2010 Annual Report
1a.Objectives (from AD-416)
The overall objective of this project is to elucidate genomic mechanisms of detoxification and tolerance of ethanologenic yeast to biomass conversion inhibitors furfural and 5-hydroxymethylfurfural (HMF), and thereafter to genome-wise manipulate and engineer more robust strains for low-cost biomass conversion to ethanol. This study will identify and characterize genes involved in pathways relevant to detoxification, biotransformation, and tolerance to furfural and HMF involved in biomass conversion to ethanol; and elucidate regulatory mechanisms of major gene interactions in relevant pathways involved in furfural and HMF detoxification and tolerance using computational prediction and mathematical modeling.
1b.Approach (from AD-416)
We plan to study genomic regulatory mechanisms of inhibitor detoxification by yeast during ethanol production from dilute acid-hydrolyzed biomass. We propose to characterize the genomic transcriptional profiling of wild-type and several improved, more inhibitor-tolerant strains in response to furfural and 5-hydroxymethylfurfural (HMF) supplied in a defined culture medium. To accomplish this, yeast cells will be sampled in a time-course study to isolate total ribonucleic acid (RNA) and conduct microarray experiments using two-color microarray with spiking universal external RNA quality controls. Inhibitor and inhibitor-conversion products, glucose consumption, ethanol production, and other byproducts generated during the fermentation process will also be monitored during the time-course study to establish metabolic profiles for wild-type and more tolerant strains involved in detoxification of biomass conversion inhibitors. Based on data from culture time-course studies, we will propose computational models to predict the behavior of the gene function and expression of natural and genetically engineered networks under furfural and HMF stress. A dynamic mathematical model using difference equations and estimate parameters will be applied and tested for its ability to describe gene regulatory network behavior. Based on these approaches, we will form testable hypotheses to explain molecular and genomic mechanisms of yeast detoxification and tolerance to furfural and HMF.
The yeast Saccharomyces cerevisiae is a superb ethanol producer yet it is sensitive to high concentrations of ethanol. Using ethanol-tolerant yeast for higher yield ethanol fermentation is desirable for cost-efficient ethanol production. However, mechanisms of ethanol tolerance are not well known and ethanol-tolerant yeast strains are not readily available. As a part of stress tolerance research, we have investigated and illustrated molecular mechanisms of ethanol tolerance for the yeast. Using a newly developed in-house quantitative real-time polymerase chain reaction (qRT-PCR) array, we found that an ethanol-tolerant yeast strain exhibited enhanced gene expression of ethanol-tolerance genes associated with heat shock proteins, trehalose-glycolysis-pentose phosphate pathways and pleiotropic drug resistance gene family are associated with the ability to withstand the ethanol stress, maintain active metabolism, and complete ethanol fermentation under the ethanol stress. Transcription factor Msn4p appeared to be a key regulator of gene interactions for the ethanol-tolerance in yeast. Results of our study directly aid metabolic engineering efforts for more tolerant strain development. We have published two research articles on this topic.
In collaboration with scientists from New Mexico State University, we developed a novel computation program, twzPEA, for enriched pathway analysis using advanced topology strategy and microarray data and available database resources. This program allows more sensitive detection of pathways affected by fermentation stress for yeast. An abstract reporting this development has been published and currently, a manuscript is under preparation.
The Authorized Departmental Officer's Designated Representative monitored the activities of this agreement via e-mail contacts and monthly teleconferences.