GENOMICS AND ENGINEERING OF STRESS TOLERANT MICROBES FOR LOWER COST PRODUCTION OF ETHANOL FROM LIGNOCELLULOSE
Location: Bioenergy Research Unit
Title: Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag
Submitted to: Biotechnology and Bioengineering
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 22, 2011
Publication Date: March 2, 2011
Citation: Slininger, P.J., Thompson, S.R., Weber, S.A., Liu, Z., Moon, J. 2011. Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag. Biotechnology and Bioengineering. 108(8):1801-1815.
Interpretive Summary: To support the expansion of the biofuels industry, efficient fermentation processes are sought to produce ethanol from low-cost plant biomass (i.e. from crop and forest residues, energy crops, or municipal wastes). One challenge of making biofuels from lignocellulose is the production of economically recoverable ethanol from the mixture of free sugars present in hydrolyzates. Scheffersomyces (Pichia) stipitis and Pachysolen tannophilus are natural yeast strains best able to ferment the five-carbon sugar xylose. However, xylose conversion to ethanol is repressed by the more abundant preferred sugar glucose. Often xylose fermentation stalls for many hours, or even completely, as yeast metabolism must switch from glucose to xylose usage. In this research, we discovered that moderately high ethanol concentrations (circa 4-5% on a weight per volume basis) completely repressed the formation of key enzymes required for xylose utilization by S. (P.) stipitis. With this new understanding of ethanol as a regulator of xylose enzyme induction, fermentation process strategies can be designed to foster more efficient production of biofuel from plant biomass. Our research showed that recycling cell populations grown on xylose resulted in faster fermentation rates without stalling during mixed sugar conversion by S. (P.) stipitis and P. tannophilus, allowing ethanol accumulations in the economically recoverable 6 to 7% range. This process strategy was successful because specific enzymes required for xylose metabolism could be induced before repressive levels of ethanol accumulated. This new knowledge relating to the regulation of pentose-fermenting yeast metabolism will be used by others involved in research and development of new technologies to support growth of the biofuels industry. As a result, it furthers our progress toward national priorities of achieving energy independence, strengthening our rural economy, and preserving our environment.
During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. When yeast were grown on glucose and resuspended in mixed sugars, the length of this lag was observed to be a function of the glucose concentration consumed (and consequently, the ethanol concentration accumulated) prior to the switch from glucose to xylose fermentation. At glucose concentrations of 95 g/L, the switch to xylose utilization was severely stalled such that efficient xylose fermentation could not occur. Further investigation focused on the impact of ethanol on cellular xylose transport and the induction and maintenance of xylose reductase and xylitol dehydrogenase activities when large cell populations of S. (P.) stipitis NRRL Y-7124 were pre-grown on glucose or xylose and then presented mixtures of glucose and xylose for fermentation. Ethanol concentrations around 50 g/L fully repressed enzyme induction although xylose transport into the cells was observed to be occurring. Increasing degrees of repression were documented between 15 and 45 g/L ethanol. Recycled cell populations grown on xylose resulted in faster fermentation rates, particularly on xylose but also on glucose, and eliminated diauxic lag and stalling during mixed sugar conversion by P. tannophilus or S. (P.) stipitis, despite ethanol accumulations in the 60 or 70 g/L range, respectively. The process strategy of priming cells on xylose was key to the successful utilization of high mixed sugar concentrations because specific enzymes for xylose utilization could be induced before ethanol concentration accumulated to an inhibitory level.