2011 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.
ARS Bioenergy Research Unit scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, in collaboration with scientists at New Mexico State University (NMSU) under a National Institute of Food and Agriculture (NIFA) award, identified at least 3 key regulatory elements and 365 candidate genes enabling yeast adaptation and inhibitor-tolerance using systems biology approaches. Interactions and gene regulatory networks were uncovered for the most predominant genes using hydroxymethylfurfural (HMF) challenge as an example. Tolerant yeast strains of Saccharomyces cerevisiae were able to adapt and in situ detoxify lignocellulose derived inhibitors such as furfural and 5-hydroxymethylfurfural. However, mechanisms of yeast tolerance to biomass pretreatment inhibitors at the genome level were unknown. New knowledge obtained by this research at the genome level provides insight into mechanisms of yeast adaptation and tolerance to lignocellulose derived inhibitors. Findings of this research directly aid engineering efforts for development of next generation biocatalysts toward economic lignocellulose-to-ethanol production in industrial applications.
In addition to overcoming inhibitory compounds generated during biomass pretreatment, another significant technical challenge is to efficiently utilize pentose sugars such as xylose that richly harbored in plant biomass. S. cerevisiae is the classical ethanologenic yeast but limited in pentose utilization. Available engineered yeast strains often use xylose at inefficient rates. Building upon the mechanism insight into the yeast tolerance, scientists at NCAUR genetically engineered an industrial yeast strain to improve its pentose utilization capability. The newly created yeast strain is tolerant to hydrolysate inhibitors and able to utilize C-5 and C-6 sugars for biofuel conversion applying systems biology approaches. New knowledge and strain materials generated by this research will benefit economic lignocellulose-to-ethanol production and potential industrial applications. An ARS patent application is currently in process.
This represents the final report of reimbursable agreement 3620-41000-147-01R between ARS and NRI. The Authorized Departmental Officer’s Designated Representative (ADODR) monitored the activities of this agreement through monthly teleconferences and frequent e-mail contacts.