Engineering of complex polyketide biosynthesis — insights from sequencing of the monensin biosynthetic gene cluster

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 360–367. Received 18 March 2001/ Accepted in revised form 09 July 2001     

Structure and mechanism of chalcone synthase-like polyketide synthases

Polyketide synthases (PKS) produce an array of natural products with different biological activities and pharmacological properties by varying the starter and extender molecules that form the final polyketide. Recent studies of the simplest PKS, the chalcone synthase (CHS)-like enzymes involved in the biosynthesis of flavonoids, anthocyanin pigments, and antimicrobial phytoalexins, have yielded insight on the molecular basis of this biosynthetic versatility. Understanding the structure–function relationship in these PKS provides a foundation for manipulating polyketide formation and suggests strategies for further increasing the scope of polyketide biosynthetic diversity. Journal of Industrial Microbiology & Biotechnology (2001) 27, 393–398. Received 14 June 2001/ Accepted in revised form 15 July 2001     

Organization of biosynthetic gene cluster for avermectin in Streptomyces avermitilis: analysis of enzymatic domains in four polyketide synthases

The analysis of the incorporation of ¹³C-labeled precursors into avermectins indicates that the avermectin aglycons are synthesized by head-to-tail condensation of various acyl groups, which is similar to the biosynthesis of other polyketides. Polyketide synthases (PKS) use the appropriate CoA ester as a primer and add acetate units from malonyl-CoA and propionate units from methylmalonyl-CoA to assemble the polyketides. Avermectin aglycons are formed by addition to the starter unit (2-methylbutyrate or isobutyrate) of 12 acyl condensations in the order P–A–A–A–A–P–P–A–P–A–P–A (P, propionyl; A, acetyl). Within the 90-kb gene cluster for avermectin biosynthesis, the central 65-kb segment was found to be required for aglycon biosynthesis by phenotypic analysis of strains containing deletion or insertion mutations in this region. A complete sequence analysis of the 65-kb segment indicated that this segment encodes avermectin PKS. The avermectin PKS genes are organized into two converging blocks of ORFs. From the results of sequencing analysis, a feature of the two regions, aveA1/aveA2 and avea3/aveA4, is that they encode four kinds of large multifunctional polypeptides containing 55 domains which possess putative fatty acid synthase-like activities. The avermectin PKS (AVES 1–4) appear to contain two, three, or four modules. AVES 1 and 2 contain two and four modules, respectively, whereas AVES 3 and AVES 4 each contains three modules. The 12 modules correspond to the 12 cycles required for synthesis of the avermectin aglycon. Journal of Industrial Microbiology & Biotechnology (2001) 27, 170–176. Received 21 September 1999/ Accepted in revised form 14 September 2000     

三烷基取代芳香聚酮gombapyrones生物合成基因簇的确认及其生物合成推导

【目的】本研究旨在确认链霉菌Streptomyces rubellomurinus ATCC 31215来源芳香聚酮化合物(gombapyrones, GOMs)的生物合成基因簇(biosynthetic gene cluster, BGC),并对其生物合成途径进行推导。【方法】对链霉菌S. rubellomurinus ATCC 31215进行大规模发酵及提取分离,得到GOM-B和GOM-D;以三烷基取代芳香聚酮生物合成途径保守存在的P450单氧化酶的蛋白序列作为探针,在GOMs产生菌S. rubellomurinus基因组中进行BLAST搜索获得潜在的GOMs生物合成基因簇(gom BGC);通过对gom BGC中的聚酮合成酶(polyketide synthase, PKS)结构基因进行同框缺失突变,对突变株发酵产物进行高效液相色谱-质谱(highperformanceliquidchromatography-massspectrometry,HPLC-MS)分析以确认gomBGC与GOMs的产生相关;基于生物信息学分析,推导GOM-B的生物合成途径。【结果】从S. rubell...     

