Effect of variation of Klebsiella pneumoniae acetolactate synthase expression on metabolic flux redistribution in Escherichia coli.
Escherichia coli strains carrying the Bacillus subtilis acetolactate synthase (ALS) gene were previously shown to produce less acetate with higher ATP yields. Metabolic flux analysis was used to show that excess pyruvate was channeled into the less inhibitory product, acetoin. To further understand the role of intrinsic enzymatic properties and the effect of variations in enzyme levels in the alternation of metabolic fluxes, we constructed a chromosomal integrant of the Klebsiella pneumoniae ALS gene. The reported in vitro Michaelis-Menten constants (K(m)) for the Bacillus and the Klebsiella ALS are 13.0 mM and 8.0 mM, respectively. Furthermore, expression of the Klebsiella ALS is under the control of an inducible trp promoter system. Shake-flask experiments showed a linear induction response (the ALS activity changes from about 9 to 223 U/mg of protein when the inducer concentration [IAA] varied from 0 to 40 mg/L). Chemostat experiments showed a similar induction response. Interactions between the branched reactions catalyzed by the PFL, LDH, and the ALS enzymes at the pyruvate node were examined. The results indicate the importance of in vivo enzyme activities in the redistribution of metabolic fluxes.
Yang
Y T
YT
Departments of Bioengineering and Chemical Engineering Institute of Biosciences and Bioengineering, Rice University, 6100 Main Street, Houston, Texas 77005-1892, USA.
Peredelchuk
M
M
Bennett
G N
GN
San
K Y
KY
D000095:Acetolactate Synthase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D019149:Bioreactors; D001709:Biotechnology; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007210:Indoleacetic Acids / Q000494:pharmacology; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D007210:Indoleacetic Acids; D011994:Recombinant Proteins; C030737:indoleacetic acid; D000095:Acetolactate Synthase