Objective: Plant-derived terpenoid ginkgolides exhibit significant pharmacological efficacy, however, their extraction remains costly. Given that levopimaradiene is a key biosynthetic precursor to ginkgolides, its high-yield production via heterologous gene expression therefore establishes a critical foundation for scaling up their manufacture. This study primarily aims to enhance the yield of levopimaradiene in Escherichia coli (LB medium) by remodeling the unnatural isopentenol utilization pathway (IUP). Methods: Plasmid construction was driven by the mechanism of homologous recombination, which utilizes recombinase to facilitate the ligation process. The expression of kinase was carried out using type 7 (T7) promoter and isopropyl b-D-1-thiogalactopyranoside (IPTG) as the inducer. The quantification of levopimaradiene produced by E. coli was determined by comparison with a standard curve that we constructed. Results: Combining the selection of kinases, ribosome-binding site (RBS) screening, protein directed evolution and optimization of fermentation parameters, the production of levopimaradiene in E. coli was ultimately enhanced to 2691.3 mg/L, surpassing the highest reported titers of levopimaradiene with 6-fold in E. coli to date. Additionally, the engineered E. coli was designed to collaborate with farnesyl pyrophosphate (FPP) synthase and geranylfarnesyl pyrophosphate (GFPP) synthase to efficiently produce FPP and GFPP for sesquiterpene and sesterterpene synthesis. Conclusion: Our work showcases a combinatorial engineering strategy that employs an IUP-enhanced E. coli chassis for the microbial production of levopimaradiene, as well as other natural terpenoids.
关键词:
定向进化;异戊二烯利用途径;左旋海松二烯;代谢工程;核糖体结合位点筛选
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Project Supported:
This research was co-financed by the Young Scientists Fund of the National Natural Science Foundation of China (NSFC) (No. 82003608), the Key Project of NSFC (No. 81991524), the Innovation Projects of State Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture (No. NZYSKL240109), the Interdisciplinary Project of Nanjing University of Chinese Medicine (No. ZYXJC2024-003 and No.JC202402), the Youth Project of Jiangsu Commission of Health (No. QN202405), and the Jiangsu Annual Basic Science Institutions (No. 25KJB350006).
Chenhao Jia a, b,, Junying Ma a, b,, Lingli Fang a, b, Huanzi Guo a, b, Meiling Ding a, b, Yishuang Wang a, b,?, Renxiang Tan c. Enhancement of levopimaradiene production in Escherichia coli via engineering isopentenol utilization pathway[J]. Chinese Herbal Medicines (CHM),2026,18(1):89-96
