Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome

Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications.     

Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions

Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way.     

Draft genome of Streptomyces tsukubaensis NRRL 18488, the producer of the clinically important immunosuppressant tacrolimus (FK506)

The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacrolimus biosynthesis gene cluster.     

苏云金素合成基因簇中非核糖体肽合成酶基因thu2功能的初步分析

对苏云金素生物合成基因簇中编码非核糖体肽合成酶基因thu2进行基因缺失插入失活的研究。用温敏型质粒pHT304-TS构建基因thu2的插入缺失质粒pEMB1434,电转化苏云金芽胞杆菌菌株CT-43后,通过抗性筛选和PCR验证得到thu2基因同源双交换基因敲除突变株CT-43-22。HPLC(高效液相色谱,High Performance Liquid Chromatography)检测发现CT-43-22没有苏云金素特征吸收峰;用pHT304构建得到含有完整thu2基因的回补质粒pEMB1435,电转化CT-43-22后得到互补重组菌CT-43-22b,发现其恢复了苏云金素的产生。显微镜观察突变株和互补重组菌均能产生正常的晶体和芽胞。thu2的基因敲除和基因互补实验证明,thu2基因为CT-43苏云金素生物合成的必需基因,但对晶体和芽胞的形成没有影响。     

New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.     

Metagenomic Analysis Reveals Diverse Polyketide Synthase Gene Clusters in Microorganisms Associated with the Marine Sponge Discodermia dissoluta

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up <1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.     

聚酮类化合物生物合成基因簇与药物筛选

由微生物和植物产生的聚酮类化合物的数量极其庞大,是一大类结构多样化和生物活性多样性的天然产物,已经成为新药的重要来源.介绍了3种类型聚酮类化合物生物合成基因簇的特点,即以模块形式存在的I型聚酮合酶,包含一套可重复使用结构域的Ⅱ型聚酮合酶以及不需要ACP参与,以植物中的查耳酮合酶为代表的Ⅲ型聚酮合酶.同时,还介绍了基于3种类型聚酮类化合物生物合成基因的特点,利用分子生物学方法构建筛选探针,进行当前药物基因筛选的进展.     

Biosynthesis of Novel Pyoverdines by Domain Substitution in a Nonribosomal Peptide Synthetase of Pseudomonas aeruginosa

Pyoverdine is a fluorescent nonribosomal peptide siderophore made by fluorescent pseudomonads. The Pseudomonas aeruginosa nonribosomal peptide synthetase (NRPS) PvdD contains two modules that each incorporate an l-threonine residue at the C-terminal end of pyoverdine. In an attempt to generate modified pyoverdine peptides, we substituted alternative-substrate-specifying adenylation (A) and peptide bond-catalyzing condensation (C) domains into the second module of PvdD. When just the A domain was substituted, the resulting strains produced only wild-type pyoverdine—at high levels if the introduced A domain specified threonine or at trace levels otherwise. The high levels of pyoverdine synthesis observed whenever the introduced A domain specified threonine indicated that these nonnative A domains were able to communicate effectively with the PvdD C domain. Moreover, the unexpected observation that non-threonine-specifying A domains nevertheless incorporated threonine into pyoverdine suggests that the native PvdD C domain exhibited stronger selectivity than these A domains for the incorporated amino acid substrate (i.e., misactivation of a threonine residue by the introduced A domains was more frequent than misincorporation of a nonthreonine residue by the PvdD C domain). In contrast, substitution of both the C and A domains of PvdD generated high yields of rationally modified pyoverdines in two instances, these pyoverdines having either a lysine or a serine residue in place of the terminal threonine. However, C-A domain substitution more commonly yielded a truncated peptide product, likely due to stalling of synthesis on a nonfunctional recombinant NRPS template.     

