Whereas cell concentrations of 5-10 grams dry cell weight per liter (gDCW/l) are typical of batch cultures, fed-batch techniques can be used to achieve concentrations greater than 50 gDCW/l. Feeding strategies for fed-batch cultures include feed-back control as well as pre-determined feeding profiles. The volumetric yield of recombinant products can be improved by controlling the specific growth rate and the substrate concentration. Furthermore, inhibitory by-product formation can be minimized in fed-batch cultures. This review focuses on the use of fed-batch techniques to produce recombinant products in Escherichia coli. The modes of nutrient feeding that have been employed are discussed, and the factors important in attaining high cell concentrations as well as high specific yields of recombinant product are described.

Fed-batch techniques were employed to obtain high cell density cultures (92-100 g DCW/L) of Escherichia coli strain X90 producing a recombinant serine protease, rat anionic trypsin, secreted to the periplasm. The specific growth rate was controlled to minimize growth-inhibiting acetate formation by utilizing an exponential feeding profile determined from mass balance equation. The volumetric yield of recombinant rat anionic trypsin was 56 mg/L, and the final cell density was 92 g DCW/L when the culture was induced in the late logarithmic phase. However, when the culture was induced in the early logarithmic phase, the volumetric yield was 13 mg/L and the final cell density was 14 g DCW/L. Thus, the induction timing is shown to have a significant effect on the final cell density as well as the overall volumetric yield of the recombinant protease.

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An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose-limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h(-1) for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose-limited chemostat culture.

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