Dual-stage fermentation

A method for continuously fermenting bacterial cells to form a product, said method comprising:

• (a) providing at least a first and a second continuous fermentation unit and a means connected between said units to communicate therebetween;
• (b) providing a strain of recombinant bacterial cells capable of being induced to undergo genetic rearrangement from a first genetic configuration to a second genetic configuration, said genetic rearrangement being lambdoid bacteriophage site-specific recombination mediated by lambdoid bacteriophage xis and int genes in response to an environmental shift from a first environmental condition, which is non-permissive for said genetic rearrangement, to a second environmental condition, which permits genetic rearrangement to form said second genetic configuration, said genetic rearrangement juxtaposing two inactive DNA segments to create an actively-expressed gene encoding a protein or polypeptide which is said product, or an intermediate or enzyme in a synthetic pathway of said product, said two inactive DNA segments being heterologous with respect to said lambdoid bacteriophage xis and int genes;
• (c) continuously growing said bacterial cells in said first fermentation unit under said first environmental condition to establish a microorganism mass;
• (d) transferring at least a portion of said microorganism mass from said first continuous fermentation unit through said means connecting said units to said second continuous fermentation unit;
• (e) continuously culturing said bacterial cells in said second fermentation unit under production-favoring conditions, said second fermentation unit being maintained at said second environmental condition, whereby said bacterial strain in said second unit is subject to said genetic rearrangement effecting expression of said actively-expressed gene and production of said desired product in said second unit;
• (f) removing an exudate of said microorganism mass from said second unit and removing said product from said exudate;
• and
• (g) recycling at least a portion of the cell mass of said exudate of step (f) into said second unit, said portion being sufficient to maintain a desired cell density in said second unit.