iso-Migrastatin, Migrastatin, and Dorrigocin Production in Streptomyces platensis NRRL 18993 Is Governed by a Single Biosynthetic Machinery Featuring an Acyltransferase-less Type I Polyketide Synthase

iso-Migrastatin and related glutarimide-containing polyketides are potent inhibitors of tumor cell migration and their implied potential as antimetastatic agents for human cancers has garnered significant attention. Genome scanning of Streptomyces platensis NRRL 18993 unveiled two candidate gene clusters (088D and mgs); each encodes acyltransferase-less type I polyketide synthases commensurate with iso-migrastatin biosynthesis. Both clusters were inactivated by λ-RED-mediated PCR-targeting mutagenesis in S. platensis; iso-migrastatin production was completely abolished in the ΔmgsF mutant SB11012 strain, whereas inactivation of 088D-orf7 yielded the SB11006 strain that exhibited no discernible change in iso-migrastatin biosynthesis. These data indicate that iso-migrastatin production is governed by the mgs cluster. Systematic gene inactivation allowed determination of the precise boundaries of the mgs cluster and the essentiality of the genes within the mgs cluster in iso-migrastatin production. The mgs cluster consists of 11 open reading frames that encode three acyltransferase-less type I polyketide synthases (MgsEFG), one discrete acyltransferase (MgsH), a type II thioesterase (MgsB), three post-PKS tailoring enzymes (MgsIJK), two glutarimide biosynthesis enzymes (MgsCD), and one regulatory protein (MgsA). A model for iso-migrastatin biosynthesis is proposed based on functional assignments derived from bioinformatics and is further supported by the results of in vivo gene inactivation experiments.Cell migration is essential for invasion of the extracellular matrix and for cell dissemination during tumor metastasis (1). The glutarimide-containing polyketides iso-migrastatin (iso-MGS),² migrastatin (MGS), the dorrigocins (DGNs), and lactimidomycin (LTM) (Fig. 1) are potent inhibitors of human tumor cell migration and thus represent novel leads for anticancer drug discovery (2–5). Synthetic analogs of these natural products have also been investigated and found to retain potent activity despite significant structural truncation (6–8). Retention of activity by such analogs supports the effectiveness of the privileged scaffolds highlighted by iso-MGS, MGS, LTM, and related natural products. Complementary to organic synthesis, combinatorial biosynthesis offers an alternative means of accessing natural product structural diversity. We have previously studied the biosynthetic pathway of these polyketides and shown that MGS and the DGNs are shunt metabolites of iso-MGS (9). As has been demonstrated in Streptomyces platensis NRRL 18993, one of the known iso-MGS producers, iso-MGS, MGS, and the DGNs are produced by a single biosynthetic machinery and iso-MGS undergoes H₂O-mediated, non-enzymatic ring-expansion, and ring-opening rearrangements to afford MGS and the DGNs as shunt metabolites. We have also isolated iso-MGS congeners as minor fermentation products, produced a small library of glutarimide-containing polyketides featuring the iso-MGS, LTM, MGS, and DGN scaffolds, and found selected analogs with biological activity to far surpass those of MGS (10–14). In so doing, we have shown the general superiority of 12-membered macrolides over the 14-membered analogs as glutarimide-containing polyketide inhibitors of cell migration. Yet, the ability to readily convert members of the 12-membered macrolides into their corresponding 14-membered and acyclic congeners synthetically presents us with novel structural diversity capabilities not commonly encountered biosynthetically. Cloning and characterization of the gene cluster governing iso-MGS biosynthesis, therefore, represents a significant undertaking with many foreseeable ramifications in drug discovery.Open in a separate windowFIGURE 1.Glutarimide-containing polyketides iso-MGS, MGS, DGN A, 13-epi-DGN A, DGN B produced by S. platensis NRRL 18993 and LTM produced by S. amphibiosporus ATCC53964.Here we report: (i) development of a genetic system for S. platensis NRRL 18993, (ii) identification and cloning of two acyltransferase (AT)-less type I polyketide synthase (PKS)-encoding clusters (088D and mgs) in S. platensis NRRL 18993 along with assignment of one (088D) as a cryptic cluster and the other (mgs) as being exclusively accountable for iso-MGS, thereby MGS and DGN, biosynthesis, and (iii) an experimentally supported proposal for a single biosynthesis machinery governing the production of iso-MGS, MGS, and the DGNs.     

