Rational engineering of cofactor specificity of glutamate dehydrogenase for poly-γ-glutamic acid synthesis in Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is a multifunctional biopolymer mainly produced by Bacillus. The cofactor specificity of enzymes plays a critical role in regulating metabolic process and metabolite production. Here, we present a novel approach for switching cofactor specificity of glutamate dehydrogenase RocG from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) to improve γ-PGA production. Firstly, 3D structural modeling and molecular docking were performed to predict the binding modes of NADH and NADPH. Several site-specific mutants based on the conventional and Random Accelerated Molecular Dynamics simulations were obtained to alter cofactor specificity. Then, the effects of RocG variants overexpressions on γ-PGA production were evaluated. Compared to the wild-type, the mutant RocG showed highest increase in γ-PGA yield, increased by 40.50%. Meanwhile, yields of main by-products acetoin and 2,3-butandieol were decreased by 21.70% and 16.53%, respectively. Finally, the results of enzymatic properties confirmed that glutamate dehydrogenase mutant RocG exhibited the higher affinity for NADH, caused a shift in coenzyme preference from NADPH to NADH, with a catalytic efficiency comparable with NADPH-dependent RocG. Taken together, this research demonstrated that switching the cofactor preference of glutamate dehydrogenase via rational design was an effective strategy for high-level production of γ-PGA in Bacillus licheniformis.