Molecular evolution of aromatic polyketides and comparative sequence analysis of polyketide ketosynthase and 16S ribosomal DNA genes from various streptomyces species

A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the gamma-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes.     

Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2).

The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor. Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy. These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined.     

Isolation and characterization of the naphthocyclinone gene cluster from Streptomyces arenae DSM 40737 and heterologous expression of the polyketide synthase genes

Streptomyces arenae produces the aromatic polyketide naphthocyclinone, which exhibits activity against Gram-positive bacteria. A cosmid clone containing the putative naphthocyclinone gene cluster was isolated from a genomic library of S. arenae by hybridization with a conserved region from the actinorhodin PKS of S. coelicolor. Sequence analysis of a 5.5-kb DNA fragment, which hybridizes with the actI probe, revealed three open reading frames coding for the minimal polyketide synthase. A strong sequence similarity was found to several previously described ketosynthases, chain length factors and acyl carrier proteins from other polyketide gene clusters. An additional open reading frame downstream of the PKS genes of S. arenae showed 53% identity to act VII probably encoding an aromatase. Another open reading frame was identified in a region of 1.436 bp upstream of the PKS genes, which, however, had no similarity to known genes in the database. Approximately 8 kb upstream of the PKS genes, a DNA fragment was identified that hybridizes to an actVII--actIV specific probe coding for a cyclase and a putative regulatory protein, respectively. Disruption of the proposed naphthocyclinone gene cluster by insertion of a thiostrepton resistance gene completely abolished production of naphthocyclinones in the mutant strain, showing that indeed the naphthocyclinone gene cluster had been isolated. Heterologous expression of the minimal PKS genes in S. coelicolor CH999 in the presence of the act ketoreductase led to the production of mutactin and dehydromutactin, indicating that the S. arenae polyketide synthase forms a C-16 backbone that is subsequently dimerized to build naphthocyclinone. The functions of the proposed cyclase and aromatase were examined by coexpression with genes from different polyketide core producers.     

Cloning and characterization of a bacterial iterative type I polyketide synthase gene encoding the 6-methylsalicyclic acid synthase

Unusual polyketide synthases (PKSs), that are structurally type I but act in an iterative manner for aromatic polyketide biosynthesis, are a new family found in bacteria. Here we report the cloning of the iterative type I PKS gene chlB1 from the chlorothricin (CHL) producer Streptomyces antibioticus DSM 40725 by a rapid PCR approach, and characterization of the function of the gene product as a 6-methylsalicyclic acid synthase (6-MSAS). Sequence analysis of various iterative type I PKSs suggests that the resulting aromatic or aliphatic structure of the products might be intrinsically determined by a catalytic feature of the paired KR-DH domains in the control of the double bond geometry. The finding of ChlB1 as a 6-MSAS not only enriches the current knowledge of aromatic polyketide biosynthesis in bacteria, but will also contribute to the generation of novel polyketide analogs via combinatorial biosynthesis with engineered PKSs.     

Insight into the molecular basis of aromatic polyketide cyclization: crystal structure and in vitro characterization of WhiE-ORFVI

Aromatic polyketides are biologically active natural products. Many important pharmaceuticals are derived from aromatic polyketides. Especially important in aromatic polyketide biosynthesis is the regiospecific cyclization of a linear, preassembled polyketide chain catalyzed by aromatase/cyclase (ARO/CYC), which serves as a key control point in aromatic ring formation. How different ARO/CYCs promote different cyclization patterns is not well understood. The whiE locus of Streptomyces coelicolor A3(2) is responsible for the biosynthesis of an aromatic polyketide precursor to the gray spore pigment. The WhiE ARO/CYC catalyzes the regiospecific C9-C14 and C7-C16 cyclization and aromatization of a 24-carbon polyketide chain. WhiE ARO/CYC shares a high degree of similarity to another nonreducing PKS ARO/CYC, TcmN ARO/CYC. This paper presents the apo crystal structure of WhiE ARO/CYC, and cocrystal structures of WhiE and TcmN ARO/CYCs bound with polycyclic aromatic compounds that mimic the respective ARO/CYC products. Site-directed mutagenesis coupled with in vitro PKS reconstitution assays was used to characterize the interior pocket residues of WhiE ARO/CYC. The results confirmed that the interior pocket of ARO/CYCs is a critical determinant of polyketide cyclization specificity. A unified ARO/CYC-mediated cyclization mechanism is proposed on the basis of these structural and functional results.     

