Parameters influencing the productivity of recombinant E. coli cultivations

In the past 10 to 15 years, many of the promises of microbial genetic engineering have been realized: the use of recombinant Escherichia coli has moved from the laboratory to the production facility, and the manufacture of therapeutic recombinant proteins such as human growth hormone and interleukins is a rapidly growing industry. Along with this progress, however, have come new problems to solve: bioreactor operators have discovered that large-scale cultivations of plasmid-containing bacteria do not behave in exactly the same way as those of plasmid-free cells, plasmid stability has been recognized as a major hurdle, and the protein product might not be present in a soluble form but rather as intracellular granules that resist solubilization. These and other difficulties represent a new generation of challenges for genetic engineering. However, genetic engineering can do more than solve these problems. Molecular biological techniques also have the ability to create new opportunities: to produce new compounds, to use cheaper substrates, to facilitate downstream processing, and to optimize production in new ways. The productivity of a cultivation can generally be expressed as the product of the cell density and the specific biological activity. Both of these parameters are influenced by a variety of factors. For recombinant cultivations, though, the level of biological activity, a reflection of the plasmid copy number and expression efficiency, is the more interesting and important consideration and will therefore be given more attention in our review. In this contribution, our general goal is to discuss the factors that influence the productivity of recombinant E. coli cultivations, covering parameters relating to DNA; parameters relating to protein synthesis; parameters relating to proteins; and parameters relating to downstream processing. The object is not to tell the reader how to choose the perfect plasmid, host, and cultivation conditions, but to make known the many variables involved in designing a recombinant process and to point out recent and potential advances made possible by genetic engineering. The discussion focuses on the production of a protein, but many of the same concepts apply to other cultivations of recombinant E. coli, including cases in which the desired product is not a protein or the cells have been designed for a special metabolic capability such as pollutant biodegradation.