芍药内生真菌的鉴定及产生活性次生代谢产物的评估

【目的】研究药用植物芍药(Paeonia lactiflora Pall.)内生真菌的种群多样性,同时对其可能存在的聚酮合酶(Polyketide synthase,PKS)和非核糖体多肽合成酶(Non-ribosomal peptide synthetase,NRPS)基因多样性进行评估,预测芍药内生真菌产生活性次生代谢产物的潜力。【方法】采用组织分离法获得芍药根部内生真菌菌株,结合形态学特征和ITS序列分析,进行鉴定;利用兼并性引物对内生真菌中存在的聚酮合酶(PKS)基因和非核糖体多肽合成酶(NRPS)基因进行PCR扩增及序列测定分析,构建系统发育树,明确芍药内真菌PKS基因序列和NRPS基因序列的系统进化地位。【结果】从芍药组织块中共分离得到105株内生分离物,去重复后获得52株内生真菌,菌株ITS基因序列信息显示,52株芍药内生真菌隶属于7目、13科、15属,其中小球腔菌属(Leptosphaeria)、土赤壳属(Ilyonectria)和镰孢属(Fusarium)为优势种群;从52株内生真菌中筛选获得13株含PKS基因片段的菌株,8株含NRPS基因片段的菌株,部分菌株功能基因的氨基酸序列与Gen Bank中已知化合物的合成序列具有一定的同源性,预示芍药根部内生真菌具有合成丰富多样的次生代谢产物的潜力。【结论】药用植物芍药根部具有丰富的内生真菌资源,且具有产生活性次生代谢产物的潜力,值得进一步开发研究和应用。     

顺式和反式-AT型聚酮合酶研究进展及应用

聚酮类化合物因广泛应用于医药等方面而被大家所熟知,Ⅰ型聚酮合酶（Polyketide Synthase,PKS）在催化聚酮类化合物的生物合成中起着重要的作用。根据不同的酰基转移酶（Acyltransferase,AT）结构域,I型PKS可分为顺式-AT （cis-Acyltransferase,cis-AT）型PKS和反式-AT （trans-Acyltransferase,trans-AT）型PKS,目前cis-AT型PKS研究得比较透彻,trans-AT型PKS相关研究成为当今热点。本文总结了cis-AT型PKS和trans-AT型PKS的联系与区别、工程进展、相关应用以及目前存在的问题,以期为了解cis-AT型PKS和trans-AT型PKS在聚酮化合物合成中的作用提供参考。     

A giant type I polyketide synthase participates in zygospore maturation in Chlamydomonas reinhardtii

Polyketide synthases (PKSs) occur in many bacteria, fungi and plants. They are highly versatile enzymes involved in the biosynthesis of a large variety of compounds including antimicrobial agents, polymers associated with bacterial cell walls and plant pigments. While harmful algae are known to produce polyketide toxins, sequences of the genomes of non‐toxic algae, including those of many green algal species, have surprisingly revealed the presence of genes encoding type I PKSs. The genome of the model alga Chlamydomonas reinhardtii (Chlorophyta) contains a single type I PKS gene, designated PKS1 (Cre10.g449750), which encodes a giant PKS with a predicted mass of 2.3 MDa. Here, we show that PKS1 is induced in 2‐day‐old zygotes and is required for their development into zygospores, the dormant stage of the zygote. Wild‐type zygospores contain knob‐like structures (~50 nm diameter) that form at the cell surface and develop a central cell wall layer; both of these structures are absent from homozygous pks1 mutants. Additionally, in contrast to wild‐type zygotes, chlorophyll degradation is delayed in homozygous pks1 mutant zygotes, indicating a disruption in zygospore development. In agreement with the role of the PKS in the formation of the highly resistant zygospore wall, mutant zygotes have lost the formidable desiccation tolerance of wild‐type zygotes. Together, our results represent functional analyses of a PKS mutant in a photosynthetic eukaryotic microorganism, revealing a central function for polyketides in the sexual cycle and survival under stressful environmental conditions.     

Phylogenetic analysis of polyketide synthase genes fromAspergillus ochraceus

A number of polyketide synthase gene sequences fromAspergillus ochraceus were isolated by both SSH-PCR and degenerate PCR. The deduced amino acid sequences of the corresponding clonedpks DNA fragments were then aligned with the amino acid sequences of other polyketide synthase enzymes. One of thesepks genes is essential for ochratoxin A biosynthesis (OTA-PKS). The OTA-PKS was most similar to methylsalicylic acid synthase (MSAS) type PKS proteins based on the alignment of the ketosynthase domains while if the acyl transferase domains were aligned it appeared to be more similar to PKS enzymes fromCochliobolus heterostrophus. The three PKS proteins identified by degenerate PCR were all from different PKS types, one was a MSAS type enzyme, the second was similar to the PKS proteins involved in lovastatin biosynthesis while the third was not similar to any of the other phylogenetic groupings. Data is presented which suggests that the use of phylogenetic analysis to predict the function of PKS proteins/genes is likely to be significantly enhanced by analyzing more than one domain of the protein. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Irish Government under the National Development Plan 2000–2006     

