Computational-guided discovery of UDP-glycosyltransferases for lauryl glucoside production using engineered E.coli  

作　　者：Kasimaporn Promubon Kritsada Tathiya Aussara Panya Wasu Pathom-Aree Pachara Sattayawat 

机构地区：[1]Department of Biology,Faculty of Science,Chiang Mai University,Chiang Mai 50200,Thailand [2]Cell Engineering for Cancer Therapy Research Group,Faculty of Science,Chiang Mai University,Chiang Mai 50200,Thailand [3]Master of Science Program in Applied Microbiology(International Program),Faculty of Science,Chiang Mai University,Chiang Mai 50200,Thailand

出　　处：《Bioresources and Bioprocessing》2024年第1期1340-1352,共13页生物资源与生物加工(英文)

基　　金：This work(Grant No.RGNS 64-069)was financially supported by Office of the Permanent Secretary,Ministry of Higher Education,Science,Research and Innovation;partially supported by Chiang Mai University.

摘　　要：Defining suitable enzymes for reaction steps in novel synthetic pathways is crucial for developing microbial cell factories for non-natural products.Here,we developed a computational workflow to identify C12 alcohol-active UDP-glycosyltransferases.The workflow involved three steps:(1)assembling initial candidates of putative UDP-glycosyltransferases,(2)refining selection by examining conserved regions,and(3)3D structure prediction and molecular docking.Genomic sequences from Candida,Pichia,Rhizopus,and Thermotoga,known for lauryl glucoside synthesis via whole-cell biocatalysis,were screened.Out of 240 predicted glycosyltransferases,8 candidates annotated as glycosyltransferases were selected after filtering out those with signal peptides and identifying conserved UDP-glycosyltransferase regions.These proteins underwent 3D structure prediction and molecular docking with 1-dodecanol.RO3G,a candidate from Rhizopus delemar RA 99-880 with a relatively high ChemPLP fitness score,was selected and expressed in Escherichia coli BL21(DE3).It was further characterized using a feeding experiment with 1-dodecanol.Results confirmed that the RO3G-expressing strain could convert 1-dodecanol to lauryl glucoside,as quantified by HPLC and identified by targeted LC-MS.Monitoring the growth and fermentation profiles of the engineered strain revealed that RO3G expression did not affect cell growth.Interestingly,acetate,a major fermentation product,was reduced in the RO3G-expressing strain compared to the GFP-expressing strain,suggesting a redirection of flux from acetate to other pathways.Overall,this work presents a successful workflow for discovering UDP-glycosyltransferase enzymes with confirmed activity toward 1-dodecanol for lauryl glucoside production.

关 键 词：UDP-glycosyltransferase Lauryl glucoside Computational method 1-Dodecanol Engineered E.coli Novel biosynthetic pathway Genome mining 

分 类 号：TP3[自动化与计算机技术—计算机科学与技术]