Location: Crop Bioprotection Research
2005 Annual Report
This program is important to strengthening the security of our nation by lessening dependence on foreign sources of fuel, preserving our environment and natural resources, and boosting our economy (especially in rural America). It specifically addresses the fermentative conversion of sugars from renewable biomass to ethanol, a process that will be required to surpass the nation's $5 billion gallon ethanol/year production goal set for 2012. Under National Program 307, Bioenergy and Energy Alternatives, this research is expected to contribute to methods to increase the efficiency and significantly reduce the costs of conversion of biomass to liquid fuel and feedstock chemicals through the development of microbial catalysts capable of surviving a variety of industrial stresses which currently limit performance. Under National Program 306, Quality and Utilization of Agricultural Products, the research will contribute to new uses for agricultural products and byproducts through the development of the knowledge base and novel techniques needed for design and application of commercially viable microbial processes for bioconversion of agricultural products to new bioproducts. Research to determine how cells survive such stresses and how to engineer their survival will provide the knowledge to allow us great flexibility and power in the design of cost-effective, highly productive microbial bioprocesses for the next decade. Success with this project will be far reaching and have impact on improving the commercialization prospects of many bioproducts produced from agricultural crops and residues by microbial catalysis, such as feedstock chemicals, specialty products such as thickeners, sweeteners, as well as bioactive products such as antibiotics and biological control inocula to control agricultural pests, including diseases, insects, and weeds.
Year 2005-2006 (FY 06) - 1.A.2. Identify phenotype differences in tolerance--develop adapted strains. - 1.A.3. Identify phenotype differences in tolerance--screen Saccharomyces cerevisiae disruption library. - 1.A.5. Identify phenotype differences in tolerance--screen cultivation environment. - 1.B.1. Identify genes/regulatory networks/function--develop quality-controlled arrays.
Year 2006-2007 (FY 07) - 1.B.4. Identify genes/regulatory networks/function--characterize cDNA library stress genes.
Year 2005-2008 (FY 08) - 1.B.2. Identify genes/regulatory networks/function--genomic expression of stress tolerance. Year 2006-2008 (FY 08) - 1.B.3. Identify genes/regulatory networks/function--verify disruption library stress genes. - 1.B.5 Identify genes/regulatory networks/function--characterize protective gene function. - 1.B.6 Identify genes/regulatory networks/function--generate tolerance-specific gene array.
Objective 2 - Engineer improved strains/processes: Year 2007-2009 (FY 09) - 2.A. Engineer commercial yeast strains. Year 2008-2009 (FY 09)
- 2.B. Optimize bioreactor design.
Development of a quality-controlled gene microarray for ethanologenic yeast study: In order to develop a quality microarray with high levels of reproducibility and reliability, we developed six external RNA controls this year and fabricated a yeast gene chip microarray with three replications on one slide. We also created a novel component of PreScan mini arrays to incorporate into the conventional microarray design. It provides a reference to adjust laser scanner voltage for both dye channels and avoids multiple scanning of the entire array and thereafter to reserve the quality and fidelity of microarray data acquisition. It serves as an unbiased reference for data normalization. This new design contributes to the microarray technology development. Furthermore, we developed the external RNA controls as a standard universal control for gene expression analysis across different platforms including microarray and real time quantitative RT-PCR. Previously there was no control of this nature available. This accomplishment constitutes an important scientific advancement of the methodology of gene expression analysis that will first benefit the development of stress tolerant microbes for production of low cost biofuels and bioproducts. This work was published in a book chapter and conference proceedings and will be presented in 2005. In 2006, we will further perfect the method and its applications.
Identification of the pleiotropic drug resistance gene family having a significant role in HMF stress tolerance: During the course of study on genomic expression in response to inhibitors involved in biomass conversion, we identified and first reported that a group of genes belonging to the pleiotropic drug resistance gene family played a significant role to cope with HMF stress involved in biomass conversion. These genes function to transport ATP-binding cassette proteins and are encoded for plasma membrane proteins. Many PDR genes mediate membrane translocation of ions and have a wide range of substrates. It appeared that expression of multiple numbers of the PDR gene family are necessary for cell survival and detoxification of the biomass fermentation inhibitors such as furfural and HMF. This work was presented and abstract published in the conference proceedings. In 2006, work will be continued to carry out for further illustration of these gene functions.
