Research Project: Biochemical Technologies to Enable the Commercial Production of Biofuels from Lignocellulosic Biomass

Location: Bioenergy Research

2018 Annual Report

Objectives
Objective 1. Develop technologies that enable the commercial production of marketable lipid-based advanced biofuels from lignocellulosic biomass hydrolyzates. Objective 2. In collaboration with industrial biorefiners, develop technologies that enable widespread commercial production of cellulosic ethanol from lignocellulosic biomass. Objective 3. Develop technologies that serve as co-products for lignocelluloses based refineries and as antibiotic alternatives for use in agriculture and animal production.

Approach
Goal 1. Develop oleaginous yeast and associated processes for converting hydrolyzates of lignocellulosic biomass to lipids for biodiesel and valuable co-products for other uses. Goal 2. Apply novel patented stress-tolerant yeast strains under commercial conditions to convert hydrolyzates of lignocellulosic biomass to ethanol. Goal 3. Develop protective soil bacteria and yeast engineered with bacterial AMP genes for cultivation on biorefinery substrates to supply new antimicrobials for animals and agriculture.

Progress Report
Progress has been made on all three Objectives of this project, all of which fall under National Program 306, Component 3, Biorefining. Specific examples of progress in FY 2018 include: Objective 1: • Yarrowia lipolytica is the only oleaginous (oil-producing) yeast used industrially. Molecular engineering and analytical tools were developed and used to engineer Yarrowia strains for enhanced growth on hydrolyzates and increased lipid production. This is the first time that Yarrowia robustness for growth on hydrolysate has been improved by expression of a single gene. The same strategy is expected to be applicable to other Y. lipolytica strains. • A method was developed that streamlined the process for selecting oil-producing yeasts and the new yeast strains identified were characterized for lipid production on various sugars and hydrolysate. Three of the strains isolated from nature accumulated greater than 30% lipids when grown on lipid production medium. • Lipid production was improved for four of our most robust oil-producing yeast strains that were discovered from our ARS culture collection. • A new strategy was developed to introduce foreign genes into commercially relevant Yarrowia strains. • A new medium for cell culture was developed that allows for growth of Yarrowia strains on crude glycerol and thin stillage, two agricultural low-value process streams. Objective 2: • Using an ARS-patented yeast strain, inhibitor tolerance pathways were identified that are important for the strains increased resistance to fermentation inhibitors present in lignocellulosic hydrolysates. This work suggested new strategies to develop yeast strains with greater resistance to toxic chemical stress conditions. Objective 3: • In consultation with ARS poultry disease researchers in Ames, Iowa, progress was made expressing antimicrobial agents in Yarrowia lipolyitca. The new yeast strains will be tested as a poultry feed supplement to improve animal health and productivity without the need for antibiotics. • Alternative antifungal microbial products are being developed to replace thiabendazole, which is now ineffective against postharvest diseases of potatoes due to resistance of the causative pathogens. Biological control agent (BCA) strains were developed that had increased tolerance of drying and dry storage. This phenotype is desired for more economical and convenient storage, allowing application of BCAs on a large scale. Studies are underway to optimize formulation compositions to enhance rehydration and rapid reactivation of cells, thereby boosting efficacy once delivered to potatoes.

Accomplishments
1. Novel gene expression system for Yarrowia. The Yarrowia yeast genus is the sole oleaginous (i.e., oil producing) yeast used commercially. However, tools for engineering this yeast are limited. To facilitate the use of this organism for the production of bio-renewable products, ARS scientists in Peoria, Illinois, developed a novel molecular method that allows simultaneous expression of multiple foreign genes, allows use of commercially desirable genetic backgrounds, and does not propagate antibiotic resistance genes. This new method is ideally suited to biotechnology companies working with this organism and will be of interest to anyone considering Yarrowia as a yeast strain for producing food, feed, chemical, or biofuel.

2. Dry tolerant anti-fungal biological control strains as biorefinery products to protect postharvest potatoes. Over 80% of fungal strains that cause potato dry rot are now resistant to thiabendazol (TBZ) and there is growing pressure to develop non-azole alternatives. However, chemical substitutes are limited, especially in postharvest potatoes destined for food use. ARS scientists in Peoria, Illinois, developed bacterial strains that are antifungal biocontrol agents active against dry rot, late blight, and pink rot, and also reduce sprouting. The new bacteria strains can be grown using switchgrass hydrolyzate and show excellent survival during dry storage conditions. Production of a broad-spectrum antifungal agent on low cost renewable substrates and improved dry storage formulations of the biocontrol product are expected to lower costs and expedite application by growers. This new technology benefits agriculture by providing an antifungal microbial alternative to azole chemicals and by serving as a potential co-product of a renewable lignocellulose biorefinery with the effect of boosting economic feasibility.