Structural analysis of actinorhodin polyketide ketoreductase: cofactor binding and substrate specificity

Aromatic polyketides are a class of natural products that include many pharmaceutically important aromatic compounds. Understanding the structure and function of PKS will provide clues to the molecular basis of polyketide biosynthesis specificity. Polyketide chain reduction by ketoreductase (KR) provides regio- and stereochemical diversity. Two cocrystal structures of actinorhodin polyketide ketoreductase (act KR) were solved to 2.3 A with either the cofactor NADP(+) or NADPH bound. The monomer fold is a highly conserved Rossmann fold. Subtle differences between structures of act KR and fatty acid KRs fine-tune the tetramer interface and substrate binding pocket. Comparisons of the NADP(+)- and NADPH-bound structures indicate that the alpha6-alpha7 loop region is highly flexible. The intricate proton-relay network in the active site leads to the proposed catalytic mechanism involving four waters, NADPH, and the active site tetrad Asn114-Ser144-Tyr157-Lys161. Acyl carrier protein and substrate docking models shed light on the molecular basis of KR regio- and stereoselectivity, as well as the differences between aromatic polyketide and fatty acid biosyntheses. Sequence comparison indicates that the above features are highly conserved among aromatic polyketide KRs. The structures of act KR provide an important step toward understanding aromatic PKS and will enhance our ability to design novel aromatic polyketide natural products with different reduction patterns.     

Candicidin biosynthesis in <Emphasis Type="Italic">Streptomyces griseus</Emphasis>

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.     

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.     

链霉菌来源的苯并异色烷醌家族抗生素的生物合成

苯并异色烷醌(benzoisochromanequinones,BIQs)家族抗生素是由链霉菌产生的聚酮类抗生素,其芳香聚酮母核结构中含有并联的两个芳香环和一个吡喃环,具有抗菌、抗肿瘤等多种生物学活性。BIQ抗生素聚酮链的早期生物合成过程代表了芳香聚酮抗生素母核的典型合成机制,而不同的后期修饰则决定了它们结构和生物学活性的多样性。在过去的二十几年中,以放线紫红素和美达霉素为研究重点,BIQ家族抗生素的生物合成机制逐渐得到揭示,但在后期结构修饰方面仍有许多问题有待解决。本文对BIQ家族抗生素的生物合成机制研究进行了综述,比较了不同BIQ家族抗生素结构特点、生物学活性,并重点阐述了它们生物合成中的后期结构修饰和调控过程的研究进展,并对BIQ抗生素在代谢工程方面的研究进行了展望。     

芳香聚酮早期后修饰环化酶的结构分类和功能分析

聚酮是一类结构和生物活性多样的天然产物,根据结构特点可以分为芳香聚酮和复合聚酮两大类。芳香聚酮环化酶是芳香聚酮生物合成过程中一种非常重要的早期后修饰酶,是决定芳香聚酮骨架结构的主要影响因素。根据序列和结构的相似性,芳香聚酮环化酶可以分为不同的种类。本文主要对其中3类芳香聚酮环化酶结构和功能进行了简要总结,从晶体结构、催化反应和催化机制等方面对它们进行了分类描述和功能分析,并结合自己实验室工作介绍了杰多霉素B环化酶催化机制的研究方法。     

Identification and characterization of the pyridomycin biosynthetic gene cluster of Streptomyces pyridomyceticus NRRL B-2517

Pyridomycin is a structurally unique antimycobacterial cyclodepsipeptide containing rare 3-(3-pyridyl)-l-alanine and 2-hydroxy-3-methylpent-2-enoic acid moieties. The biosynthetic gene cluster for pyridomycin has been cloned and identified from Streptomyces pyridomyceticus NRRL B-2517. Sequence analysis of a 42.5-kb DNA region revealed 26 putative open reading frames, including two nonribosomal peptide synthetase (NRPS) genes and a polyketide synthase gene. A special feature is the presence of a polyketide synthase-type ketoreductase domain embedded in an NRPS. Furthermore, we showed that PyrA functioned as an NRPS adenylation domain that activates 3-hydroxypicolinic acid and transfers it to a discrete peptidyl carrier protein, PyrU, which functions as a loading module that initiates pyridomycin biosynthesis in vivo and in vitro. PyrA could also activate other aromatic acids, generating three pyridomycin analogues in vivo.     

