Location: Bioenergy Research
2022 Annual Report
Objectives
Objective 1: Dissect molecular mechanisms underlying yeast tolerance against toxic chemicals present in lignocellulosic sugars, to enable engineering of biocatalysts for efficient biorefinery processes.
Objective 2: Discover genes and pathways activated in response to lignocellulosic hydrolysates in an inhibitor-tolerant fungus, to generate less-toxic feedstocks for producing bioproducts.
Objective 3: Develop new gene regulation technologies and engineer metabolic pathways for increased yield of bio-based products.
Approach
Renewable biofuels have the potential to reduce U.S. dependency on imported oil, lower greenhouse gas emissions, and enhance rural economies. It is estimated that biomass availability can exceed one billion tons per year. Although technologically proven, commercialization of lignocellulosic biomass biorefining has been slowed by technical risks and unfavorable operating and capital costs. A major limitation that remains, as an issue for biorefineries, is the lack of suitable biocatalysts tolerant to inhibitors generated during the production of fermentable sugars. Efficient fermentation of these biomass-derived sugars into bioproducts at high yields is also an ongoing challenge. To address these issues, this project plans to identify genes/alleles, regulatory sequences, and pathways that are required for tolerance to the major inhibitory compounds found in lignocellulosic hydrolysates. Additionally, the inhibitor-tolerant biocatalysts will be used as platform microorganisms for synthesis of multiple bioproducts.
Progress Report
Objective 1: Advanced methods (next generation sequencing technology) were used to determine and analyze the genome sequence of derivative strains of Saccharomyces cerevisiae strain YB-2625. This yeast is of interest because it is better at growing on unrefined cellulosic sugars than commonly used industrial Brewers’ yeast strains. The draft genome of the strain having increased inhibitor tolerance has been assembled and annotation completed. These results are being made available to other scientists: specifically, the sequencing reads have been deposited in the Genbank Sequence Read Archive (SRA) and the assembled annotated genome will be deposited into the National Center for Biotechnology Information (NCBI) Genbank genome database.
When diploid, or 2X genome, Saccharomyces strains sporulate they form ascii containing four haploid (1X genome) spores. This property is often used to identify the genetic mechanism determining various traits. Haploid spores isolated from the same Saccharomyces YB-2625 ascus demonstrate growth differences in acid hydrolyzed corn stover containing high concentrations of inhibitors. Among the four spores, or tetrads, isolated from individual ascii, one spore demonstrates tolerance essentially identical to the parental YB-2625 strain, one spore has extremely poor tolerance and/or growth, and the other two spores demonstrate intermediate inhibitor tolerance.
This pattern has been shown to follow for additional tetrads isolated from strain YB-2625. Genome analysis has shown differences in the genes present, particularly at the ends of a few chromosomes. The differences among the genomes could be impacting gene expression in the haploid strains leading to greater or lesser inhibitor tolerance. To understand at a more detailed level what was controlling varied inhibitor tolerance, differences in gene expression were compared for the highly tolerant and weakly tolerant haploid strains isolated from strain YB-2625. Gene expression was followed by using the method of RNA-Seq. Experiments were performed using a concentration of inhibitors that elicits a stress response but does not impact growth. This is important as we are only interested in aspects that aid in inhibitor tolerance and want to separate these from secondary effects that result from reduced growth. Analysis of the RNA-Seq data is ongoing and upon completion, the RNA-Seq data will be archived in NCBI databases for use by other researchers. This work provides a more thorough understanding of inhibitor tolerance in a widely used yeast and engineered yeast strains with improved tolerance to inhibitors leading to more efficient and cost-effective production of fuels and chemicals from biomass.
We are also developing a non-Saccharomyces yeast strain for biofuels production that operated at reduced enzyme costs because it makes one of the enzymes needed for breakdown of biomass to sugars. Earlier, a mutant of this yeast was evolved for growth on unrefined biomass sugars. It was discovered that the yeast was, in part, more robust than the unadapted yeast because of increased biosynthesis of certain amino acids, which are the building blocks for proteins. This year the adapted and unadapted yeast were compared for genetic changes and 34 mutations were discovered. Reintroducing these mutations into the control yeast will allow for us to identify the minimal number of changes needed to make this yeast more robust. This will allow us to design better yeast strains for producing cellulosic biofuels that operate with reduced enzyme costs.
Objective 2: This research exploits an inhibitor-tolerant filamentous fungus as a new source of tolerance genes. Two genes activated in the presence of inhibitors that putatively reduce the toxicity of furans, the major inhibitor found in cellulosic sugars were cloned and the enzymes expressed and purified. Both are flavin proteins; temperature and pH optima were determined, and enzyme activity was demonstrated for both enzymes in the presence of furan inhibitors. The reaction products are yet to be determined, and expression of one of the genes in yeast to test for enhanced inhibitor tolerance showed no effect on growth in the presence of inhibitors.
