Direct fermentation of 2-keto-L-gulonic acid in recombinant Gluconobacter oxydans.
We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from D-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of L-sorbosone and 2-KLGA, L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine resulted in improvement of the production level.
Saito
Y
Y
Biotechnology Division, Pharmacological Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kashima, Yodogawa-ku, Osaka 532, Japan.
Ishii
Y
Y
Hayashi
H
H
Yoshikawa
K
K
Noguchi
Y
Y
Yoshida
S
S
Soeda
S
S
Yoshida
M
M
D016947:Acetobacteraceae / Q000235:genetics / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005285:Fermentation; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D013400:Sugar Acids / Q000378:metabolism*
D004269:DNA, Bacterial; D013400:Sugar Acids; D002237:Carbohydrate Dehydrogenases; C104557:sorbose dehydrogenase; D000445:Aldehyde Oxidoreductases; C095707:L-sorbosone dehydrogenase; C011491:provitamin C