To improve the rate and yield of ethanol production from hemicellulose hydrolysates

	Identify the principal enzymes necessary for fermentation of D-xylose and L-arabinose
	Express those enzymes at appropriate levels for fermentation
	Employ expression analysis to characterize the responses of cells to the engineered substrate
	Use mutagenesis and targeted knock-outs to alter expression of undesired pathways
	Optimize expression levels of multiple genes to relieve rate limiting effects and avoid toxic responses
	Employ transposon mutagenesis and library expression analysis to identify mutational events that can enhance growth and fermentation

	Constructed strains of Saccharomyces cerevisiae that will grow on and ferment xylose and glucose
	Down-regulated respiration in both the native xylose fermenting yeast, Pichia stipitis and in engineered strains of S. cerevisiae
	Showed that respiration deficient mutants convert xylose to ethanol in higher yield
	Demonstrated that over expression of D-xylulokinase is toxic in S. cerevisiae
	Demonstrated that engineered S. cerevisiae attempts to respire rather than ferment on xylose
	Identified two key transposon mutagenesis events that will relieve the toxic effect of xylulokinase over expression
	Constructed strains of Saccharomyces cerevisiae that will grow on and ferment xylose and glucose
	Identified several additional genes that relieve the toxic effect when they are over expressed in the engineered cells
	In cooperation with DOE Joint Genome Institute, completed sequencing of the 15 Mbp P. stipitis genome
	Developed an efficient transformation and marker recovery system for P. stipitis that is based on modified ble/CRE
	Identified missing genes in P. stipitis that are essential for anaerobic growth and ethanol production

	Disruption of CYC1 in P. stipitis increased the specific ethanol production rate by 50% and increased the yield 15%
	Increased the rate and doubled the yield of ethanol produced from xylose by respiration deficient mutants of engineered S. cerevisiae
	Integration of these technologies with optimal expression of essential genes in a modified genetic background will lead to commercial yeasts for the fermentation of hemicellulosic hydrolysates
	Successful implementation and commercialization of this technology along with cellulose saccharification could produce 80 x 109 liters of ethanol per year from domestic resources, which is equivalent to about 20% of U.S. gasoline consumption.

Abstract:

Biorefining of lignocellulose seeks to recover both hemicellulosic sugars and fibers from the processing of hardwood into paper.  The hemicellulosic sugars contribute little to the fiber that is obtained from kraft pulping, and if they can be converted into ethanol, this co-product could double the profitability of pulp manufacture.  Agricultural residues also present a large renewable resource for ethanol production, but the economics of this process depend completely on the recovery and conversion of all major sugar components. Xylose is the second most abundant carbohydrate in terrestrial plants and it is particularly abundant in the lignocellulose of angiosperms, which comprise the majority of agricultural and silvicultural crops.  Xylose is relatively easy to recover from agricultural lignocellulose, but its bioconversion to ethanol is difficult. Only a handful of native yeasts, the best studied of which is Pichia stipitis, will produce significant amounts of ethanol from xylose.  A few bacteria – most notably Escherichia coli and Zymomonas mobilis – have been genetically engineered for xylose fermentation along with the yeast, Saccharomyces cerevisiae.  Each organism exhibits particular advantages and disadvantages, but industrial practices favor the development of xylose fermenting yeasts. The larger cells and thick cell walls make yeasts easier to harvest and recycle; they do not succumb to bacteriophage infections, their nutritional requirements are minimal, and they tolerate low pH.

Metabolic engineering of S. cerevisiae for xylose fermentation has been underway for almost 20 years.  Heterologous expression of xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) along with D-xylulokinase (XYL3) will impart the capacity for growth on xylose, but the resulting cells do not recognize xylose as a fermentable carbon source, and they attempt to oxidize it. Only recently have we recognized that it is necessary to introduce appropriate regulatory mechanisms along with the genes for xylose metabolism in order to ferment this sugar to ethanol. Also over expression of some genes, particularly kinases, can have unexpected and detrimental effects.  Alteration of expression of various genes can overcome toxicity, and obtaining optimal expression for maximal fermentation rates and yields presents a significant challenge. 

Our current research is focused on identifying genes that are essential for the efficient fermentation of xylose and expressing them at optimal levels.

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