Research Project: Develop Technologies for Production of Platform Chemicals and Advanced Biofuels from Lignocellulosic Feedstocks

Location: Bioenergy Research

2018 Annual Report

Objectives
Objective 1. Develop platform yeast technology to enable commercial conversion of lignocellulose-derived xylose to chemicals such as triacetic acid lactone (4-hydroxy-6-methyl-2-pyrone). Sub-objective 1.A. Develop an expanded xylose inducible expression system with various expression levels for tunable control of gene expression in Saccharomyces yeasts. Sub-objective 1.B. Generate a xylose-specific transporter that is not significantly inhibited by glucose. Sub-objective 1.C. Engineer industrial Saccharomyces cerevisiae strains to produce triacetic acid lactone from xylose. Objective 2. Develop technologies that enable the commercial production of itaconic acid (methylene succinic acid) from all the carbohydrates in lignocellulosic feedstocks. Sub-objective 2.A. Screen Aspergillus terreus strains for itaconic acid production from xylose and arabinose. Sub-objective 2.B. Adapt the best performing itaconic acid producing A. terreus strain to (i) dilute acid pretreated wheat straw hydrolyzate for inhibitor tolerance and (ii) high concentrations of itaconic acid for itaconic acid tolerance. Sub-objective 2.C. Optimize process parameters for batch production of itaconic acid from dilute acid pretreated wheat straw hydrolyzate by (i) separate hydrolysis and fermentation (SHF) and (ii) simultaneous saccharification and fermentation (SSF). Sub-objective 2.D. Demonstrate the batch itaconic acid production from wheat straw at a pilot scale (100 L). Objective 3. Develop technologies that enable the commercial production of xylitol from lignocellulosic hydrolyzates. Sub-objective 3.A. Optimize xylitol production by Coniochaeta ligniaria C8100, a fungal strain that produces xylitol from xylose but does not grow on xylose. Sub-objective 3.B. Clone and express heterologous xylose reductase in C. ligniaria C8100. Objective 4. Develop technologies that enable the commercial production of butanol from sweet sorghum bagasse. Sub-objective 4.A. Develop efficient pretreatment and enzymatic saccharification processes for generation of fermentable sugars from sweet sorghum bagasse. Sub-objective 4.B. Integrate enzymatic hydrolysis, fermentation and product recovery schemes for conversion of pretreated sweet sorghum bagasse to butanol. Sub-objective 4.C. Evaluate process economics of butanol production from sweet sorghum bagasse.

Approach
Hypothesis 1.A. Expressing xylose metabolism genes from tunable xylose-inducible expression modules will improve yield and productivity from both glucose and xylose. Hypothesis 1.B. Enhanced co-utilization of xylose and glucose will increase the xylose utilization rate. Goal 1.C. Integrate the genes required for triacetic acid lactone (TAL) production from xylose into an industrial S. cerevisiae strain and produce TAL from lignocellulosic feedstocks. Goal 2.A. Through screening of Aspergillus terreus strains from varied sources, identify a strain that effectively produces itaconic acid from all sugars typically present in a lignocellulosic hydrolyzate. Goal 2.B. Determine if the mixed sugar utilizing and itaconic acid (IA) producing A. terreus strain will be able to tolerate the common fermentation inhibitors typically present in dilute acid hydrolyzates of lignocellulosic feedstock and high concentrations of IA through adaptive evolution. Goal 2.C. Develop efficient SHF or SSF process for itaconic acid production from pretreated lignocellulosic feedstocks. Goal 2.D. Scale up the itaconic acid production process from one L to 100 L. Goal 3.A. Optimize xylitol production from hemicellulosic hydrolyzates by the inhibitor-tolerant fungus C. ligniaria C8100. Hypothesis 3.B. Increasing xylose reductase activity in C. ligniaria strain 8100 will enhance xylitol production from xylose by the recombinant fungal strain. Goal 4.A. Develop an optimized process of sweet sorghum bagasse pretreatment and enzymatic hydrolysis to release sugars that are efficiently fermented to butanol by Clostridium beijerinckii P260. Goal 4.B. Develop an integrated process for butanol production from pretreated sweet sorghum bagasse by combining enzymatic saccharification, fermentation, and product recovery. Goal 4.C. Perform economic analysis of conversion of sweet sorghum bagasse to butanol.

