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  • 1
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The operon encoding aspartokinase and aspartate semialdehyde dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask −) and E. coli X6118 (asd −), respectively. The A. mediterranei U32 aspartokinase and aspartate semialdehyde dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and aspartate semialdehyde dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and aspartate semialdehyde dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has K m values of 3.4 mM for aspartate and 2.3 mM for ATP, while aspartate semialdehyde dehydrogenase has K m values of 1.25 mM for dl-aspartate semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by l-threonine, l-lysine, and l-methionine, but not by l-isoleucine and diaminopimelate. Aspartate semialdehyde dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that aspartokinase was subject to allosteric control by l-threonine. Repression of both aspartokinase and aspartate semialdehyde dehydrogenase gene transcription in A. mediterranei U32 by l-lysine, l-methionine, l-threonine, and l-isoleucine were found. The network of regulation of aspartokinase and aspartate semialdehyde dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.
    Type of Medium: Electronic Resource
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