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 I FO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with t he mobilizable cosmid vector pVK102 (mob(+)) in Escherichia coil S17-1 (Tra(+)). The library was transferred by conjugal mating into Glucono bacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L -sorbosone in the presence of L-sorbose. The transconjugants were scre ened for SNDH activity by performing a direct expression assay. One cl one harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic a cid (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 p7 A6 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 mol ecular weight of 48,222. The SNDH gene ,vas introduced into the 2KGA-p roducing strain G. oxydans IFO 3293 and its derivatives, which contain ed membrane bound L-sorbose dehydrogenase. The cloned SNDH was correct ly located in the membrane of the host. The membrane fraction of the c lone exhibited almost stoichiometric formation of 2KGA from L-sorboson e and L-sorbose. Resting cells of the clones produced 2KGA very effici ently from L-sorbosone and L-sorbose, but not from D sorbitol; the con version yield from L-sorbosone was improved from approximately 25 to 8 3%, whereas the yield from L-sorbose was increased from 68 to 81%. Und er fermentation conditions, cloning did not obviously improve the yiel d of 2KGA from L-sorbose.