A new plasmid construct has been used in conjunction with selective recycle to successfully maintain otherwise unstable plasmid‐bearing E. coli cells in a continuous bioreactor and to produce significant amounts of the plasmid‐encoded protein β‐lactamase. The plasmid is constructed so that pilin expression, which leads to bacterial flocculation, is under control of the tac operon. The plasmid‐bearing cells are induced to flocculate in the separator, whereas cell growth and product synthesis occur in the main fermentation vessel without the inhibiting effects of pilin production. Selective recycle allows for the maintenance of the plasmid‐bearing cells by separating flocculent, plasmid‐bearing cells from nonflocculent, segregant cells in an inclined settler, and recycling only the plasmid‐bearing cells to the reactor. As a result, product expression levels are maintained that are more than ten times the level achieved without selective recycle. All experimental data agree well with theoretical predictions.
- fermentations, continuous
- protein overproduction
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology