Biosynthesis of poly(3-hydroxybutyrate-co-lactate) using an NADH regeneration strategy in E. coli

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 <i>Escherichia coli</i>. 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 <i>ptxD</i> gene was integrated into the genome of strain WJPCTP-01, which successfully synthesized 3.24 ​g/L P(3HB-<i>co</i>-23.0 ​mol% LA) and 2.23 ​g/L P(3HB-<i>co</i>-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-<i>co</i>-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-<i>co</i>-LA).