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ARS Home Midwest Area Peoria, Illinois National Center for Agricultural Utilization Research Bioenergy Research Research





- Nichols, Nancy
- Hector, Ronald - Ron
- 2023
- 2022
- 2021
- Publications
- Product Quality and New Uses (306)

Research Project: Technologies to Improve Conversion of Biomass-Derived Sugars to Bioproducts **Location:
                            Bioenergy Research** 2023 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: An obstacle in converting biomass to fuels and chemicals is the presence of inhibitory compounds present in biomass sugar liquors that interfere with microbial growth and fermentation. Research in support of Objective 1 previously showed that individual spores, isolated from a single Saccharomyces cerevisiae yeast spore structure (ascus), demonstrate differences in tolerance to inhibitors when their growth is compared. Among the four spores, or tetrads, isolated from an ascus, one spore demonstrates tolerance essentially identical to the parental strain, one has extremely poor tolerance and/or growth, and the other two demonstrate intermediate inhibitor tolerance. This year, to further investigate the genetic components leading to varying inhibitor tolerance, differences in gene expression between the highly tolerant and weakly tolerant haploid strains were examined. The initial strategy for RNA Sequencing (RNA-Seq) experiments was to grow the strains in chemostat cultures using a concentration of inhibitors that was high enough to elicit a cellular response, but not high enough to prevent growth. Surprisingly, under these growth conditions, little difference was observed in gene expression, including genes known to increase or decrease in response to inhibitors.

Due to the unexpected results from the chemostat approach, a smaller number of growth experiments were then performed using a very high concentration of furan inhibitors, with comparisons at multiple time points over a shorter time course. In this second approach, RNA-Seq analysis uncovered significant differences in expression levels of aryl alcohol dehydrogenase and medium chain alcohol dehydrogenase enzymes between the tolerant and sensitive strains. These enzymes are recognized to convert toxic furan inhibitors to alcohols. Additional genes, both positively and negatively impacted by the furan inhibitors, also show expression differences among the tolerant and sensitive haploid strains. Because a smaller number of samples and strains were tested by this method, additional RNA-Seq experiments will be performed to narrow the number of genes identified at high statistical confidence. Already, analysis of the RNA-seq results together with the previously determined genome sequences has yielded a few genes that can be confidently targeted. These targets will be used to engineer S. cerevisiae haploid strains, to systematically identify genes that must be present (or absent) in yeast to improve their inhibitor tolerance. The RNA-Seq results and the analysis of gene expression will be submitted to a public database to enable freely available access to the data associated with this work. Development of tolerant strains for biomass conversion stands to improve efficiency and cost of producing renewable products from lignocellulosic biomass.

Objective 2: Conversion of biomass to fuels or chemicals requires liberation of biomass sugars from the chains (polymers) that make up plant fibers. In this conversion, acting on the complex fibers are a suite of enzymes, which, other than feedstock, are the costliest input. ARS researchers in Peoria, Illinois, discovered enzyme activities that are missing from commercial formulations. This year in support of Objective 2, ARS researchers developed systems to produce four candidate enzymes and expressed them in native and heterologous platforms. Activity was detected from two of these, and also led to investigation of export signal sequences for systems in which no activity was found. This work generated enzymes that can be used to characterize activities on carbohydrate polymers. Supplementation of commercial enzyme formulations, to release sugars more completely from fiber, could increase efficiency of biomass conversion to fuels and chemicals.

Other research in support of Objective 2 examined the response of a microbe to inhibitors that are found in sugars obtained from biomass; these inhibitors interfere with efficient conversion of biomass to products. This work exploited an inhibitor-tolerant microbe as a new source of genes important for hardiness. Two enzymes, identified as being “turned on” in the presence of inhibitors, were expressed and purified. The enzymes were both shown to act on microbial inhibitors and both use the same source of electrons to do so. The production of these enzymes in response to the presence of inhibitors points to their possible use to transfer “hardiness” traits to useful fermenting microbes. This work is important because the ability of microbes to withstand a challenging fermentation environment directly impacts the rate and yield, and therefore cost, of products formed.

