||dc.creator||Aristidou, Aristos Andrea
This study focuses on the application of metabolic engineering techniques to diminish acetate excretion, as a means to enhance recombinant protein productivities. The recombinant model system is based on the formation of plasmid encoded cadA-LacZ fusion protein with $\beta$-Galactosidase activity under the control of the pH-regulated promoter. This recently developed expression system has the potential for very high gene expression, of 30 to 40% of the total soluble cell protein. Three approaches are presented.
E. coli acetate mutants were isolated and characterized as recombinant hosts in relatively dense cultures. An acetate kinase mutant (ack) was found to excrete less acetate and in the meantime produce significantly higher amounts of recombinant protein, compared with the parent strain. In addition, this strain was observed to be less susceptible to fermentor dissolved oxygen deficiencies.
The possibility of replacing the common carbon source glucose with its isomer fructose was further investigated. Fructose is a feasible alternative since its uptake is more tightly regulated, and also because it is a less effective catabolic repressor. Comparative fermentation studies indicate that acetate levels were reduced to less than 6 mM from more than 90 mM, while in the meantime a 35% improvement in biomass yields was achieved. High cell density batch fermentations using fructose resulted in volumetric $\beta$-Galactosidase activities of 2.2 million U/ml, which represents a 65% improvement compared with similar glucose cultures.
The third method focuses on minimizing the metabolic imbalances through the genetic manipulation of the host organism. The alsS gene from B. Subtilis encoding the acetolactate synthase enzyme was successfully expressed in E. coli. This enzyme redirects pyruvate away from acetate, towards a non-inhibitory byproduct, acetoin. Acetate excretion can be maintained below 20 mM even in dense cultures employing rich glucose media. Moreover the engineered strain is a more efficient host for the production of recombinant proteins. The volumetric expression of $\beta$-Galactosidase was found to increase by about 50% in batch cultivations and by about 220% in high cell density fed-batch cultivations.
Application of metabolic and biochemical engineering techniques for the enhancement of recombinant protein production in Escherichia coli
Doctor of Philosophy
Aristidou, Aristos Andrea. "Application of metabolic and biochemical engineering techniques for the enhancement of recombinant protein production in Escherichia coli." (1995) Diss., Rice University. https://hdl.handle.net/1911/16799.