Advances in synthetic biology enable microbial hosts to synthesize valuable natural products in an efficient, cost-competitive and safe manner. However, current engineering endeavors focus mainly on enzyme engineering and pathway optimization, leaving the role of cofactors in microbial production of natural products and cofactor engineering largely ignored. Here we systematically engineered the supply and recycling of three cofactors (FADH2, S-adenosyl-L-methion and NADPH) in the yeast Saccharomyces cerevisiae, for high-level production of the phenolic acids caffeic acid and ferulic acid, the precursors of many pharmaceutical molecules. Tailored engineering strategies were developed for rewiring biosynthesis, compartmentalization and recycling of the cofactors, which enabled the highest production of caffeic acid (5.5 ± 0.2 g l-1) and ferulic acid (3.8 ± 0.3 g l-1) in microbial cell factories. These results demonstrate that cofactors play an essential role in driving natural product biosynthesis and the engineering strategies described here can be easily adopted for regulating the metabolism of other cofactors.
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