Coculture engineering for efficient production of vanillyl alcohol in Escherichia coli

Abstract

Vanillyl alcohol is a precursor of vanillin, which is one of the most widely used flavor compounds. Currently, vanillyl alcohol biosynthesis still encounters the problem of low efficiency. In this study, coculture engineering was adopted to improve production efficiency of vanillyl alcohol in E. coli. First, two pathways were compared for biosynthesis of the immediate precursor 3, 4-dihydroxybenzyl alcohol in monocultures, and the 3-dehydroshikimate-derived pathway showed higher efficiency than the 4-hydroxybenzoate-derived pathway. To enhance the efficiency of the last methylation step, two strategies were used, and strengthening S-adenosylmethionine (SAM) regeneration showed positive effect while strengthening SAM biosynthesis showed negative effect. Then, the optimized pathway was assembled in a single cell. However, the biosynthetic efficiency was still low, and was not significantly improved by modular optimization of pathway genes. Thus, coculturing engineering strategy was adopted. At the optimal inoculation ratio, the titer reached 328.9 mg/L. Further, gene aroE was knocked out to reduce cell growth and improve 3,4-DHBA biosynthesis of the upstream strain. As a result, the titer was improved to 559.4 mg/L in shake flasks and to 3.89 g/L in fed-batch fermentation. These are the highest reported titers of vanillyl alcohol so far. This work provides an effective strategy for sustainable production of vanillyl alcohol.