2009 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.
In collaboration with New Mexico State University under National Research Institute funding, we identified genes and gene regulatory networks, established discrete dynamic system models, and continued the characterization of gene interactions in response to stress and diauxic sugar utilization challenges. The collaborative efforts were monitored by frequent emails, phone conversations, and occasional visits. Using newly developed enhanced rigorous gene expression technology, we discovered reprogrammed pathways by tolerant ethanologenic yeast strain NRRL Y-50049 that allow the yeast to in situ detoxify biomass conversion inhibitors while producing ethanol. Inhibitory compounds generated during hydrolysis pretreatment of lignocellulosic biomass inhibit cell growth and interfere with subsequent fermentation of fermentative microbes. Currently, there is no tolerant yeast available for cellulosic ethanol production at an industrial scale. Development of a tolerant yeast to overcome inhibitor stress is needed for a sustainable biomass-to-ethanol industry. Using the tolerant yeast strain Y-50049 developed at ARS, we identified numerous genes, new elements, and alternative pathways responsible for the detoxification of the inhibitors and ethanol production. The tolerant yeast can be applied for two-stage fermentation using hydrolysates without additional treatment, such as overliming to remove inhibitors. Thus, processing procedures can be simplified and cost reduced. Knowledge obtained from this study contribute to the understanding of the mechanisms of stress tolerance and guides future, more robust strain development in the bioenergy field.