Progress 08/29/14 to 07/24/19
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 2019 include: Objective 1: The genome of an inhibitor-tolerant fungus was previously sequenced and analyzed. The fungus, discovered by an ARS scientist in Peoria, Illinois, is able to consume the major inhibitors in lignocellulosic hydrolysates. Using the genome and gene expression data, ARS scientists identified new sugar transporters for cell uptake of xylose, a biomass-derived sugar. New transporters were tested for their ability to increase uptake of xylose in Saccharomyces yeasts. Two of the newly discovered transporters showed xylose transport ability. From three new classes of promoters that function at different phases of cell metabolism, one promoter was previously shown to increase production of a platform chemical (triacetic acid lactone). Gene expression from the promoters was further characterized, showing that increased production was due to more efficient timing of enzyme expression to achieve maximum activity of the enzyme. A tunable set of xylose inducible gene expression tools was further developed this year. The toolset was characterized to analyze xylose- induced gene expression throughout a batch culture. Stable gene expression was observed throughout the cell growth phase of the culture, after which, gene expression started to decrease. It was also shown that extremely low levels of xylose were needed to fully induced gene expression, indicating that xylose could be used as an inexpensive inducer at industrial scale. Objective 2: Itaconic acid is one of the 12 identified building block platform chemicals that can be produced by fermentation. We made substantial progress in removing the specific components of wheat straw hydrolyzate that prevent its production using an itaconic acid producing fungus. The levels of the inhibitory components removed were also determined, providing information needed to develop a commercial process using the fungus. In addition to typical fermentation inhibitory compounds, a metal (manganese) present in wheat straw hydrolyzate was found to be a strong inhibitor of itaconic acid production. A novel medium was then developed for production of itaconic acid which alleviated the strong inhibitory effect of manganese. The fungus was able to tolerate manganese very well in the newly developed medium for efficient production of itaconic acid. Egg shell powder, a food industrial waste, was used successfully as a detoxifying and buffering agent for efficient production of polymalic acid by a fungal strain from barley straw hydrolyzate. Polymalic acid and its component L-malic acid have a wide range of applications in the food, pharmaceutical, agriculture and chemical industries. Objective 3: Coniochaeta ligniaria strain C8100 does not grow on xylose, instead converting xylose to xylitol which is secreted. Culture conditions were investigated to identify important factors for xylitol production. Appropriate temperature and pH ranges were identified and xylitol production was measured both in culture medium and in sugars obtained by hydrolysis of corn stover. The addition of an aromatic chemical increased xylitol production. The xylose reductase gene from another fungus was cloned into the strain and increased yield by 20% in rich medium and 11% in corn stover hydrolysate. Objective 4: The production of butanol requires 3 operations, namely feedstock (sweet sorghum bagasse) hydrolysis to sugars, fermentation of sugars to butanol, and butanol (product) recovery from the fermentation broth. During the prior years we developed two processes: Process 1, where 2 operations were combined; and Process 2, where three operations were combined. Process 1 involves separate hydrolysis, combined with fermentation and product recovery. Process 2 involves simultaneous saccharification, fermentation, and product recovery. For these two processes, the cost of butanol production from sweet sorghum bagasse was estimated. For both processes, economic analysis was performed for a plant capacity of 100 thousand tons of butanol production per year. The cost of production of butanol was projected to be $3.48 per gallon for Process 1. Sweet sorghum bagasse cost was considered to be $50 per ton. The production cost is affected by feedstock price, enzymes (to hydrolyze feedstock), fermentation and recovery process used. For this process, the total cost of feedstock, enzymes, and other chemicals was $46.6 million per year. The utilities and other operating costs were at $50.1, and $46. 