GENE STRUCTURE AND EXPRESSION OF THE CORYNEBACTERIUM-FLAVUM N-13 ASK-ASD OPERON

Two promoters required for expression of the ask-asd genes, encoding a spartokinase (AK) and aspartate-semialdehyde dehydrogenase (ASD), in C orynebacterium flavum N13, askP1 and askP2, have been identified by de letion analysis and S1 nuclease mapping. Transcription from askP1 init iates 35 and 38 bp upstream of the ask structural gene. A second promo ter, askP2, lies within the ask coding region, upstream of the transla tion start site of the AKbeta subunit and can direct the expression of AKbeta and ASD. Western immunoblot analysis and heterologous expressi on in Escherichia coli demonstrate that two separate polypeptides, a 4 4.8-kDa alpha subunit and an 18.5-kDa 13 subunit, are expressed from t he C. flavum N13 ask gene from distinct, in-frame translation initiati on sites. A second AK mutation, G345D, which reduces the sensitivity o f AK to concerted feedback inhibition by threonine plus lysine, was id entified.