Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control
A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h−1, respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.
KeywordsFed-batch technique Acetate formation High cell density Recombinant product formation Phosphotransferase system PTS mutations
- 3.Sandén AM, Prytz I, Tubulekas I, Förberg C, Le H, Hektor A, Neubauer P, Pragai Z, Harwood C, Ward A, Picon A, Teixeira de Mattos J, Postma P, Farewell A, Nyström T, Reeh S, Pedersen S, Larsson G (2003) Limiting factors in Escherichia coli fed-batch production of recombinant proteins. Biotech Bioeng 81:158–166CrossRefGoogle Scholar
- 5.Linn T, St.Pierre R (1990) Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ. J Bacteriol 172:1077–1084Google Scholar
- 9.Doelle H, Ewings K, Hollywood N (1982) Regulation of glucose metabolism in bacterial systems. Adv Biochem Eng 23:1–35Google Scholar
- 12.Curtis S, Epstein W (1975) Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase. J Bacteriol 122:1189–1199Google Scholar
- 18.De Anda R, Lara A, Hernandez V, Hernandez-Montalvo V, Gosset G, Bolivar F, Ramirez O (2006) Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate. Metabolic Eng 8:281–290CrossRefGoogle Scholar