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

Abstract

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.

Fermentation performance of engineered and evolved xylose‐fermenting Saccharomyces cerevisiae strains

“…Clearly, the choice of the starting strain(s) for such random procedures is critical, and the polyploid industrial isolates were more resistant to the presence of lignocellulose hydrolysate. Overall, the mutagenized and selected polyploid strain TMB3400 (Wahlbom et al, 2003) appears to be the most promising because it was by far the most rapid in converting sugars to ethanol, reducing the total fermentation time from the typical 90 -150 h to below 60 h. Moreover, it was somewhat resistant to the presence of hydrolysate. These properties would lead to commercially attractive short fermentation periods.…”

“…The successful improvement of desirable process properties from various pentose-utilizing strains by different random approaches (Becker and Boles, 2003;Sonderegger and Sauer, 2003;van Maris et al, 2004;Wahlbom et al, 2003) suggests that further improved process strains will be generated in this fashion. By altering the expression pattern of several genes involved in redox metabolism, evolutionary strategies have significantly reduced xylitol formation in the TMB3001-derivatives C1 and C5 (Sonderegger and Sauer, 2003;Sonderegger et al, 2004).…”

“…Isolated after mutagenesis and selection of TMB3399 (Wahlbom et al 2003) flasks at 140 rpm at 30jC. The minimal inhibitory concentration was defined as the lowest hydrolysate concentration that prevented exponential growth for at least 48 h after inoculation from a freshly streaked YPD-plate (Nilvebrant and Persson, 2003).…”

“…Nevertheless, the strategy is useful because it improved final ethanol titers of a polyploid industrial S. cerevisiae strain on mixtures of glucose and xylose (Zaldivar et al, 2002). Further S. cerevisiae strains of laboratory or industrial background with significantly improved xylose fermentation properties were recently isolated from evolutionary (Sonderegger and Sauer, 2003) or mutation and selection strategies (Wahlbom et al, 2003). Since all those reports focused on particular aspects of xylose metabolism and on one or few strains, it is difficult to evaluate the capabilities of the presently available variants.…”

Sequential Targeting of Xylose and Glucose Conversion in Fed-Batch Simultaneous Saccharification and Co-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

“…The strain was developed by evolutionary engineering [34] of the industrial strain S. cerevisiae TMB3400 [35] to improve its tolerance to inhibitors and xylose conversion capacity. Stock culture aliquots contained a mass fraction of 20% glycerol in water and were stored at −80°C.…”