[The selection of fluoroacetate-resistant mutant from E. coli MMR204 and its influence on the expression of heterologous GL-7ACA acylase].
In the cultivation of gene engineered strain of Escherichia coli on glucose medium, excretion and accumulation of acetic acid inhibit not only cell growth but also the the expression of heterologous protein. It is obvious that the desirable host strain maintaining acetate at a low level is one of the approaches to increase the production of recombinant protein. The present article deals with the selection of mutants of E. coli DP19, DP8, which grow on the medium containing pyruvate as the sole carbon source in the presence of 50 mmol/L fluoroacetic acid. It is shown that mutant DP19 is defective in its phosphotransacetylase(PTA) activity and accumulates less acetate in the medium, while DP8 is defective in acetate kinase (ACK) and accumulates similar level of acetate comparing with its parent. Using pta- mutant E. coli DP19 as host, the expression of GL-7ACA acylase gene on the recombinant plasmid pMR24 is improved, and the yield of enzyme activity in flask fermentation is about twice as much as its parent.
Zhu
T
T
Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai 200032.
Yang
Y
Y
Jiao
R
R
D000084:Acetate Kinase / Q000378:metabolism; D019342:Acetic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005463:Fluoroacetates / Q000378:metabolism*; D015870:Gene Expression; D009154:Mutation; D010399:Penicillin Amidase / Q000235:genetics* / Q000378:metabolism; D010709:Phosphate Acetyltransferase / Q000378:metabolism; D012641:Selection, Genetic
D005463:Fluoroacetates; C014815:fluoroacetic acid; D010709:Phosphate Acetyltransferase; D000084:Acetate Kinase; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase; D019342:Acetic Acid