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1.
Ketosynthase Domain Probes Identify Two Subclasses of Fungal Polyketide Synthase Genes 总被引:12,自引:0,他引:12
Lewis E. H. Bingle Thomas J. Simpson Colin M. Lazarus 《Fungal genetics and biology : FG & B》1999,26(3):209-223
Analysis of fungal polyketide synthase gene sequences suggested that these might be divided into two subclasses, designated WA-type and MSAS-type. Two pairs of degenerate PCR primers (LC1 and LC2c, LC3 and LC5c) were designed for the amplification of ketosynthase domain fragments from fungal PKS genes in each of these subclasses. Both primer pairs were shown to amplify one or more PCR products from the genomes of a range of ascomycetous Deuteromycetes and Southern blot analysis confirmed that the products obtained with each pair of primers emanated from distinct genomic loci. PCR products obtained from Penicillium patulum and Aspergillus parasiticus with the LC1/2c primer pair and from Phoma sp. C2932 with both primer pairs were cloned and sequenced; the deduced protein sequences were highly homologous to the ketosynthase domains of other fungal PKS genes. Genes from which LC1/2c fragments were amplified (WA-type) were shown by a phylogenetic analysis to be closely related to fungal PKS genes involved in pigment and aflatoxin biosynthetic pathways, whereas the gene from which the LC3/5c fragment was amplified (MSAS-type) was shown to be closely related to genes encoding 6-methylsalicylic acid synthase (MSAS). The phylogenetic tree strongly supported the division of fungal PKS genes into two subclasses. The LC-series primers may be useful molecular tools to facilitate the cloning of novel fungal polyketide synthase genes. 相似文献
2.
Ji-Long Liao Ka-Lai Pang Guang-Huan Sun Tun-Wen Pai Pang-Hung Hsu Jyuan-Siou Lin Kuang-Hui Sun Chii-Cheng Hsieh Shye-Jye Tang 《Microbial biotechnology》2019,12(5):920-931
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. 相似文献
3.
Wang Y Kim JA Cheong YH Joshi Y Koh YJ Hur JS 《Journal of microbiology (Seoul, Korea)》2011,49(3):473-480
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. 相似文献
4.
【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。 相似文献
5.
Lichenized and non-lichenized filamentous ascomycetes produce a great variety of polyketide secondary metabolites. Some polyketide synthase (PKS) genes from non-lichenized fungi have been characterized, but the function of PKS genes from lichenized species remains unknown. Phylogenetic analysis of keto synthase (KS) domains allows prediction of the presence or absence of particular domains in the PKS gene. In the current study we screened genomic DNA from lichenized fungi for the presence of non-reducing and 6-methylsalicylic acid synthase (6-MSAS)-type PKS genes. We developed new degenerate primers in the acyl transferase (AT) region to amplify a PKS fragment spanning most of the KS region, the entire linker between KS and AT, and half of the AT region. Phylogenetic analysis shows that lichenized taxa possess PKS genes of the 6-MSAS-type. The extended alignment confirms overall phylogenetic relationships between fungal non-reducing, 6-MSAS-type and bacterial type I PKS genes. 相似文献
6.
J M Jez J-L Ferrer M E Bowman M B Austin J Schröder R A Dixon J P Noel 《Journal of industrial microbiology & biotechnology》2001,27(6):393-398
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 相似文献
7.
