Caffeic acid is an important phenolic acid compound widely used in food, biomedical, and nutraceutical industries.Its microbial biosynthesis receives increasing attention, and the highest production titer is achieved in recombinant Escherichia coli.However, in all the reported E.coli systems, the pathway genes for caffeic acid biosynthesis are expressed episomally, requiring antibiotic supplementation during fermentation.These lead to strain instability, high production costs, and dissemination of antibiotic-resistance genes.To this end, the biosynthetic pathway genes were inserted into E.coli genome for the construction of an integrative strain capable of de novo biosynthesis of caffeic acid from glucose.Via comparison of hydroxylases from various sources, comparison of various gene integration sites, and optimization of gene copy number, a three-copy strain CA8 was obtained via gene integration in three sites, and the corresponding titer of caffeic acid in shake flasks reached 488 mg/L.Further optimization of fermentation media, inoculation size, and inducer concentration promoted the titer to 598 mg/L in shake flasks.Under the optimal condition, this recombinant strain produced 1.55 g/L of caffeic acid in a 5 L bioreactor using fed-batch fermentation.This study provides guidance to efficient microbial biosynthesis of caffeic acid and other plant natural products.
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