Discovery of furan tolerance genes from C. ligniaria--a natural strain able to grow on furfural and HMF--that can provide S. cerevisiae the ability to grow in previously lethal concentrations of furfural: In efforts to engineer yeast strains that are tolerant to various fermentation inhibitors, we have looked beyond S. cerevisiae's genome for help. A wild growing fungus, C. ligniaria, has been identified that grows on furfural and HMF as its sole carbon source. We have attempted to find genes from C. ligniaria that, when placed in S. cerevisiae, will confer a growth advantage to S. cerevisiae when furfural or HMF is present. Currently, we have screened over 10,000 genes from C. ligniaria and have identified at least 3 genes that do indeed appear to allow S. cerevisiae to grow in otherwise lethal concentrations of furfural. This finding is a promising step toward engineering inhibitor tolerant yeast strains capable of lower-cost conversion of biomass sugars to bioproducts such as ethanol for biofuels.
Optimization of a culture medium for kinetic evaluation of stress tolerant strains and design of nutritional and environmental conditions to foster the expression of tolerance during ethanol fermentation of sugars from lignocellulose: Pichia stipitis NRRL Y-7124 is a natural yeast able to convert xylose to ethanol, a capability not possessed by traditional Saccharomyces strains used to produce ethanol from corn starch. To investigate nutrition-based strategies for improving xylose to ethanol conversion by P. stipitis, kinetic studies were conducted on cultures provided a defined medium which was varied in nitrogen, vitamin, mineral, and purine/pyrimidine content. Mineral and nitrogen source optimization significantly enhanced D-xylose conversion to ethanol by the yeast P. stipitis. Certain amino acids, especially proline, arginine and histidine significantly improved both ethanol and cell yields. Results further showed that ethanol production from 150 g/L xylose was significantly enhanced by optimizing the combination of urea and amino acids to supply 50-80% nitrogen from urea and 50-20% from casamino acids. When either urea or casamino acids was used as sole nitrogen source, ethanol accumulation dropped to 11 or 24 g/L, respectively, from the maximum of 53 g/L for the optimal nitrogen combination. Magnesium, iron, and zinc sulfates significantly improved ethanol production supported by casamino acids, urea, or their combination, while manganese chloride reduced it. The striking influence of nitrogen source and mineral composition on ethanol yield will be further investigated to study impact on yeast cell viability and inhibitor tolerance mechanisms. Efficient fermentation processes that foster inhibitor tolerance and allow ethanol production from both hexose and pentose sugars released by dilute acid hydroysis of low-cost lignocellulosic biomass are sought to support the expansion of the biofuels industry.
To further explore natural furan and ethanol tolerance mechanisms, an S. cerevisiae disruption library was screened to identify mutants that failed to grow or grew better than wild type in the presence of furfural or ethanol. Over 100 disruption mutants were identified that grew better than wild type and another 100 that failed to grow in the presence of 15 mM/L furfural. Furthermore, this screen identified 40 disruption mutants that grew better and 55 that grew slower or not at all compared to wild type in 8 or 10% ethanol. The identified gene mutants implicated several pathways in ethanol tolerance, including macromolecule modification and biosynthesis, organelle dynamics, plasma membrane and cell wall maintenance, fermentation, cell cycle, endocytosis, metabolite biosynthesis, and membrane transport mechanisms. Genes influencing furfural tolerance have been verified to be involved in pathways including transcription, translation, metabolite biosynthesis, cytoskeletal dynamics, and organelle function (vacuole, mitochondria, and peroxisome). Notably, our work provides the first evidence that the pentose phosphate pathway (long known for converting xylose to ethanol) plays a critical role in protecting yeast against furfural stress perhaps via generation of NADPH, a cofactor that is necessary for protein, nucleic acid, and lipid biosynthesis, and for protection from oxidative stress.
Once again, members of our NCAUR Crop Bioprotection research team combined forces with Fermentation Biotechnology scientists to discover furan tolerance genes from C. ligniaria--a natural strain able to grow on furfural and HMF--that can provide S. cerevisiae the ability to grow in previously lethal concentrations of furfural. This finding is a promising step toward engineering inhibitor tolerant yeast strains capable of lower-cost conversion of biomass sugars to bioproducts such as ethanol for biofuels.