3. Sensor genes to engineer enhanced cell protection mechanisms in yeast. When renewable plant biomass is hydrolyzed to simple sugars, furfural and hydroxymethyl furfural (HMF) form as byproducts which inhibit microbial conversion to fuels and chemicals. Using an inhibitor tolerant yeast strain, ARS scientists in Peoria, Illinois, identified a gene responsible for sensing inhibitors that had a significantly higher expression level in the presence of furfural and HMF. This feature is advantageous to stimulating cell protection mechanisms, and represents a genetic strategy to engineer yeast for cellulosic biomass conversion with improved resistance to inhibitors. Robust industrial yeast strains are vital to low cost fuels and chemicals from renewable plant biomass and the advancement of national energy independence, strong rural economy, and preservation of the environment.

Review Publications
Quanzhou, F., Liu, Z.L., Weber, S.A., Li, S. 2018. Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae. PLoS One. 13(4):e0195633. doi: 10.1371/journal.pone.0195633.
Liu, Z.L., Wang, X., Weber, S.A. 2018. Tolerant industrial yeast Saccharomyces cerevisiae posses a more robust cell wall integrity signaling pathway against 2-furaldehyde and 5-(hydroxymethyl)-2-furaldehyde. Journal of Biotechnology. 276-277:15-24. doi: 10.1016/j.jbiotec.2018.04.002.
Nichols, N.N., Quarterman, J.C., Frazer, S.E. 2018. Use of green fluorescent protein to monitor fungal growth in biomass hydrolysate. Biology Methods and Protocols. 3(1)bpx012. doi: 10.1093/biomethods/bpx012.
Vogel, K.P., Casler, M.D., Dien, B.S. 2017. Switchgrass biomass composition traits and their effects on its digestion by ruminants and bioconversion to ethanol. Crop Science. 57(1):275-281. https://doi.org/10.2135/cropsci2016.07.0625.
Dias-Lopes, D., Rosa, C.A, Hector, R.E., Dien, B.S., Mertens, J.A., Ayub, M.A.Z. 2017. Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates. Journal of Industrial Microbiology and Biotechnology. 44(11):1575-1588. doi: 10.1007/s10295-017-1979-z.
Wang, Z., Sharma, V., Dien, B.S., Singh, V. 2018. High-conversion hydrolysates and corn sweetener production in dry-grind corn process. Cereal Chemistry. 95:302-311. https://doi.org/10.1002/cche.10030.
Liu, Z.L. 2018. Understanding the tolerance of the industrial yeast Saccharomyces cerevisiae against a major class of toxic aldehyde compounds. Applied Microbiology and Biotechnology. 102(13):5369-5390. doi: 10.1007/s00253-018-8993-6.
Wang, Z., Dien, B.S., Rausch, K.D,. Tumbleson, M.E., Singh, V. 2018. Fermentation of undetoxified sugarcane bagasse hydrolyzates using a two stage hydrothermal and mechanical refining pretreatment. Bioresource Technology. 261:313-321. https://doi.org/10.1016/j.biortech.2018.04.018.
Seshadri, R., Leahy, S.C., Attwood, G.T., Hoong Teh, K., Lambie, S.C., Eloe-Fadrosh, E.A., Pavlopoulos, G.A, Hadjithomas, M., Varghese, N.J., Paez-Espino, D., Hungate1000 Project Collaborators*: Palevich, N., Janssen,P.H.,Ronimus, R.S., Noel, S., Soni, P., Reilly, K., Atherly, T., Ziemer, C., Wright, A.D., Ishaq, S., Cotta, M., Thompson, S.R., Crosley, K., Mckain, N., Wallace, R.J., Flint, H.J., Martin, J.C., Forster, R.J., Gruninger, R.J., McAllister, T., Gilbert, R., OuwerkerK, D., Klieve, A., Jassim, R.A., Denman, S., McSweeney, C., Rosewarne, C., Koike, S., Kobayashi, Y., Mitsumori, M., Shinkai, T., Cravero, S., Ceron Cucchi, M.*, Perry, R., Henderson, G., Creevey, C.J., Tarrapon, N., Lapebie, P., Drula, E., Lombard, V., Rubin, E., Kyrpides, N.C., Henrissat, B., Woyke, T., Ivanova, N.N, Kelly, W.J. 2018. Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection. Nature Biotechnology. 36:359-367. doi: 10.1038/nbt.4110.