A comprehensive and engaging overview of the type III family of polyketide synthases

Customizing biosynthesis of natural products to yield biologically active derivatives has captivated scientists in the field of biosynthetic research. To substantiate this goal, there are scores of obstacles to consider. To create novel metabolites by mutating amino acid residues in wild-type enzymes, a researcher must broaden the range of the enzymes substrate tolerance and increase its turnover rate during reaction catalysis. In the past decade, numerous gene clusters responsible for the biosynthesis of notable natural products have been identified from a variety of organisms. Several genes coding for type III polyketide synthases, particularly the chalcone synthase superfamily enzymes, were recently uncovered and expressed in E. coli. Furthermore, it was observed and reported how these recombinant enzymes are capable of producing essential metabolites in vitro. Three of the type III polyketide synthases, chalcone synthase, octaketide synthase and pentaketide chromone synthase, have been characterized and their active sites subjected to rational engineering for biosynthetic production of their analogs. Because they are encoded in a single open reading frame and are post-translationally small in size, type III polyketide synthases are ideal targets for protein engineering. The relative ease with which these genes are expressed makes molecular biological manipulation to obtain mutated enzymes more procurable, ameliorating analysis of its biosynthetic pathway. In summary, time devoted to modification of biosynthetic proteins and unravelling of the detailed reaction mechanisms involved in biosynthesis will be shortened, paving the way for a much wider scope for metabolic engineers in future. This review focuses on the use of chalcone synthase, octaketide synthase and pentaketide chromone synthase for rational biosynthetic engineering to generate molecular diversity and pursue innovative, biologically potent compounds.     

Characterization of two polyketide synthase genes involved in zearalenone biosynthesis in Gibberella zeae

Zearalenone, a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. Zearalenone is a polyketide derived from the sequential condensation of multiple acetate units by a polyketide synthase (PKS), but the genetics of its biosynthesis are not understood. We cloned two genes, designated ZEA1 and ZEA2, which encode polyketide synthases that participate in the biosynthesis of zearalenone by Gibberella zeae (anamorph Fusarium graminearum). Disruption of either gene resulted in the loss of zearalenone production under inducing conditions. ZEA1 and ZEA2 are transcribed divergently from a common promoter region. Quantitative PCR analysis of both PKS genes and six flanking genes supports the view that the two polyketide synthases make up the core biosynthetic unit for zearalenone biosynthesis. An appreciation of the genetics of zearalenone biosynthesis is needed to understand how zearalenone is synthesized under field conditions that result in the contamination of grain.     

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.     

Biochemical and Structural Study of the Atypical Acyltransferase Domain from the Mycobacterial Polyketide Synthase Pks13

Pks13 is a type I polyketide synthase involved in the final biosynthesis step of mycolic acids, virulence factors, and essential components of the Mycobacterium tuberculosis envelope. Here, we report the biochemical and structural characterization of a 52-kDa fragment containing the acyltransferase domain of Pks13. This fragment retains the ability to load atypical extender units, unusually long chain acyl-CoA with a predilection for carboxylated substrates. High resolution crystal structures were determined for the apo, palmitoylated, and carboxypalmitoylated forms. Structural conservation with type I polyketide synthases and related fatty-acid synthases also extends to the interdomain connections. Subtle changes could be identified both in the active site and in the upstream and downstream linkers in line with the organization displayed by this singular polyketide synthase. More importantly, the crystallographic analysis illustrated for the first time how a long saturated chain can fit in the core structure of an acyltransferase domain through a dedicated channel. The structures also revealed the unexpected binding of a 12-mer peptide that might provide insight into domain-domain interaction.     

A fungal ketoreductase domain that displays substrate-dependent stereospecificity

Iterative highly reducing polyketide synthases from filamentous fungi are the most complex and enigmatic type of polyketide synthase discovered to date. Here we uncover an unusual degree of programming by the hypothemycin highly reducing polyketide synthase, in which a single ketoreductase domain shows stereospecificity that is controlled by substrate length. Mapping of the structural domains responsible for this feature allowed for the biosynthesis of an unnatural diastereomer of the natural product dehydrozearalenol.     

