Enhancement of levopimaradiene production in Escherichia coli via engineering isopentenol utilization pathway

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).

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 β-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.

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.

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.