Cloning of a gluconate/polyol dehydrogenase gene from Gluconobacter suboxydans IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant Escherichia coli strain

Abstract

A 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.

l-Tartaric acid synthesis from vitamin C in higher plants

“…This hypothesis was subsequently refuted when 18 O tracer experiments suggested the cleavage mechanism to involve a putative hydrolase (18). More recently (19), it has been shown that, in Gluconobacter suboxidans, 5-keto D-gluconic acid can be used as the substrate for a dedicated transketolase. These authors speculated that further oxidation of the resulting four-carbon semialdehyde by a succinate semialdehyde dehydrogenase homolog could result in formation of TA.…”

Membrane-Bound, 2-Keto- d -Gluconate-Yielding d -Gluconate Dehydrogenase from “ Gluconobacter dioxyacetonicus ” IFO 3271: Molecular Properties and Gene Disruption

“…The K m values of PQQ-GLDH are rather high (20,40, and 420 mM for D-arabitol, D-sorbitol, and GA, respectively [10]). 5KGA is a useful compound as a precursor of tartaric acid, xylaric acid, or a number of flavor compounds (18); thus, several attempts to produce 5KGA have been made with Glu-conobacter strains, and recently, exclusive production of 5KGA was accomplished with an FAD-GADH-defective mutant of Gluconobacter oxydans 621H (5). On the other hand, 2KGA itself is also a useful chemical and is applicable as a building block in the chemical synthesis of heterocyclic compounds and for regioselective and stereoselective chemical reactions (23).…”

Screening of Thermotolerant Gluconobacter Strains for Production of 5-Keto- d -Gluconic Acid and Disruption of Flavin Adenine Dinucleotide-Containing d -Gluconate Dehydrogenase

“…5KGA is a useful raw material for the production of tartaric acid and xylaric acid and is used as a precursor for the synthesis of a number of flavor compounds, including 4-hydroxy-5-methyl-2,3-dihydrofuranone-3 (15). Moreover, it has been reported that 5KGA can be used to produce vitamin C by Gray's method (6,7), which is different from Reichstein's method, which is now commonly used in industry.…”