SEARCHPKS: A program for detection and analysis of polyketide synthase domains

SEARCHPKS is a software for detection and analysis of polyketide synthase (PKS) domains in a polypeptide sequence. Modular polyketide synthases are unusually large multi-enzymatic multi-domain megasynthases, which are involved in the biosynthesis of pharmaceutically important natural products using an assembly-line mechanism. This program facilitates easy identification of various PKS domains and modules from a given polypeptide sequence. In addition, it also predicts the specificity of the potential acyltransferase domains for various starter and extender precursor units. SEARCHPKS is a user-friendly tool for correlating polyketide chemical structures with the organization of domains and modules in the corresponding modular polyketide synthases. This program also allows the user to extensively analyze and assess the sequence homology of various polyketide synthase domains, thus providing guidelines for carrying out domain and module swapping experiments. SEARCHPKS can also aid in identification of polyketide products made by PKS clusters found in newly sequenced genomes. The computational approach used in SEARCHPKS is based on a comprehensive analysis of various characterized clusters of modular polyketide synthases compiled in PKSDB, a database of modular polyketide synthases. SEARCHPKS can be accessed at http://www.nii.res.in/searchpks.html.     

An acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata

A cDNA encoding a novel plant type III polyketide synthase was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae). The deduced amino acid sequence of Hu. serrata polyketide synthase 1 showed 44-66% identity to those of other chalcone synthase superfamily enzymes of plant origin. Further, phylogenetic tree analysis revealed that Hu. serrata polyketide synthase 1 groups with other nonchalcone-producing type III polyketide synthases. Indeed, a recombinant enzyme expressed in Escherichia coli showed unusually versatile catalytic potential to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, it is remarkable that the enzyme accepted bulky starter substrates such as 4-methoxycinnamoyl-CoA and N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 4-methoxy-2',4',6'-trihydroxychalcone and 1,3-dihydroxy-N-methylacridone, respectively. In contrast, regular chalcone synthase does not accept these bulky substrates, suggesting that the enzyme has a larger starter substrate-binding pocket at the active site. Although acridone alkaloids have not been isolated from Hu. serrata, this is the first demonstration of the enzymatic production of acridone by a type III polyketide synthase from a non-Rutaceae plant. Interestingly, Hu. serrata polyketide synthase 1 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).     

Cloning a part of the ochratoxin A biosynthetic gene cluster of Penicillium nordicum and characterization of the ochratoxin polyketide synthase gene

Penicillium nordicum is a fungal species able to produce high amounts of ochratoxin A. A 10kb genomic DNA fragment of P. nordicumn has been cloned which carries three long open reading frames. One open reading frame (otapksPN) has homology to fungal polyketide synthases. The second open reading frame (npsPN) has homology to non-ribosomal peptide synthetases and the third open reading frame (aspPN) has homology to fungal alkaline serine proteinases. The non-ribosomal peptide synthetase and the polyketide synthase are convergently transcribed. Interestingly, the polyketide synthase can be identified by PCR only in P. nordicum strains and not in the related species Penicillium verrucosum or in ochratoxigenic Aspergillus species, indicating that the ochratoxin polyketide synthases are different in the important ochratoxigenic species. In contrast, the non-ribosomal peptide synthetase can be identified in P. nordicum and P. verrucosum, but not in other species. An inactivation of the polyketide synthase resulted in strains with abolished capacity to produce ochratoxin A. Expression of the polyketide synthase correlates with ochratoxin A biosynthesis.     

Characterization of Two Polyketide Synthase Genes Involved in Zearalenone Biosynthesis in Gibberella zeae

Zearalenone, a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. Zearalenone is a polyketide derived from the sequential condensation of multiple acetate units by a polyketide synthase (PKS), but the genetics of its biosynthesis are not understood. We cloned two genes, designated ZEA1 and ZEA2, which encode polyketide synthases that participate in the biosynthesis of zearalenone by Gibberella zeae (anamorph Fusarium graminearum). Disruption of either gene resulted in the loss of zearalenone production under inducing conditions. ZEA1 and ZEA2 are transcribed divergently from a common promoter region. Quantitative PCR analysis of both PKS genes and six flanking genes supports the view that the two polyketide synthases make up the core biosynthetic unit for zearalenone biosynthesis. An appreciation of the genetics of zearalenone biosynthesis is needed to understand how zearalenone is synthesized under field conditions that result in the contamination of grain.     

