Organization of the biosynthetic gene cluster for the polyketide antitumor macrolide, pladienolide, in Streptomyces platensis Mer-11107

Pladienolides are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107. They show strong antitumor activity and are a potential lead in the search for novel antitumor agents. We sequenced the 65-kb region covering the biosynthetic gene cluster, and found four polyketide synthase genes (pldAI-pldAIV) composed of 11 modules, three genes involved in post-modifications (pldB-D), and a luxR-family regulatory gene (pldR). The thioesterase domain of pldAIV was more dissimilar to that of polyketide synthase systems synthesizing 12/14-membered macrolide polyketides than to that of systems synthesizing other cyclic polyketides. The pldB gene was identified as a 6-hydroxylase belonging to a cytochrome P450 of the CYP107 family. This was clarified by a disruption experiment on pldB, in which the disruptant produced 6-dehydroxy pladienolide B. Two genes located downstream of pldB, designated pldC and pldD, are thought to be a probable genes for 7-O-acetylase and 18, 19-epoxydase respectively.     

Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae

Development of host microorganisms for heterologous expression of polyketide synthases (PKS) that possess the intrinsic capacity to overproduce polyketides with a broad spectrum of precursors supports the current demand for new tools to create novel chemical structures by combinatorial engineering of modular and other classes of PKS. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors—malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA—used by modular PKS, and is a host that is amenable to genetic manipulation. We have expanded the utility of an overproducing S. fradiae strain for engineered biosynthesis of polyketides by engineering a biosynthetic pathway for methoxymalonyl-ACP, a fourth precursor used by many 16-membered macrolide PKS. This was achieved by introducing a set of five genes, fkbG–K from Streptomyces hygroscopicus, putatively encoding the methoxymalonyl-ACP biosynthetic pathway, into the S. fradiae chromosome. Heterologous expression of the midecamycin PKS genes in this strain resulted in 1 g/l production of a midecamycin analog. These results confirm the ability to engineer unusual precursor pathways to support high levels of polyketide production, and validate the use of S. fradiae for overproduction of 16-membered macrolides derived from heterologous PKS that require a broad range of precursors.     

Genome mining reveals the evolutionary origin and biosynthetic potential of basidiomycete polyketide synthases

Numerous polyketides are known from bacteria, plants, and fungi. However, only a few have been isolated from basidiomycetes. Large scale genome sequencing projects now help anticipate the capacity of basidiomycetes to synthesize polyketides. In this study, we identified and annotated 111 type I and three type III polyketide synthase (PKS) genes from 35 sequenced basidiomycete genomes. Phylogenetic analysis of PKS genes suggests that all main types of fungal iterative PKS had already evolved before the Ascomycota and Basidiomycota diverged. A comparison of genomic and metabolomic data shows that the number of polyketide genes exceeds the number of known polyketide structures by far. Exploiting these results to design degenerate PCR primers, we amplified and cloned the complete sequence of armB, a PKS gene from the melleolide producer Armillaria mellea. We expect this study will serve as a guide for future genomic mining projects to discover structurally diverse mushroom-derived polyketides.     

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.     

Expression of polyketide biosynthesis and regulatory genes in heterologous streptomycetes

Summary There are now several examples showing that hybrid secondary metabolites can be produced as a result of interspecies cloning of antibiotic biosynthesis genes in streptomycetes. This paper reviews examples of hybrid secondary metabolite production, and examines the underlying biochemical and regulatory principles leading to the formation of hybrid anthraquinones by recombinant anthracycline-producing streptomycetes carrying actinorhodin biosynthesis genes. An anthraquinone, aloesaponarin II, was produced by cloning theactI, actIII, actIV, andactVII genes (pANT12) of actinorhodin biosynthesis pathway fromStreptomyces coelicolor in anthracycline producing streptomycetes.Streptomyces galilaeus strains 31 133 and 31 671, aclacinomycin and 2-hydroxyaklavinone producers, respectively, formed aloesaponarin II as their major polyketide product when transformed with pANT12. Subcloning experiments indicated that a 2.8-kbXhoI fragment containing only theactI andactVII loci was necessary for aloesaponarin II biosynthesis byS. galilaeus 31 133. WhenS. galilaeus 31 671 was transformed with theactI, actVII, andactIV genes, however, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin and 1-0-methyldesoxyerythrolaccin. WhenS. galilaeus 31671 was transformed with only the intactactIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for theactIII gene, which is the reduction of the keto group at C-9 from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The effects of three regulatory loci,dauG, dnrR1, andasaA, on the production of natural and hybrid polyketides were also shown.     

