2007 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 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.
This report documents accomplishments under a Reimbursable Agreement
between the National Research Institute (NRI) and Agricultural Research Services (ARS). Additional details of research can be found in the parent project 3620-41000-123-00D entitled "Genomics and Engineering of Stress-tolerant Microbes for Lower Cost Production of Biofuels and Bioproducts." Since the NRI grant award in September 2006, we have studied mechanisms of in situ detoxification of biomass conversion inhibitors by the tolerant ethanologenic yeast Saccharomyces cerevisiae. We discovered and characterized a group of enzymes potentially responsible for the detoxification of furfural and HMF that involves nicotinamide dinucleotide phosphate-dependent aldehyde reduction. This detoxification function is mediated by aldehyde reductase encoded by multiple genes but not a single gene. We identified responsible genes and cloned major functional genes; characterization of these genes is underway. A manuscript entitled “Multiple gene mediated aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae” was completed and submitted to Applied Environmental Microbiology and is under review. In conjunction with quantitative gene expression analysis, we further developed a robust standard for quantitative real time polymerase chain reaction. This development provided a consistent quality control and normalization standard that replaces unreliable housekeeping genes used for such assays. As a result, two Material Transfer Agreements with researchers from Germany and France have been put in place. For this project, we had two conference presentations this year to report our research results. Research is being monitored by annual reporting to NRI.
5.Significant Activities that Support Special Target Populations