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.     

真菌聚酮合酶基因分离方法的研究进展

真菌聚酮合酶在代谢中可催化合成多种具有重要生物学活性的次级代谢物,所以真菌聚酮合酶正逐渐成为药学、食品科学和农学等领域的研究热点。本文综述了近五年来建立的几种分离真菌聚酮合酶基因的方法。这些方法解决了真菌中聚酮合酶基因簇难以分离的问题,为改造和利用真菌聚酮合酶以及发掘真菌聚酮化合物资源提供了强有力的手段。     

真菌聚酮化合物生物合成研究进展

具有广泛生物活性的真菌聚酮化合物因具有复杂的化学结构,其生物合成途径一般包含多样且新颖的酶催化反应。文中主要综述了2013-2016年来源于还原性聚酮合成酶(HR-PKSs)、非还原性聚酮合成酶(NR-PKSs)、聚酮-非核糖体多肽合成酶(PKS-NRPSs)和还原性-非还原性聚酮合成酶(HR-NR PKSs)杂合型等四大类型的真菌聚酮类化合物的生物合成研究进展。众多真菌聚酮类化合物生物机理的阐明,为未来新型真菌聚酮类天然产物生物合成基因簇的挖掘、新结构化合物的发现及其类似物的研究提供了方向和理论基础。     

A Sorangium cellulosum (myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes.

A 40-kb region of DNA from Sorangium cellulosum So ce26, which contains polyketide synthase (PKS) genes for synthesis of the antifungal macrolide antibiotic soraphen A, was cloned. These genes were detected by homology to Streptomyces violaceoruber genes encoding components of granaticin PKS, thus extending this powerful technique for the identification of bacterial PKS genes, which has so far been applied only to actinomycetes, to the gram-negative myxobacteria. Functional analysis by gene disruption has indicated that about 32 kb of contiguous DNA of the cloned region contains genes involved in soraphen A biosynthesis. The nucleotide sequence of a 6.4-kb DNA fragment, derived from the region with homology to granaticin PKS genes, was determined. Analysis of this sequence has revealed the presence of a single large open reading frame beginning and ending outside the 6.4-kb fragment. The deduced amino acid sequence indicates the presence of a domain with a high level of similarity to beta-ketoacyl synthases that are involved in polyketide synthesis. Other domains with high levels of similarity to regions of known polyketide biosynthetic functions were identified, including those for acyl transferase, acyl carrier protein, ketoreductase, and dehydratase. We present data which indicate that soraphen A biosynthesis is catalyzed by large, multifunctional enzymes analogous to other bacterial PKSs of type I.     

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.     

Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis.

Key information about the biosynthesis of polyketide metabolites has been uncovered by sequence analysis of the tetracenomycin C polyketide synthase genes (tcml) from Streptomyces glaucescens GLA.0. The sequence data revealed the presence of three complete open reading frames (ORFs). ORF1 and ORF2 appear to be translationally coupled and would encode proteins containing 426 and 405 amino acids, respectively. The two deduced proteins are homologous to known beta-ketoacyl synthases. ORF3 begins 70 nucleotides after the stop codon of ORF2 and would code for an 83 amino acid protein with a strong resemblance to known bacterial, animal and plant acyl-carrier proteins (ACP). The presence of an ACP gene within the tcm gene cluster suggests that different ACPs are used in fatty acid and polyketide biosynthesis in Streptomyces. We conclude from these data and earlier information that polyketide biosynthesis in S. glaucescens, and most likely in other bacteria, involves a multienzyme complex consisting of at least five types of enzymes: acylCoA transferases that load the acyl and 2-carboxyacyl precursors onto the ACP; a beta-ketoacyl synthase that, along with the acylated ACP, forms the poly-beta-ketoacyl intermediates; a poly-beta-ketone cyclase that forms carbocyclic structures from the latter intermediates; a beta-ketoacyl oxidoreductase that forms beta-hydroxyacyl intermediates or reduces ketone groups in fully formed polyketides; and a thioesterase that releases the assembled polyketide from the enzyme.     

