Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production.
The cloning of a bifunctional FAD synthetase gene, which shows flavokinase and FMN adenylyltransferase activities, from Corynebacterium ammoniagenes was tried by hybridization with synthetic DNAs corresponding to the N-terminal amino acid sequence. The cloned PstI-digested 4.4 x 10(3)-base (4.4-kb) fragment could not express the FAD synthetase activity in E. coli, but could increase the two activities by the same factor of about 20 in C. ammoniagenes. The FAD-synthetase-gene-amplified C. ammoniagenes cells were applied to the production of FAD from FMN or riboflavin. The productivity of FAD from FMN was increased four to five times compared with the parent strain, and reached a 90% molar yield. The productivity of FAD from riboflavin was increased about eight times, with a 50% molar yield. The addition of Zn2+ to the reaction mixtures for the conversion from riboflavin to FAD brought about the specific inhibition of adenylyl-transferase activity and resulted in the accumulation of FMN.
Hagihara
T
T
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Japan.
Fujio
T
T
Aisaka
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000235:genetics* / Q000378:metabolism*; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005486:Flavin Mononucleotide / Q000096:biosynthesis*; D005182:Flavin-Adenine Dinucleotide / Q000096:biosynthesis*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D012256:Riboflavin / Q000378:metabolism
D015342:DNA Probes; D004269:DNA, Bacterial; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase; D012256:Riboflavin