Elucidation of the Function of Domains of Dual Acyl Carrier Protein and Methyltransferase from Chimeric 6-Methylsalicyclic Acid Synthase in Biosynthesis of 2-Hydroxy-2-(Propan-2-Yl) Cyclobutane-1,3-Dione

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Details

Project title

Code/計畫編號

MOST109-2313-B019-008

Translated Name/計畫中文名

解析重組6MSAS生物合成2-hydrox-2-(propan-2-yl) cyclobutane-1,3-dione雙重ACP和甲基化酵素區域之功能

Project Coordinator/計畫主持人

Shye-Jye Tang

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=13535043

Year

2020

Start date/計畫起

01-08-2020

Expected Completion/計畫迄

31-07-2021

Bugetid/研究經費

1190千元

ResearchField/研究領域

生物技術(農)

Description

Abstract

Secondary metabolites that are obtained from microbes are widely applied in medicine, agriculture, environmental treatment and industrial processes. Devising a feasible strategy for synthesis of secondary metabolites is important to contribute to further biological applications. Biosynthesis of polyketides is carried out by single polyketide synthase (PKS) or multiple PKSs in successive elongations of the enzyme-bound intermediates likely to fatty acid biosynthesis. PKSs containing domains of ketosynthase (KS), acyltransferase (AT), dehydratase (DH), ketoreductase (KR), enoyl reductase (ER), thioesterase (TE) and acyl carrier protein (ACP) execute series of decarboxylative Claisen condensations to synthesize polyketides. Moreover, phosphopantetheinyl transferase (PPTase) uses a phosphopantethein to modify a serine residue in the ACP and activated ACP may harbor acyl group intermediates in the biosynthesis. The first identified PKS gene, 6-methylsalicylic acid synthase (6MSAS), produces 6-methylsalicylic acid (6MSA). The PKS gene harboring KS-AT-DH-KR-ACP domains was successfully and heterologously expressed with PPTase in Escherichia coli and Saccharomyces cerevisiae to produce 6MSA. Pseudallescheria boydii NTOU2362 was isolated from mangrove endophytic fungi and identified to produce polyketides. PKSs obtained from P. boydii were not detected novel compounds in the extract of the S. cerevisiae transformants analyzed by HPLC. Chimeric 6MSAS, R6MSAS-306-ACP-MT, was created by the fusion of the domains of KS, AT, DH, and KR from 6MSAS with ACP and methyltransferase (MT) from P. boydii NTOU2362, and the enzyme produced a novel compound, 2-hydroxy-2-(propan-2-yl) cyclobutane-1,3-dione. Significantly, the chimeric PKS harboring ACP and MT from PKS306 may contribute to biosynthesize the novel compound. ACPs consist of 70-100 amino acids typically and participate in the biosynthesis of polyketides to relay growing acyl chains through the assemble line of PKS. Compared with the protein sequence of ACP from 6MSAS, a low similarity of dual ACPs from PKS306 is shown except at the phosphopantetheine attachment site, which is recognized by phosphopantetheinyl transferase. Methyltransferase in PKS functional domains may perform iterative C-methylation of fungal nonreduced polyketides using S-adenosylmethionine as the donor of methyl groups one or more times to a tetra- or pentaketide. The novel compound containing an isopropyl group demonstrates activity of the MT domain that is essential for biosynthesis because removal of the domain abolishes the synthesis of the compound. To characterize the novel R6MSAS-306-ACP-MT, this project will perform mutagenesis, purification and identification of novel compounds in recombinant expression in S. cerevisiae or in vitro biosynthesis, and identification of intermediates linked into ACP using mass spectrometry. We will explore: 1. The role of dual ACP domains in R6MSAS-306-ACP-MT in the biosynthesis of the novel compound. 2. The function of methyltransferase domain in R6MSAS-306-ACP-MT in the biosynthesis of the novel compound. 3. Domains of DH and KR from 6MSAS in the biosynthesis of the novel compound. Although 6MSAS is the first PKS discovered in the biosynthesis of secondary metabolites, its use in gene engineering to create the novel compound remains valuable. It is possible to establish the workflow to produce novel compounds that involve in the generation of bioactive compounds that have the medical application potentials and may be used for patent application.微生物產生的二次代謝物，廣用於醫藥、農業、環境處理、和工業等用途。因此建立可生產二次代謝物的策略, 可提昇未來的應用發展。聚酮的生物合成是由聚酮合成酵素(PKS)，經一連續式的酵素及decarboxylative Claisen condensation與中間產物連接而成。PKS含有的功能區域acyl carrier protein (ACP)、acyl transferase、ketosynthase、ketoreductase、dehydratase、 enoyl reductase 經 thioesterase釋放。此外，在ACP 上需經phosphopantetheinyl transferase在Ser上接入phosphopantethein用於中間體接在其上。 6-methylsalicylic acid synthase (6MSAS) 具有KS-AT-DH-KR-ACP domain 合成6-methylsalicylic acid。先前從紅樹林共生黴菌Pseudallescheria boydii NTOU2362中獲得一系列的PKS，但皆無法獲得生物活性。為增進PKS多樣性建構combinatorial PKS R6MSAS-306-ACP-MT，使用6MSAS之KS-AT-DH-KR與PKS306之ACP-MT (methyltransferase)可合成新化合物，結構分析得到2-hydroxy-2-(propan-2-yl) cyclobutane-1,3-dione。比較6MSAS與這PKS，顯現含兩ACP及MT，是否這變化負與PKS新活性。在計畫中將使用定位突變、表現在S. cerevisiae或in vitro biosynthesis後純化和鑑定新化合物、使用質譜鑑定結合在ACP上的中間體。在計畫中將探討:1. Dual ACP domains在R6MSAS-306-ACP-MT對2-hydroxy-2-(propan-2-yl) cyclobutane-1,3-dione生物合成之作用。2. Methyltransferase domain 在對新化合物合成之作用。3. DH和KR在對新化合物合成之作用。PKS涉及未來的藥物合成，在化學和生技領域皆極為重要，本計畫的執行，有助於這領域的開發。

Keyword(s)

二次代物
聚酮酵素
聚酮
生物合成
6-methylsalicylic acid synthase
2-hydroxy-2-(propan-2-yl) cyclobutane-1
3-dione
6-Methylsalicylic acid synthase
6MSAS
Pseudallescheria boydii NTOU2362
Polyketide synthase
Chimeric PKS
2-Hydroxy-2-(propan-2-yl) cyclobutane-1
3-dione； Biosynthesis