Metabolic Engineering Of Escherichia Coli For Production Of Nerol And(+)-Borneol

Acyclic nerol and bicyclic（+）-borneol are two valuable monoterpene alcohols with wide applications in food,cosmetics,household products,and pharmaceutical industries.Plant extraction can only provide a small amount of nerol and（+）-borneol due to the restrictions of plant resources and extraction technologies,while large-scale production of monoterpene alcohols by chemical synthesis results in multiple byproducts and is uneco-friendly and uneconomical.However,the emerging synthetic biology enables engineered microorganisms such as Escherichia coli to be a more efficient and stable alternative for the production of nerol and（+）-borneol.We reconstructed the biosynthetic pathway of monoterpene alcohols in E.coli,and thus we improved the de novo production of nerol and（+）-borneol.By introducing an optimized exogenous mevalonate pathway into E.coli BW25113（DE3）strain,we first constructed the terpenoid production platform of EIP81 strain.Neryl diphosphate（NPP）synthase and nerol synthase（Gm NES）were coexpressed in strain EIP81,and 55.37mg/L nerol was synthesized.We screened different combinations of bornyl diphosphate synthases（BPPSs）with cis-precursor NPP or trans-precursor geranyl diphosphate（GPP）in strain EIP81.Ldt BPPS from Lippia dulcis using NPP as precursor performed best,with a（+）-borneol titer of 4.26 mg/L.Further through engineering Ldt BPPS based on the structural simulation,we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic（+）-borneol,of which the Ldt BPPS^S488T mutant outperformed the wild-type Ldt BPPS with the（+）-borneol titer being increased by 140%to 10.27 mg/L and the Ldt BPPS^F612A variant was superior to Gm NES with a 24%higher nerol titer of 68.77mg/L.Subsequently,we screened 11 endogenous or heterologous phosphatases to facilitate the hydrolysis of NPP or BPP.The overexpression of the endogenous Nudix hydrolase Nud J boosted the production of nerol and（+）-borneol from glucose by 281%and 175%,synthesizing 261.88 mg/L nerol and 28.26 mg/L（+）-borneol,respectively.Finally,after optimizing the induction conditions and initial glucose concentration,engineered strain ENO2 produced 966.55 mg/L nerol under two-phase fermentation in78 h,and strain ENB57 synthesized 87.20 mg/L（+）-borneol in 114-h two-phase fermentation.In this study,the highest microbial production titers of nerol and（+）-borneol are achieved by metabolically engineered E.coli.It also sets a good example for engineering plant monoterpene synthases and producing terpene alcohols in microbes.