Systematic identification of Ocimum sanctum sesquiterpenoid synthases and (-)-eremophilene overproduction in engineered yeast
文献类型： 外文期刊
作者： Deng, Xiaomin 1 ; Shi, Bin 2 ; Ye, Ziling 2 ; Huang, Man 5 ; Chen, Rong 2 ; Cai, Yousheng 2 ; Kuang, Zhaolin 2 ; Sun, Xiang 2 ; Bian, Guangkai 2 ; Deng, Zixin 2 ; Liu, Tiangang 2 ;
作者机构： 1.Chinese Acad Trop Agr Sci, State Key Lab Breeding Base Cultivat & Physiol Tr, Key Lab Biol & Genet Resources Rubber Tree, Minist Agr,Rubber Res Inst, Haikou 571101, Hainan, Peoples R China
2.Wuhan Univ, Minist Educ, Key Lab Combinatorial Biosynthesis & Drug Discove, Sch Pharmaceut Sci, Wuhan 430071, Hubei, Peoples R China
3.Wuhan Inst Biotechnol, Wuhan 470074, Hubei, Peoples R China
4.Wuhan Vocat Coll Software & Engn, Wuhan Drug Solubilizat & Delivery Technol Res Ctr, Wuhan 430205, Hubei, Peoples R China
5.J1 Biotech Co Ltd, Wuhan 430075, Hubei, Peoples R China
关键词： Saccharomyces cerevisiae; Metabolic engineering; Plant sesquiterpene; (-)-Eremophilene; beta-caryophyllene; Ocimum sanctum
期刊名称：METABOLIC ENGINEERING （ 影响因子：8.829； 五年影响因子：9.073 ）
ISSN： 1096-7176
年卷期： 2022 年 69 卷
页码：
收录情况： SCI
摘要： Plant-derived natural active products have attracted increasing attention for use in flavors and perfumes. These compounds also have applications in insect pest control because of their environment-friendly properties. Holy basil (Ocimum sanctum), a famous herb used in Ayurveda in India, is a natural source of medical healing agents and insecticidal repellents. Despite the available genomic sequences and genome-wide bioinformatic analysis of terpene synthase genes, the functionality of the sesquiterpene genes involved in the unique fragrance and insecticidal activities of Holy basil are largely unknown. In this study, we systematically screened the sesquiterpenoid biosynthesis genes in this plant using a precursor-providing yeast system. The enzymes that synthesize beta-caryophyllene and its close isomer alpha-humulene were successfully identified. The enzymatic product of OsaTPS07 was characterized by in vivo mining, in vitro reaction, and NMR detection. This product was revealed as (-)-eremophilene. We created a mutant yeast strain that can achieve a high-yield titer by adjusting the gene copy number and FPP precursor enhancement. An optimized two-stage fed-batch fermentation method achieved high biosynthetic capacity, with a titer of 34.6 g/L cyclic sesquiterpene bioproduction in a 15-L bioreactor. Further insect-repelling assays demonstrated that (-)-eremophilene repelled the insect pest, fall leafworm, suggesting the potential of (-)-eremophilene as an alternative to synthetic chemicals for agricultural pest control. This study highlights the potential of our microbial platform for the bulk mining of plant-derived ingredients and provides an impressive cornerstone for their industrial utilization.
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