Progress Report
The overall goal of this project is to develop commercially targeted, integrated bioprocess technologies for production of platform chemicals (triacetic acid lactose, xylitol and itaconic acid) and advanced biofuel (butanol) from lignocellulosic feedstocks. The project emphasizes microbiologically based approaches to overcome technical constraints that impede industrial applications. It addresses and facilitates the elimination of microbial and fermentation related challenges associated with the production of platform chemicals and advanced biofuels from lignocellulose based feedstocks. Significant progress was made on all objectives, all of which fall under National Program 306, Component 3: Biorefining. Specific examples of progress in FY 2018 include: Objective 1: • Adaptation of an engineered industrial yeast strain expressing a USDA patented enzyme for increased sugar utilization was successful. The patented enzyme bypasses two steps of the cells metabolism, allowing more efficient sugar utilization for the yeast strain. • The genome of an inhibitor-tolerant fungus was sequenced and analyzed. The fungus, discovered by an ARS scientist, is able to consume the major inhibitors in lignocellulosic hydrolysates. Using the genome and gene expression data, ARS scientists identified new sugar transporters and enzymes for utilizing biomass-derived sugars. The new transporters and enzymes are important for increasing sugar utilization in industrial yeasts that are used to produce renewable fuels and chemicals. • Correct timing of enzyme production in the cell is often critical to achieving maximum activity of the enzyme. Three new classes of promoters that function at different phases of cell metabolism were tested for their ability to increase production of a platform chemical (triacetic acid lactone). One class of the new promoters resulted in a significant increase in product. Objective 2: • Itaconic acid is one of the 12 identified building block platform chemicals that can be produced by fermentation. We made significant progress identifying the specific components of wheat straw hydrolysate that prevent its production using an itaconic acid producing fungus. The levels to which these components are inhibitory were also determined, providing information needed to develop a commercial process using this organism. Objective 3: • A fungal strain was engineered to express an extra protein called xylose reductase, which is needed for producing xylitol, a naturally occurring sweetener. The new strain increased xylitol production by 22%. The strain used is resistant to inhibitory compounds present in sugars obtained from fibrous biomass, a trait that makes the strain useful for biomass-based processes. • We identified genes from an inhibitor-resistant fungus that are activated when it is grown on the biomass-derived sugar xylose. These new genes will be useful in engineering more efficient xylitol producing microbes and for increasing profitability in existing industrial biorefineries. Objective 4: • Sweet sorghum bagasse was pretreated with liquid hot water at high temperature at high solid loading. Generation of sugars from pretreated bagasse using commercial enzymes, fermentation of the generated sugars to acetone butanol ethanol (ABE) by a bacterium and recovery of ABE were performed simultaneously in a single process operation. Combining two or more process steps to run simultaneously results in lower production cost. • A new process for recovering acetone butanol ethanol (ABE) from fermentation broth was developed. This new extraction technology uses carbon dioxide, an abundant and inexpensive chemical.

Accomplishments
1. Expanded toolset for metabolic engineering of industrial yeasts. The majority of tools currently available for expressing new proteins (e.g., enzymes and sugar transporters) in cells produce a lot of protein, all of the time. However, constant high-level protein production is not always beneficial when it comes to rewiring cell metabolic pathways. ARS scientists in Peoria, Illinois, recently patented a technology for use in industrial Brewer’s yeast that only turns on protein expression when the biomass-derived sugar xylose is available. Based on this original technology, they created an expanded set of tools that also control the amount of protein made. The ability to control both the amount of protein produced, and when it is produced, allows fine-tuning of cellular pathways, resulting in more efficient sugar metabolism. Utilizing all of the sugars available in agricultural materials as efficiently as possible increases product yield and profitability. This technology is critical to developing engineered microorganisms and bioprocesses for converting agricultural wastes and energy crops into biofuels, chemicals, and polymers, which will expand domestic and export markets for American agriculture.

2. Itaconic acid production from agricultural residues. Itaconic acid has gained importance as a fully sustainable building block platform chemical for wide applications for the manufacture of various resins, coatings, polymers and clear plastics. It is currently produced industrially from corn-derived glucose by fermentation with a fungus. However, the production cost must be lowered in order to expand its market. Agricultural residues such as corn stover and wheat straw can be used as source of sugars for production of itaconic acid by the fungus. ARS scientists in Peoria, Illinois, found that the fungus could not grow and produce itaconic acid from wheat straw hydrolysate. Inhibitory components of the hydrolysate were identified and quantified. Tolerable levels of inhibitors for itaconic acid production by the fungus were established. The results will be highly useful for developing a process technology for itaconic acid production from agricultural residues and energy crops.

Review Publications
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.
Saha, B.C., Kennedy, G.J. 2017. Mannose and galactose as substrates for production of itaconic acid by Aspergillus terreus. Letters in Applied Microbiology. 66(6):527-533. https://doi.org/10.1111/lam.12810.
Saha, B.C., Kennedy, G.J. 2017. Ninety six well microtiter plate as microbioreactors for production of itaconic acid by six Aspergillus terreus strains. Journal of Microbiological Methods. 144:53-59. doi: 10.1016/j.mimet.2017.11.002.
Cortivo, P.R.D., Hickert, L.R.H, Hector, R., Ayub, M.A.Z. 2018. Fermentation of oat and soybean hull hydrolysates into ethanol and xylitol by recombinant industrial strains of Saccharomyces cerevisiae under diverse oxygen environments. Industrial Crops and Products. 113:10-18. doi: 10.1016/j.indcrop.2018.01.010.
Lopes, D.D., Cibulski, S.P., Mayer, F.Q., Siqueira, F.M., Rosa, C.A., Hector, R.E., Ayub, M.A.Z. 2017. Draft genome sequence of the D-Xylose-Fermenting yeast Spathaspora xylofermentans UFMG-HMD23.3. Genome Announcements. 5(33):e00815-17.
Klasson, K.T., Qureshi, N., Powell, R., Heckemeyer, M., Eggleston, G. 2018. Fermentation of sweet sorghum syrup to butanol in the presence of natural nutrients and inhibitors. Sugar Tech. 20(3):224-234.
Liu, S., Duncan, S., Qureshi, N., Rich, J.O. 2018. Fermentative production of butyric acid from paper mill sludge hydrolysates using Clostridium tyrobutyricum NRRL B-67062/RPT 4213. Biocatalysis and Agricultural Biotechnology. 14:48-51. https://doi.org/10.1016/j.bcab.2018.02.002.
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.