Targeting cofactors regeneration in methylation and hydroxylation for high level production of Ferulic acid.

Ferulic acid (FA) is a natural methylated phenolic acid which represents various bioactivities. Bioproduction of FA suffers from insufficient methyl donor supplement and inefficient hydroxylation. To overcome these hurdles, we first activate the S -adenosylmethionine (SAM) cycle in E. coli by using endogenous genes to supply sufficient methyl donor. Then, a small protein Fre is introduced into the pathway to efficiently regenerate FADH 2 for the hydroxylation. Remarkably, regeneration of these two cofactors dramatically promotes FA synthesis. Together with decreasing the byproducts formation and boosting precursor supply, the titer of FA reaches 5.09 g/L under fed-batch conditions, indicating a 20-fold improvement compared with the original producing E. coli strain. This work not only establishes a promising microbial platform for industrial level production of FA and its derivatives, but also highlights a convenient and effective strategy to enhance the biosynthesis of chemicals requiring methylation and FADH 2 -dependent hydroxylation. • Regeneration of cofactors promoted the efficiency of methylation and hydroxylation. • A highly specific HpaBC was screened for reducing the byproduct formation. • The engineered strain produced highest titer with 5.09 g/L FA in fed-batch fermenter. [ABSTRACT FROM AUTHOR]