Cloning and nucleotide sequence of the glutamate decarboxylase-encoding gene gadA from Aspergillus oryzae.
We cloned a genomic DNA encoding the glutamate decarboxylase (GAD) from Aspergillus oryzae using a 200-bp DNA fragment as the probe. This DNA fragment was amplified by the reverse transcription polymerase chain reaction with mRNA of A. oryzae as the template and degenerate primers designed from the conserved amino acid sequence of Escherichia coli GAD and Arabidopsis thaliana GAD. Nucleotide sequencing analysis showed that the cloned gene (designated gadA) encoded 514 amino acid residues and contained three introns. Southern hybridization showed that the gadA gene was on a 6.0-kb SacI fragment and that there was a single copy in the A. oryzae chromosome. The cloned gene was functional, because one transformant of A. oryzae containing multiple copies of the gadA gene had 10-fold the GAD activity and a 12-fold increase in gamma-aminobutyric acid production compared with the control strain.
Kato
Yuki
Y
General Research Laboratories of Kiku-Masamune Sake Brewing Co., Ltd., 1-8-6 Uozakinishi-machi, Higashinada-ku, Kobe, Hyogo 658-0026, Japan. y-kato@kikumasamune.co.jp
Kato
Yoko
Y
Furukawa
Keiji
K
Hara
Shodo
S
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018628:Gene Dosage; D015870:Gene Expression; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis
D018076:DNA, Complementary; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase