Metabolic characterisation of E. coli citrate synthase and phosphoenolpyruvate carboxylase mutants in aerobic cultures.
E. coli is still one of the most commonly used hosts for protein production. However, when it is grown with excess glucose, acetate accumulation occurs. Elevated acetate concentrations have an inhibitory effect on growth rate and recombinant protein yield, and thus elimination of acetate formation is an important aim towards industrial production of recombinant proteins. Here we examine if over-expression of citrate synthase (gltA) or phosphoenolpyruvate carboxylase (ppc) can eliminate acetate production. Knock-out as well as over-expression mutants were constructed and characterized. Knocking out ppc or gltA decreased the maximum cell density by 14% and increased the acetate excretion by 7%, respectively decreased it by 10%. Over-expression of ppc or gltA increased the maximum cell dry weight by 91% and 23%, respectively. No acetate excretion was detected at these increased cell densities (35 and 23 g/l, respectively).
De Maeseneire
S L
SL
Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure links 653, Gent, Belgium, Sofie.DeMaeseneire@UGent.be
De Mey
M
M
Vandedrinck
S
S
Vandamme
E J
EJ
D000085:Acetates / Q000737:chemistry; D001709:Biotechnology / Q000379:methods*; D002950:Citrate (si)-Synthase / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005821:Genetic Techniques; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008660:Metabolism; D008828:Microbiological Techniques; D008954:Models, Biological; D009154:Mutation; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry* / Q000235:genetics
D000085:Acetates; D002950:Citrate (si)-Synthase; D010730:Phosphoenolpyruvate Carboxylase; D005947:Glucose