Biosynthesis of cinnamyl acetate by three plant-derived benzyl acyltransferases in engineered Escherichia coli

Abstract:

[Background] Cinnamyl acetate is an important flavor compound and widely used in cosmetics and food industries. The traditional production methods include direct extraction from plants and chemical synthesis. [Objective] In this work, we aim to achieve de novo biosynthesis of cinnamyl acetate in Escherichia coli by screening benzyl alcohol O-acyl transferases from different plants and constructing biosynthetic pathway for cinnamyl acetate. [Methods] First, a biosynthetic pathway of aglycon cinnamyl alcohol from phenylalanine was constructed in the high-phenylalanine-producing E. coli strain named BPHE by expressing the enzymes phenylalanine ammonialyase (PAL), hydroxycinnamate:CoA ligase (4CL), and cinnamyl-CoA reductase and endogenous alcohol dehydrogenases or aldo-keto reductases in E. coli. Subsequently, the benzyl alcohol O-benzoyltransferase from Nicotiana tabacum (ANN09798) or benzyl alcohol O-benzoyltransferase from Clarkia breweri (ANN09796) or benzyl alcohol acetyltransferase from Clarkia breweri (BEAT) were introduced into the above recombinant E. coli strain to produce cinnamyl acetate. We further improved the acetyl-CoA production by overexpressing endogenous acetyl-CoA synthetase (ACS) in E. coli. [Results] We investigated the ability of three plant-derived benzyl alcohol acyltransferase to biosynthesize cinnamyl acetate, which were further applied for synthesizing cinnamyl acetate in E. coli. The production of cinnamyl acetate by the engineered E. coli reached 166.9±6.6 mg/L. [Conclusion] Plant derived benzyl alcohol acyltransferase demonstrate flexibility to a wide range of substrates and can catalyze the synthesis of cinnamyl acetate by using cinnamyl alcohol as substrate. This study provides a foundation for microbial production of cinnamyl acetate and its derivatives using glucose as the renewable carbon source.

Key words:cinnamyl acetate;biosynthesis;Escherichia coli;benzyl acyltransferases;fermentation