Our optimization of culture nutritional factors has revealed another lead to follow in our investigation of inhibitor stress tolerance mechanisms and maintenance of yeast cell viability during fermentation--the striking influence of nitrogen source and mineral composition on ethanol yield in the fermentation of high xylose concentrations to ethanol by the natural pentose-fermenting strain P. stipitis. The design of culture nutritional and environmental conditions is expected to be key in fostering stress tolerance and rapid fermentation rates needed for efficient conversion of biomass-derived sugars to ethanol and other bioproducts.
Thus, according to our proposed plan, phenotype differences in natural and mutant yeast strains were demonstrated when inhibitor stress factors were applied. Our development of a unique quality-controlled gene microarray for ethanologenic yeast study has now allowed these phenotype differences to be exploited to track down key genes and gene systems for planned genomics studies that will elucidate stress tolerance mechanisms and how they are regulated and networked. For example, we have identified the pleiotropic drug resistance gene family as having a significant role in HMF stress tolerance. Genomics data will help to provide a genetic blueprint that will let us develop and deploy viable strategies for engineering industrial yeast strains and processes that foster inhibitor tolerance and enhance the profitability of biomass to ethanol conversion.
Gorsich, S.W. The physiological damage in yeast caused by furfural and the hunt for furfural tolerant genes in S. cerevisiae and other organisms. Department of Chemical Engineering at Lund University, Lund Sweden, August 25, 2005.
Liu, Z., Duboy, R.T., Press, C., Loper, J.E., Paulsen, I.T., Slininger, P.J. A new DNA oligo microarray for complete genome of Pseudomonas fluorescens pf-5. International Union of Microbiological Societies (IUMS 2005), San Francisco, CA, July 13-28, 2005.
Liu, Z.L., Slininger, P.J. Universal external RNA controls for gene expression analysis using microbial gene microarray and real time quantitative RT-PCR. 8th International Meeting of the Microarray Gene Expression Data Society, Bergen, Norway, September 11-13, 2005.
Gorsich, S.W., Slininger, P.J., Liu, Z.L. Physiological responses to furfural and HMF and the link to other stress pathways. 12th European Congress on Biotechnology, Copenhagen, Denmark, August 21-24, 2005.
Liu, Z., Slininger, P.J., Gorsich, S.W. 2005. Enhanced biotransformation of furfural and 5-hydroxymethylfurfural by newly developed ethanologenic yeast strains. Applied Biochemistry and Biotechnology. 121-124: 451-460.
Gorsich, S.W., Liu, Z., Slininger, P.J. 2005. The role of the pentose phosphate pathway in fermentation inhibitor tolerance. Biotechnology for Fuels and Chemicals Symposium Proceedings. Abstract No. 5-33.
Liu, Z., Slininger, P.J. 2005. Transcriptome dynamics of ethanologenic yeast in response to 5-hydroxymethylfurfural stress related to biomass conversion to ethanol [abstract]. Abstract Book. p. 991.
Liu, Z. 2005. Genomic adaptive response of yeast to biofuel fermentation inhibitors [abstract]. Society of Industrial Microbiology Annual Meeting. Abstract No. S37.
Slininger, P.J., Dien, B.S., Gorsich, S.W., Liu, Z. 2005. Mineral and nitrogen source optimization enhance d-xylose conversion to ethanol by the yeast Pichia stipitis [abstract]. Society of Industrial Microbiology Annual Meeting. Paper No. P07.
Gorsich, S.W. 2005. Stress tolerance in Saccharomyces cerevisiae cells overexpressing furfural-stress genes [abstract]. Gordon Research Conference Proceedings. Abstract No. 19.
Liu, Z., Slininger, P.J. 2005. Development of genetically engineered stress tolerant ethanologenic yeasts using integrated functional genomics for effective biomass conversion to ethanol. In: Collins, K., Duffield, J., Outlaw, J., editors. Agriculture as a Producer and Consumer of Energy. Wallingford, UK: CAB International, p. 283-294.