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.     

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.     

Biosynthesis of Polyketide Antibiotics by Various StreptomycesSpecies that Produce Actinomycins

A collection of actinomycin-producing Streptomycesstrains, their variants with different levels of antibiotic biosynthesis, and recombinant strains were screened in order to select new strains that produce polyketide antibiotics. Screening with the use of the cloned actgene encoding a component of actinorhodin polyketide synthase (PKS) multienzyme complex from Streptomyces coelicolorrevealed that many strains tested can synthesize polyketide antibiotics along with actinomycins. A relationship between the biosynthetic pathways of actinomycins and polyketides is discussed.     

A Polyketide Synthase Gene Required for Biosynthesis of the Aflatoxin-like Toxin, Dothistromin

Dothistromin is a polyketide toxin, produced by a fungal forest pathogen, with structural similarity to the aflatoxin precursor versicolorin B. Biochemical and genetic studies suggested that there are common steps in the biosynthetic pathways for these metabolites and showed similarities between some of the genes. A polyketide synthase gene (pksA) was isolated from dothistromin-producing Dothistroma septosporum by hybridization with an aflatoxin ortholog from Aspergillus parasiticus. Inactivation of this gene in D. septosporum resulted in mutants that could not produce dothistromin but that could convert exogenous aflatoxin precursors, including norsolorinic acid, into dothistromin. The mutants also had reduced asexual sporulation compared to the wild type. So far four other genes are known to be clustered immediately alongside pksA. Three of these (cypA, moxA, avfA) are predicted to be orthologs of aflatoxin biosynthetic genes. The other gene (epoA), located between avfA and moxA, is predicted to encode an epoxide hydrolase, for which there is no homolog in either the aflatoxin or sterigmatocystin gene clusters. The pksA gene is located on a small chromosome of ~1.3 Mb in size, along with the dothistromin ketoreductase (dotA) gene.     

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 Single Module Type I Polyketide Synthase Directs de Novo Macrolactone Biogenesis during Galbonolide Biosynthesis in Streptomyces galbus

Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-¹³C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed β-keto group modifications.     

Identification of the Fluvirucin B2 (Sch 38518) Biosynthetic Gene Cluster from Actinomadura fulva subsp. indica ATCC 53714: substrate Specificity of the β-Amino Acid Selective Adenylating Enzyme FlvN

Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antivirus activities. Fluvirucins have the β-alanine starter unit at their polyketide skeletons. To understand the construction mechanism of the β-alanine moiety in fluvirucin biosyntheses, we have identified the biosynthetic cluster of fluvirucin B₂ produced from Actinomadura fulva subsp. indica ATCC 53714. The identified gene cluster contains three polyketide synthases, four characteristic β-amino acid-carrying enzymes, one decarboxylase, and one amidohydrolase. We next investigated the activity of the adenylation enzyme FlvN, which is a key enzyme for the selective incorporation of a β-amino acid substrate. FlvN showed strong preference for l-aspartate over other amino acids such as β-alanine. Based on these results, we propose a biosynthetic pathway for fluvirucin B₂.     

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C₁₈ chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides.     

