2010 Annual Report
1a.Objectives (from AD-416)
1) Determine the key metabolic, physiologic, transport, genetic, and regulatory mechanisms underlying stress tolerance and adaptation in ethanologenic yeast when they convert lignocellulosic hydrolyzates. .
2)Via directed evolution, genetic engineering, and/or adaptation, create new commercially preferred yeast strains for converting lignocellulose hydrolyzates to ethanol. .
3)In collaboration with Cooperative Research and Development (CRADA) partner(s), optimize fermentation process conditions so as to (1) leverage advantages of stress tolerant strains developed in Objective 2 and (2) minimize the cost of fermenting lignocellulosic hydrolyzates to fuel-grade ethanol.
1b.Approach (from AD-416)
The lignocellulose-to-ethanol process involves pretreatment of biomass to predispose it to chemical and enzymatic hydrolysis, saccharification of sugar polymers to simple sugars, and fermentation of the sugars to ethanol. The hydrolysis product is difficult to ferment because inhibitory byproducts are produced, and the resulting sugar mixture contains both hexose and pentose sugars, the latter not fermentable by traditional brewing yeasts. Needed are improved yeast strains which will ferment both types of sugars and are able to withstand, survive, and function in the presence of inhibitors (including furfural and hydroxymethyl furfural), high ethanol concentration and osmotic pressure, and sufficiently elevated temperatures for simultaneous saccharification-fermentation processes. In the research proposed, fermentation hurdles will be overcome by combining process optimization strategies and strain improvements aided by new molecular biology tools allowing high throughput screening of whole genomes to identify key genes and gene networks involved in stress tolerance and sugar utilization. Products of the research will be stress-tolerant yeasts capable of resisting and detoxifying inhibitors and efficiently fermenting hexose and pentose sugars to ethanol, a genetic blueprint describing tolerance mechanisms and metabolic pathways, and optimal culture conditions and process configurations to lower costs by maximizing yeast stress resistance, ethanol productivity, and yield.
Project 3620-41000-147-00D started October 2009 and continues from Project 3620-41000-123-00D. Gene expression and metabolic profiling of an evolved tolerant Saccharomyces cerevisiae strain NRRL Y-50049 and its parent revealed reprogrammed glucose metabolic pathways and interactive gene networks underlying tolerance to stress challenges by furfural and hydroxymethylfurfural (HMF).
Genetic constructs suitable for transformation were developed, and initial xylose utilizing recombinants with inhibitor-tolerance background were generated for future gene expression profiling.
Using evolutionary engineering, a more ethanol-tolerant strain (patent pending) was derived from inhibitor-tolerant Y-50049. Dynamic transcription analysis of tolerant derivative and parent identified candidate genes and key regulators enhancing ethanol tolerance.
In collaborative studies, first-year improved strains of the native pentose-fermenting yeast Scheffersomyces (Pichia) stipitis NRRL Y-7124 were obtained through targeted adaption in stressed environments with reduced oxygen, elevated temperature, elevated ethanol concentration, and high solids hydrolyzates (20-25%). Comparative kinetics documented significant improvements in adapted strains over parents.
Using directed enzyme evolution, we engineered the genetic code of a reductase gene commonly up-regulated during inhibitor stress. Clones with beneficial mutations established viable cultures under high HMF (30mM) stress and displayed engineered reductase activities toward HMF and furfural that were several fold higher compared with the parent gene. Derivatives of Y-50049 transformed with mutated genes showed modified enzyme cofactor preference and significantly higher levels of inhibitor detoxification.
Several putative xylose transporter genes present in the native pentose-fermenting S. stipitis genome were sequenced and cloned. Transformation of Y-7124 and an inhibitor-tolerant derivative of Y-50049 with cloned genes allowed assessment of xylose utilization and identification of transporter genes useful for future engineering of inhibitor–tolerant xylose fermenting strains.
Commercially preferred low-cost nitrogen sources were identified in laboratory media experiments for optimal ethanol production by Y-7124, inhibitor-tolerant Y-50049, and its parent. Studies to optimize nitrogen source composition for fermentation of dilute acid switchgrass hydrolyzates are underway and scheduled to finish by October 2010.
