A polyketide synthase gene, ACRTS2, is responsible for biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including β-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, β-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.     

Polyketide biosynthesis beyond the type I,II and III polyketide synthase paradigms

Recent literature on polyketide biosynthesis suggests that polyketide synthases have much greater diversity in both mechanism and structure than the current type I, II and III paradigms. These examples serve as an inspiration for searching novel polyketide synthases to give new insights into polyketide biosynthesis and to provide new opportunities for combinatorial biosynthesis.     

Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster

A generic design of Type I polyketide synthase genes has been reported in which modules, and domains within modules, are flanked by sets of unique restriction sites that are repeated in every module [1]. Using the universal design, we synthesized the six-module DEBS gene cluster optimized for codon usage in E. coli, and cloned the three open reading frames into three compatible expression vectors. With one correctable exception, the amino acid substitutions required for restriction site placements were compatible with polyketide production. When expressed in E. coli the codon-optimized synthetic gene cluster produced significantly more protein than did the wild-type sequence. Indeed, for optimal polyketide production, PKS expression had to be down-regulated by promoter attenuation to achieve balance with expression of the accessory proteins needed to support polyketide biosynthesis.     

p-Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii.

Chalcone synthase and stilbene synthase are plant-specific polyketide synthases. They catalyze three common consecutive decarboxylative condensations and specific cyclization reactions. They are highly homologous to each other, and are likely to fall into a family of polyketide synthases along with acridone synthase and bibenzyl synthase. Two cDNA clones (named HmC and HmS), both of which show high homology to the known chalcone synthases, were obtained from leaves of Hydrangea macrophylla var. thunbergii. They were expressed in Escherichia coli in order to determine their enzyme functions. Detection of chalcone formation clearly indicated that HmC encoded chalcone synthase, while HmS protein catalyzed the formation of neither chalcone nor stilbene. However, a novel pyrone, a lactonization product of a linear tetraketide was detected in reaction products of HmS protein. This proves that HmS encodes a novel polyketide synthase that catalyzes only chain elongation without cyclization.     

Convergence in the biosynthesis of acetogenic natural products from plants,fungi, and bacteria

This review deals with polyketides to which nature has developed different biosynthetic pathways in the course of evolution. The anthraquinone chrysophanol is the first example of an acetogenic natural product that is, in an organism-specific manner, formed via more than one polyketide folding mode: In eukaryotes, like e.g., in fungi, in higher plants, and in insects, it is synthesized via folding mode F, while in prokaryotes it originates through mode S. It has, more recently, even been found to be synthesized by a third pathway, named mode S′. Thus, chrysophanol is the first polyketide synthase product that originates through a divergent–convergent biosynthesis (depending on the respective producing organisms). A second example of a striking biosynthetic convergence is the isoquinoline alkaloids. While all as yet investigated representatives of this large family of plant-derived metabolites (more than 2500 known representatives!) are formed from aromatic amino acids, the biosynthetic origin of naphthylisoquinoline alkaloids like dioncophylline A is unprecedented in following a route to isoquinolines in plants: we have shown that such naphthylisoquinolines represent the as yet only known polyketidic di- and tetrahydroisoquinolines, originating from acetate and malonate units, exclusively. Both molecular halves, the isoquinoline part and the naphthalene portion, are even synthesized from a joint polyketide precursor, the first proven case of the F-type folding mode in higher plants. The biosynthetic origins of the natural products presented in this paper were elucidated by feeding ¹³C₂-labeled acetate (or advanced precursors) to the respective producing organisms, with subsequent NMR analysis of their ¹³C₂ incorporation patterns using the potent cryoprobe methodology, thus making the full polyketide folding pattern visible.     

Cloning, analysis, and heterologous expression of a polyketide synthase gene cluster of Streptomyces curacoi.