Combinatorial biosynthesis for new drug discovery

Combinatorial biosynthesis involves interchanging secondary metabolism genes between antibiotic-producing microorganisms to create unnatural gene combinations or hybrid genes if only part of a gene is exchanged. Novel metabolites can be made by both approaches, due to the effect of a new enzyme on a metabolic pathway or to the formation of proteins with new enzymatic properties. The method has been particularly successful with polyketide synthase (PKS) genes: derivatives of medically important macrolide antibiotics and unusual polycyclic aromatic compounds have been produced by novel combinations of the type I and type II PKS genes, respectively. Recent extensions of the approach to include deoxysugar biosynthesis genes have expanded the possibilities for making new microbial metabolites and discovering valuable drugs through the genetic engineering of bacteria.     

Incorrectly folded aromatic polyketides from polyketide reductase deficient mutants.

Compounds produced by the polyketide ketoreductase deficient Streptomyces mutants HO61 and P67 are described. The structures of the compounds indicate that ketoreductase activity is required for correct condensation of the polyketide chain in the biosynthesis of aromatic polyketides.     

真菌芳香聚酮化合物的合成生物学研究进展*

真菌芳香聚酮化合物是由真菌非还原聚酮合酶(NR-PKSs)催化形成的具有广泛生物活性的一类天然产物。大部分内源真菌菌株存在难培养、致病性或产率低等问题,从根本上限制了真菌芳香聚酮化合物的开发和应用。随着合成生物学和代谢工程的发展,很多具有生物活性的聚酮产物实现了在工业微生物(如酿酒酵母、构巢曲霉等)中的异源生产,相关研究逐渐成为热点。从合成途径解析与挖掘、底盘细胞的构建与改造等方面综述了近年来真菌芳香聚酮化合物的合成生物学研究进展,为未来真菌芳香聚酮化合物人工代谢途径的高效构建和实现工业化生产奠定基础。     

The biosynthesis of the aromatic myxobacterial electron transport inhibitor stigmatellin is directed by a novel type of modular polyketide synthase.

Deductions from the molecular analysis of the 65,000-bp stigmatellin biosynthetic gene cluster are reported. The biosynthetic genes (stiA-J) encode an unusual bacterial modular type I polyketide synthase (PKS) responsible for the formation of this aromatic electron transport inhibitor produced by the myxobacterium Stigmatella aurantiaca. Involvement of the PKS gene cluster in stigmatellin biosynthesis is shown using site-directed mutagenesis. One module of the PKS is assumed to be used iteratively during the biosynthetic process, which seems to involve an unusual transacylation of the biosynthetic intermediate from an acyl carrier protein domain back to the preceding ketosynthase domain. Finally, the polyketide chain which is presumably catalyzed by a novel C-terminal domain in StiJ that does not resemble thioesterases, is cyclized and aromatized. The presented results of feeding experiments are in good agreement with the proposed biosynthetic scheme. In contrast to all other PKS type I systems reported to date, each module of StiA-J is encoded on a separate gene. The gene cluster contains a "stand alone" O-methyltransferase and two unusual O-methyltransferase domains embedded in the PKS. In addition, inactivation of a cytochrome P450 monooxygenase-encoding gene involved in post-PKS hydroxylation of the aromatic ring leads to the formation of two novel stigmatellin derivatives.     

Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases

Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs). The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.     

A hydroxylase-like gene product contributes to synthesis of a polyketide spore pigment in Streptomyces halstedii.

A gene, schC, adjacent to the sch gene cluster encoding the biosynthesis of a polyketide spore pigment in Streptomyces halstedii was sequenced. Its deduced product resembled flavin adenine nucleotide-containing hydroxylases involved in the biosynthesis of polycyclic aromatic polyketide antibiotics and in catabolic pathways of aromatic compounds. When schC was disrupted, the normally green spores of S. halstedii became lilac. An schC-like gene was located in an equivalent position next to a large gene cluster (whiE) known to determine spore pigment in Streptomyces coelicolor A3(2).     

Polyketide synthase complexes: their structure and function in antibiotic biosynthesis.

This paper gives an overview of existing knowledge concerning the structure and deduced functions of polyketide synthases active in antibiotic-producing streptomycetes. Using monensin A as an example of a structurally complex polyketide metabolite, the problem of understanding how individual strains of microorganism are 'programmed' to produce a given polyketide metabolite is first outlined. The question then arises, how is the programming of polyketide assembly related to the structural organization of individual polyketide synthase complexes at the biochemical and genetic levels? Experimental results that help to illuminate these relations are described, in particular, those giving information about the structures and deduced functions of polyketide synthases involved in aromatic polyketide biosynthesis (actinorhodin, granaticin, tetracenomycin, whiE spore pigment and an act homologous region from the monensin-producing organism), as well as the macrolide polyketide synthase active in the biosynthesis of 6-deoxyerythronolide A.