Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose

Abstract

Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose.

FIG. 1.

Evolution of S. cerevisiae TMB3001 in carbon-limited chemostat cultures at a D of 0.05 h⁻¹ under aerobic conditions with 5 g of xylose per liter and 1 g of glucose per liter (A); under aerobic, microaerobic (light gray background), and anaerobic (dark gray background) conditions with 5 g of xylose per liter (B); and under anaerobic conditions with 5 g of xylose per liter (C). Arrow 1 indicates the time when the airflow was reduced from 0.3 liters min⁻¹ to <1 ml min⁻¹; arrow 2 indicates the time when the airflow was shut off; and arrow 3 indicates the time when anaerobiosis was initiated by sparging with technical N₂. The evolving population was subjected to EMS mutagenesis prior to inoculation of the chemostats.

FIG. 2.

Fermentation profiles for TMB3001 (A), the 460-generation population (B), clone TMB3001C5 representing the first phenotypic class (C), and clone TMB3001C1 representing the second phenotypic class (D) during anaerobic growth on 50 g of glucose per liter and 50 g of xylose per liter. The glucose and xylose consumption phases are indicated by I and II, respectively. Gray shading indicates the time when there was simultaneous consumption of glucose and xylose.

FIG. 3.

Physiological parameters during anaerobic growth on 50 g of glucose per liter and 50 g of xylose per liter for TMB3001, the 460-generation population, and 15 clones isolated from this population. (A) Maximum growth rate and biomass yield during exponential growth on glucose. (B) Specific xylose uptake rate and xylitol yield on xylose between the time that glucose was depleted and 100 h of fermentation. (C) Final ethanol concentration at 180 h. Values for TMB3001 and the populations are averages from duplicate experiments. The horizontal lines indicate the reference values for TMB3001.

FIG. 4.

Yields of acetate (A) and glycerol (B) on glucose (solid bars) and xylose (open bars) during anaerobic growth on 50 g of glucose per liter and 50 g of xylose per liter for TMB3001 and selected clones from both phenotypic classes. Yields on glucose were determined between the time of inoculation and the beginning of the xylose uptake phase. Yields on xylose were determined between the time that glucose was depleted and 130 h. Values were determined in single experiments.

FIG. 5.

OD₆₀₀ and xylose concentration during strictly anaerobic growth of TMB3001C1 in minimal medium with xylose as the sole carbon source.

FIG. 6.

Strictly anaerobic growth rates on xylose minimal medium for 20 clones that were isolated after seven serial anaerobic batch cultures on xylose. The horizontal line indicates the growth rate of parental strain TMB3001C1 before selection.