Acetate-inducible protein overexpression from the glnAp2 promoter of Escherichia coli.
The Ntr regulon in Escherichia coli has previously been engineered to control the expression of a heterologous metabolic pathway. In this study, we reengineered the same system for protein production. In the absence of NRII (glnL gene product), we showed that glnAp2 can be an effective promoter for protein production that is inducible by exogenous acetate, but both the induction ratio and the range of modulation are low. To deal with this issue, we inactivated phosphotransacetylase (pta gene product), which disrupts the acetate pathway and denies the cell the ability to synthesize acetate. With this additional modification, gene expression from glnAp2 can be controlled by directly adding acetate into the growth medium. Using a lacZ reporter fusion, we found that glnAp2 induction was modulatable over a range of potassium acetate concentrations, and the induction/noninduction ratio increased to 77 in the absence of pta. The extracellular acetate required for maximal induction is lower than the concentration that causes toxicity, and thus growth inhibition by acetate addition was not a matter of concern. Furthermore, compared to the P(tac) promoter, overexpression of a model protein using the modified glnAp2 promoter system did not cause significant growth inhibition, although a higher level of protein expression was achieved.
Farmer
W R
WR
Department of Chemical Engineering, 5531 Boelter Hall, University of California Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095, USA. liaoj@ucla.edu
Liao
J C
JC
D019342:Acetic Acid / Q000494:pharmacology*; D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D009586:Nitrogen Fixation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D015854:Up-Regulation
D011506:Proteins; D019342:Acetic Acid