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.     

Amplification of DNA Encoding Entire Type I Polyketide Synthase Domains and Linkers from Streptomyces Species

Polyketides are a group of bioactive compounds from bacteria, plants, and fungi. To increase the availability of analogs for testing, the active sites of polyketide synthases are often substituted with homologous domains having altered substrate specificities. This study reports the design of polymerase chain reaction primers that enables isolation of entire active site domains from type I polyketide synthases with native interdomain linkers. This bypasses the need for further genetic screening to obtain functional units for use in genetic engineering. This is especially important in bioprospecting projects exploring new environments for bioresources.     

Molecular Cloning of the Actinomycin Synthetase Gene Cluster from Streptomyces chrysomallus and Functional Heterologous Expression of the Gene Encoding Actinomycin Synthetase II

The actinomycin synthetases ACMS I, II, and III catalyze the assembly of the acyl peptide lactone precursor of actinomycin by a nonribosomal mechanism. We have cloned the genes of ACMS I (acmA) and ACMS II (acmB) by hybridization screening of a cosmid library of Streptomyces chrysomallus DNA with synthetic oligonucleotides derived from peptide sequences of the two enzymes. Their genes were found to be closely linked and are arranged in opposite orientations. Hybridization mapping and partial sequence analyses indicate that the gene of an additional peptide synthetase, most likely the gene of ACMS III (acmC), is located immediately downstream of acmB in the same orientation. The protein sequence of ACMS II, deduced from acmB, shows that the enzyme contains two amino acid activation domains, which are characteristic of peptide synthetases, and an additional epimerization domain. Heterologous expression of acmB from the mel promoter of plasmid PIJ702 in Streptomyces lividans yielded a functional 280-kDa peptide synthetase which activates threonine and valine as enzyme-bound thioesters. It also catalyzes the dipeptide formation of threonyl–l-valine, which is epimerized to threonyl–d-valine. Both of these dipeptides are enzyme bound as thioesters. This catalytic activity is identical to the in vitro activity of ACMS II from S. chrysomallus.The actinomycins are a class of chromopeptide lactones produced by various Streptomyces strains. They contain two pentapeptide lactone rings attached to chromophoric 4,6-dimethylphenoxazinone-1,9-dicarboxylic acid (actinocin) in an amide-like fashion. Actinocin is formally derived from the compound 4-methyl-3-hydroxyanthranilic acid (4-MHA), but actually the bicyclic actinomycins arise from the oxidative condensation of two preformed monocyclic 4-MHA pentapeptide lactones (12). Previous investigations have revealed that the formation of the 4-MHA pentapeptide lactones is catalyzed by three actinomycin synthetases (ACMS I, II, and III) (13, 15). ACMS I (45 kDa) is a 4-MHA–AMP ligase which activates 4-MHA as adenylate. The five amino acids of the pentapeptide lactone ring of actinomycin (NH₂-cyclo[Thr–d-Val–Pro–N-methyl-Gly–N-methyl-Val] for actinomycin D) are assembled by ACMS II (280 kDa) and ACMS III (480 kDa) which from their properties belong to the class of peptide synthetases (13, 26, 27). ACMS II catalyzes the activation of threonine and valine. In the presence of ACMS I, which supplies 4-MHA–adenylate, 4-MHA–threonine and 4-MHA–threonyl–d-valine (via 4-MHA–threonyl–l-valine) are formed on the surface of ACMS II. In the absence of 4-MHA or ACMS I, purified ACMS II can synthesize both threonyl–l-valine and threonyl–d-valine, though to a lesser extent than the corresponding 4-MHA dipeptides can. The epimerization of valine is catalyzed by ACMS II at the acyl-dipeptide stage. An analysis of ACMS III suggests that it elongates the 4-MHA–Thr–d-Val dipeptide by successive incorporation of proline, N-methylglycine (sarcosine), and N-methyl-l-valine into the growing peptide chain (13). N-methylation is an additional feature of ACMS III. A total cell-free system for 4-MHA pentapeptide lactone synthesis is not available yet. Thus, it is not known how 4-MHA dipeptide transfer from ACMS II to ACMS III is accomplished, nor is the mechanism of lactone formation and release from the 4-MHA pentapeptide known.The available data indicate that ACMS II and ACMS III contain two- and three-amino-acid activation domains, respectively. It is known that activation domains of peptide synthetases are highly conserved in their sequences and are composed of a segment for amino acid adenylation and a segment for binding the activated amino acid as a thioester (17, 24, 25, 32). Thioester formation occurs via the thiol group of 4′-phosphopantetheine, which is a covalently bound cofactor of the activation domain. ACMS II and III both contain 4′-phosphopantetheine. In contrast, ACMS I has no 4′-phosphopantetheine cofactor, consistent with the finding that it does not form a thioester with 4-MHA. Data from previous work pointed instead to the formation of a 4-MHA thioester with ACMS II (26). In order to investigate the modular structure of the ACMSs and the reaction mechanisms in more detail, we set out to clone the ACMS genes from Streptomyces chrysomallus with oligonucleotide probes derived from partial sequences of ACMS I and II. We show that the genes of ACMS I and II and of a third peptide synthetase, most probably the gene of ACMS III (acmA, acmB, and acmC, respectively) are closely linked, forming a gene cluster. A total sequence determination of acmB and the characterization of the heterologously expressed functional active gene product confirm the significance of this peptide synthetase gene cluster.     

