Journal
ACS SYNTHETIC BIOLOGY
Volume 10, Issue 6, Pages 1531-1544Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.1c00081
Keywords
nerol; borneol; enzyme engineering; synthetic biology; Escherichia coli
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Funding
- Key-Area Research and Development Program of Guangdong Province [2020B0303070002]
- National Natural Science Foundation of China [32070062]
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This study successfully improved the de novo production of nerol and borneol in E. coli by combining metabolic and plant monoterpene synthase engineering, achieving the highest reported titers of nerol and borneol in microbes to date.
The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the de novo production of nerol and borneol in prene-overproducing Escherichia coli. We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from Glycine max to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from Lippia dulcis for borneol production. Further, through site-directed mutation of LdtBPPS based on the structural simulation, we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic borneol, of which the LdtBPPS(S488T) mutant outperformed the wild-type LdtBPPS on borneol synthesis and the LdtBPPS(F612A) variant was superior to GmNES on nerol production. Subsequently, we overexpressed the endogenous Nudix hydrolase NudJ to facilitate the dephosphorylation of precursors and boosted the production of nerol and borneol from glucose. Finally, after the optimization of the fermentation process, the engineered strain ENO2 produced 966.55 mg/L nerol, and strain ENB57 generated 87.20 mg/L borneol in a shake flask, achieving the highest reported titers of nerol and borneol in microbes to date. This work shows a combinatorial engineering strategy for microbial production of natural terpene alcohols.
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