Streptomyces curacoi produces curamycin, an antibiotic based on a modified orsellinic acid skeleton that is synthesized by the polyketide pathway. We have cloned, characterized, and partly sequenced a polyketide synthase gene cluster of S. curacoi. The sequence data reveal an organization of open reading frames that is similar to those of other polyketide synthetic clusters, although the biosynthetic products differ considerably in size and structure. We propose that one of the predicted open reading frames (curA) encodes polykeptide synthase, on the basis of its homology with other enzymes with similar functions. Expression of the cloned chromosomal fragment in the heterologous host S. lividans leads to the production of a brown pigment in large quantities. The analysis and expression of the cur genes for detailed molecular studies of the mechanism of polyketide biosynthesis is discussed.     

MapsiDB: an integrated web database for type I polyketide synthases

Polyketides have diverse biological activities, including pharmacological functions such as antibiotic, antitumor and agrochemical properties. They are biosynthesized from short carboxylic acid precursors by polyketide synthases (PKSs). As natural polyketide products include many clinically important drugs and the volume of data on polyketides is rapidly increasing, the development of a database system to manage polyketide data is essential. MapsiDB is an integrated web database formulated to contain data on type I polyketides and their PKSs, including domain and module composition and related genome information. Data on polyketides were collected from journals and online resources and processed with analysis programs. Web interfaces were utilized to construct and to access this database, allowing polyketide researchers to add their data to this database and to use it easily. MapsiDB is available at .     

Classification,Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

The fungal iterative nonreducing polyketide synthases (NRPKSs) synthesize aromatic polyketides, many of which have important biological activities. The product template domains (PT) embedded in the multidomain NRPKSs mediate the regioselective cyclization of the highly reactive polyketide backbones and dictate the final structures of the products. Understanding the sequence-activity relationships of different PT domains is therefore an important step toward the prediction of polyketide structures from NRPKS sequences and can enable the genome mining of hundreds of cryptic NRPKSs uncovered via genome sequencing. In this work, we first performed phylogenetic analysis of PT domains from NRPKSs of known functions and showed that the PT domains can be classified into five groups, with each group corresponding to a unique product size or cyclization regioselectivity. Group V contains the formerly unverified PT domains that were identified as C6-C11 aldol cyclases. The regioselectivity of PTs from this group were verified by product-based assays using the PT domain excised from the asperthecin AptA NRPKS. When combined with dissociated PKS4 minimal PKS, or replaced the endogenous PKS4 C2-C7 PT domain in a hybrid NRPKS, AptA-PT directed the C6-C11 cyclization of the nonaketide backbone to yield a tetracyclic pyranoanthraquinone 4. Extensive NMR analysis verified that the backbone of 4 was indeed cyclized with the expected regioselectivity. The PT phylogenetic analysis was then expanded to include ∼100 PT sequences from unverified NRPKSs. Using the assays developed for AptA-PT, the regioselectivities of additional PT domains were investigated and matched to those predicted by the phylogenetic classifications.     

Biosynthesis,regulation, and engineering of a linear polyketide tautomycetin: a novel immunosuppressant in <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CK4412

Tautomycetin (TMC) is a natural product with a linear structure that includes an ester bond connecting a dialkylmaleic moiety to a type I polyketide chain. Although TMC was originally identified as an antifungal antibiotic in the late 1980s, follow-up studies revealed its novel immunosuppressant activity. Specifically, TMC exhibited a mechanistically unique immunosuppressant activity about 100 times higher than that of cyclosporine A, a widely used immunosuppressant drug. Interestingly, a structurally close relative, tautomycin (TTM), was reported to not possess TMC-like immunosuppressant activity, suggesting that a distinctive polyketide moiety of TMC plays a critical role in immunosuppressant activity. Cloning and engineering of a TMC polyketide biosynthetic gene cluster generated several derivatives showing different biological activities. TMC was also found to be biosynthesized as a linear structure without forming a lactone ring, unlike the most polyketide-based compounds, implying the presence of a unique polyketide thioesterase in the cluster. Although TMC biosynthesis was limited due to its tight regulation by two pathway-specific regulatory genes located in the cluster, its production was significantly stimulated through homologous and heterologous expression of its entire biosynthetic gene cluster using a Streptomyces artificial chromosome vector system. In this mini-review, we summarize recent advances in the biosynthesis, regulation, and pathway engineering of a linear polyketide, TMC, in Streptomyces sp. CK4412.     