Studies showed that priming Y-7124 on xylose was key to successful use of mixed sugars because specific enzymes for xylose metabolism could be induced before repressive levels of ethanol accumulated. Studies to apply this strategy and cell recycling to hydrolyzate fermentation (utilizing a separable pentose stream) are underway and scheduled to complete by October 2010.
Ethanol inhibits induction of enzymes for xylose utilization: impact on process strategy. One challenge of making biofuels from lignocellulose is the production of economically recoverable ethanol from pentoses, such as xylose, which comprise about one-third of the available sugar. Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, discovered that moderately high ethanol concentrations (circa 4-5% on a weight per volume basis) completely repressed key enzymes required for xylose utilization by a natural pentose-fermenting yeast Scheffersomyces (Pichia) stipitis. By recycling cell populations grown on xylose, faster fermentation rates during mixed sugar conversion by S. stipitis occurred, allowing for ethanol accumulations in the 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. By understanding the mechanism of ethanol’s impact on the fermentation of xylose in lignocellulose hydrolyzates, fermentation process technologies can be designed to foster more efficient production of biofuel from plant biomass, a low-cost renewable energy source expected to reduce United States dependence on foreign petroleum.
Mechanism of furfural-induced yeast damage elucidated. One challenge of producing biofuels from plant lignocellulosic biomass is the cultivation of a robust fermentative microorganism that can tolerate the inhibitors, such as furfural, which are produced during biomass hydrolysis to fermentable sugars. Scientists at Central Michigan University, Johns Hopkins University, and the Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, collaborated to demonstrate that furfural induces the internal accumulation of compounds called “reactive oxygen species,” or ROS. As a result, furfural causes damage via ROS to internal yeast structures (such as vacuole membranes, actin, and chromatin) rather than the external cell wall. During furfural exposure, yeast were found to not only detoxify furfural into furfuryl alcohol, but also to protect themselves from its cellular effects and to repair damage. By understanding the mechanisms of inhibitor damage to cells, technologies can be developed to engineer both fermentation processes and robust yeast strains to foster yeast survival and more efficient production of ethanol from plant biomass, a renewable source of bioenergy expected to reduce United States dependence on foreign petroleum.
Molecular mechanisms of ethanol tolerance identified in the yeast Saccharomyces cerevisiae. The yeast S. cerevisiae is a superb ethanol producer which has been widely applied in production of biofuel from starch and more recently from lignocellulose hydrolyzates. However, it is sensitive to inhibitors present in hydrolyzates as well as to the ethanol product of fermentation. Applying a newly developed quantitative real time polymerase chain reaction (qRT-PCR) array and a S. cerevisiae derivative with tolerance to both ethanol and hydrolyzate inhibitors, Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, identified important genes, pathways, and regulatory genes responsible for ethanol tolerance. Knowledge of molecular mechanisms of ethanol tolerance resulting from this study will directly aid metabolic engineering efforts for more tolerant yeast development. More stress-tolerant yeast are desirable to rapidly ferment and accumulate high titers of ethanol in inhibitory hydrolyzates, reduce recovery costs, and deliver to consumers low cost biofuel from renewable plant biomass.
Ma, M., Liu, Z. 2010. Quantitative Transcription Dynamic Analysis Reveals Candidate Genes and Key Regulators for Ethanol Tolerance in Saccharomyces cerevisiae. Biomed Central (BMC) Genomics. 10:169.
Allen, S.A., Clark, W., Mccaffery, J.M., Cai, Z., Lanctot, A., Slininger, P.J., Liu, Z., Gorsich, S.W. 2010. Furfural Induces Reactive Oxygen Species Accumulation and Cellular Damage in Saccharomyces cerevisiae. Biotechnology for Biofuels. 3(2):1-10.
Liu, Z., Moon, J. 2009. A novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from Lignocellulosic biomass conversion. Gene. 446(1):1-10. DOI: 10.1016/j.gene.2009.06.018
Liu, Z., Menggen, M., Song, M.J. 2009. Evolutionarily Engineered Ethanologenic Yeast Detoxifies Lignocellulosic Biomass Conversion Inhibitors by Reprogrammed Pathways. Molecular Genetics and Genomics. 282(3):233-244.
Ma, M., Liu, Z. 2010. Mechanisms of Ethanol Tolerance in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 87(3)829-845.