Two NADPH-dependent 2-ketogluconate reductases involved in 2-ketogluconate assimilation in sp. strain CHM43.

UNLABELLED

Incomplete oxidation of glucose by sp. strain CHM43 produces gluconic acid and then 2- or 5-ketogluconic acid. Although 2-keto-D-gluconate (2KG) is a valuable compound, it is sometimes consumed by itself via an unknown metabolic pathway. We anticipated that 2KG reductase (2KGR) would be a key enzyme in 2KG metabolism. and were identified in the genome of strain CHM43, which encode proteins with 70% and 48% amino acid sequence identity, respectively, to the 2KGR of strain 621H. Constructed mutant derivatives of strain CHM43 lacking , , or both were examined for their 2KG consumption ability. Strains ∆ and ∆ consumed 2KG like the parental strain. However, the double-deletion (∆∆) strain did not consume 2KG at all, although it produced 2KG like the parental strain. Strains ∆ and ∆ each showed decreased 2KGR activity compared with the parental strain, and strain ΔΔ lost 2KGR activity. These results suggest that reduction of 2KG catalyzed by GLF_0478 and GLF_1777 is the committed step in 2KG metabolism in sp. strain CHM43. The two 2KGRs showed high activity at neutral pH and lower values for NADPH than NADH. Results of induction experiments suggest that GLF_0478 is constitutively expressed at a low level but induced by 2KG, and GLF_1777 is also inducible by 2KG but repressed in the absence of an inducer. Our study that characterizes the key genes for 2KG consumption in gives insights for improvement of biological 2KG production systems.

IMPORTANCE

2-Keto-D-gluconate (2KG), a product of incomplete oxidation of glucose by acetic acid bacteria including spp., is used for various purposes, including in the food industry. also consumes 2KG via an unclear metabolic pathway. It is reported that spp. and phosphorylate 2KG in the first step of 2KG metabolism, but some enteric bacteria including reduce 2KG. This study evaluated the 2KG consumption ability of a mutant derivative of a strain of that lacks two putative 2KGR-encoding genes. The mutant strain did not consume 2KG at all; the two 2KGRs were each found to catalyze 2KG reduction. It is concluded that reduction of 2KG is the committed step in 2KG metabolism in . The results presented here give insights that might facilitate improvement of 2KG production systems that use .