Abstract
Coniferyl alcohol is a common monolignol, which is a precursor of lignin. Coniferyl alcohol is rarely found as free molecules in plants based on its conversion to lignins by peroxidases or laccases. It can be used for biosynthesis of silybin and a lot of pinoresinol derivatives. Although increased output of coniferyl alcohol has been attempted, few methods are effective. In this study, we evaluated 20 promoters for their ability to express genes in Penicillium simplicissimum encoding vanillyl alcohol oxidase (PsVAO) and catalase (ScCTA1) for producing coniferyl alcohol, while eliminating harmful H2O2. We constructed 100 combinations of constitutive promoters in E. coli to enhance gene expression. Then orthogonal experiments, fed-batch kinetic model and CRISPR/Cas9 system were followed to improve the yield of coniferyl alcohol, while eliminate the pinoresinol. Finally, the coniferyl alcohol titers, bioconversion rates, and productivity levels reaching 53.90 g/L, 86.72 %, and 2.45 g/(L × h), respectively.
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