PMID:2178553
| Citation |
Bartsch, K, Dichmann, R, Schmitt, P, Uhlmann, E and Schulz, A (1990) Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli. Appl. Environ. Microbiol. 56:7-12 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Aminobutyrates/metabolism; Ammonium Chloride/metabolism; Base Sequence; Cloning, Molecular; Cosmids; Culture Media; DNA, Bacterial/genetics; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Fermentation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Molecular Sequence Data; Oligonucleotide Probes; Oligonucleotides/genetics; Plasmids; Restriction Mapping; Transaminases/biosynthesis; Transaminases/genetics; Transaminases/metabolism; gamma-Aminobutyric Acid/metabolism |
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