Effects of lysine-sensitive aspartokinase III on lysine biosynthesis in Escherichia coli K-12.
A promoterless lysC gene, coding for Escherichia coli aspartokinase III (AKase III), has been cloned by phenotypic complementation using plasmid pUC19 as the vector. The hybrid plasmid obtained, pUC19AK3, preserved the ribosome binding site and transcriptional termination signal of the gene but with a lac promoter. E. coli strains containing the recombinant plasmid had high levels of AKase III activity. AKase III activity from expressing strains was inhibited by lysine, leucine, and S-(2-aminoethyl)-L-cysteine (AEC) but not by threonine and methionine. The overexpressed AKase III enzyme had a molecular weight of about 50 kD from SDS-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis confirmed that the product from the hybrid plasmid was identical to native AKase III rather than a fusion protein. Moreover, overexpression of AKase III significantly increased lysine excretion in the plasmid-harboring E. coli strain DH1. This increase in the level of AKase III activity also affected other metabolites than lysine. Addition of aspartate to the medium brought about significant increases in lysine excretion. A maximum increase (about 8-fold) in lysine accumulation was observed 45 minutes after incubation in minimal medium containing 0.2% aspartate as compared to aspartate-free medium.
Huang
K J
KJ
Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, ROC.
Hseu
T H
TH
D000595:Amino Acid Sequence; D001222:Aspartate Kinase / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000494:pharmacology; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008239:Lysine / Q000096:biosynthesis* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D014169:Transformation, Bacterial
D003470:Culture Media; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001222:Aspartate Kinase; D008239:Lysine