Engineered Coenzyme A Biosynthesis and Butyrate Transporter Drives High‐Efficient Butyrate Synthesis in Escherichia coli

ABSTRACT Butyric acid is a four‐carbon fatty acid with a range of applications in chemical, food and pharmaceutical industries. In this study, a heterologous butyrate biosynthetic pathway was engineered in Escherichia coli , which was afflicted by low titer and low yield. To address these issues, two key strategies for metabolic engineering of E. coli were implemented by enhancing coenzyme A (CoA) biosynthesis and optimizing butyrate transport. First, the CoA biosynthesis pathway was engineered through alleviating CoA‐mediated inhibition, and enhancing the supply of pantothenate and cysteine precursors. Second, a TolC‐associated MdtEF efflux pumps was identified and optimized to mitigate butyrate reuptake. The combined implementation in strain JH016 led to 11.1‐fold and 86% increase of butyrate titer and yield, resulting in production of 21.12 g/L butyrate with a yield of 0.95 mol/mol. Our results suggested that CoA engineering and butyrate transporter optimization had a synergistic effect on butyrate production. Furthermore, these strategies could be broadly utilized for the production of various other useful chemicals in the fields of metabolic engineering and synthetic biology.