Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli.
We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.
Bartsch
K
K
Hoechst AG, Frankfurt, Federal Republic of Germany.
Dichmann
R
R
Schmitt
P
P
Uhlmann
E
E
Schulz
A
A
D000613:Aminobutyrates / Q000378:metabolism*; D000643:Ammonium Chloride / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids; D015183:Restriction Mapping; D000637:Transaminases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000613:Aminobutyrates; D003470:Culture Media; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D000643:Ammonium Chloride; C003121:phosphinothricin; D005680:gamma-Aminobutyric Acid; D000637:Transaminases