Optimization of recombinant Escherichia coli fermentation processes

Tai Hyun Park, Purdue University

Abstract

An optimal operating strategy to maximize the production of cloned-gene proteins in recombinant Escherichia coli fermentation was developed. Regulated promoters were used to separate host cell growth from cloned-gene expression for better performance. Two kinds of regulated systems were studied. Those are trp promoter cloned on plasmids and temperature-sensitive bacteriophage $\lambda$ cloned on host chromosome. Fermentation processes using regulated systems were optimized through theoretical and experimental analyses. In the plasmid-containing system, model equations for cell growth and cloned-gene expression have been formulated and used to evaluate process performances under different operating modes. It was found that the optimal switching time exists for a given total operation period in a batch and fed-batch modes. For a two-stage continuous fermentation system, the productivity is more sensitive to the combination of the dilution rates than the volume ratio of two reactors. As long as the down time is less than the total operation time in the fed-batch mode, the fed-batch mode gives higher productivity using a same amount of the substrate based on unit volume and time than the two-stage continuous system. Practically, however, the productivity in the fed-batch mode would be limited by the maximum packing density of the cell, product degradation, other nutrients or waste metabolites. Plasmids in this system were experimentally found to be stably maintained in batch operation; however, plasmid instability was a problem in continuous operation, especially in long-term operation. Plasmids have been extensively studied as a vector for the cloned-gene expression. On the other hand, bacteriophage $\lambda$ has not been studied for large scale production of cloned-gene proteins, although it has been used as a cloning vehicle. The potential of bacteriophage $\lambda$ as an expression vector for large scale production of cloned-gene proteins was experimentally evaluated. Induction kinetics of the $\lambda$ system was experimentally and theoretically studied. In two-stage continuous operation, the first reactor is maintained in the lysogenic state at a lower temperature to stably maintain the foreign DNA in the host cell, while the second reactor is maintained in the lytic state to force replication of the cloned-gene and overproduction of its products. Experimental results showed that the cell density and cloned-gene product concentration were constantly maintained in lysogenic state. This implies that the bacteriophage $\lambda$ containing cloned-gene is stably maintained in lysogenic state in continuous fermentation. This means that this $\lambda$ system may solve plasmid instability problem in recombinant cell fermentation for long-term operation. The experimental results are promising but suggest a greater potential for a mutant which lacks ${\cal Q}$ gene which is responsible for host cell lysis and packaging of phage particles.

Degree

Ph.D.

Advisors

Lim, Purdue University.

Subject Area

Chemical engineering

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