Augusto Etchegaray Edenilson Rabello Ralf Dieckmann David H. Moon Marli F. Fiore H. von Döhren Siu M. Tsai Brett A. Neilan 《Journal of applied phycology》2004,16(3):237-243
The biosynthesis of algicides produced by a novelFischerellastrain was investigated. Two allelochemicals were identified, the aminoacylpolyketide fischerellin A (FsA) and the alkaloid 12-epi-hapalindole F (HapF). Based on the structure of FsA, genes that could be involved in its biosynthesis, including those encoding nonribosomal peptide synthetases (NRPSs) and a polyketide synthase (PKS), were identified by the polymerase chain reaction (PCR). By showing that the expression of NRPSs and PKSs is concomitant with algicide production we suggest that the identified genes may be involved in algicide biosynthesis. Analysis of an algicide preparation of the Brazilian-Amazonian strainFischerellasp. CENA 19 revealed the production of FsA,m/z409 (MH+), HapF,m/z370 (MH+), and other potential isoforms of the latter compounds, which were identified by high-performance liquid chromatography (HPLC) and matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass-spectrometry. The production of HapF was confirmed after purification by HPLC, analysis by NMR, and high-resolution mass-spectrometry (HRMS). Two-NRPS and a PKS gene were identified after specific amplification using a degenerate PCR. The expression of these synthetases was confirmed by Western blot analysis employing enzyme family-specific antibodies. These analyses revealed the presence of three NRPSs and a single PKS inFischerellasp. CENA 19. The structure of FsA indicates both aminoacyl- and polyketide moeities, suggesting that its biosynthesis may require an integrated NRPS/PKS enzyme system, possibly involving the genes and the synthetases identified. 相似文献
8.
K. Ylihonko J. Tuikkanen S. Jussila L. Cong P. Mäntsälä 《Molecular & general genetics : MGG》1996,251(2):113-120
We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS. 相似文献
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10.
Guang-Rong Zhao Ting Luo Yong-Jin Zhou Xin Jiang Bin Qiao Feng-Ming Yu Ying-Jin Yuan 《Applied microbiology and biotechnology》2009,83(2):305-313
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. 相似文献
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13.
Rojas JD Sette LD de Araujo WL Lopes MS da Silva LF Furlan RL Padilla G 《Microbial ecology》2012,63(3):565-577
The chemical ecology and biotechnological potential of metabolites from endophytic and rhizosphere fungi are receiving much
attention. A collection of 17 sugarcane-derived fungi were identified and assessed by PCR for the presence of polyketide synthase
(PKS) genes. The fungi were all various genera of ascomycetes, the genomes of which encoded 36 putative PKS sequences, 26
shared sequence homology with β-ketoacyl synthase domains, while 10 sequences showed homology to known fungal C-methyltransferase
domains. A neighbour–joining phylogenetic analysis of the translated sequences could group the domains into previously established
chemistry-based clades that represented non-reducing, partially reducing and highly reducing fungal PKSs. We observed that,
in many cases, the membership of each clade also reflected the taxonomy of the fungal isolates. The functional assignment
of the domains was further confirmed by in silico secondary and tertiary protein structure predictions. This genome mining
study reveals, for the first time, the genetic potential of specific taxonomic groups of sugarcane-derived fungi to produce
specific types of polyketides. Future work will focus on isolating these compounds with a view to understanding their chemical
ecology and likely biotechnological potential. 相似文献
14.
P F Leadlay J Staunton M Oliynyk C Bisang J Cortés E Frost Z A Hughes-Thomas M A Jones S G Kendrew J B Lester P F Long H AI McArthur E L McCormick Z Oliynyk C BW Stark C J Wilkinson 《Journal of industrial microbiology & biotechnology》2001,27(6):360-367
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 相似文献
15.
B Shen L Du C Sanchez D J Edwards M Chen J M Murrell 《Journal of industrial microbiology & biotechnology》2001,27(6):378-385
The hybrid peptide–polyketide backbone of bleomycin (BLM) is assembled by the BLM megasynthetase that consists of both nonribosomal
peptide synthetase (NRPS) and polyketide synthase (PKS) modules. BlmIX/BlmVIII/BlmVII constitute a natural hybrid NRPS/PKS/NRPS
system, serving as a model for both hybrid NRPS/PKS and PKS/NRPS systems. Sequence analysis and functional comparison of domains
and modules of BlmIX/BlmVIII/BlmVII with those of nonhybrid NRPS and PKS systems suggest that (1) the same catalytic sites
appear to be conserved in both hybrid NRPS–PKS and nonhybrid NRPS or PKS systems, with the exception of the KS domains in
the hybrid NRPS/PKS systems that are unique; (2) specific interpolypeptide linkers may play a critical role in intermodular
communication to facilitate transfer of the growing intermediates between the interacting NRPS and/or PKS modules; and (3)
posttranslational modification of the BLM megasynthetase has been accomplished by a single PPTase with a broad substrate specificity
toward the apo forms of both acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). Journal of Industrial Microbiology & Biotechnology (2001) 27, 378–385.
