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

Abstract

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. 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 0.05mM IPTG, induction for 18h 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 0.2mM IPTG, induction for 19h 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 200mM D-PPT was biocatalyzed by E. coli DAAO-CAT for 4h with space-time yield of 9.0g·L-1 ·h-1 and conversion rate of over 99.0%. Then 220mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3h with space-time yield of 14.5g·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. 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.