6533b857fe1ef96bd12b4f3a

RESEARCH PRODUCT

Cloning of branched chain amino acid biosynthesis genes and assays of alpha-acetolactate synthase activities in Leuconostoc mesenteroides subsp. cremoris.

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Abstract A genomic library from Leuconostoc mesenteroides subsp. cremoris (Lmc) in Escherichia coli was screened for α-acetolactate synthase (ALS) activity using a phenotypic test detecting the production of acetolactate or related C 4 derivatives (diacetyl, acetoin or 2,3-butanediol) in the culture. Four recombinant E. coli clones, with plasmids containing overlapping DNA fragments and displaying anabolic ALS activity, were selected. This activity is encoded by an ilvB gene belonging to a putative operon which contains genes highly similar to the genes of the branched chain amino acid (BCAA) operon of Lactococcus lactis subsp. lactis. This putative BCAA operon is not functional as the ilvA gene is interrupted by a single mutation and the strain is auxotrophic for the three BCAAs. Only a very low anabolic ALS activity was present in cell-free extracts of Lmc and no transcript from the ilvB gene could be detected. Instability of ilvB expression in E. coli wasthe consequence of a frequent IS 5 insertion sequence in this gene. Despite the detection of a high catabolicALS activity in Lmc, no catabolic ALS activity gene could be found in the BCAA gene locus, indicating the presence of a catabolic als gene in the Lmc chromosome that could be absent or not expressed in the screened library.