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. This year, work continued identifying genetic changes responsible for improved xylose utilization in an adapted Brewer’s yeast strain that grows well using xylose. Seven genes with mutations were previously identified from genome sequencing of the adapted strain. Each gene was analyzed to determine if the mutation was responsible for improved growth on xylose. From this analysis, two gene mutations were identified that, when present, improved xylose utilization. These mutations are being transferred to additional industrial yeast strains to show their efficacy in multiple different strains. This year, 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 the mutation identified for improving growth on xylose. This strain grows very well on xylose and is also tolerant to lignocellulosic inhibitors. 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 product 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. If this is accurate, 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, a gene for producing triacetic acid lactone (TAL) was placed into the new vectors, representing high, medium, and low expression levels for each phase of the metabolic cycle. TAL production requires expression of a single gene, and it is made from a metabolite in Brewer’s yeast that has been shown to fluctuate through the yeast metabolic cycle. TAL is a potential platform chemical that can be converted to other chemical precursors that are normally petroleum-based and can be used in multiple commercial applications. Its production is one example of a bioproduct that can be produced in a biorefinery. However, any improvement in TAL production will be potentially applicable to all products in this class of chemicals known as polyketides. All vectors containing the gene for TAL production were transformed into Brewer’s yeast and are being analyzed for increased efficiency when producing TAL. **Accomplishments**
                                

1. Improving ethanol production by identifying transferable mutations that increase xylose utilization. Yeast have been engineered to consume xylose and make ethanol but suffer from numerous problems that limit their ability to convert xylose to ethanol. A yeast that efficiently ferments xylose and is tolerant to inhibitors present in biomass-derived sugar feedstocks is critical to developing bioprocesses for converting agricultural wastes and energy crops into biofuels, chemicals, and polymers, which will expand domestic and export markets for American agriculture. ARS researchers in Peoria, Illinois, generated an adapted yeast strain with increased ability to convert xylose to ethanol and identified the mutations in the adapted strain leading to increased productivity. They then demonstrated that the mutations could be transferred to other more industrially friendly yeast strains and continue to improve conversion of xylose to ethanol in a more robust strain. As an example of the potential impact, improving the fermentation rate by 10% will allow a 40 million gallon per year ethanol plant to produce 4 million extra gallons. Increasing production directly benefits the farmers by creating demand for their unused agricultural residues. 2. Improved inhibitor tolerance of yeast may lead to more efficient fuel production from agricultural waste. Plant residues, often called biomass, are an important source of simple sugars from which yeast make fuels and chemicals. Unfortunately, the process of obtaining sugars from biomass comes with a drawback: undesirable chemicals form that interfere with the ability of yeast to convert those simple sugars into fuels and value-added chemicals. ARS scientists in Peoria, Illinois, isolated a yeast strain that is more tolerant to inhibitors than commercial strains currently in use. ARS scientists analyzed the strains’ genomes and gene expression profiles to identify genetic aspects leading to improved tolerance. For example, some are involved in converting toxic compounds to more tolerable substances. This work expands understanding of genetic traits important for inhibitor tolerance and provides a path to improved productivity and cost-effectiveness of using biomass as a feedstock for production of fuels and chemicals. **Review Publications**
                            *de Godoi Silva, F., Dias Lopes, D., Hector, R.E., do Nascimento, M., de Avila Miguel, T., Kuroda, E., Andrade de Nobreag, G., Harada, K., Hirooka, E. 2023. Microcystin-detoxifying recombinant Saccharomyces cerevisiae expressing the mlrA gene from Sphingosinicella microcystinivorans B9. Microorganisms. 11(3). Article 575. https://doi.org/10.3390/microorganisms11030575.*
                                *Hector, R.E., Mertens, J.A., Nichols, N.N. 2022. Identification of mutations responsible for improved xylose utilization in an adapted xylose isomerase expressing Saccharomyces cerevisiae strain. Fermentation. 8(12). Article 669. https://doi.org/10.3390/fermentation8120669.*
                                *Nichols, N.N., Mertens, J.A., Frazer, S.E., Hector, R.E. 2022. Growth of Coniochaeta species on acetate in biomass sugars. Fermentation. 8(12). Article 721. https://doi.org/10.3390/fermentation8120721.*


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