Metabolic engineering of Halomonas bluephagenesis for production of five carbon molecular chemicals derived from L-lysine

Abstract

5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB-co-5HV) of 3-hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117ΔgabT₁₊₂, which produced 16.4 g L⁻¹ and 67.4 g L⁻¹ 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on P_porin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-P_{porin 42}-yqhD_EC produced 6 g L⁻¹ 5HV in shake flask growth, while H. bluephagenesis YF117 harboring pSEVA341-P_{porin 42}-yqhD_EC-P_{porin 278}-phaC_RE-abfT synthesized 42 wt% P(3HB-co-4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB-co-5HV) utilizing L-lysine as the substrate.