Stability of Continuous Culture with Recombinant Organisms

Abstract

It was found that both poor selection pressure and a variable rate of plasmid loss were present in the system studied and that both have significant effects on continuous reactor operation. At least some of these effects were analyzed by a simple model. At this point, experimental analysis for extracellular levels of tryptophan sufficient to support X- growth (1-4 mg/l) has given contradictory results. This has at least partially indicated the effect may be an intracellular one, and thus the culture history would be critical in such experiments. Since the system studied is not atypical of recombinant cultures, it leads one to speculate on the generality of the phenomena and its extent in other cultures. If important, the use of double auxotrophs or auxotrophs that are mutant in a metabolite for which the cell has a greater growth requirement should be used. Additionally, the presence of higher copy numbers in yeast at lower growth rates also leads one to speculate on how these apparently contradictory phenomena are related.

Consequences of phosphoglycerate kinase overproduction for the growth and physiology of Saccharomyces cerevisiae

“…In general, these genes are cloned o n high-copy episomal plasmids (Botstein and Fink 1988;Kingsman et al 1987). However, during long-term growth on a non-selective medium most of those plasmids, together with the ability of overproduction, are lost (Futcher and Cox 1984;DiBiasio and Sardonni 1986;Parker and DiBiasio 1987). Therefore we decided to use a multi-copy integration system for integration of the PGK1 gene in the yeast genome in order to achieve strong overproduction.…”

Effects of phosphoglycerate kinase overproduction in Saccheromyces cerevisiae on the physiology and plasmid stability

“…This stabilization could be due to an equilibrium between plasmid loss and an increased growth yield of the plasmid-containing cells, caused by the efficient glucose metabolism of the PGKoverproducing strains. Many groups Hassan, 1987, 1988;DiBiasio and Sardonni, 1986;Kleinman et al, 1986;Parker and DiBiasio, 1987) have reported that the genetic stability of some epi-soma1 plasmids is influenced by the growth rate: the segregation rate decreases at high growth rates. As a possible explanation, Kleinrnan et al (1986) suggest that at low specific growth rates, the daughter cells are smaller, which will reduce their chance of receiving plasmids.…”

“…While recent advances in recombinant DNA technology have made it possible to obtain high copy numbers of recombinant plasmids in a variety of micro-organisms, genetic instability under nonselective conditions remains a serious problem (Hsiao and Carbon, 1979;Walmsley et al, 1983;DiBiasio and Sardonni, 1986;Lee et al, 1986; tAddressee for correspondence. 0749-503)3/92/0 10047-09 $05.00 0 1992 by John Wiley & Sons Ltd Kleinman et al, 1986;Mead et al, 1986;Schwartz et al, 1987).…”

A fermentation strategy for production of recombinant protein subjected to plasmid instability

“…Another selective pressure method is to cultivate an auxotrophic host that requires the plasmid to synthesize essential amino acids in an incomplete selective medium. However, the plasmidlosing cells can still survive, because they can ingest the particular amino acid produced by plasmid-carrying cells [7]. In addition, several engineering approaches have also been proposed for preventing plasmid instability, including cell immobilization [8], cycling the dilution rate for delaying the washout of plasmid-carrying cells in a chemostat culture [9,10], and selective recycling of plasmidcarrying cells [11].…”