2007 Annual Report 1a.Objectives (from AD-416) Determine the metabolic, physiologic, and genetic fundamentals underlying stress tolerance of ethanologenic yeast strains and other microbes. Using this fundamental stress tolerance knowledge, engineer improved strains and/or design process conditions that foster stress tolerance and functionality of microbes for production of ethanol and bioproducts from corn fiber and other lignocellulosic materials, despite exposure to harsh environments. 1b.Approach (from AD-416) Screening for phenotype differences in stress tolerance of yeast strains will be the first step in the search for genes and gene systems fostering resilience to stress factors. Stress factors common to fermentations of lignocellulose hydrolzates will be the focus of this research, including various chemical fermentation inhibitors (furans, phenolics, organic acids, etc.), high ethanol concentration, wide pH and osmotic shifts, and the high temperatures needed for simultaneous saccharification-fermentation processes. Cultures screened will include natural ethanologenic yeasts capable of hexose and pentose fermentation, strains adapted to increasing levels of stress, an S. cerevisiae disruption mutant library, and S. cerevisiae transformants prepared using a cDNA library from a furan-resistant/detoxifying yeast. Genes responsible for observed changes in stress tolerance will be identified and characterized with respect to sequence homology, protein structural domains, and protective function. Cultural conditions (nutrition, carbon and nitrogen source/ratio/concentration, physiological cell age, osmotic pressure, pH, temperature, dissolved oxygen, and others) will also be varied to screen for shifts in stress tolerance. Quality-controlled microarrays will be designed and applied to identify genes associated with stress responses and to study the genomic response of cultures to applied stress factors during fermentation time courses in order to identify gene networks and control mechanisms involved in stress tolerance and structural domain. Genetic tools and information gained will be applied to engineer improved strains and optimize the fermentation process to foster improved stress tolerance for lower cost production of ethanol from hydrolyzates of lignocellulosic biomass. 3.Progress Report Although they were not scheduled to complete this year, the following milestones are currently in progress and expected to be met as planned by the scheduled completion year (FY in parentheses): 1.B.2. Identify genes/regulatory networks/function--genomic expression of stress tolerance (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 (FY 08); 1.B.6 Identify genes/regulatory networks/function--generate tolerance-specific gene array (FY 08); 2.A. Engineer commercial yeast strains (FY09); 2.B. Optimize bioreactor design (FY 09) 4.Accomplishments Tolerant ethanologenic yeast. Furfural and hydroxymethylfurfural (HMF) are key byproducts of dilute-acid hydrolysis, currently the most cost-effective pretreatment precursing the conversion of lignocellulose to ethanol. Most yeasts are unable to withstand and grow in the presence of biomass conversion inhibitors such as furfural and HMF. By repeated exposure to increasing inhibitor concentrations, we developed tolerant strains of xylose- and pentose-fermenting yeasts which can be used as inocula without initial acclimatization to transform furfural and HMF to less toxic compounds such as furan methanol and furan dimethanol, and to produce a normal yield of ethanol from sugars. Thus, no detoxification of inhibitors is needed such that cost is reduced, process procedures are simplified and additional wastes are eliminated. Genomic analysis of such adapted strains will provide a genetic basis of the practice for selection under directed pressure. Enhanced directed adaptation under laboratory conditions will significantly reduce the time needed to obtain desirable strains that can be applied to a broad range of fields. This research is conducted under National Program 307 Bioenergy and Energy Alternatives; Component 1--Ethanol; Problem Area: Process Efficiencies; and National Program 306 Quality and Utilization of Agricultural Products; Component 2--New Processes, New Uses, and Value-Added Foods and Biobased Products; Problem Area 2c: New and Improved Processes and Feedstocks. Development of universal ribonucleic acid (RNA) control. Housekeeping genes within a genome of interest have been used as controls for quantitative gene expression, but they have problems with bias due to variation resulting from different environmental stimuli. In order to study gene expressions associated with stress tolerance mechanisms in ethanologenic yeast and plant-protective bacteria, exogenous control genes insensitive to experimental stress stimuli were needed to standardize gene expressions and allow comparison of expression data between experiments. Our work resulted in the development of the first universal external RNA controls for microbial gene expression analysis using two different high- throughput assay platforms (microarray and quantitative real time polymerase chain reaction (qRT-PCR)). Application of the controls guards data reliability and reproducibility, and allows comparison of expression data across different platforms and from different experiment sets. This is the first time such controls are available since the high-throughput gene expression analysis techniques appeared; and this development contributes to the entire gene expression research community around the world, including broad U.S. agricultural research on stress-tolerant microbes for production of low-cost biofuels and biocontrol of crop diseases. A journal article was published and listed in the progress report for CRIS 3620-22410-007-00D to which this accomplishment equally pertains (Z. L. Liu and P. J. Slininger. 2007. Universal external RNA controls for microbial gene expression analysis using microarray and qRT-PCR. Journal of Microbiological Methods 68:486-496). This research addresses National Program 307 Bioenergy and Energy Alternatives; Component 1--Ethanol; Problem Area: Process Efficiencies; National Program 306 Quality and Utilization of Agricultural Products; Component 2--New Processes, New Uses, and Value-Added Foods and Biobased Products; Problem Area 2c: New and Improved Processes and Feedstocks; National Program 303 Plant Diseases; Component 4--Biological and Cultural Strategies for Sustainable Disease Management, Problem Statement 4c: Application of Sustainable Disease Management Tools; and National Program 306 Quality and Utilization of Agricultural Products; Component 1--Quality, Characterization, Preservation, and Enhancement; Problem Statement 1d: Preservation and/or Ehnancement of Quality and Marketability. Culture Conditions Optimizing Inhibitor Tolerance. The economics of converting biomass to ethanol biofuel are currently unfavorable. To expand the biomass to fuel ethanol industry, microorganisms are needed which ferment available sugars while surviving exposure to inhibitors (such as ethanol, furfural, and HMF). Furfural and HMF are key byproducts of dilute-acid hydrolysis, currently the most cost-effective pretreatment. The ability of Pichia stipitis (a natural xylose-fermenting yeast) to survive exposure to ethanol and hydrolyzate inhibitors and to detoxify inhibitors was significantly improved by optimizing the nitrogen source composition (urea and amino acids) and the levels of minerals supplied during cell production relative to the available sugar (glucose or xylose). Culture physiology also had significant impact on inhibitor resistance: Stationary-phase cells were found to be far more resistant to inhibitors than were growth-phase cells. These findings will help to shape process-strategies for most efficient conversion of lignocellulose to ethanol. This research addresses National Program 307 Bioenergy and Energy Alternatives; Component 1--Ethanol, Problem Area: Process Efficiencies; and National Program 306 Quality and Utilization of Agricultural Products, Component 2 New Processes, New Uses, and Value-Added Foods and Biobased Products, Problem Area 2c: New and Improved Processes and Feedstocks. Computational modeling for microbial stress tolerance. To improve economics and allow expansion of the biomass to fuel ethanol industry, stress-tolerant yeast are needed which can survive and ferment available sugars while exposed to inhibitors (such as ethanol, furfural, and HMF). Genomic studies of the stress response of yeast to fermentation inhibitors will be advanced by computational modeling of gene regulatory networks, but few systems biology models describe the genetic information flow over time in a large network. Under a National Research Institute grant awarded to the Agricultural Research Services (ARS) National Center for Agricultural Utilization Research (NCAUR) scientists, collaboration with New Mexico State University led to development of a preliminary dynamic system model inferring time-dependent gene interaction and regulation. The temporal dependencies described by this study have potential to provide key information accounting for the genomic mechanism of inhibitory HMF detoxification and tolerance in ethanologenic yeast. Continued refinement and use of this modeling technique will aid genomic analysis of inhibitor-tolerant adapted strains to provide a genetic basis of the tolerance mechanism and is expected to significantly reduce the time needed to develop desirable strains for lower-cost production of biofuels and bioproducts. This research addresses National Program 307 Bioenergy and Energy Alternatives; Component 1--Ethanol; Problem Area: Process Efficiencies; and National Program 306 Quality and Utilization of Agricultural Products; Component 2--New Processes, New Uses, and Value-Added Foods and Biobased Products; Problem Area 2c: New and Improved Processes and Feedstocks. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of new CRADAs and MTAs 2 Number of invention disclosures submitted 1 Number of patent granted 1 Number of non-peer reviewed presentations and proceedings 20 Review Publications Liu, Z. 2006. Genomic adaptation of ethanologenic yeast to biomass conversion inhibitors. Applied Microbiology and Biotechnology. 73(1):27-36. Song, M., Liu, Z. 2007. A linear discrete dynamic system model for temporal gene interaction and regulatory network influence in response to bioethanol conversion inhibitor HMF for ethanologenic yeast. Lecture Notes in Bioinformatics. 4532:77-95.