Cloning and expression of Methylovorus mays No. 9 gene encoding gamma-glutamylmethylamide synthetase: an enzyme usable in theanine formation by coupling with the alcoholic fermentation system of baker's yeast.
Gamma-glutamylmethylamide synthetase (GMAS), found in an obligate methylotroph, Methylovorus mays No. 9, can form theanine from glutamic acid and ethylamine in a mixture in which yeast sugar fermentation is coupled for ATP regeneration. The internal and N-terminal amino acid sequences of GMAS had certain similarities to putative glutamine synthetase type III (GS III) of Methylobacillus flagellatus KT. From the M. mays No. 9 genomic DNA library, a clone containing a 6.5-kbp insertional DNA fragment was selected by the PCR screening technique with oligonucleotide primers specific for the GMAS gene. The fragment had an open reading frame of the GMAS gene encoding a protein of 444 amino acids (molecular mass, 49 kDa). The deduced amino acid sequence showed significant identity with that of Met. flagellatus KT GS III (78%). The isolated gene was ligated into an expression vector (pET21a) and expressed in Escherichia coli AD494 (DE3). Enzyme productivity in the expression system was about 23-fold higher than that in M. mays No. 9. Recombinant GMAS had the same properties as intrinsic GMAS, and it formed theanine by coupling the reaction with the ATP-regeneration system of yeast sugar fermentation.
Yamamoto
Sachiko
S
Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo, Japan.
Wakayama
Mamoru
M
Tachiki
Takashi
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D020562:Betaproteobacteria / Q000201:enzymology / Q000235:genetics*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005971:Glutamates / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D001426:Bacterial Proteins; D005971:Glutamates; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000431:Ethanol; C026166:theanine; D019731:Carbon-Nitrogen Ligases; C020661:gamma-glutamylmethylamide synthetase