The Polyketide Synthase Gene pks4 of Trichoderma reesei Provides Pigmentation and Stress Resistance

Species of the fungal genus Trichoderma (Hypocreales, Ascomycota) are well-known for their production of various secondary metabolites. Nonribosomal peptides and polyketides represent a major portion of these products. In a recent phylogenomic investigation of Trichoderma polyketide synthase (PKS)-encoding genes, the pks4 from T. reesei was shown to be an orthologue of pigment-forming PKSs involved in synthesis of aurofusarin and bikaverin in Fusarium spp. In this study, we show that deletion of this gene in T. reesei results in loss of green conidial pigmentation and in pigmentation alteration of teleomorph structures. It also has an impact on conidial cell wall stability and the antagonistic abilities of T. reesei against other fungi, including formation of inhibitory metabolites. In addition, deletion of pks4 significantly influences the expression of other PKS-encoding genes of T. reesei. To our knowledge, this is the first indication that a low-molecular-weight pigment-forming PKS is involved in defense, mechanical stability, and stress resistance in fungi.     

Structure of a Eukaryotic Nonribosomal Peptide Synthetase Adenylation Domain That Activates a Large Hydroxamate Amino Acid in Siderophore Biosynthesis

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N^δ-cis-anhydromevalonyl-N^δ-hydroxy-l-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.     

紫菜外生细菌抑菌活性及其多聚酮合酶（PKS I） 基因筛选

摘要：【目的】：基于紫菜外生细菌抑菌活性的研究,本文对具有广谱抑菌活性的菌株进行了多聚酮合酶(Polyketide synthase I, PKS I)基因的筛选,以期获得PKS I 阳性菌株及探讨紫菜藻际微生物区系细菌的拮抗机制与PKS I 途径的关系。【方法】：利用琼脂柱法筛选出具广谱抑菌活性的菌株31株,以其基因组DNA 为模板,设计引物扩增酮基合成酶(Ketosynthase, KS) 片段基因并将其克隆到 pMD19-T Vector,筛选出PKS I 阳性菌,进行16S rDNA 测序分析。【结果】：紫菜外生细菌表现出广谱抑菌性。从温州病烂紫菜外生菌中筛选出3株具强抑菌活性的PKS I 阳性菌,BLAST 比对结果显示：菌株WPhG3、WPySw1和WPySw2 扩增得到PKS I 的KS 结构域核苷酸序列所对应的氨基酸序列与Bacillus subtilis subsp. subtilis str. 168（NP_389602）、Bacillus subtilis (ABR19776)和Aspergillus carbonarius (AAZ99721) 的PKS I 的KS 结构域的同源性分别达到98%、99%和98%;16S rDNA 系统发生学分析显示它们均与Bacillus 的同源性最高。【结论】：紫菜藻际微生物群落组成复杂,通过多条途径调节藻际微生物区系的平衡。PKS I 途径可能是温州病烂紫菜外生菌Bacillus 表现抑菌活性的一种方式。     

Identification and Regulation of fusA,the Polyketide Synthase Gene Responsible for Fusarin Production in Fusarium fujikuroi

Fusarins are a class of mycotoxins of the polyketide family produced by different Fusarium species, including the gibberellin-producing fungus Fusarium fujikuroi. Based on sequence comparisons between polyketide synthase (PKS) enzymes for fusarin production in other Fusarium strains, we have identified the F. fujikuroi orthologue, called fusA. The participation of fusA in fusarin biosynthesis was demonstrated by targeted mutagenesis. Fusarin production is transiently stimulated by nitrogen availability in this fungus, a regulation paralleled by the fusA mRNA levels in the cell. Illumination of the cultures results in a reduction of the fusarin content, an effect partially explained by a high sensitivity of these compounds to light. Mutants of the fusA gene exhibit no external phenotypic alterations, including morphology and conidiation, except for a lack of the characteristic yellow and/or orange pigmentation of fusarins. Moreover, the fusA mutants are less efficient than the wild type at degrading cellophane on agar cultures, a trait associated with pathogenesis functions in Fusarium oxysporum. The fusA mutants, however, are not affected in their capacities to grow on plant tissues.     

Insect-Specific Polyketide Synthases (PKSs), Potential PKS-Nonribosomal Peptide Synthetase Hybrids,and Novel PKS Clades in Tropical Fungi