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.     

Computational analysis of phenotypic space in heterologous polyketide biosynthesis—Applications to Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae

Polyketides represent a class of natural product small molecules with an impressive range of medicinal activities. In order to improve access to therapeutic polyketide compounds, heterologous metabolic engineering has been applied to transfer polyketide genetic pathways from often fastidious native hosts to more industrially-amenable heterologous hosts such as Escherichia coli, Saccharomyces cerevisiae, or Streptomyces coelicolor. Efforts thus far have resulted in titers either inferior to the native host and significantly below the theoretical yield, emphasizing the need to computationally investigate and engineer the interaction between native and heterologous metabolism for the improved production of heterologous polyketide compounds. In this work, we applied flux balance analysis on genome-scale models to simulate cellular metabolism and 6-deoxyerythronolide B (the cyclized polyketide precursor to erythromycin) production in three common heterologous hosts (E. coli, Bacillus subtilis, and S. cerevisiae) under a variety of carbon-source and medium compositions. We then undertook minimization of metabolic adjustment optimization to identify single and double gene-knockouts that resulted in increased polyketide production while maintaining cellular growth. For the production of 6-deoxyerythronolide B, the results suggest B. subtilis and E. coli are better heterologous hosts when compared to S. cerevisiae and that several single and multiple gene-knockout mutants are computationally predicted to improve specific production, in some cases, over 25-fold.     

Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases

Modular polyketide biosynthesis can be harnessed to generate rationally designed complex natural products through bioengineering. A detailed understanding of the features that govern transfer and processing of polyketide biosynthetic intermediates is crucial to successfully engineer new polyketide pathways. Previous studies have shown that substrate stereochemistry and protein-protein interactions between polyketide synthase modules are both important factors in this process. Here we investigated the substrate tolerance of different polyketide modules and assessed the relative importance of inter-module chain transfer versus chain elongation activity of some of these modules. By constructing a variety of hybrid modular polyketide synthase systems and assaying their ability to generate polyketide products, it was determined that the substrate tolerance of each individual ketosynthase domain is an important parameter for the successful recombination of polyketide synthase modules. Surprisingly, however, failure by a module to process a candidate substrate was not due to its inability to bind to it. Rather, it appeared to result from a blockage in carbon-carbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzyme-catalyzed decarboxylative condensation reaction.     

Mupirocin: biosynthesis,special features and applications of an antibiotic from a Gram-negative bacterium

Mupirocin is a polyketide antibiotic produced by Pseudomonas fluorescens. The biosynthetic cluster encodes 6 type I polyketide synthase multifunctional proteins and 29 single function proteins. The biosynthetic pathway belongs to the trans-AT group in which acyltransferase activity is provided by a separate polypeptide rather than in-cis as found in the original type I polyketide synthases. Special features of this group are in-cis methyltransferase domains and a trans-acting HMG-CoA synthase-cassette which insert α- and β- methyl groups respectively while enoyl reductase domains are absent from the condensing modules. In addition, for the mupirocin system, there is no obvious loading mechanism for initiation of the polyketide chain and many aspects of the pathway remain to be elucidated. Mupirocin inhibits isoleucyl-tRNA synthetase and has been used since 1985 to help prevent infection by methicillin-resistant Staphylococcus aureus, particularly within hospitals. Resistance to mupirocin was first detected in 1987 and high-level resistance in S. aureus is due to a plasmid-encoded second isoleucyl-tRNA synthetase, a more eukaryotic-like enzyme. Recent analysis of the biosynthetic pathway for thiomarinols from marine bacteria opens up possibilities to modify mupirocin so as to overcome this resistance.     

