A Probing Feeding Strategy for Recombinant E. coli Cultivations

*Summary: The bacterium Escherichia coli is a common host organism for recombinant protein production. A problem encountered is the accumulation of the by-product acetate, which tends to reduce growth and protein production (1). Formation of acetate can be avoided by a proper substrate feeding strategy, but most strategies require considerable process knowledge to work well. The main problem is that many important process variables cannot be measured on-line. This paper presents a feedback strategy for substrate feeding that is based on standard measurements and that only requires a minimum of process knowledge. The validity of the approach is demonstrated by both simulations and experiments. *Materials and Methods: Fed-batch cultivations of E. coli BL21(DE3) with different plasmids was made in a 3 L bioreactor using a defined medium. The temperature was 37 C and pH was kept at 7.0 by titration of ammonia. DO was kept around 30% saturation by manipulation of the stirrer speed. Expression of recombinant protein was initiated by addition of IPTG. OD was measured at 620 nm. Glucose and acetate concentration was determined using enzymatic kits. *Overview and discussion of results: The key idea of the novel feedback strategy is to exploit a characteristic change in the cell metabolism at the onset of acetate formation. By superimposing short pulses in the substrate feed rate, on-line detection of acetate formation can be made using a standard dissolved oxygen sensor (2). A simple feedback algorithm is then used to adjust the feed rate to avoid acetate formation while maintaining a high cell growth rate. The feasibility of the feeding strategy is demonstrated by simulations and tuning rules that require a minimum of process specific information are derived. The strategy was used in fed-batch cultivations of recombinant E. coli. Acetate accumulation was avoided and a good production of the recombinant protein was achieved. *Conclusions: A feedback strategy for substrate feeding in E. coli cultivations has been developed. An attractive feature is that only a minimum of process specific knowledge is required which makes it a valuable tool for cutting the development time for new processes. *References: (1) Bauer et al (1990) Appl. Env. Microbiol. 56, 1296-1302 (2) Akesson (1998) Licentiate Thesis ISRN LUTFD2/TFRT--3220--SE, Dept. of Automatic Control, Lund Inst. of Technology, Lund, Swede