymes of the acetate pathway. However, one significant and persistent challenge in such analyses is resolution of the acyl carrier protein (ACP), a small (～9 kDa) protein to which biosynthetic intermediates are tethered throughout the biosynthetic cycle. Here we report a chemoenzymatic crosslinking strategy in which the installation of a historical suicide substrate scaffold upon the 4′-phosphopantetheine (PPant) arm of the ACP is used to capture the active site of acyl carrier protein dehydratase (DH) domains in FAS. Through the synthesis of a small panel of related probes we identify structural features essential for ACP–DH crosslinking, and apply gel-based assays to demonstrate the stability as well as purification strategies for isolation of the chemoenzymatically modified ACP. Applying these carrier protein crosslinking techniques to the structural analysis of FAS and PKS complexes has the potential to provide snapshots of these biosynthetic assembly lines at work.     

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.     

Chimeric 6-methylsalicylic acid synthase with domains of acyl carrier protein and methyltransferase from Pseudallescheria boydii shows novel biosynthetic activity

Polyketides are important secondary metabolites, many of which exhibit potent pharmacological applications. Biosynthesis of polyketides is carried out by a single polyketide synthase (PKS) or multiple PKSs in successive elongations of enzyme-bound intermediates related to fatty acid biosynthesis. The polyketide gene PKS306 from Pseudallescheria boydii NTOU2362 containing domains of ketosynthase (KS), acyltransferase (AT), dehydratase (DH), acyl carrier protein (ACP) and methyltransferase (MT) was cloned in an attempt to produce novel chemical compounds, and this PKS harbouring green fluorescent protein (GFP) was expressed in Saccharomyces cerevisiae. Although fluorescence of GFP and fusion protein analysed by anti-GFP antibody were observed, no novel compound was detected. 6-methylsalicylic acid synthase (6MSAS) was then used as a template and engineered with PKS306 by combinatorial fusion. The chimeric PKS containing domains of KS, AT, DH and ketoreductase (KR) from 6MSAS with ACP and MT from PKS306 demonstrated biosynthesis of a novel compound. The compound was identified with a deduced chemical formula of C₇H₁₀O₃, and the chemical structure was named as 2-hydroxy-2-(propan-2-yl) cyclobutane-1,3-dione. The novel compound synthesized by the chimeric PKS in this study demonstrates the feasibility of combinatorial fusion of PKS genes to produce novel polyketides.     

Architectures of Whole-Module and Bimodular Proteins from the 6-Deoxyerythronolide B Synthase

The 6-deoxyerythronolide B synthase (DEBS) is a prototypical assembly line polyketide synthase produced by the actinomycete Saccharopolyspora erythraea that synthesizes the macrocyclic core of the antibiotic erythromycin 6-deoxyerythronolide B. The megasynthase is a 2-MDa trimeric complex composed of three unique homodimers assembled from the gene products DEBS1, DEBS2, and DEBS3, which are housed within the erythromycin biosynthetic gene cluster. Each homodimer contains two clusters of catalytically independent enzymatic domains, each referred to as a module, which catalyzes one round of polyketide chain extension and modification. Modules are named sequentially to indicate the order in which they are utilized during synthesis of 6-deoxyerythronolide B. We report small-angle X-ray scattering (SAXS) analyses of a whole module and a bimodule from DEBS, as well as a set of domains for which high-resolution structures are available. In all cases, the solution state was probed under previously established conditions ensuring that each protein is catalytically active. SAXS data are consistent with atomic-resolution structures of DEBS fragments. Therefore, we used the available high-resolution structures of DEBS domains to model the architectures of the larger protein assemblies using rigid-body refinement. Our data support a model in which the third module of DEBS forms a disc-shaped structure capable of caging the acyl carrier protein domain proximal to each active site. The molecular envelope of DEBS3 is a thin elongated ellipsoid, and the results of rigid-body modeling suggest that modules 5 and 6 stack collinearly along the 2-fold axis of symmetry.     

草菇聚酮合酶编码基因vv-alb的克隆及其表达的初步分析

聚酮化合物（polyketides）是一类庞大的次级代谢家族,聚酮合酶（polyketide synthase,PKS）是介导聚酮化合物生物合成的关键酶。通过巢氏简并PCR与染色体步行的方法,获得了草菇中的编码PKS的基因vv-alb的全长序列,并通过荧光实时定量RT-PCR方法对vv-alb基因在草菇不同生长阶段与不同部位的表达情况进行了初步分析,为进一步研究PKS在草菇和其他食用真菌生物代谢过程中的作用奠定了一定的基础。     

Enzymatic assembly of epothilones: the EpoC subunit and reconstitution of the EpoA-ACP/B/C polyketide and nonribosomal peptide interfaces

The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value. Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli. EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis. The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction. The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB. The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain. The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible. These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.     

