Efficient L-phosphinothricin production by engineered Escherichia coli co-expressing glutamate dehydrogenase, glucose dehydrogenase and NAD kinase with NADPH regeneration

L-Phosphinothricin (L-PPT) is the active compound of a broad-spectrum herbicide. In this study, we biosynthesized L-PPT via the asymmetric reduction of 2-carboxy-4-(hydroxymethylphosphoryl) butyric acid (PPO) using a whole-cell biocatalyst. Glutamate dehydrogenase from Pseudomonas moorei (PmGluDH), Glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH) and NAD kinase from Corynebacterium glutamicum (CgNADK) were coexpressed in E. coli, significantly increasing the catalytic efficiency by regenerating more NADPH to promote PPO reduction. Following the optimization of balancing the co-expression of the three enzymes in pETDuet-1 and pCDFDuet-1, the coexpressing strain increases in the intracellular NADP(H) concentration to 54.9 μmol/g DCW, which was 3.8-fold higher than the control. The co-expression system exhibited optimal activity at a temperature of 35°C, pH 7.5, and the glucose to PPO molar ratio of 1.4:1. In a 30 L bioconversion system the final concentration and productivity of L-PPT reached 79.8 g/L (>99% ee) and 1440 g/(L·d), respectively. These results will contribute to the industrial biological production of L-PPT. •NAD kinase enhances NADPH regeneration and increases intracellular NADP(H) levels.•Regulate protein expression by optimizing gene copy number in co-expression systems.•E. coli GluDH-GDH-NADK strain effectively synthesized L-PPT at 30 L scale.