Multi-enzyme cascade strategy for efficient biosynthesis of 5-hydroxypipecolic acid from L-lysine

Multi-enzyme cascade designed for 5-hydroxypipecolic acid synthesis from low-cost substrate. • Self-sufficient cofactor recycling achieved without expensive exogenous coenzymes. • Under glucose-controlled feeding, the system achieved a product titer of 23.65 g L -1 . 5-Hydroxypipecolic acid (5-HPA) is a chiral building block widely employed in pharmaceutical synthesis. Conventional chemical synthesis of 5-HPA is limited by poor stereoselectivity and environmental burdens. Herein, we developed a biosynthetic method for 5-HPA production from L-lysine (L-Lys) through a multi-enzyme cascade. Firstly, a lysine 6-aminotransferase SkL6AT from Stenotrophomonas koreensis and an imine reductase HeP5C from Halomonas elongata were screened and introduced into recombinant strains capable of converting L-Lys to L-pipecolic acid (L-PA). After optimization of plasmid copy number and ribosome-binding site strength, as well as establishment of the NADH regeneration system, the L-PA titer reached 7.50 g L⁻¹. Subsequently, a hydroxylase XdPH from Xenorhabdus doucetiae was introduced to construct a 5-HPA-producing strain. Fermentation conditions were optimized in shake flasks and subsequently scaled up to a 5 L bioreactor. Under a fed-batch strategy, the final engineered strain produced 23.65 g L -1 5-HPA with a yield of 0.587 g g⁻¹ from L-Lys. Download: Download high-res image (192KB) Download: Download full-size image