Development of efficient 5-ketogluconate production system by Gluconobacter japonicus.

5-Ketogluconate (5KGA) is a precursor for synthesizing tartrate, a valuable compound used in several industries. In a previous study, Gluconobacter japonicus NBRC 3271 mutant strain D2, which lacks two membranous gluconate 2-dehydrogenases, was shown to produce 5KGA but not 2-ketogluconate from a mixture of glucose and gluconate. In this study, we aimed to develop an efficient 5KGA production system using G. japonicus D2 as the parental strain. D2 produced 5KGA from glucose in a jar fermentor culture; however, 5KGA levels were reduced during the late phase of cultivation. To increase the potential of D2 for 5KGA production, the cytoplasmic metabolism related to the utilization of 5KGA and gluconate was modified; the gno and gntK genes encoding 5KGA reductase and gluconokinase, respectively, were deleted from D2, generating D4. Improved 5KGA production was observed in D4 compared to that in D2, but a significant amount of gluconate remained at the end of cultivation, leading to an unsatisfied yield of 0.83 mol (mol glucose)-1. The conversion of gluconate to 5KGA is catalyzed by pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH), which easily forms an apoenzyme by releasing PQQ and calcium ions. Thus, the effects of CaCl2 addition to the culture medium on 5KGA production by D4 were investigated. We demonstrated that 1 mM CaCl2 addition positively affected the maintenance of the PQQ-GLDH activity toward gluconate and consequently enhanced 5KGA production, and the yield reached 0.97 mol (mol glucose)-1. KEY POINTS: • An efficient 5KGA production system was developed with Gluconobacter japonicus. • Deleting the gno and gntK genes blocked the catabolism of 5KGA and gluconate. • The addition of 1 mM CaCl2 efficiently improved the conversion of glucose to 5KGA.

Characterization of a cryptic, pyrroloquinoline quinone-dependent dehydrogenase of Gluconobacter sp. strain CHM43.

We characterized the pyrroloquinoline quinone (PQQ)-dependent dehydrogenase 9 (PQQ-DH9) of Gluconobacter sp. strain CHM43, which is a homolog of PQQ-dependent glycerol dehydrogenase (GLDH). We used a plasmid construct to express PQQ-DH9. The expression host was a derivative strain of CHM43, which lacked the genes for GLDH and the membrane-bound alcohol dehydrogenase and consequently had minimal ability to oxidize primary and secondary alcohols. The membranes of the transformant exhibited considerable d-arabitol dehydrogenase activity, whereas the reference strain did not, even if it had PQQ-DH9-encoding genes in the chromosome and harbored the empty vector. This suggests that PQQ-DH9 is not expressed in the genome. The activities of the membranes containing PQQ-DH9 and GLDH suggested that similar to GLDH, PQQ-DH9 oxidized a wide variety of secondary alcohols but had higher Michaelis constants than GLDH with regard to linear substrates such as glycerol. Cyclic substrates such as cis-1,2-cyclohexanediol were readily oxidized by PQQ-DH9.