Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans

Abstract

Cloning and expression of the gene encoding Acetobacter liquefaciens IFO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with the mobilizable cosmid vector pVK102 (mob ؉) in Escherichia coli S17-1 (Tra ؉). The library was transferred by conjugal mating into Gluconobacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L-sorbosone in the presence of L-sorbose. The transconjugants were screened for SNDH activity by performing a direct expression assay. One clone harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic acid (2KGA) more rapidly than its host did and also converted L-sorbose to 2KGA with no accumulation of L-sorbosone. The insert (25 kb) of p7A6 was shortened to a 3.1-kb fragment, in which one open reading frame (1,347 bp) was found and was shown to encode a polypeptide with a molecular weight of 48,222. The SNDH gene was introduced into the 2KGA-producing strain G. oxydans IFO 3293 and its derivatives, which contained membrane-bound L-sorbose dehydrogenase. The cloned SNDH was correctly located in the membrane of the host. The membrane fraction of the clone exhibited almost stoichiometric formation of 2KGA from L-sorbosone and L-sorbose. Resting cells of the clones produced 2KGA very efficiently from L-sorbosone and L-sorbose, but not from D-sorbitol; the conversion yield from L-sorbosone was improved from approximately 25 to 83%, whereas the yield from L-sorbose was increased from 68 to 81%. Under fermentation conditions, cloning did not obviously improve the yield of 2KGA from L-sorbose.

Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain

“…The SNDH from G. oxydans is a cytoplasmic protein (molecular mass, approximately 50 kDa) that requires NAD(P) as an electron acceptor (10). To carry out the two-step conversion on the membrane of G. oxydans cells, Shinjoh et al cloned the gene for the membrane-bound SNDH from Acetobacter liquefaciens and introduced the gene into 2-KLGA-producing G. oxydans (23). However, the yield of 2-KLGA in the recombinant strain under fermentation conditions was not improved compared to that by the wild strain G. oxydans UV10 (22,23).…”

“…To carry out the two-step conversion on the membrane of G. oxydans cells, Shinjoh et al cloned the gene for the membrane-bound SNDH from Acetobacter liquefaciens and introduced the gene into 2-KLGA-producing G. oxydans (23). However, the yield of 2-KLGA in the recombinant strain under fermentation conditions was not improved compared to that by the wild strain G. oxydans UV10 (22,23). Our approach was to construct a recombinant strain that ferments 2-KLGA from D-sorbitol by (i) cloning both genes for SDH and SNDH from G. oxydans T-100 that produces 2-KLGA from D-sorbitol and (ii) transforming G. oxydans G624, which accumulates L-sorbose by introducing these dehydrogenase genes.…”

Production of Vitamin E in Erwinia herbicola Bearing the Vitreoscilla Hemoglobin Gene (vgb+)

“…For these processes, microbiological processes are available and are progressing rapidly. Different methods such as genetic engineering and biocatalyst conversion are used to improve vitamin production [31,32]. Advances in genetic and genomic engineering accelerate the optimization of metabolic flows to increase the production of target biomass products.…”

Antioxidant effect of glutathione on promoting 2-keto-l-gulonic acid production in vitamin C fermentation system

“…), including the construction of engineered strains (Shinjoh et al . ; Saito et al . , ), the mutagenesis of high 2‐KGA production strains (Yan et al .…”