In addition to isolating the two genes from the filamentous fungus, researchers in Peoria, Illinois assembled a set of 21 fungi that are intrinsically tolerant to fermentation inhibitors. These were tested and select fungi were found to also tolerate acetate. Acetate is found in plant cell walls and when solubilized, acts as a microbial inhibitor that interferes with producing cellulosic biofuels by slowing fermentation. Four of the most resistant strains were further characterized for tolerance to combinations of inhibitors under relevant conditions for unrefined biomass sugars. At the highest acetate concentrations, only one strain consumed acetate when mixed with the furan furfural, but all four consumed acetate with added glucose. The best strains could consume the added furans within 24 hours, followed by acetate within 40 hours when grown in dilute acid pretreated rice hulls. This work identifies candidate strains that show potential for abating non-desirable compounds found in biomass hydrolysates and enabling conversion of sugars to bioproducts.
Objective 3: Last year ARS researchers in Peoria, Illinois, found a novel method to greatly improve the brightness of a fluorescent reporter gene that is used to monitor gene expression in yeast. This year, the same strategy was applied to both a luminescence and a new fluorescence-based reporting system to also improve their brightness and sensitivities.
These findings will be used to engineer yeast that are better able to produce chemicals and biofuels by providing knowledge of gene regulation. More broadly, this will help other yeast researchers using this commonly used technique to follow expression of various genes.
Improving the conversion rate and efficiency of cellulosic sugars to product is required for industrial adoption. A novel strategy has been developed that may allow for improved conversion rates in yeast. Typically, new products are engineered in yeast by expressing new genes at a constant (often high) rate of synthesis. However, there is evidence that yeast modulate their metabolic rate throughout their growth cycle. If this is accurate, tuning gene expressing to match this cycle will create less stress in the individual yeast cells and lead to enhanced cell health and rates of production. To test this hypothesis, a set of 24 tunable expression vectors was created for gene expression in Brewer’s yeast. A gene for utilizing the biomass-derived sugar xylose was placed into twelve of the new expression vectors, representing high, medium, and low expression levels for each phase of the metabolic cycle. All vectors containing the gene were transformed into Brewer’s yeast. The cells will next be analyzed for increased efficiency of xylose utilization. Work also continued to identify genetic changes responsible for improved xylose utilization in an adapted Brewer’s yeast strain that grows well using xylose. Seven genes identified from genome sequencing contained mutations.
Each gene is being analyzed to determine if the mutation is responsible for improved growth on xylose. Any industry using Brewer’s yeast to convert biomass-derived sugars to fuels or chemicals will benefit from the above research.
A common problem in corn ethanol fermentations is production of lactic acid by spoilage bacteria. If concentrated enough, the lactate slows down the rate and reduces the yield of ethanol. We have adapted a genetic tool developed earlier by ARS that triggers gene expression only in the presence of xylose to sense lactic acid instead. In cooperation with other ARS researchers in Peoria, Illinois, we are engineering yeast that will sense lactate and respond by releasing a biological active product that attacks the spoilage bacteria, thereby, saving the ethanol fermentation.
Accomplishments
1. Improved quality of unrefined biomass sugars for fermentation to biofuels. Unrefined sugars extracted from agricultural residues are difficult to ferment industrially because they contain numerous other chemicals that inhibit yeast growth. One of the most problematic of these is acetic acid (a component in vinegar) because it is persistent throughout the fermentation and dramatically lowers production at modest concentrations. It is also expensive to remove using present day technologies. ARS researchers in Peoria, Illinois, developed a process to conveniently remove acetate and other chemical inhibitors. The process is advantageous because it does not require additional equipment compared to other detoxification methods. The heart of the process is a fungus that is especially good at growing on acetate. The process was also successful for fermentation using sugars prepared from acid treatment of rice hulls, which is notoriously difficult to ferment because of its high acetate content. The fermentation treated with this fungus proceeded at high yield. While of special interest to rice farmers looking for a new market for their hulls, this work also directly benefits agricultural processors interested in biofuel production.
Review Publications
Liu, Z., Dien, B.S. 2022. Cellulosic ethanol production using a dual function novel yeast. International Journal of Microbiology. 2022: Article 7853935. https://doi.org/10.1155/2022/7853935.
Hector, R.E., Mertens, J.A., Nichols, N.N. 2021. Increased expression of the fluorescent reporter protein ymNeonGreen in Saccharomyces cerevisiae by reducing RNA secondary structure near the start codon. Biotechnology Reports. 33: Article e00697. https://doi.org/10.1016/j.btre.2021.e00697.
Cortivo, P.R.D, Aydos, L.F., Hickert, L.R., Rosa, C.A., Hector, R.E., Mertens, J.A., Ayub, M.A.Z. 2021. Performance of xylose-fermenting yeasts in oat and soybean hulls hydrolysate and improvement of ethanol production using immobilized cell systems. Biotechnology Letters. 43:2011-2026. https://doi.org/10.1007/s10529-021-03182-2.
