Amplification of three threonine biosynthesis genes in Corynebacterium glutamicum and its influence on carbon flux in different strains.
The hom-thrB operon (homoserine dehydrogenase/homoserine kinase) and the thrC gene (threonine synthase) of Corynebacterium glutamicum ATCC 13,032 and the homFBR (homoserine dehydrogenase resistant to feedback inhibition by threonine) alone as well as homFBR-thrB operon of C. glutamicum DM 368-3 were cloned separately and in combination in the Escherichia coli/C. glutamicum shuttle vector pEK0 and introduced into different corynebacterial strains. All recombinant strains showed 8- to 20-fold higher specific activities of homoserine dehydrogenase, homoserine kinase, and/or threonine synthase compared to the respective host. In wild-type C. glutamicum, amplification of the threonine genes did not result in secretion of threonine. In the lysine producer C. glutamicum DG 52-5 and in the lysine-plus-threonine producer C. glutamicum DM 368-3 overexpression of hom-thrB resulted in a notable shift of carbon flux from lysine to threonine whereas cloning of homFBR-thrB as well as of homFBR in C. glutamicum DM 368-3 led to a complete shift towards threonine or towards threonine and its precursor homoserine, respectively. Overexpression of thrC alone or in combination with that of homFBR and thrB had no effect on threonine or lysine formation in all recombinant strains tested.
Institut für Biotechnologie 1, Forschungszentrums Jülich GmbH, Federal Republic of Germany.
B J
BJ
Eikmanns
M
M
Metzger
D
D
Reinscheid
M
M
Kircher
H
H
Sahm
D002244:Carbon / Q000378:metabolism*; D019757:Carbon-Oxygen Lyases; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D015870:Gene Expression; D005798:Genes, Bacterial; D006715:Homoserine Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D008190:Lyases / Q000096:biosynthesis / Q000235:genetics*; D009876:Operon; D010770:Phosphotransferases / Q000096:biosynthesis / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor); D015183:Restriction Mapping; D013379:Substrate Specificity
D002244:Carbon; D006715:Homoserine Dehydrogenase; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021370:homoserine kinase; D008190:Lyases; D019757:Carbon-Oxygen Lyases; C023160:threonine synthase