Type II Thioesterase ScoT, Associated with Streptomyces coelicolor A3(2) Modular Polyketide Synthase Cpk, Hydrolyzes Acyl Residues and Has a Preference for Propionate

Type II thioesterases (TE IIs) were shown to maintain the efficiency of polyketide synthases (PKSs) by removing acyl residues blocking extension modules. However, the substrate specificity and kinetic parameters of these enzymes differ, which may have significant consequences when they are included in engineered hybrid systems for the production of novel compounds. Here we show that thioesterase ScoT associated with polyketide synthase Cpk from Streptomyces coelicolor A3(2) is able to hydrolyze acetyl, propionyl, and butyryl residues, which is consistent with its editing function. This enzyme clearly prefers propionate, in contrast to the TE IIs tested previously, and this indicates that it may have a role in control of the starter unit. We also determined activities of ScoT mutants and concluded that this enzyme is an α/β hydrolase with Ser90 and His224 in its active site.     

Biosynthesis of depudecin, a metabolite of Nimbya scirpicola

Feeding experiments of labeled acetates to Nimbya scirpicola proved the carbon origin of the straight-chain polyketide, depudecin. Differential hydrogen exchange of the 2H label originating from 2H labeled acetate along the polyketide chain occurred. In particular, the deuterium of an epoxide methine at C-3 was lost to a substantial extent in the formation of depudecin.     

Solution NMR structure of CGL2373, a polyketide cyclase-like protein from Corynebacterium glutamicum

Protein CGL2373 from Corynebacterium glutamicum was previously proposed to be a member of the polyketide_cyc2 family, based on amino-acid sequence and secondary structure features derived from NMR chemical shift assignments. We report here the solution NMR structure of CGL2373, which contains three α-helices and one antiparallel β-sheet and adopts a helix-grip fold. This structure shows moderate similarities to the representative polyketide cyclases, TcmN, WhiE, and ZhuI. Nevertheless, unlike the structures of these homologs, CGL2373 structure looks like a half-open shell with a much larger pocket, and key residues in the representative polyketide cyclases for binding substrate and catalyzing aromatic ring formation are replaced with different residues in CGL2373. Also, the gene cluster where the CGL2373-encoding gene is located in C. glutamicum contains additional genes encoding nucleoside diphosphate kinase, folylpolyglutamate synthase, and valine-tRNA ligase, different from the typical gene cluster encoding polyketide cyclase in Streptomyces. Thus, although CGL2373 is structurally a polyketide cyclase-like protein, the function of CGL2373 may differ from the known polyketide cyclases and needs to be further investigated. The solution structure of CGL2373 lays a foundation for in silico ligand screening and binding site identifying in future functional study.     

除虫链霉菌10个聚酮类抗生素生物合成基因簇的敲除对阿维菌素产量的影响

【目的】考察除虫链霉菌基因组中其它聚酮合成酶类(Polyketide synthase,PKS)抗生素生物合成基因簇的敲除突变对于阿维菌素产量的影响。【方法】构建了11个PKS基因簇的打靶Cosmid和质粒载体,导入除虫链霉菌中筛选突变株。【结果】在工业菌株MMR630中成功敲除了10个PKS基因簇。发酵结果显示7个PKS基因簇敲除突变株中阿维菌素的产量均有不同程度的提高,而2个突变株不能产生阿维菌素。然而,在3个连续敲除2个PKS基因簇的突变株中阿维菌素产量没有能够超过单个PKS敲除突变株的提升幅度。【结论】除虫链霉菌基因组的一些PKS基因簇的敲除可以提高阿维菌素的产量,同时暗示同一类次生代谢产物的代谢流之间存在复杂的相互作用关系。     

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。     

链霉菌中高效生产聚酮化合物的研究方法及进展北大核心CSCD

聚酮化合物是通过聚酮合成途径产生的一大类结构和生物活性多样的次级代谢产物,是链霉菌产生的主要次级代谢产物,具有重要的经济价值。为了在链霉菌中提高聚酮化合物产量,以满足工业生产需求,近年来,代谢工程的方法被广泛应用,例如,过表达合成途径中限速酶或途径特异性激活蛋白、强化前体供应、去除产物反馈抑制、合成基因簇异源表达等。本文将从代谢工程改造实例入手,全面综述链霉菌中聚酮化合物高效生物合成的研究方法及进展,并对利用合成生物学策略智能动态适配各个相关途径,进而提高该类化合物产量的研究思路进行展望。