Development of a 2-hydroxyglutarate production system by Corynebacterium glutamicum.

2-Oxoglutarate (2-OG) is a tricarboxylate cycle intermediate that can be biologically converted into several industrially important compounds. However, studies on the fermentative production of compounds synthesized from 2-OG, but not via glutamate (defined as 2-OG derivatives), have been limited. Herein, a system that can efficiently produce 2-hydroxyglutarate (2-HG), a 2-OG derivative biosynthesized by the hgdH-encoded NADH-dependent 2-HG dehydrogenase of Acidaminococcus fermentans, was developed as a model using Corynebacterium glutamicum. First, the D3 strain, which lacked the two NADH-consuming enzymes, lactate dehydrogenase and malate dehydrogenase, as well as isocitrate lyase, was constructed as a starting strain. Next, the growth conditions that induced the accumulation of 2-OG were investigated, and it was found that the biotin- and nitrogen-limited (B/N-limited) aerobic growth conditions were suitable for this purpose. Finally, the hgdH gene of A. fermentans became overexpressed in the D3 strain by inserting it into the intergenic regions with the strong constitutive promoter of the tuf gene of C. glutamicum; the engineered strain was cultured under the B/N-limited aerobic growth conditions. The engineered strain produced 80.1 mM 2-HG with a yield of 0.390 mol/mol glucose, which are the highest titer and yield reported thus far, to the best of our knowledge. Furthermore, reverse genetics showed that the produced 2-HG was partially exported via the YggB protein (NCgl1221 protein, a mechanosensitive channel) known as an exporter for glutamate under the conditions used herein. KEY POINTS: • An efficient 2-HG production system was developed with Corynebacterium glutamicum. • Biotin- and nitrogen-limited aerobic growth conditions induced 2-OG production. • Produced 2-HG was partially excreted via the glutamate exporter, YggB.