Use of a glycerol-limited, long-term chemostat for isolation of Escherichia coli mutants with improved physiological properties

Abstract

The evolution of Escherichia coli MG1655 mutants was followed over 126 d in a glycerol-limited chemostat at a dilution rate of 095 h'l. This corresponds to a total of 217 generations at a doubling time of 1 3 9 h. After this time, nearly 90% of the chemostat population consisted of evolved mutant strains as determined by their altered colony morphologies on plates. Two mutants were isolated that exhibited generally improved growth phenotypes in batch cultivations on glycerol, glucose or the gluconeogenic substrate acetate. Higher specific growth rates and increased biomass yields were found for both mutants. For one mutant, this behaviour was combined with significantly reduced secretion of overflow metabolites when either glycerol or glucose was the carbon source. Additionally, during all growth phases of a batch cultivation, this mutant exhibited increased resistance to a variety of adverse conditions including heat shock, osmotic stress and nutrient deprivation. It also displayed significantly shorter lag phases.

Development of Efficient Xylose Fermentation in Saccharomyces cerevisiae: Xylose Isomerase as a Key Component

“…Soon after the invention of the chemostat it was already established that prolonged cultivation in nutrient-limited chemostats leads to selection of spontaneous mutants with an improved affinity for the growth-limiting nutrient [52,53]. This principle, which has since been demonstrated for many micro-organisms and nutrients [40,58,72,73] was applied to improve the affinity of S. cerevisiae RWB 217 for d-xylose [44].…”

Chemostat Approach for the Directed Evolution of Biodesulfurization Gain-of-Function Mutants

“…Chemostats have been designed to select for mutants that produce enzymes with increased activity rates (10,32,33) or with altered substrate specificity (29). The latter type have been termed gain-of-function mutants.…”

Prolonged selection in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae causes a partial loss of glycolytic capacity

“…In addition, studies on mutative adaptation of micro-organisms in chemostat cultures have demonstrated changes in cellular activity (Novick & Szilard, 1950a;van Schie et al, 1989;Weikert et al, 1997) and morphology (Adams et al, 1985;Brown & Hough, 1965). The main challenge now resides in the identification of the molecular basis for the adaptation.…”