Cloning and expression of Pseudomonas taetrolens Y-30 gene encoding glutamine synthetase: an enzyme available for theanine production by coupled fermentation with energy transfer.
Glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 can form theanine from glutamic acid and ethylamine in a mixture where yeast fermentation of sugar is coupled for ATP regeneration (coupled fermentation with energy transfer). From a genomic DNA library of P. taetrolens Y-30, a clone containing 6 kbp insertional DNA fragment was selected by the PCR screening technique with specific oligonucleotide primers for the GS gene. The fragment had an open reading frame of the GS gene encoding a protein of 468 amino acids (molecular mass, 52 kDa). The deduced amino acid sequence showed a significant homology with that of P. syringae pv. tomato GS (97%), and all the amino acid residues were fully conserved, which concern with catalytic activity in other bacterial GS. A tyrosine residue for adenylylation of GS was also found, and in vivo adenylylation was confirmed in P. taetrolens Y-30. The isolated GS gene was ligated into an expression vector (pET21a), and expressed in Escherichia coli AD494 (DE3). The enzyme productivity in the expression system was 30-fold higher than that in P. taetrolens Y-30. Recombinant GS had the same properties as those of unnadenylylated intrinsic GS, and formed theanine in the mixture of coupled fermentation with energy transfer.
Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Shiga, Japan.
Sachiko
S
Yamamoto
Mamoru
M
Wakayama
Takashi
T
Tachiki
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004735:Energy Transfer; D005285:Fermentation; D015870:Gene Expression / Q000235:genetics*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005971:Glutamates / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D005971:Glutamates; D011994:Recombinant Proteins; C026166:theanine; D005974:Glutamate-Ammonia Ligase