Location: Bioenergy Research
2024 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: Previous work on this objective identified several unique brewer’s yeast strains that are tolerant to high levels of inhibitory compounds present in lignocellulosic materials that have been processed to release fermentable sugars. These strains were analyzed to identify genetic targets that are responsible for the tolerance levels seen. Several target genes were identified that are present only in the tolerant strains.
Objective 2: Production of fuels or chemicals from biomass requires use of enzymes to break up the sugar chains (polymers) that make up plant fibers. Numerous enzymes act in concert to break down the complex fibers. The enzymes are regarded as the second most costly input to bioproducts, after the biomass, and ARS researchers in Peoria, Illinois, previously identified enzyme activities that are missing from commercial enzyme mixtures. This year, in support of Objective 2, ARS researchers in Peoria, Illinois, developed a platform for convenient production of an enzyme to fill the gap and found that placement of a protein marker typically used for enzyme recovery and purification positively impacted the enzyme’s activity. This result is important for making enough of the protein to work with and determining how the enzyme acts on biomass polymers. Then, the team began mining additional genomes for enzymes including some that are active at increased temperature. This work yielded a system that can be used to produce enzymes in a microbe that is naturally hardy and has useful characteristics for working in a biomass environment. Production of a “missing” enzyme to supplement commercial enzymes would allow for more complete release of sugars from fiber, yielding increased efficiency of producing biofuels and biochemicals.
Other research related to Objective 2 examined the use of modified grasses to increase production of ethanol as a potential alternative fuel. Working with a collaborator at Delaware State University, model grasses bearing alterations in genes responsible for the grasses structure were processed to release available sugars and then shown to be amenable to producing ethanol from these sugars. The conversion results were shared with the collaborator for identification of trends in yields consistent with the nature of the altered grass varieties.
Objective 3: Engineering microorganisms like Brewer’s yeast (Saccharomyces cerevisiae) to produce biobased products faces multiple challenges. One challenge addressed by Objective 3 focuses on improving use of the biomass sugar xylose. Complete consumption of xylose will result in significant increases in product yield from lignocellulosic hydrolysates, which can contain up to 40% xylose. Previously, one of the inhibitor-tolerant brewer’s yeast strains identified in Objective 1 was engineered to express the genes required for xylose utilization, in addition to a mutation identified for improving growth on xylose. This strain grows very well on xylose and is also tolerant to lignocellulosic inhibitors. This year, to better understand xylose uptake into the cell, which normally occurs through glucose transporters, seven of the main glucose transport proteins were deleted in this strain. Surprisingly, the strain lacking the main transport proteins retains its ability to grow well on both glucose and xylose. These results indicate that a novel sugar transporter is uniquely expressed in the strain. Other remaining glucose transporters are currently being investigated in the strain to discover the transporter’s identity. A yeast strain that efficiently ferments xylose and is tolerant to inhibitors present in biomass- derived sugar feedstocks is critical to developing bioprocesses for converting agricultural wastes into biofuels, chemicals, and polymers, which will expand domestic and export markets for American agriculture.
Another challenge addressed by Objective 3 is related to improving the rate and efficiency of converting biomass- derived sugars to products in engineered Brewer’s yeast strains. Expressing new enzymes and metabolic pathways to produce fuels and chemicals often leads to poor conversion to the final product. Poor conversion to product leads to increased time for the process as well as unutilized sugar at the end of the process, decreasing profitability. Improving the conversion rate and efficiency of cellulosic sugars to product is required for industrial adoption. To improve product yield, a new method of gene expression control is being used to coordinate enzyme expression with natural cell metabolism cycles. 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. Tuning gene expression to match this metabolite cycle will create less stress in the individual yeast cells and lead to enhanced cell health and increased production rates. To test this hypothesis, a set of 12 tunable expression vectors was previously created for gene expression in Brewer’s yeast. This year, genes for producing xylitol or itaconic acid were placed into the new vectors, representing high, medium, and low expression levels for each phase of the metabolic cycle. Xylitol and itaconic acid production each require expression of a single gene, and they both require a metabolite in Brewer’s yeast that has been shown to fluctuate through the yeast metabolic cycle. These are two examples of bioproducts that can be produced in a biorefinery. All vectors containing the genes for xylitol and itaconic acid were transformed into Brewer’s yeast and are being analyzed for increased efficiency.
Accomplishments
1. Improved brewer’s yeast for advanced biofuels. There is an unmet demand for second generation “cellulosic” ethanol to be catalytically upgraded to sustainable aviation fuel (SAF). Finding yeast hardy enough to grow and ferment unrefined cellulosic sugars is a major barrier to processing cellulosic agricultural residues into ethanol and other bio-based products. Previously, ARS scientists in Peoria, Illinois, identified an exceptionally hardy distiller’s yeast that performed well when challenged on unrefined cellulosic sugars. However, distillers’ yeast does not ferment a sugar, xylose, that happens to be 20-30% of the sugar present in sugar syrups manufactured from agricultural residues. The discovered yeast was also shown to stick to itself and to bioreactor walls, making it difficult to work with. Now, the ARS researchers in Peoria, Illinois, solved this problem by developing a new yeast that maintained the original yeast’s hardiness and has improved growth characteristics. The new yeast was successfully engineered to ferment xylose and produced 5-17 times more ethanol than other yeast strains when fermenting cellulosic sugars. The newly developed yeast will advance efforts to produce second generation ethanol because increased production directly increases plant revenue. Ethanol producers are keen to produce second generation ethanol because the SAF market is targeted to expand from the first commercial (9 million gallon) production facility, opened early in 2024, to 3 billion gallons by 2030.
Review Publications
Hector, R.E., Mertens, J.A., Nichols, N.N. 2023. Metabolic engineering of a stable haploid strain derived from lignocellulosic inhibitor tolerant Saccharomyces cerevisiae natural isolate YB-2625. Biotechnology for Biofuels and Bioproducts. https://doi.org/10.1186/s13068-023-02442-9.
Dias Lopes, D., Dien, B.S., Hector, R.E., Singh, V., Thompson, S.R., Slininger, P.J., Boundy-Mills, K., Jagtap, S.S., Rao, C.V. 2023. Determining mating type and ploidy in Rhodotorula toruloides and its effect on growth on sugars from lignocellulosic biomass. Journal of Industrial Microbiology and Biotechnology. https://doi.org/10.1093/jimb/kuad040.
Qureshi, N., Ashby, R.D., Nichols, N.N., Hector, R.E. 2024. Novel technologies for butyric acid fermentation: use of cellulosic biomass, rapid bioreactor, and efficient product recovery. Fermentation. https://doi.org/10.3390/fermentation10030142.
Liu, A., Machas, M., Mhatre, A., Hajinajaf, N., Sarnaik, A., Nichols, N.N., Frazer, S.E., Wang, X., Varman, A.M., Nielsen, D.R. 2023. Synergistic co-utilization of biomass-derived sugars enhances aromatic amino acid production by engineered Escherichia coli. Biotechnology and Bioengineering. https://doi.org/10.1002/bit.28585.