The Use of PCR for Detecting Genes That Encode Type I Polyketide Synthases in Genomes of Actinomycetes

PCR screening of type I polyketidesynthase genes (PKS) was conducted in genomes of actinomycetes, producers of antibiotics. Some DNA fragments from the Streptomyces globisporus 1912 strain, a producer of a novel angucycline antibiotic landomycin E, were amplified. These fragments shared appreciable homology with type I PKS controlling the biosynthesis of polyene antibiotics (pymaricin and nistatin). The cloned regions were used to inactivate putative type I PKS genes in S. globisporus 1912. Strains with inactivated genes of PKS modular do not differ from the original strain in the spectrum of synthesized polyketides. Apparently, these are silent genes, which require specific induction for their expression. The method of PCR screening can be used in a large-scale search for producers of new antibiotics.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 595–600.Original Russian Text Copyright © 2005 by Ostash, Ogonyan, Luzhetskyy, Bechthold, Fedorenko.     

Structural insights into biosynthesis of resorcinolic lipids by a type III polyketide synthase in Neurospora crassa

Microbial type III polyketide synthases (PKSs) have revealed remarkable mechanistic as well as functional versatility. Recently, a type III PKS homolog from Azotobacter has been implicated in the biosynthesis of resorcinolic lipids, thus adding a new functional significance to this class of proteins. Here, we report the structural and mutational investigations of a novel type III PKS protein from Neurospora crassa involved in the biosynthesis of resorcinolic metabolites by utilizing long chain fatty acyl-CoAs. The structure revealed a long hydrophobic tunnel responsible for its fatty acyl chain length specificity resembling that of PKS18, a mycobacterial type III PKS. Structure-based mutational studies to block the tunnel not only altered the fatty acyl chain specificity but also resulted in change of cyclization pattern affecting the product profile. This first structural characterization of a resorcinolic lipid synthase provides insights into the coordinated functioning of cyclization and a substrate-binding pocket, which shows mechanistic intricacy underlying type III PKS catalysis.     

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.     

Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incanaR. Br. (Brassicaceae)

For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.     

洛伐他汀工业菌株土曲霉HZ01的次级代谢产物合成分析

【目的】分析洛伐他汀工业生产菌株土曲霉HZ01的次级代谢产物合成能力,为后期的遗传改造、次级代谢产物及其基因簇挖掘提供指导。【方法】对洛伐他汀发酵条件下的样品进行了转录组分析,同时运用色谱分离技术及波谱学方法对主要次级代谢产物进行了分离和结构鉴定。【结果】洛伐他汀合成相关基因转录水平非常高,还有4个聚酮合酶(PKS)、6个非核糖体多肽合成酶(NRPS)和1个PKS-NRPS杂合酶基因进行了转录,其他PKS和NRPS基因都处于沉默状态。此外,从该菌的发酵产物中分离鉴定了10个主要副产物并确定了其结构。【结论】土曲霉HZ01是一株优良的洛伐他汀生产菌株,在构建次级代谢产物异源合成细胞工厂和鉴定次级代谢产物生物合成途径方面具有很好的应用潜力。     

<Emphasis Type="Italic">fabC</Emphasis> of <Emphasis Type="Italic">Streptomyces lydicus</Emphasis> involvement in the biosynthesis of streptolydigin

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography–mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and β-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.     

Targeting polyketide synthase gene pool within actinomycetes: new degenerate primers

Natural products provide a unique element of molecular diversity and biological functionality and they are still indispensable for drug discovery. The polyketides, comprising a large and structurally diverse family of bioactive natural products, have been isolated from a group of mycelia-forming Gram-positive microorganisms, the actinomycetes. Relatively high amino acid sequence identity of the actinomycetes type I polyketide synthases (PKSs) was used to design three degenerate primer pairs for homology-based PCR detection of novel PKS genes, with particular interest into PKSs involved in biosynthesis of immunosuppressive-like metabolites. The stepdown PCR method, described here, enables fast insight into the PKS arsenal within actinomycetes. Designed primers and stepdown PCR were applied for the analysis of two natural isolates, Streptomyces sp. strains NP13 and MS405. Sequence analysis of chosen clones revealed the presence of two distinctive sequences in strain Streptomyces sp. NP13, but only one of these showed homology to PKS-related sequences. On analysing PCR amplicons derived from Streptomyces sp. strain MS405, three different PKS-related sequences were identified demonstrating a potential of designed primers to target PKS gene pool within single organism.     

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.     

Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase

Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.     

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.