Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans.
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.
Shinjoh
M
M
Department of Applied Microbiology, Nippon Roche Research Center, Kanagawa, Japan.
Tomiyama
N
N
Asakura
A
A
Hoshino
T
T
D000091:Acetobacter / Q000201:enzymology* / Q000235:genetics*; D016947:Acetobacteraceae / Q000235:genetics*; D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D013012:Sorbitol / Q000378:metabolism; D013013:Sorbose / Q000031:analogs & derivatives / Q000378:metabolism; D013400:Sugar Acids / Q000378:metabolism
D004269:DNA, Bacterial; D013400:Sugar Acids; C013686:sorbosone; D013012:Sorbitol; D000445:Aldehyde Oxidoreductases; C095707:L-sorbosone dehydrogenase; C011491:provitamin C; D013013:Sorbose