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Updated: Oct 13, 2025
A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products
Published on: October 4, 2019
Systematic identification of Ocimum sanctum sesquiterpenoid synthases and (-)-eremophilene overproduction in
Xiaomin Deng1, Bin Shi2, Ziling Ye3
1Ministry of Agriculture Key Laboratory of Biology and Genetic Resources of Rubber Tree/State Key Laboratory Breeding Base of Cultivation and Physiology for Tropical Crops, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, Hainan, People's Republic of China; Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, Hubei, People's Republic of China; Wuhan Institute of Biotechnology, Wuhan, 470074, Hubei, People's Republic of China.
Abstract:
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 β-caryophyllene and its close isomer α-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.
