l -Sorbose Reductase and Its Transcriptional Regulator Involved in l -Sorbose Utilization of Gluconobacter frateurii

Abstract

Upstream of the gene for flavin adenine dinucleotide (FAD)-dependent D-sorbitol dehydrogenase (SLDH),sldSLC, a putative transcriptional regulator was found in Gluconobacter frateurii THD32 (NBRC 101656). In this study, the whole sboR gene and the adjacent gene, sboA, were cloned and analyzed. sboR mutation did not affect FAD-SLDH activity in the membrane fractions. The SboA enzyme expressed and purified from an Escherichia coli transformant showed NADPH-dependent L-sorbose reductase (NADPH-SR) activity, and the enzyme was different from the NADPH-SR previously reported for Gluconobacter suboxydans IFO 3291 in molecular size and amino acid sequence. A mutant defective in sboA showed significantly reduced growth on L-sorbose, indicating that the SboA enzyme is required for efficient growth on L-sorbose. The sboR mutant grew on L-sorbose even better than the wild-type strain did, and higher NADPH-SR activity was detected in cytoplasm fractions. Reverse transcription-PCR experiments indicated that sboRA comprises an operon. These data suggest that sboR is involved in the repression of sboA, but not in the induction of sldSLC, on D-sorbitol and that another activator is required for the induction of these genes by D-sorbitol or L-sorbose.

Characterization of Genes Involved inD-Sorbitol Oxidation in ThermotolerantGluconobacter frateurii

“…In contrast, the activities of NAD-dependent D-sorbitol dehydrogenase (NAD-SLDH) and NADPH-dependent L-sorbose reductase (NADPH-SR) in the cytoplasm fraction of the mutant were not significantly different as compared to the wild-type strain (Fig. 4), indicating that SldR did not regulate the expression of the genes for NAD-SLDH and NADPH-SR. As described in a previous report, 10) disruption of sboR resulted in enhancement of the induction of sboA, the gene for NADPH-SR, by Dsorbitol, and induction was not affected by the disruption of sldR (Fig. 4).…”

“…10) We expected that the gene(s) nearby this sboRA gene cluster might be involved in the regulation of sldSLC expression. We attempted to obtain a gene fragment downstream of the sboRA operon by inverse PCR, as described above in ''Materials and Methods.''…”

“…No corresponding gene with high identity has been found in the genome of G. oxydans ATCC621H, although non-functional sldSLC exists. 10) We have reported that PQQ-GLDH, the other enzyme for D-sorbitol oxidation, might be mainly responsible for Gly, 1% glycerol-containing medium Sor, 1% sorbitol-containing medium * * the oxidation of D-sorbitol to L-sorbose at the beginning of cultivation, since it is constitutively expressed. 10) After oxidation by PQQ-GLDH proceeds and enough membrane potential is generated, the oxidation process with PQQ-GLDH can be suppressed through excess membrane potential.…”

“…10) We have reported that PQQ-GLDH, the other enzyme for D-sorbitol oxidation, might be mainly responsible for Gly, 1% glycerol-containing medium Sor, 1% sorbitol-containing medium * * the oxidation of D-sorbitol to L-sorbose at the beginning of cultivation, since it is constitutively expressed. 10) After oxidation by PQQ-GLDH proceeds and enough membrane potential is generated, the oxidation process with PQQ-GLDH can be suppressed through excess membrane potential. 24) Under such conditions, an alternative oxidation pathway with FAD-SLDH might be induced to oxidize the remaining D-sorbitol further, which generates lower membrane potential and uses the alternative terminal oxidase more preferably than cytochrome bo 3 , which produces higher energy.…”

“…We expected it to be involved in the induction of sldSLC, but it is not, but is rather a repressor of the NADPH-dependent L-sorbose reductase gene (sboA), located downstream of sboR and essential for efficient utilization of L-sorbose. 10) Thus the gene required for induction of sldSLC is not yet identified.…”