Detection of nonribosomal peptide synthetase genes in Xylaria sp. BCC1067 and cloning of XyNRPSA

Nonribosomal peptides, synthesized by nonribosomal peptide synthetases (NRPS), are an important group of diverse bioactive fungal metabolites. Xylaria sp. BCC1067, which is known to produce a variety of biologically active metabolites, was studied for gene encoding NRPS by two different PCR-based methods and seven different NRPS fragments were obtained. In addition, screening a genomic library with an amplified NRPS fragment as a probe identified a putative NRPS gene named XyNRPSA. The functionality of XyNRPSA for the production of a corresponding metabolite was probed by gene insertion inactivation. Comparing the disrupting metabolite profile with that of the wild type led to the identification of a speculated metabolite. The crude extract of Xylaria sp. BCC1067 also exhibits antifungal activity against the human pathogens Candida albicans and Trichophyton mentagrophytes. However, the evaluation of biological activity of the XyNRPSA product suggests that it is neither a compound with antifungal activity nor a siderophore. In the vicinity of XyNRPSA, a second gene (named XyPtB) was identified. Its localization and homology to orfB of the ergot alkaloid biosynthetic gene cluster suggests that XyPtB may be involved in XyNRPSA product biosynthesis.     

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.     

四种红树植物根际土壤微生物Ⅰ型和Ⅱ型PKS基因的检测与多样性分析

[目的]探究红树林土壤中聚酮合酶(Polyketide synthase,PKS)基因的多样性和新颖性.[方法]用Ⅰ型和Ⅱ型PKS基因酮基合成酶(Ketosynthase,KS)域的简并引物对海南清澜港红树林海莲、黄槿、银叶、老鼠簕4种红树根际土壤样品中DNA进行PCR扩增,之后利用PCR-限制性酶切片段多样性(PCR-RFLP)和测序分析法对Ⅰ型和Ⅱ型PKS基因的多样性进行探讨.[结果]对得到的72条Ⅰ型PKS基因的酮基合成酶(Ketosynthase,KS)域DNA序列进行PCR-RFLP分析,共得到51个可操作分类单元(Operational taxonomic unit,OTUs),其中37个OTUs为单克隆产生,没有明显的优势OTU.选取了26个代表不同OTU的克隆进行测序分析,这些序列与GenBank中已知序列的最大相似率均未超过85％. KS域氨基酸序列的系统发育分析显示,所得KS域来源广泛,包括蓝细菌门(Cyanobacteria)、变形杆菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和一些未可培养细菌;对55条PKSⅡ基因KS域DNA序列的PCR-RFLP分析后共得到25个OTUs,有两个明显的优势OTUs,代表的克隆子数所占比例超过10％.[结论]PCR-RFLP分析表明红树林根际土壤中存在着丰富多样的Ⅰ型和Ⅱ型PKS基因,且前者多样性更高;低的序列相似度表明所获得的PKSⅠ基因KS域序列独特;系统发育分析表明得到的PKSⅠ基因来源广泛.     

Cloning and characterization of a type III polyketide synthase from Aspergillus niger

Type III polyketide synthases (PKSs) are the condensing enzymes that catalyze the formation of a myriad of aromatic polyketides in plant, bacteria, and fungi. Here we report the cloning and characterization of a putative type III PKS from Aspergillusniger, AnPKS. This enzyme catalyzes the synthesis of alkyl pyrones from C2 to C18 starter CoA thioesters with malonyl-CoA as an extender CoA through decaboxylative condensation and cyclization. It displays broad substrate specificity toward fatty acyl-CoA starters to yield triketide and tetraketide pyrones, with benzoyl-CoA as the most preferred starter. The optimal temperature and pH of AnPKS are 50°C and 8, respectively. Under optimal conditions, the enzyme shows the highest catalytic efficiency (k(cat)/K(m)) of 7.4×10(5)s(-1)M(-1) toward benzoyl-CoA. Homology modeling and site-directed mutagenesis were used to probe the molecular basis of its substrate specificity. This study should open doors for further engineering of AnPKS as a biocatalyst for synthesis of value-added polyketides.     