Chemical investigation of novel ascomycetes using PCR based screening approaches

Fungi are well known for a wealth of pharmacologically important activities and agrochemical properties. Polyketides that are widely found in fungi, are a large group of secondary metabolites which exhibit diversity in their function and structure. Here we described an investigation of three fungal strains which were prospected for production of polyketides. The aim of this work was to employ the diversity of reducing type I polyketide synthase genes in these fungi using a molecular and bioinformatics approaches as a mini tool. A degenerate primer pair for highly reduced PKSs was newly designed and used together with ketosynthase primers for amplification. One hundred and thirty-eight clones were sequenced. Ten KS domain sequences were isolated, using two primer pairs specific for highly reduced type PKSs. This study revealed four sequences from Emarcea castanopsidicola, four ketosynthase sequences from Gaeumannomyces amomi and two sequences from Leiosphaerella amomi, respectively. Bioinformatic techniques were employed to identify a group of these KS domain sequences. Based on these sequences suggested that rapid screening provided the potential to explore significant PKS structural diversity. Hence chemical investigation had been conducted and exhibited nine compounds. The endophytic fungus L. amomi was cultivated and elucidated linoleic acid, ergosterol and an unidentified sterol in the extracts. Linoleic acid, sitosterol, and p-hydroxybenzoic acid were isolated from the saprobic fungus E. castanopsidicola. We first isolated a new polyketide, stemphol 1-O-β-D-galactopyranoside together with four known metabolites; stemphol, kojic acid, ergosterol, indole-3-carboxylic acid from an ethyl acetate extract of the cultures of G. amomi. Stemphol was classified as a phenolic lipid or resorcinolic lipid, which have biopharmacological, biomedical, and biotechnological importance. However, recent researches have revealed that these molecule types are synthesized by 2′-oxoalkylresorcinolic acid synthase. The prospective KS domain sequences from this study will be used as probes to isolate putative PKS genes. A gene cluster responsible for PK biosynthesis should be confirmed by determination of PK products generated by these enzymes.     

Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains

Development of natural products for therapeutic use is often hindered by limited availability of material from producing organisms. The speed at which current technologies enable the cloning, sequencing, and manipulation of secondary metabolite genes for production of novel compounds has made it impractical to optimize each new organism by conventional strain improvement procedures. We have exploited the overproduction properties of two industrial organisms—Saccharopolyspora erythraea and Streptomyces fradiae, previously improved for erythromycin and tylosin production, respectively—to enhance titers of polyketides produced by genetically modified polyketide synthases (PKSs). An efficient method for delivering large PKS expression vectors into S. erythraea was achieved by insertion of a chromosomal attachment site (attB) for φC31-based integrating vectors. For both strains, it was discovered that only the native PKS-associated promoter was capable of sustaining high polyketide titers in that strain. Expression of PKS genes cloned from wild-type organisms in the overproduction strains resulted in high polyketide titers whereas expression of the PKS gene from the S. erythraea overproducer in heterologous hosts resulted in only normal titers. This demonstrated that the overproduction characteristics are primarily due to mutations in non-PKS genes and should therefore operate on other PKSs. Expression of genetically engineered erythromycin PKS genes resulted in production of erythromycin analogs in greatly superior quantity than obtained from previously used hosts. Further development of these hosts could bypass tedious mutagenesis and screening approaches to strain improvement and expedite development of compounds from this valuable class of natural products.     

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.     

ASMPKS: an analysis system for modular polyketide synthases

Background  

Polyketides are secondary metabolites of microorganisms with diverse biological activities, including pharmacological functions such as antibiotic, antitumor and agrochemical properties. Polyketides are synthesized by serialized reactions of a set of enzymes called polyketide synthase(PKS)s, which coordinate the elongation of carbon skeletons by the stepwise condensation of short carbon precursors. Due to their importance as drugs, the volume of data on polyketides is rapidly increasing and creating a need for computational analysis methods for efficient polyketide research. Moreover, the increasing use of genetic engineering to research new kinds of polyketides requires genome wide analysis.     

Heterologous production of epothilones B and D in Streptomyces venezuelae

Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 μg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 μg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.     

Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402. Received 31 May 2001/ Accepted in revised form 09 July 2001