Expanding Upon Styrene Biosynthesis to Engineer a Novel Route to 2‐Phenylethanol

Abstract

2-Phenylethanol (2PE) is a key molecule used in the fragrance and food industries, as well as a potential biofuel. In contrast to its extraction from plant biomass and/or more common chemical synthesis, microbial 2PE production has been demonstrated via both native and heterologous expression of the yeast Ehrlich pathway. Here, a novel alternative to this established pathway is systematically engineered in Escherichia coli and evaluated as a more robust and efficient route. This novel pathway is constructed via the modular extension of a previously engineered styrene biosynthesis pathway, proceeding from endogenous l-phenylalanine in five steps and involving four heterologous enzymes. This "styrene-derived" pathway boasts nearly a 10-fold greater thermodynamic driving force than the Ehrlich pathway, and enables reduced accumulation of acetate byproduct. When directly compared using a host strain engineered for l-phenylalanine over-production, preservation of phosphoenolpyruvate, and reduced formation of byproduct 2-phenylacetic acid, final 2PE titers via the styrene-derived and Ehrlich pathways reached 1817 and 1164 mg L^-1 , respectively, at yields of 60.6 and 38.8 mg g^-1 . Following optimization of induction timing and initial glucose loading, 2PE titers by the styrene-derived pathway approached 2 g L^-1 - nearly a two-fold twofold increase over prior reports for 2PE production by E. coli employing the Ehrlich pathway.