Phylogenetic analysis of type I polyketide synthase and nonribosomal peptide synthetase genes in Antarctic sediment

The modular polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) have been found to be involved in natural product synthesis in many microorganisms. Study on their diversities in natural environment may provide important ecological insights, in addition to opportunities for antibacterial drugs development. In this study, the PKS and NRPS gene diversities in two coast sediments near China Zhongshan Station were studied. The phylogenetic analysis of amino acid (AA) sequences indicated that the identified ketosynthase (KS) domains were clustered with those from diverse bacterial groups, including Proteobacteria, Firmicutes, Planctomycetes, Cyanobacteria, Actinobacteria, and some uncultured symbiotic bacteria. One new branch belonging to hybrid PKS/NRPS enzyme complexes and five independent clades were found on the phylogenetic tree. The obtained adenylation (A) domains were mainly clustered within the Cyanobacteria and Proteobacteria group. Most of the identified KS and A domains showed below 80 and 60% identities at the AA level to their closest matches in GenBank, respectively. The diversities of both KS and A domains in natural environmental sample were different from those in sewage-contaminated sample. These results revealed the great diversity and novelty of both PKS and NRPS genes in Antarctic sediment.     

Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex

An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.     

Analysis of the enzymatic domains in the modular portion of the coronafacic acid polyketide synthase

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant pathogen Pseudomonas syringae. The CFA polyketide synthase (PKS) consists of two open reading frames (ORFs) that encode type I multifunctional proteins and several ORFs that encode monofunctional proteins. Sequence comparisons of the modular portions of the CFA PKS with other prokaryotic, modular PKSs elucidated the boundaries of the domains that are involved in the individual stages of polyketide assembly. The two β-ketoacyl:acyl carrier protein synthase (KS) domains in the modular portion of the CFA PKS exhibit a high degree of similarity to each other (53%), but are even more similar to the KS domains of DEBS, RAPS, and RIF. Cfa6 possesses two acyltransferases- AT0, which is associated with a loading domain, and AT1, which uses ethylmalonyl-CoA (eMCoA) as a substrate for chain extension. Cfa7 contains an AT that uses malonyl-CoA as a substrate for chain extension. The Cfa6 AT0 shows 35 and 32% similarity to the DEBS1 and NidA1 AT0s, respectively, and 32 and 36% similarity to the Cfa6 and Cfa7 AT1s. Sequence motifs have previously been identified that correlate with AT substrates. The motifs in Cfa6 AT1 were found to correlate reasonably well with those predicted for methylmalonyl-CoA (mMCoA) ATs. The motifs in the AT of Cfa7 correlated more poorly with those predicted for MCoA ATs. Three ACP domains occur in the modular proteins of the COR PKS. The loading domain-associated ACP0 showed 38% similarity to the loading domain ACP0s of DEBS1 and NidA1 and 32–36% similarity to the two module-associated ACPs of the COR PKS. It exhibited a higher degree of similarity to the module-associated ACPs of RAPS. The two module-associated ACPs show 39% similarity to each other, but appear more closely related to module-associated ACP domains in RAPS and RIFS. Furthermore, the DH and KR domains of Cfa6 and Cfa7 show greater similarity to DH and KR domains in RAPS and RIFS than to each other. The CFA PKS includes a thioesterase domain (TE I) that resides at the C-terminus of Cfa7 and a second thioesterase, which exists as a separate ORF (Cfa9, a TE II). Analysis of a Cfa7 thioesterase mutant demonstrated that the TE domain is required for the production of CFA. The co-existence of TE domains within modular PKSs along with physically separated, monofunctional TEs (TE IIs) has been reported for a number of modular polyketide and non-ribosomal peptide synthases (NRPS). An analysis of the two types of thioesterases using Clustal X yielded a dendrogram showing that TE IIs from PKSs and NRPSs are more closely related to each other than to domain TEs from either PKSs or NRPSs. Furthermore, the dendrogram indicates that both types of TE IIs are more closely related to TE domains associated with PKSs than to TE domains in NRPSs. Finally, the overall % G+C content and the % G+C content at the third codon for all of the PKS genes in the COR cluster suggest that these genes may have been recruited from a gram-positive bacterium.     

