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

## TL;DR

This study identifies two enzymes in Gluconobacter that are essential for consuming 2-ketogluconate, a compound used in the food industry.

## Contribution

The study identifies two NADPH-dependent 2-ketogluconate reductases in Gluconobacter sp. strain CHM43 and their role in 2KG metabolism.

## Key findings

- The double-deletion mutant of GLF_0478 and GLF_1777 failed to consume 2KG, confirming their essential role.
- Both 2KGRs showed high activity at neutral pH and lower KM values for NADPH than NADH.
- GLF_0478 is constitutively expressed and inducible by 2KG, while GLF_1777 is inducible but repressed without an inducer.

## Abstract

Incomplete oxidation of glucose by Gluconobacter 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 Gluconobacter itself via an unknown metabolic pathway. We anticipated that 2KG reductase (2KGR) would be a key enzyme in 2KG metabolism. GLF_0478 and GLF_1777 were identified in the genome of strain CHM43, which encode proteins with 70% and 48% amino acid sequence identity, respectively, to the 2KGR of Gluconobacter oxydans strain 621H. Constructed mutant derivatives of strain CHM43 lacking GLF_0478, GLF_1777, or both were examined for their 2KG consumption ability. Strains ∆GLF_0478 and ∆GLF_1777 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 ∆GLF_0478 and ∆GLF_1777 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 Gluconobacter sp. strain CHM43. The two 2KGRs showed high activity at neutral pH and lower KM 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 Gluconobacter gives insights for improvement of biological 2KG production systems.

2-Keto-D-gluconate (2KG), a product of incomplete oxidation of glucose by acetic acid bacteria including Gluconobacter spp., is used for various purposes, including in the food industry. Gluconobacter also consumes 2KG via an unclear metabolic pathway. It is reported that Pseudomonas spp. and Cupriavidus necator phosphorylate 2KG in the first step of 2KG metabolism, but some enteric bacteria including Escherichia coli reduce 2KG. This study evaluated the 2KG consumption ability of a mutant derivative of a strain of Gluconobacter 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 Gluconobacter. The results presented here give insights that might facilitate improvement of 2KG production systems that use Gluconobacter.