Received 08 June 2001/ Accepted in revised form 18 July 2001 相似文献
16.
Isolation and characterization of a non-reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima 总被引:1,自引:0,他引:1
Yi Wang Jung A Kim Yong Hwa Cheong Young Jin Koh Jae-Seoun Hur 《Mycological Progress》2012,11(1):75-83
Usnea longissima has long been used as a traditional medicine in China, India, Turkey, Canada and Europe. This lichen can produce several
bioactive compounds that primarily belong to the polyketide family. The enzymes responsible for the production of these compounds
are the polyketide synthases, but the biosynthetic processes in lichens are still unclear. In this study, a cultured mycobiont
of Usnea longissima was used to isolate and characterize a polyketide synthase gene (UlPKS1). Complete sequence information regarding UlPKS1 (6,468 bp) was obtained by screening a Fosmid genomic library using a 512-bp fragment corresponding to part of the ketosynthase
(KS) domain. Sequence analysis of UlPKS1 suggested that it contained features of a non-reducing fungal type I PKS with a starter unit of ACP transacylase (SAT), ketosynthase
(KS), product template (PT), acyl carrier protein (ACP) transacylase, acyltransferase (AT) and thioesterase (TE) domain, and
had five intervening introns. The domain organization of UlPKS1 (SAT-KS-AT-PT-ACP-ACP-TE) was quite similar to that of aromatic
PKSs, and phylogenetic analysis showed that UlPKS1 belonged to the clade of lichenized fungal non-reducing PKS. RT-PCR analyses revealed that the expression of UlPKS1 was down-regulated by glycine and high concentrations of sorbitol, inositol and fructose and up-regulated by sucrose and
glucose. Here, we introduce a non-reducing PKS gene in the lichen-forming fungus U. longissima, with a domain structure similar to the structure of orsellinic acid synthase A (OrsA) which is required for orsellinic acid biosynthesis in Aspergillus nidulans. 相似文献
17.
A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A. 总被引:7,自引:0,他引:7
R H Proctor A E Desjardins R D Plattner T M Hohn 《Fungal genetics and biology : FG & B》1999,27(1):100-112
Fumonisins are toxins associated with several mycotoxicoses and are produced by the maize pathogen Gibberella fujikuroi mating population A (MP-A). Biochemical analyses indicate that fumonisins are a product of either polyketide or fatty acid biosynthesis. To isolate a putative polyketide synthase (PKS) gene involved in fumonisin biosynthesis, we employed PCR with degenerate PKS primers and a cDNA template prepared from a fumonisin-producing culture of G. fujikuroi. Sequence analysis of the single PCR product and its flanking DNA revealed a gene (FUM5) with a 7.8-kb coding region. The predicted FUM5 translation product was highly similar to bacterial and fungal Type I PKSs. Transformation of a cosmid clone carrying FUM5 into G. fujikuroi enhanced production in three strains and restored wild-type production in a fumonisin nonproducing mutant. Disruption of FUM5 reduced fumonisin production by over 99% in G. fujikuroi MP-A. Together, these results indicate that FUM5 is a PKS gene required for fumonisin biosynthesis. 相似文献
18.
Austin MB Saito T Bowman ME Haydock S Kato A Moore BS Kay RR Noel JP 《Nature chemical biology》2006,2(9):494-502
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. 相似文献
19.
Jumpathong Juangjun Peberdy John Fujii Isao Lumyong Saisamorn 《World journal of microbiology & biotechnology》2011,27(8):1947-1953
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. 相似文献