Characterization of two polyketide synthase genes in Exophiala lecanii-corni, a melanized fungus with bioremediation potential

Exophiala lecanii-corni has significant bioremediation potential because it can degrade a wide range of volatile organic compounds. In order to identify sites for the insertion of genes that might enhance this potential, a genetic analysis of E. lecanii-corni was undertaken. Two polyketide synthase genes, ElPKS1 and ElPKS2, have now been discovered by a PCR-based strategy. ElPKS1 was isolated by a marker rescue technique. The nucleotide sequence of ElPKS1 consists of a 6576-bp open reading frame encoding a protein with 2192 amino acids, which was interrupted by a 60-bp intron near the 5' end and a 54-bp intron near the 3' end. Sequence analysis, results from disruption experiments, and physiological tests showed that ElPKS1 encoded a polyketide synthase required for melanin biosynthesis. Since ElPKS1 is non-essential, it is a desirable bioengineering target site for the insertion of native and foreign genes. The successful expression of these genes could enhance the bioremediation capability of the organism. ElPKS2 was cloned by colony hybridization screening of a partial genomic library with an ElPKS2 PCR product. ElPKS2 had a 6465-bp open reading frame that encoded 2155 amino acids and had introns of 56, 67, 54, and 71 bp. Although sequence analysis of the derived protein of ElPKS2 confirmed the polyketide synthase nature of its protein product, the function of that product remains unclear.     

Aspergillus oryzae type III polyketide synthase CsyB uses a fatty acyl starter for the biosynthesis of csypyrone B compounds

Csypyrones B1, B2 and B3 are α-pyrones that can be obtained from Aspergillus oryzae expressing CsyB, which is a type III polyketide synthase. We investigated the biosynthesis of the csypyrone B compounds using [1-¹³C] and [2-¹³C] acetate feeding experiments. ¹³C NMR analyses of the methyl esters of the csypyrone B compounds fed with the ¹³C-labeled acetates showed that the carboxyl carbons of the csypyrone B side-chains were derived from the C-2 methyl carbon of the acetate. These results indicated that fatty acyl starters are involved in the CsyB reaction and that the csypyrone B compounds are formed by the oxidation of side-chains by the host fungus.     

Widening the bottleneck: Heterologous expression,purification, and characterization of the Ktedonobacter racemifer minimal type II polyketide synthase in Escherichia coli

Enzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable clinical success, the biosynthetic prowess of these enzymes has been stymied by 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. To begin filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KS-CLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer (Kr). Using E. coli as a heterologous host, we obtained soluble proteins in titers signifying improvements over previous KS-CLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKS-CLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. These results lay important groundwork for optimizing the interaction between KrKS-CLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.     

草菇聚酮合酶编码基因vv-alb的克隆及其表达的初步分析

聚酮化合物（polyketides）是一类庞大的次级代谢家族,聚酮合酶（polyketide synthase,PKS）是介导聚酮化合物生物合成的关键酶。通过巢氏简并PCR与染色体步行的方法,获得了草菇中的编码PKS的基因vv-alb的全长序列,并通过荧光实时定量RT-PCR方法对vv-alb基因在草菇不同生长阶段与不同部位的表达情况进行了初步分析,为进一步研究PKS在草菇和其他食用真菌生物代谢过程中的作用奠定了一定的基础。     

Diversity and evolution of polyketide biosynthesis gene clusters in the Ceratocystidaceae

Polyketides are secondary metabolites with diverse biological activities. Polyketide synthases (PKS) are often encoded from genes clustered in the same genomic region. Functional analyses and genomic studies show that most fungi are capable of producing a repertoire of polyketides. We considered the potential of Ceratocystidaceae for producing polyketides using a comparative genomics approach. Our aims were to identify the putative polyketide biosynthesis gene clusters, to characterize them and predict the types of polyketide compounds they might produce. We used sequences from nineteen species in the genera, Ceratocystis, Endoconidiophora, Davidsoniella, Huntiella, Thielaviopsis and Bretziella, to identify and characterize PKS gene clusters, by employing a range of bioinformatics and phylogenetic tools. We showed that the genomes contained putative clusters containing a non-reducing type I PKS and a type III PKS. Phylogenetic analyses suggested that these genes were already present in the ancestor of the Ceratocystidaceae. By contrast, the various reducing type I PKS-containing clusters identified in these genomes appeared to have distinct evolutionary origins. Although one of the identified clusters potentially allows for the production of melanin, their functional characterization will undoubtedly reveal many novel and important compounds implicated in the biology of the Ceratocystidaceae.     

