Self-Cycling Fermentation: A Large Scale Approach to Modifying Cellular Processes for the Production of Recombinant Proteins

Self-Cycling Fermentation (SCF) is a large scale method to synchronize microbial populations in a bioreactor.  SCF is a cyclical, semi-continuous, automated process that maintains the bacterial culture in logarithmic growth.  By design, the SCF process aligns the cell doubling time with the cycling time such that cells double once per cycle with a significant level of synchronization. For example, when applying SCF to populations of Escherichia coli, cell synchrony indices of up to 0.74 were obtained — compared to 0 for a batch culture and 1 for an ideal synchronization.  Synchronized growth magnifies cellular events by temporally aligning metabolic activity.  Ideally, the behavior of a synchronized culture directly reflects that of an individual cell, enabling optimization of product formation according to cell metabolic function during the doubling time of the cell.

The maintenance of several strains of Escherichia coli using Self-Cycling Fermentation has led to some unexpected results, when compared to other strains.  Cellular metabolism, as measured by the average doubling time of the cells and by the rate of substrate consumption, slows down significantly.  In addition, the cells double in the middle of a cycle as opposed to at the end of the cycle.  In spite of this, the cell culture maintains productivity equal to or better than that of a cell culture grown under batch conditions. 

To evaluate the impact of SCF on the production of recombinant proteins using bacteriophages as vectors, two phage-host systems have been studied:  the lytic phage T4 (host - Escherichia coli) and a lysogenized strain of Escherichia coli containing a temperature sensitive mutant of phage λ modified to produce recombinant β-galactosidase.  By using SCF, protein production can be optimized with respect to not only the traditional parameters—nutrient levels, temperature, aeration, etc.—but, more importantly, with respect to the cell metabolic activity.  Experimental results indicate that phage and protein production are both significantly dependent on the time of induction within an SCF cycle.  These different times correspond to different cell age.  Data from both phage-host systems will be shown and the implications for an industrially viable set-up for recombinant protein production will be discussed.