Location: Bioenergy Research2021 Annual Report
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.
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.
Work under Objective 1 identifies mechanisms of inhibitor tolerance via genomic and transcriptomic analyses, relevant for increased efficiency and economical conversion of biomass to fuels and chemicals. Genome sequencing of a Saccharomyces cerevisiae diploid strain with increased inhibitor tolerance, was completed. The draft genome has been assembled and annotation nearly completed; some additional manual annotation will be needed to improve accuracy. Haploid spores from the several strains have been isolated for growth studies and genome sequencing to identify genes and/or genome structure associated with improved inhibitor tolerance. Spores isolated from a single strain ascus demonstrate differences in inhibitor tolerance when grown in acid-hydrolyzed corn stover. Among the four spores, or tetrads, isolated from individual ascii, one spore demonstrates tolerance essentially identical to the parental strain, one has extremely poor tolerance and/or growth, and the other two exhibit intermediate tolerance. Genomes of four spores from one ascus along with two spores from another ascus have been sequenced and assembled. Initial analysis showed that poor-growing strains contain what is commonly referred to as the “wine circle,” a complex of five genes transferred from Zygosaccharomyces bailii. A unique aspect is that this complex of genes is inserted in a region of the genome that has not been previously seen as an insertion site. Haploid spores from the other two strains were isolated, but thus far do not demonstrate the same differences in tolerance. This work is ongoing and the impact of these findings through continued study may lead to improved inhibitor tolerance and economics of biomass conversion to useful products. To further dissect molecular mechanisms of inhibitor tolerance, levels of gene expression and metabolic pathways were compared between an industrial strain and an ARS-patented strain adapted from it to tolerate fermentation stressors. A new component of transposable element (TE) genes was found to impact the adapted resistance to toxic chemicals. All these acquired tolerant characteristics of the adapted yeast were distinct from the innate stress response of its progenitor. Our study found an unexpectedly lower number of nonsynonymous SNPs (single nucleotide polymorphism or single nucleotide variation) between the two strains. Instead, a significant difference in copy number (CN) variations involving TE genes was observed between the two strains. CN variation could be an important mechanism effecting yeast adaptation of tolerance against fermentation inhibitors. New knowledge and research efforts provide insight for developing next-generation biocatalysts for a sustainable biorefinery. In Objective 2, an inhibitor-tolerant fungal strain was mined as a source of genes that are activated in response to fermentation inhibitors. One of these, a putative aldehyde dehydrogenase, was tested for phenotypic effects in yeast. Native and yeast-optimized sequences were expressed in three different Saccharomyces cerevisiae genetic backgrounds; the presence of the cloned adh gene had no discernable effect on growth rate, lag phase, and density of S. cerevisiae challenged over a range of furfural (inhibitor) concentrations. Similarly, the addition of a putative permease did not impact growth in the presence of furfural. In Objective 3, a newer fluorescent reporter protein was optimized for expression in Saccharomyces yeasts. The goal of this work is to develop tools for engineering yeast and in particular, to understand genes and proteins expressed at low levels inside yeast cells. The NeonGreen protein is a much brighter fluorescent reporter variant than the commonly used green fluorescent protein (GFP). The reported yeast version of NeonGreen showed a stable secondary structure in the intermediate transcript that the protein is made from. Removal of the secondary structure resulted in a two-fold increase in expression. Expression was also tested in a bacterial system and removal of secondary structure increased expression approximately four-fold.
1. Transposable element genes impact yeast adaptation to toxic chemicals. Conventional industrial yeast is susceptible to stresses (such as chemicals) that occur in the cellulose-to-fuels fermentation process. In practice, yeast can adapt to have increased robustness, but mechanisms of adaptation are poorly understood. ARS scientists at Peoria, Illinois, discovered multiple transposable elements (TE; i.e. “movable genes) associated with adapted yeast tolerance against the major toxic chemicals that arise when biomas is used to make bioproducts. Genome sequence analysis conducted in collaboration with a scientist at Iowa State University produced evidence of specific changes caused by TEs. This new knowledge points to genes and metabolic strategies useful for continued development of more robust and efficient industrial strains. These research efforts aid production of sustainable energy for a cleaner environment.
2. Improved tool for engineering yeast. Fluorescent markers are essential for studying cellular proteins. They make it possible to see a protein’s location in the cell and investigate the dynamics of how much of, and when, a protein is made. A newer fluorescent protein, reported to have increased brightness and maturation time compared to other reporter proteins, is useful for analyzing proteins even when they are present in small amounts. ARS scientists at Peoria, Illinois, created a version of the protein that more than doubled its brightness in yeast. This improved sensor will allow scientists to visualize essential yeast proteins that are produced at very low levels. Improving the understanding of how cells control and regulate protein expression makes it easier to engineer yeast strains to produce fuels and chemicals. This research benefits scientists using Brewer’s yeast as a model organism and producers of renewable products seeking to optimize yields.
Liu, Z., Huang, X. 2020. A glimpse of potential transposable element impact on adaptation of the industrial yeast Saccharomyces cerevisiae. Federation of European Microbiological Societies Yeast Research. 20(6). Article foaa043. https://doi.org/10.1093/femsyr/foaa043.
Mertens, J.A., Skory, C.D., Nichols, N.N., Hector, R.E. 2020. Impact of stress-response related transcription factor overexpression on lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae environmental isolates. Biotechnology Progress. 37(2). Article e3094. https://doi.org/10.1002/btpr.3094.
Jimenez, D., Wang, Y., de Mares, M., Cortes-Tolalpa, L., Mertens, J.A., Hector, R.E., Lin, J., Johnson, J., Lipzen, A., Barry, K., Mondo, S.J., Grigoriev, I.V., Nichols, N.N., Van Elsas, J.D. 2019. Defining the eco-enzymological role of the fungal strain Coniochaeta sp. 2T2.1 in a tripartite lignocellulolytic microbial consortium. FEMS Microbiology Ecology. 96(1). Article fiz186. https://doi.org/10.1093/femsec/fiz186.
Nichols, N.N., Hector, R.E., Mertens, J.A., Frazer, S.E. 2020. Abatement of inhibitors in recycled process water from biomass fermentations relieves inhibition of a Saccharomyces cerevisiae penthose phosphate pathway mutant. Fermentation. 6(4). Article 107. https://doi.org/10.3390/fermentation6040107.