A family of polyketide synthase genes expressed in ripening Rubus fruits

Quality traits of raspberry fruits such as aroma and color derive in part from the polyketide derivatives, benzalacetone and dihydrochalcone, respectively. The formation of these metabolites during fruit ripening is the result of the activity of polyketide synthases (PKS), benzalcetone synthase and chalcone synthase (CHS), during fruit development. To gain an understanding of the regulation of these multiple PKSs during fruit ripening, we have characterized the repertoire of Rubus PKS genes and studied their expression patterns during fruit ripening. Using a PCR-based homology search, a family of ten PKS genes (Ripks1-10) sharing 82-98% nucleotide sequence identity was identified in the Rubus idaeus genome. Low stringency screening of a ripening fruit-specific cDNA library, identified three groups of PKS cDNAs. Group 1 and 2 cDNAs were also represented in the PCR amplified products, while group 3 represented a new class of Rubus PKS gene. The Rubus PKS gene-family thus consists of at least eleven members. The three cDNAs exhibit distinct tissue-specific and developmentally regulated patterns of expression. RiPKS5 has high constitutive levels of expression in all organs, including developing flowers and fruits, while RiPKS6 and RiPKS11 expression is consistent with developmental and tissue-specific regulation in various organs. The recombinant proteins encoded by the three RiPKS cDNAs showed a typical CHS-type PKS activity. While phylogenetic analysis placed the three Rubus PKSs in one cluster, suggesting a recent duplication event, their distinct expression patterns suggest that their regulation, and thus function(s), has evolved independently of the structural genes themselves.     

Investigation of bacteria with polyketide synthase genes and antimicrobial activity isolated from South China Sea sponges

Aims:  To obtain bacteria with PKS (polyketide synthase) genes and antimicrobial activity from sponges.
Methods and Results:  Eighteen bacteria with KS (ketosynthase) genes were identified by polymerase chain reaction (PCR) screening of 98 isolates from South China Sea sponges, Stelletta tenuis , Halichondria rugosa , Dysidea avara and Craniella australiensis . 16S rRNA gene-based B last analysis indicated that 15 isolates belonged to the phylum Firmicutes , among which 14 isolates were closely related to genus Bacillus , and 1 to Staphylococcus lentus . Two isolates were identified as actinomycetes, and one as Alcaligenes sp. in the phylum Proteobacteria . The 18 KS domains belong to trans-AT type I PKS and match PKS of marine bacterial symbionts. The 18 bacteria exhibited broad-spectrum antimicrobial activities against fungi, gram-positive and gram-negative bacteria. A 21·8-kb PKS gene cluster fragment containing five modules was isolated from the Staphylococcus lentus isolate A75 by screening of a fosmid library.
Conclusions:  The PKS gene diversity and different antimicrobial spectra indicate the potential of bacteria associated with South China Sea sponges for diverse polyketide production.
Significance and Impact of the Study:  Combined with bioactivity assay the PKS gene-based approach can be applied to efficient screening of strains of pharmaceutical value and the prediction of related compounds.     

Expression and characterization of the type III polyketide synthase 1,3,6,8-tetrahydroxynaphthalene synthase from<Emphasis Type="Italic"> Streptomyces coelicolor</Emphasis> A3(2)

Sequence analysis of the metabolically rich 8.7-Mbp genome of the model actinomycete Streptomyces coelicolor A3(2) revealed three genes encoding predicted type III polyketide synthases (PKSs). We report the inactivation, expression, and characterization of the type III PKS homologous SCO1206 gene product as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Incubation of recombinant THNS with malonyl-CoA showed THN production, as demonstrated by UV and HPLC analyses. The K_m value for malonyl-CoA and the k_cat value for THN synthesis were determined spectrophotometrically to be 3.58±0.85 µM and 0.48±0.03 min^–1, respectively. The C-terminal region of S. coelicolor THNS, which is longer than most other bacterial and plant type III PKSs, was shortened by 25 amino acid residues and the resulting mutant was shown to be slightly more active (K_m=1.97±0.19 µM, k_cat=0.75±0.04 min^–1) than the wild-type enzyme.     

