Rapid strain improvement through optimized evolution in the cytostat |
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Authors: | Alan Gilbert Dipen P. Sangurdekar Friedrich Srienc |
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Affiliation: | 1. Department of Chemical Engineering and Materials Science, University of Minnesota, 151 Amundson Hall, 421 Washington Avenue S.E., Minneapolis, Minnesota 55455‐0312;2. BioTechnology Institute, University of Minnesota, 240 Gortner Laboratory, 1479 Gortner Avenue, St. Paul, Minnesota 55108;3. telephone: 612‐624‐9776;4. fax: 612‐625‐1700 |
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Abstract: | Acetate is present in lignocellulosic hydrolysates at growth inhibiting concentrations. Industrial processes based on such feedstock require strains that are tolerant of this and other inhibitors present. We investigated the effect of acetate on Saccharomyces cerevisiae and show that elevated acetate concentrations result in a decreased specific growth rate, an accumulation of cells in the G1 phase of the cell cycle, and an increased cell size. With the cytostat cultivation technology under previously derived optimal operating conditions, several acetate resistant mutants were enriched and isolated in the shortest possible time. In each case, the isolation time was less than 5 days. The independently isolated mutant strains have increased specific growth rates under conditions of high acetate concentrations, high ethanol concentrations, and high temperature. In the presence of high acetate concentrations, the isolated mutants produce ethanol at higher rates and titers than the parental strain and a commercial ethanol producing strain that has been analyzed for comparison. Whole genome microarray analysis revealed gene amplifications in each mutant. In one case, the LPP1 gene, coding for lipid phosphate phosphatase, was amplified. Two mutants contained amplified ENA1, ENA2, and ENA5 genes, which code for P‐type ATPase sodium pumps. LPP1 was overexpressed on a plasmid, and the growth data at elevated acetate concentrations suggest that LPP1 likely contributes to the phenotype of acetate tolerance. A diploid cross of the two mutants with the amplified ENA genes grew faster than either individual haploid parent strain when 20 g/L acetate was supplemented to the medium, which suggests that these genes contribute to acetate tolerance in a gene dosage dependent manner. Biotechnol. Bioeng. 2009;103: 500–512. © 2009 Wiley Periodicals, Inc. |
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Keywords: | robust strains evolution cytostat Saccharomyces cerevisiae lignocellulosic ethanol |
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