Polyketides draw much attention because of their potential use in pharmaceutical and biotechnological applications. This study identifies an abundant pool of polyketide synthase (PKS) genes from local isolates of tropical fungi found in Thailand in three different ecological niches: insect pathogens, marine inhabitants, and lichen mutualists. We detected 149 PKS genes from 48 fungi using PCR with PKS-specific degenerate primers. We identified and classified 283 additional PKS genes from 13 fungal genomes. Phylogenetic analysis of all these PKS sequences the comprising ketosynthase (KS) conserved region and the KS-acyltransferase interdomain region yielded results very similar to those for phylogenies of the KS domain and suggested a number of remarkable points. (i) Twelve PKS genes amplified from 12 different insect-pathogenic fungi form a tight cluster, although along with two PKS genes extracted from genomes of Aspergillus niger and Aspergillus terreus, in reducing clade III. Some of these insect-specific fungal PKSs are nearly identical. (ii) We identified 38 new PKS-nonribosomal peptide synthetase hybrid genes in reducing clade II. (iii) Four distinct clades were discovered with more than 75% bootstrap support. We propose to designate the novel clade D1 with 100% bootstrap support “reducing clade V.” The newly cloned PKS genes from these tropical fungi should provide useful and diverse genetic resources for future research on the characterization of polyketide compounds synthesized by these enzymes.One hallmark of tropical countries is the tremendous availability and diversity of natural resources. Tropical forests, freshwater reservoirs, and seas are home to an uncountable number of species, ranging from microorganisms (e.g., bacteria, fungi, and protozoa) to invertebrates to vertebrates to plants. Thailand is no exception. The country has a large collection of fungi found in different niches and habitats in its ecosystems. Interesting groups include fungi that are associated with insects, those that inhabit the sea, and those that are in lichen complexes; these are referred to here as insect fungi, marine fungi, and lichenized fungi, respectively. The first group is of particular interest because it represents a remarkable relationship (in this case, pathogenesis) between the fungi and their insect hosts. These entomopathogenic fungi were isolated from the dead insect bodies in different stages (e.g., larvae, pupae, nymphs, or adults). The marine fungi used in this study were mostly isolated from the living or dead plant parts floating at the seashore, whereas the lichen mutualistic fungi were isolated from lichen complexes on the bark of trees in tropical forests in Thailand. All these fungal isolates were deposited in National Center for Genetic Engineering and Biotechnology (BIOTEC) Culture Collection (BCC). The BCC has one of the richest collections (approximately 400 species and 5,000 isolates) of insect fungi in the world (19).Secondary metabolites may play an important role in organisms that synthesize them, for example, in spore development (7), protection, or host virulence (5). Polyketides (PKs) are natural secondary metabolite compounds derived from the condensation of acyl coenzyme A subunits in a head-to-tail manner, and they have a tremendous diversity in structure (33). Structural diversification of the PKs includes a variation in the number of subunits, types of subunits, degree of chemical reduction of the β-keto thioester, extent of stereochemistry of the α-keto group at each condensation, and subsequent processing (e.g., cyclicization) (25, 28, 33). The high therapeutic and economic value of PK compounds has attracted the interest of drug companies and government research agencies. Some PKs are commercially available for medical treatments, such as grahamimycin and patulin (antibiotics), lovastatin and compactin (cholesterol-lowering agents), griseofulvin (an antibiotic/antifungal agent), and monocerin (an antifungal agent).Enzymes that synthesize the PKs are called PK synthases (PKSs). PKSs are multifunctional enzymes that are composed of three principal domains: ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP). Fungal PKSs are type I, multifunctional large enzymes and use an iterative strategy to synthesize PKs. They can be divided into two groups, nonreducing (NR) and reducing (4), and further subdivided into NR subclades I, II, and III and reducing subclades I, II, III, and IV (26). NR PKSs include those synthesizing pigments or aflatoxin. Reducing PKSs are involved in the synthesis of PK compounds with various chemical reductions in structure. Apart from the three major domains (KS, AT, and ACP) present in all PKSs, reducing PKSs contain three additional domains, i.e., dehydratase, enoyl reductase, and ketoreductase, which are involved in the reduction of the keto group to various stages (i.e., alcohol, unsaturated thiolester, and full saturation, respectively), therefore enhancing diversity of the PK structure.Kroken et al. (26) studied putative amino acid sequences of the PKS genes previously characterized in fungi and the PKS genes discovered from the genome sequencing projects for eight fungal species in the Ascomycota. PKS genes were found only in the genomes of the Pezizomycotina and not in the sequenced genomes of either Ascomycota in the Taphrinomycota or Saccharomycotina or Basidiomycota in the Hymenomycetes. Thus, we focused our search on the fungi in this subphylum. We aimed to mine valuable PKS genes from this fungal resource. One of the main objectives is to find novel secondary metabolites useful for medical or agricultural applications. One highly regarded example is the “vegetable caterpillar,” where the fungus Cordyceps sinensis grows on Hepialidae caterpillars. The fungus has long been used in traditional Chinese medicine. Extracts of Cordyceps sinensis were reported to have a variety of therapeutic effects, for example, antitumor (6), antioxidant (42), and antiaging (24) activities. The C. sinensis-Hepialidae pair is also called the “body snatcher”. This name comes from the fact that the fungus infects and consumes the insect tissue and fills up the insect cavity with its mycelia. Thus, another objective is to find metabolites involved in interaction between fungal pathogens and their insect hosts. Insect pests pose tremendous losses to humans in regard to health issues (vectors of diseases) and economic issues (crop plant losses by insect pathogens and building structure damage by termites). Little was known regarding the roles of PKs in producing fungi on their interaction with insect host. Better understanding of this relationship might have implications for insect control.We conducted our PKS screening using PCR with the degenerate KA series primers (2). In addition to our preliminary PKS screening with these primers in a few fungi (2), the KA series primers were used to clone the reducing PKS gene for radicicol biosynthesis from the fungus Pochonia chlamydosporia, and later its whole biosynthetic cluster was revealed (37). Here, the method and the primers were also proven to be successful in finding rich resources of hidden metabolic pathways for PK biosynthesis from 48 fungi that were isolated in Thailand and, particularly, have no genome sequences determined. In addition, more than 200 PKS genes were identified from our genome analysis of 13 filamentous fungi.