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.     

Evaluating type III polyketide synthase (PKS) and terpene synthase (TPS) expression in incompatible interactions of Zingiber zerumbet to Pythium myriotylum Drechsler

Abstract

Pythium species are aggressive soil-borne necrotrophic oomycetes causing soft-rot disease of ginger. Disease severity indices determined following infection with P. myriotylum Drechsler in ginger cultivar, Zingiber officinale cv. Varada and a wild congener, Z. zerumbet at varying zoospore concentrations (10⁴–10¹² spores/ml) revealed high disease severity (100%) in ginger cultivar whereas Z. zerumbet displayed resistance. Absence of positive correlation between Z. zerumbet resistance and polyphenolic content indicates role of polyketides and zerumbone in preventing pathogen ingress, as reported earlier. Towards elucidating this, Z. zerumbet specific polyketide synthase (PKS) and terpene synthase (TPS) gene sequences designated ZzTPS and ZzPKS respectively were characterised. Phylogenetic analysis clustered ZzTPS with TPS-b sub-family and ZzPKS with non-chalcone forming PKS. ZzTPS and ZzPKS showed biphasic expression with first at 6?hours post infection (hpi) and then at 8 hpi, indicative of rapid induction followed by reinforcement to sustain resistance mechanisms in the wild taxon.     

A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium

The melanin polyketide synthase (pks) gene of Nodulisporium sp. MF5954 (ATCC74245) was cloned by exploiting its homology to the Colletotrichum lagenarium pks1 gene. Sequence analysis demonstrated that this gene is 70% identical to the C. lagenarium pks1 gene. A gene disruption construct, designed to replace both the ketoacyl synthase and acyl transferase domains with a hygromycin resistance (Hy^r) gene, was synthesized, and used to disrupt the Nodulisporium melanin pks1 gene via homologous recombination, resulting in a mel(−) phenotype. Sequence analyses of the gene and of cDNA segments generated by RT-PCR indicate that there are three introns in the 5′ half of the gene. The proposed 2159-amino acid product is 72% identical and 78% similar to the 2187-amino acid sequence deduced from the C. lagenarium pks1 gene. This similarity is notable, considering that C. lagenarium is a member of the order Phyllachoales or Sordariales, whereas Nodulisporium is generally believed to be member of the order Xylariales. However, despite the strong resemblance between the amino acid sequences in the acyl transferase domains of the two proteins, only one in five codons are conserved in the DNA sequences that encode this motif. The Nodulisporium sp. pks1 gene sequence and the amino acid sequence deduced from its coding region have been deposited in Genbank under Accession No. AF151533. Received: 15 May 1999 / Accepted: 26 July 1999     

Determination of the extent of phosphopantetheinylation of polyketide synthases expressed in Escherichia coli and Saccharomyces cerevisiae

A sensitive fluorescent assay was developed to measure the extent of phosphopantetheinylation of polyketide synthase (PKS) acyl carrier protein (ACP) domains in polyketide production strains. The in vitro assay measures PKS fluorescence after transfer of fluorescently labeled phosphopantetheine from coenzyme A to PKS ACP domains in crude protein extracts. The assay was used to determine the extent of phosphopantetheinylation of ACP domains of the erythromycin precursor polyketide synthase, 6-deoxyerythronolide B synthase (DEBS), expressed in a heterologous Escherichia coli polyketide production strain. The data showed that greater than 99.9% of DEBS is phosphopantetheinylated. The assay was also used to interrogate the extent of phosphopantetheinylation of the lovastatin nonaketide synthase (LNKS) heterologously expressed in Saccharomyces cerevisiae. The data showed that LNKS was efficiently phosphopantetheinylated in S. cerevisiae and that lack of production of the lovastatin precursor polyketide was not due to insufficient phosphopantetheinylation of the expressed synthase.