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Improvement of biomass yield and recombinant gene expression in Escherichia coli by using fructose as the primary
A A Aristidou1, K Y San, G N Bennett
1Department of Bioengineering and Chemical Engineering and Department of Biochemistry and Cell Biology, Rice University, P.O. Box 1892, Houston, Texas 77251-1892, USA.
Abstract:
The feasibility of substituting glucose with fructose as a carbon source in Escherichia coli fermentations was investigated. Glucose, the most commonly used sugar in bacterial cultivations, is well-known to pose a number of drawbacks; the most important of which is the Crabtree effect, which results in acidogenesis. Fructose, a glucose structural isomer, offers a reasonable alternative for glucose, since its uptake and utilization are more tightly regulated. Comparative fermentation studies indicate that lower acetate excretion and higher biomass yields were attained in fructose-supplemented growth media compared with those of glucose media. More specifically, cells grown in defined media supplemented with fructose do not excrete detectable amounts of acetate, while about 40 mM of acetate was detected extracellularly in similar glucose cultures. A reduction in the initial growth rate of about 20% was observed with fructose, but final cell densities were about 70% higher compared with glucose supplements. Growth in complex LB media supplemented with fructose again resulted in higher biomass yields (up to 40%) and lower acetate excretion (30-40%) than the comparable glucose media. In bioreactor studies using LB media, acetate levels were reduced from 90 to less than 6 mM, while achieving a 25% improvement in biomass yield. When using richer media, cell densities of more than 40 g L-1 dry cell weight were attained in batch cultivation using fructose compared with 30 g L-1 for glucose. These results have immense applicability in the area of recombinant protein processes. Recombinant E. coli, overexpressing beta-galactosidase under the control of the strong pH-inducible promoter, achieved a volumetric recombinant protein yield of 2.2 million U mL-1 (corresponding to approximately 1.5 g L-1) in batch fructose cultures. This represents a 65% recombinant protein yield enhancement when compared to similar glucose cultivations.