Improved production of α-ketoglutaric acid (α-KG) by a Bacillus subtilis whole-cell biocatalyst via engineering of l-amino acid deaminase and deletion of the α-KG utilization pathway

• Three rounds of ep-PCR of P. mirabilis pm1 followed by site-saturation mutation were done.
• α-KG titer of the mutant F110I/A255T/E31D/R228C/L249S/I351T increased from 4.65 g/L to 10.08 g/L.
• The deletion of sucA gene increased the α-KG titer from 10.08 g/L to 12.21 g/L.
• Significantly improved α-KG titer by engineering P. mirabilis pm1 and blocking α-KG degradation.

We previously developed a novel one-step biotransformation process for the production of α-ketoglutarate (α-KG) from l-glutamic acid by a Bacillus subtilis whole-cell biocatalyst expressing an l-amino acid deaminase (pm1) of Proteus mirabilis. However, the biotransformation efficiency of this process was low owing to low substrate specificity and high α-KG degradation. In this study, we further improved α-KG production by protein engineering P. mirabilis pm1 and deleting the B. subtilis α-KG degradation pathway. We first performed three rounds of error-prone polymerase chain reaction and identified mutations at six sites (F110, A255, E349, R228, T249, and I352) that influence catalytic efficiency. We then performed site-saturation mutagenesis at these sites, and the mutant F110I/A255T/E349D/R228C/T249S/I352A increased the biotransformation ratio of l-glutamic acid from 31% to 83.25% and the α-KG titer from 4.65 g/L to 10.08 g/L. Next, the reaction kinetics and biochemical properties of the mutant were analyzed. The Michaelis constant for l-glutamic acid decreased from 49.21 mM to 23.58 mM, and the maximum rate of α-KG production increased from 22.82 μM min−1 to 56.7 μM min−1. Finally, the sucA gene, encoding α-ketodehydrogenase, was deleted to reduce α-KG degradation, increasing the α-KG titer from 10.08 g/L to 12.21 g/L. Protein engineering of P. mirabilis pm1 and deletion of the α-KG degradation pathway in B. subtilis improved α-KG production over that of previously developed processes.