Molecular analysis of a Saccharomyces cerevisiae mutant with improved ability to utilize xylose shows enhanced expression of proteins involved in transport, initial xylose metabolism, and the pentose phosphate pathway

Differences between the recombinant xylose-utilizing Saccharomyces cerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays. Samples for analysis were withdrawn from chemostat cultures. The characteristics of S. cerevisiae TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of S. cerevisiae TMB 3400 grown on only xylose, were investigated. The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h-1, with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter-1, 10 g of glucose plus 10 g of xylose liter-1 or, for S. cerevisiae TMB 3400, 20 g of xylose liter-1. S. cerevisiae TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than S. cerevisiae TMB 3399. The biomass yields for S. cerevisiae TMB 3400 were 0.46 g of biomass g of consumed carbohydrate-1 on glucose and 0.43 g of biomass g of consumed carbohydrate-1 on xylose. A Ks value of 33 mM for xylose was obtained for S. cerevisiae TMB 3400. In general, the percentage error was <20% between duplicate microarray experiments originating from independent fermentation experiments. Microarray analysis showed higher expression in S. cerevisiae TMB 3400 than in S. cerevisiae TMB 3399 for (i) HXT5, encoding a hexose transporter; (ii) XKS1, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii) SOL3, GND1, TAL1, and TKL1, encoding enzymes in the pentose phosphate pathway. In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains. Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted. The higher expression of XKS1 in S. cerevisiae TMB 3400 than in TMB 3399 correlated with higher specific xylulokinase activity in the cell extracts. The specific activity of xylose reductase and xylitol dehydrogenase was also higher for S. cerevisiae TMB 3400 than for TMB 3399, both on glucose and on the mixture of glucose and xylose.Articl

Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054

The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding D-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on D-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only D-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h-1 for S. cerevisiae TMB 3399 to 0.14 h-1 for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h-1. All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose-1 and 0.001, 0.10, and 0.16 g ethanol g biomass-1 h-1 for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein-1, was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant. © 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.Articl

A constitutive catabolite repression mutant of a recombinant Saccharomyces cerevisiae strain improves xylose consumption during fermentation

Efficient xylose utilisation by microorganisms is of importance to the lignocellulose fermentation industry. The aim of this work was to develop constitutive catabolite repression mutants in a xylose-utilising recombinant Saccharomyces cerevisiae strain and evaluate the differences in xylose consumption under fermentation conditions. S. cerevisiae YUSM was constitutively catabolite repressed through specific disruptions within theMIG1 gene. The strains were grown aerobically in synthetic complete medium with xylose as the sole carbon source. Constitutive catabolite repressed strain YCR17 grew four-fold better on xylose in aerobic conditions than the control strain YUSM. Anaerobic batch fermentation in minimal medium with glucose-xylose mixtures and N-limited chemostats with varying sugar concentrations were performed. Sugar utilisation and metabolite production during fermentation were monitored. YCR17 exhibited a faster xylose consumption rate than YUSM under high glucose conditions in nitrogen-limited chemostat cultivations. This study shows that a constitutive catabolite repressed mutant could be used to enhance the xylose consumption rate even in the presence of high glucose in the fermentation medium. This could help in reducing fermentation time and cost in mixed sugar fermentation.Vasudevan Thanvanthri Gururajan, Marie-F. Gorwa-Grauslund, Bärbel Hahn-Hägerdal, Isak S. Pretorius and Ricardo R. Cordero Oter