The structure of a ketoreductase determines the organization of the beta-carbon processing enzymes of modular polyketide synthases

The structure of the ketoreductase (KR) from the first module of the erythromycin synthase with NADPH bound was solved to 1.79 A resolution. The 51 kDa domain has two subdomains, each similar to a short-chain dehydrogenase/reductase (SDR) monomer. One subdomain has a truncated Rossmann fold and serves a purely structural role stabilizing the other subdomain, which catalyzes the reduction of the beta-carbonyl of a polyketide and possibly the epimerization of an alpha-substituent. The structure enabled us to define the domain boundaries of KR, the dehydratase (DH), and the enoylreductase (ER). It also constrains the three-dimensional organization of these domains within a module, revealing that KR does not make dimeric contacts across the 2-fold axis of the module. The quaternary structure elucidates how substrates are shuttled between the active sites of polyketide synthases (PKSs), as well as related fatty acid synthases (FASs), and suggests how domains can be swapped to make hybrid synthases that produce novel polyketides.     

Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56

Myxobacteria belonging to the genus Sorangium are known to produce a variety of biologically active secondary metabolites. Chivosazol is a macrocyclic antibiotic active against yeast, filamentous fungi and especially against mammalian cells. The compound specifically destroys the actin skeleton of eucaryotic cells and does not show activity against bacteria. Chivosazol contains an oxazole ring and a glycosidically bound 6-deoxyglucose (except for chivosazol F). In this paper we describe the biosynthetic gene cluster that directs chivosazol biosynthesis in the model strain Sorangium cellulosum So ce56. This biosynthetic gene cluster spans 92 kbp on the chromosome and contains four polyketide synthase genes and one hybrid polyketide synthase/nonribosomal peptide synthetase gene. An additional gene encoding a protein with similarity to different methyltransferases and presumably involved in post-polyketide modification was identified downstream of the core biosynthetic gene cluster. The chivosazol biosynthetic gene locus belongs to the recently identified and rapidly growing class of trans-acyltransferase polyketide synthases, which do not contain acyltransferase domains integrated into the multimodular megasynthetases.     

Molecular biology of peptide and polyketide biosynthesis in cyanobacteria

Cyanobacteria produce numerous and structurally diverse secondary metabolites, in particular nonribosomal peptide and polyketide structures. Various bioactivities could be assigned to these compounds, and some may prove useful either for development into commercial drugs or as biochemical research tools. Microcystin, a worldwide common cyanobacterial hepatotoxin, was the first metabolite whose nonribosomal biosynthesis could be confirmed by knock-out mutagenesis. The microcystin synthetase complex consists of peptide synthetases, polyketide synthases, and hybrid enzymes, and reveals a number of novel enzymatic features, signifying the potential of cyanobacterial biosynthetic systems for combinatorial biochemistry. Recent studies have shown the presence of peptide synthetase genes and polyketide synthase genes within a number of cyanobacterial genomes. This knowledge may be very valuable for future screening projects aimed at the detection of new bioactive compounds.     

A photocrosslinking assay for reporting protein interactions in polyketide and fatty acid synthases

Understanding protein-protein interactions that occur between ACP and KS domains of polyketide synthases and fatty acid synthases is critical to improving the scope and efficiency of combinatorial biosynthesis efforts aimed at producing non-natural polyketides. Here, we report a facile strategy for rapidly reporting such ACP-KS interactions based on the incorporation of an amino acid with photocrosslinking functionality. Crucially, this photocrosslinking strategy can be applied to any polyketide or fatty acid synthase regardless of substrate specificity, and can be adapted to a high-throughput format for directed evolution studies.