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.     

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.     

Solution structure of Asl1650, an acyl carrier protein from Anabaena sp. PCC 7120 with a variant phosphopantetheinylation-site sequence

Cyanobacteria, such as Anabaena, produce a variety of bioactive natural products via polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS), and hybrid peptide/polyketide pathways. The protein Asl1650, which is a member of the acyl carrier protein family from the cyanobacterium Anabaena sp. PCC 7120, is encoded in a region of the Anabaena genome that is rich in PKS and NRPS genes. To gain new insight into the physiological role of acyl carriers in Anabaena, the solution structure of Asl1650 has been solved by NMR spectroscopy. The protein adopts a twisted antiparallel four-helix bundle fold, with a variant phosphopantetheine-attachment motif positioned at the start of the second helix. Structure comparisons with proteins from other organisms suggest a likely physiological function as a discrete peptidyl carrier protein.     

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     

Approaches to stabilization of inter-domain recombination in polyketide synthase gene expression plasmids

Regions of extremely high sequence identity are recurrent in modular polyketide synthase (PKS) genes. Such sequences are potentially detrimental to the stability of PKS expression plasmids used in the combinatorial biosynthesis of polyketide metabolites. We present two different solutions for circumventing intra-plasmid recombination within the megalomicin PKS genes in Streptomyces coelicolor. In one example, a synthetic gene was used in which the codon usage was reengineered without affecting the primary amino acid sequence. The other approach utilized a heterologous subunit complementation strategy to replace one of the problematic regions. Both methods resulted in PKS complexes capable of 6-deoxyerythronolide B analogue biosynthesis in S. coelicolor CH999, permitting reproducible scale-up to at least 5-l stirred-tank fermentation and a comparison of diketide precursor incorporation efficiencies between the erythromycin and megalomicin PKSs. Electronic Publication     

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.     

Structural and functional studies on SCO1815: a beta-ketoacyl-acyl carrier protein reductase from Streptomyces coelicolor A3(2)

Aromatic polyketides are medicinally important natural products produced by type II polyketide synthases (PKSs). Some aromatic PKSs are bimodular and include a dedicated initiation module which synthesizes a non-acetate primer unit. Understanding the mechanism of this initiation module is expected to further enhance the potential for regiospecific modification of bacterial aromatic polyketides. A typical initiation module is comprised of a ketosynthase (KS), an acyl carrier protein (ACP), a malonyl-CoA:ACP transacylase (MAT), an acyl-ACP thioesterase, a ketoreductase (KR), a dehydratase (DH), and an enoyl reductase (ER). Thus far, the identities of the ketoreductase, dehydratase, and enoyl reductase remain a mystery because they are not encoded within the PKS biosynthetic gene cluster. Here we report that SCO1815 from Streptomyces coelicolor A3(2), an uncharacterized homologue of a NADPH-dependent ketoreductase, recognizes and reduces the beta-ketoacyl-ACP intermediate from the initiation module of the R1128 PKS. SCO1815 exhibits moderate specificity for both the acyl chain and the thiol donor. The X-ray crystal structure of SCO1815 was determined to 2.0 A. The structure shows that SCO1815 adopts a Rossmann fold and suggests that a conformational change occurs upon cofactor binding. We propose that a positively charged patch formed by three conserved residues is the ACP docking site. Our findings provide new engineering opportunities for incorporating unnatural primer units into novel polyketides and shed light on the biology of yet another cryptic protein in the S. coelicolor genome.     

Identification of two polyketide synthase gene clusters on the linear plasmid pSLA2-L in Streptomyces rochei

The 200kb linear plasmid pSLA2-L was suggested to be involved in the production of two macrolide antibiotics, lankamycin (Lm) and lankacidin (Lc), in Streptomyces rochei 7434AN4. Hybridization experiments with the polyketide synthase (PKS) genes for erythromycin and actinorhodin identified two eryAI-homologous regions and an actI-homologous region on pSLA2-L. The nucleotide sequence of a 3.6kb SacI fragment carrying one of the eryAI-homologs revealed that it codes for part of a large protein with four domains for ketoreductase, acyl carrier protein, ketosynthase, and acyltransferase. Gene disruption confirmed that the two eryAI-homologs are parts of a large type-I PKS gene cluster for Lm. A 4.8kb DNA carrying the actI-homologous region contains four open reading frames (ORF1-ORF4) as well as an additional ORF, i.e. ORF5, which might code for a thioesterase. Deletion of the ORF2-ORF4 region showed that it is not involved in the synthesis of Lm or Lc. Thus, it was confirmed that pSLA2-L contains two PKS gene clusters for Lm and an unknown type-II polyketide.     

Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant.

Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.