文献类型：期刊文献

英文题名：Combination of protein engineering and metabolic engineering to enhance (+)-nootkatone production in Saccharomyces cerevisiae

作者：Guo, Jingyan[1];Zhou, Wei[1];Li, Yuanyi[1];Qin, Weichuang[1];Wei, Dongzhi[1];Wang, Fengqing[1];Gao, Bei[1]

机构：[1]East China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, POB 311,130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2022

卷号：1

期号：2

起止页码：192

外文期刊名：FOOD BIOENGINEERING

收录：EI(收录号：20232414215027);WOS:【ESCI(收录号:WOS:001332185100008)】；

基金：National Natural Foundation of China, Grant/Award Number: 31772007; Natural Science Foundation of Shanghai, Grant/Award Number: 21ZR1416500

语种：英文

外文关键词：(+)-nootkatone; (+)-valencene; metabolic engineering; protein engineering; Saccharomyces cerevisiae

摘要：The introduction of heterologous pathways into microorganisms for high-level synthesis of valuable compounds would be extremely meaningful. However, the endogenous heterologous pathway conversion point frequently serves as a bottleneck that limits product synthesis. (+)-Nootkatone is an important functional sesquiterpenoid with a unique grapefruit flavor, which is frequently used as a fragrance. The low catalytic efficiency of the valencene synthase CnVS, the first heterologous enzyme in the biosynthesis of (+)-nootkatone, is the major challenge that restricts the production of (+)-nootkatone in Saccharomyces cerevisiae. In this study, we modified CnVS on the basis of the catalytic environment and mechanism of the enzyme. The residue M560 involving in alkyl transfer during substrate cyclization was found to affect activity. By tuning the residue, mutant M560L exhibited a 60% increase in the unit cell yield of (+)-valencene, which is the precursor of (+)-nootkatone. Furthermore, multistep strategies of metabolic engineering were employed that transferred the metabolic flux to the target product synthesis pathway and displayed the superimposed effect of metabolic and protein engineering on (+)-nootkatone yield. Finally, multicopy integration of tHMG1 and ERG20ERG20-GSG-CnVS(M560L) boosted the production of (+)-nootkatone to 85.43mg/L in flask and 804.96mg/L (5.29mg/[Lh]) in bioreactor. In conclusion, this study presents a paradigm for constructing microbial cell factories using a combination of protein and metabolic engineering in S. cerevisiae.