IPTG-induced high protein expression for whole-cell biosynthesis of L-phosphinothricin

Background Biocatalytic production of L-phosphinothricin (L-PPT) is currently the most promising method. In this work, we use an Escherichia coli strain coexpressing of D-amino acid oxidase and catalase ( E. coli DAAO-CAT) to oxidation biocatalytic D-PPT to PPO, then use the second E. coli strain coexpressing glutamate dehydrogenase and formate dehydrogenase ( E. coli GluDH-FDH) to reduce biocatalytic PPO to L-PPT. Main Methods and Major Results We compared the effects of different concentrations of IPTG or lactose on protein expression and enzyme activity in 5 L fermenter. The best induction conditions for E. coli DAAO-CAT were 0.05 mM IPTG, induction for 18 h at 28°C. The specific enzyme activities of DAAO and CAT were 153.20 U g −1 and 896.23 U g −1 , respectively. The optimal induction conditions for E. coli GluDH-FDH were 0.2 mM IPTG, induction for 19 h at 28°C. The specific enzyme activities of GluDH and FDH were 41.72 U g −1 and 109.70 U g −1 , respectively. The 200 mM D-PPT was biocatalyzed by E. coli DAAO-CAT for 4 h with space-time yield of 9.0 g·L −1 ·h −1 and conversion rate of over 99.0%. Then 220 mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3 h with space-time yield of 14.5 g·L −1 ·h −1 and conversion rate of over 99.0%. To our knowledge, this is the most efficient biocatalytic reaction for L-PPT production. Conclusions and Implications We found that IPTG has advantages compared with lactose in the enzyme activity and biomass of E. coli DAAO-CAT and E. coli GluDH-FDH, and IPTG is more environmentally friendly. Our data implicated that IPTG can replace lactose in terms of economic feasibility and effectiveness for scaled-up industrial fermentations. Graphical and Lay Summary A two-step enzymatic method was developed for the biological preparation of L-PPT from D-PPT. In the first step, we constructed a recombinant E. coli BL21(DE3)/pCDFDuet-DAAO-CAT that utilized D-amino acid oxidase (DAAO) to catalyze the conversion of D-PPT to PPO, and catalase to degrade the hydrogen peroxide (H 2 O 2 ) produced during the reaction. Subsequently, in the second step, we constructed another recombinant E. coli BL21(DE3)/pETDuet-LcGluDH-PseFDH that employed glutamate dehydrogenase (GluDH) for the reductive amination of PPO to L-PPT, with NADH supplied by formate dehydrogenase (FDH).