Genome-Wide Survey and Characterization of Polyketide Synthases and Nonribosomal Peptide Synthetases in Pseudallescheria boydii NTOU2362 from Endophytic Fungi of Mangrove Plants

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Project title

Code/計畫編號

NSC102-2313-B019-013-MY3

Translated Name/計畫中文名

紅樹林共生黴菌Pseudallescheria boydii NTOU2362全基因體分析與polyketide synthase和nonribosomal peptide synthetase鑑定

Project Coordinator/計畫主持人

Shye-Jye Tang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=8107423

Year

2014

Start date/計畫起

01-08-2014

Expected Completion/計畫迄

01-07-2015

Bugetid/研究經費

1250千元

ResearchField/研究領域

生物技術(理)
生物科學

"微生物全基因體序列的獲得已十分簡易可行，因此利用生物資訊的方式分析微生物基因體，可直接獲得與二次代謝物生物合成相關的基因序列。黴菌可產生多種二次代謝物，包括polyketides (PKs)和non-ribosomal peptides (NRPs)，其生物合成主要經由polyketide synthases (PKS)和non-ribosomal peptide synthetases (NRPS)所產生。先前從紅樹林共生黴菌Pseudallescheria boydii #NTOU2362中測得具抗病毒的生物活性化合物，經使用Next generation sequencing 和生物資訊策略，顯示這菌具有42.5x106個鹼基對，包含可合成二次代謝物的9個polyketide synthase (PKS)，8個nonribosomal peptide synthetase (NRPS)，和3個PKS/NRPS hybrid基因序列，預測這些基因大小從4809到16788鹼基對，可轉譯出蛋白的長度為1603 ~ 5596胺基酸，顯示蛋白質非常巨大，屬於megaenzyme，也包含各種functional domain，應與PK和NRP的生成有關，此與先前純化的二次代謝物結構上具有polyketide和peptide bond的結果相符，可推知這些化合物的產生應與PKS和NRPS的表現與活性相關。但這些基因的酵素活性與他們合成之化合物為何，則從未被探討過，我們因此將選殖PKS和NRPS基因，預期可用於產生具有新化學結構的新穎化合物。於此計畫中，將使用long rang PCR擴增PKS和NRPS基因，選殖入Gateway cloning system，接入Saccharomyces cerevisiae表現載體，該載體在蛋白質C端具有綠螢光蛋白質(GFP)和tandem-affinity purification (TAP) tags，GFP可用於標誌與追蹤蛋白，而TAP可用於親和性純化。首先以質譜分析比較帶有PKS和NRPS基因之S. cerevisiae與原菌種生成之化合物的差異。經多樣性組合(combinatorial) PKS和NRPS表現載體，使用in vivo方式合成這些二次化謝物，鑑定化合物的可能合成途徑。計畫中將完成下列目標： 1. 分析PKS/NRPS在不同培養條件下的基因表現。 2. 選殖PKS和NRPS，利用S. cerevisiae表現系統表現，並分析產生的二次代謝物。 3. 經多樣性組合(combinatorial) PKS和NRPS表現載體，製備PKS/NRPS組成式生物合成策略。 4. 鑑定產生的組成式生物活性物質，並分析抗病毒活性。 5. 計畫中產生的具醫用價值的PKS/NRPS及生物活性物質將可申請專利。三年也可提出三篇期刊論文。" "The recent increase and availability of whole genome sequences allow mining of genes that are involved in the metabolism of microorganisms. A large number of orphan biosynthesis pathways have been identified by bioinformatics, showing that microbial genomes contain gene clusters responsible for biosynthesis of secondary metabolites. Fungi are known to produce several classes of secondary metabolites, including polyketides (PKs) and non-ribosomal peptides (NRPs). These natural products are synthesized by polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS), respectively. Pseudallescheria boydii #NTOU2362 isolated from endophytic fungi associated with mangrove plants was demonstrated the antiviral activities in its culture medium. Recently, the fungus was performed next generation sequencing to obtain 42.5x106 base pairs. By genome-wide data mining, the strain was found to contain 8 NRPS, 9 PKS, and 3 PKS/NRPS hybrid genes. The sizes of open reading frame are from 4809 to 16788 base pairs, and the predicted proteins contain amino acids from 1603 to 5596. Our findings indicate that the strain contains mega-enzymes that involve in the biosynthesis of the second metabolites. Moreover, these second metabolites containing functional groups of polyketide and peptide bonds indicate that genes of PKS and NRPS are active to involve in the biosynthesis of secondary metabolite. Therefore, there are still a vast number of novel compounds with new chemical structures that are able to be produced by the cloned PKS and NRPS. In this project, PKS and NRPS genes will be amplified by long rang PCR and cloned into Gateway cloning system and Saccharomyces cerevisiae expression vectors that contain tag of green fluorescent protein (GFP) or tandem-affinity purification (TAP) in the C-terminus. The green fluorescence may show the expression of the full-length protein, and TAP will be used in affinity purification using IgG antibody matrix. The production of secondary metabolites by overexpression of biosynthetic genes in S. cerevisiae will be identified by mass spectrometry (MS). The combinatorial PKS/NRPS enzymes are able to be expressed in S. cerevisiae to performed in vivo biosynthesis. Moreover, combinatorial biosynthesis strategies are expected to create diversity of secondary metabolites from combinatorial PKS/NRPS. Major anticipated achievements - 1. The gene expression of PKS/NRPS will be characterized for gene regulation of the PKSs and NRPSs in various culture conditions. . 2. The genes of PKS and NRPS will be cloned, and the bioactive compounds synthesized by these PKS- or NRPS-mediated pathways will be characterized and identified by comparative MS analysis. 3. The combinatorial PKS/NRPS will be expressed in S. cerevisiae to performed in vivo biosynthesis. 4. The combinatorial bioactive compounds will be produced and be screened for their anti-viral activity. 5. The novel PKS/NRPS and novel bioactive compounds, with medical application potentials, may be used for patent application. Moreover, at least 3 papers/3 years will be published in international refereed journal from the results of this project."

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Genome-Wide Survey and Characterization of Polyketide Synthases and Nonribosomal Peptide Synthetases in Pseudallescheria boydii NTOU2362 from Endophytic Fungi of Mangrove Plants

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