Abstract
In this study, a platform was constructed for the efficient biosynthesis of a lactate-based copolymer using a phosphite dehydrogenase (PtxD)-based NADH regeneration strategy. PtxD catalyzes the conversion of phosphite to phosphate while reducing NAD (+) to NADH. The latter is an essential cofactor for lactate synthesis in Escherichia coli. This strategy allows the decoupling of NADH regeneration from carbon metabolism flow, providing sufficient NADH for lactate synthesis. Different concentrations of isopropyl β-d-1-thiogalactopyranoside (IPTG) were used to control the intensity of PtxD expression, and the lactate fraction in the copolymer synthesized by the engineered strain ranged from 6.2 to 16.7 mol%. The ptxD gene was integrated into the genome of strain WJPCTP-01, which successfully synthesized 3.24 g/L P(3HB-co-23.0 mol% LA) and 2.23 g/L P(3HB-co-39.0 mol% LA) using glucose and xylose as substrates, respectively, in shake flask cultures. In 5 L bioreactor fermentations, the titer of P(3HB-co-41.3 mol% LA) reached 8.57 g/L, with a synthesis rate of 0.12 g/L/h when xylose was used as a substrate. These findings indicate that the PtxD-based NADH regeneration strategy enhances lactate synthesis without any significant negative impact on bacterial growth or the synthesis of P(3HB-co-LA).