3 million per year, respectively. For Process 2 in which all three operations were combined, the production cost was estimated to be $3.28 per gallon. In this process, the total cost of feedstock, enzymes and other chemicals was $46.6 million per year. Utilities were at $50.1 million per year, and the other operating costs were $43.4 million per year. The operating cost was reduced by $3 million per year. Effect of sweet sorghum bagasse price on butanol production cost was also projected. If sweet sorghum bagasse price is reduced to $30 per ton, butanol production cost can be reduced to $2.89 per gallon using Process 2. Accomplishments 01 Efficient production of itaconic acid by relieving the strong inhibitory effect of a metal. Itaconic acid (a building block platform chemical with a variety of industrial applications) is currently produced industrially from glucose by a fungal fermentation. In order to expand the use of itaconic acid, its production cost must be lowered. Waste agricultural residues have the potential to serve as a low cost source of sugars for itaconic acid production. ARS researchers in Peoria, Illinois, found that a specific metal typically present in agricultural residues inhibits the itaconic acid production by the fungus completely. A novel growth medium was then developed for production of itaconic acid which relieved the strong inhibitory effect of the metal. The fungus was able to tolerate the metal very well in the newly developed medium. This new medium is expected to perform well for production of itaconic acid in the presence of the metal. 02 Production of xylitol by a novel inhibitor tolerant microbe. Production of xylitol by a novel inhibitor-tolerant microbe. Xylitol is a naturally-occurring sweetener that has 40% fewer calories than table sugar and has been shown to improve dental health and prevent ear infections. These desirable traits support use of xylitol in pharmaceutical and personal-care products, and as an alternative sweetener in gums and mints. Xylitol is difficult to extract from natural sources and, because the current chemical method of production has high energy and cost demands, a biological route to xylitol is desirable. ARS scientists in Peoria, Illinois, measured the effect of several factors on xylitol production using a microbe that makes xylitol from a sugar, xylose, found in biomass. The microbe has intrinsic resistance to inhibitors of the types encountered in converting biomass to fuels and chemicals. Production of xylitol from fibrous biomass would be a new use for agricultural residues that are typically viewed as low-value.
Impacts
(N/A)
Publications
- Jin, Q., Qureshi, N., Wang, H., Huang, H. 2019. Acetone-butanol-ethanol (ABE) fermentation of soluble and hydrolyzed sugars in apple pomace by Clostridium beijerinckii P260. Fuel. 244:536-544.
- Yegin, S., Saha, B.C., Kennedy, G.J., Leathers, T. 2019. Valorization of egg shell as a detoxifying and buffering agent for efficient polymalic acid production by Aureobasidium pullulans NRRL Y⿿2311⿿1 from barley straw hydrolysate. Bioresource Technology. 278: 130-137.
- Cheng, C., Tang, R., Xiong, L., Hector, R.E., Bai, F., Zhao, X. 2018. Association of improved oxidative stress tolerance and alleviation of glucose repression with superior xylose-utilization capability by a natural isolate of Saccharomyces cerevisiae. Biotechnology for Biofuels. 11:28.
- Qureshi, N., Klasson, K.T., Saha, B.C., Liu, S. 2018. Butanol production from sweet sorghum bagasse (SSB) with high solids content: Part I ⿿ comparison of liquid hot water pretreatment with dilute sulfuric acid. Biotechnology Progress. 34(4):960-966.
- Qureshi, N., Klasson, K.T., Saha, B.C., Liu, S. 2018. High solid fed-batch butanol fermentation with simultaneous product recovery: Part II - process integration. Biotechnology Progress. 34(4):967-972.
- Saha, B.C., Kennedy, G.J., Bowman, M.J., Qureshi, N., Dunn, R.O. 2018. Factors affecting production of itaconic acid from mixed sugars by Aspergillus terreus. Applied Biochemistry and Biotechnology. 187(2):449- 460.
- Mertens, J.A., Kelly, A., Hector, R.E. 2018. Screening for inhibitor tolerant Saccharomyces cerevisiae strains from diverse environments for use as platform strains for production of fuels and chemicals from biomass. Bioresource Technology. 3:154-161.
- Quarterman, J.C., Slininger, P.J., Hector, R.E., Dien, B.S. 2018. Engineering Candida phangngensis ⿿ an oleaginous yeast from the Yarrowia clade ⿿ for enhanced detoxification of lignocellulose-derived inhibitors and lipid overproduction. Federation Of European Microbiological Societies Yeast Research. 18(8):foy102.
- Nichols, N.N., Hector, R.E., Frazer, S.E. 2019. Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium. Biology Methods and Protocols. 4:1-5.
- Nichols, N.N., Hector, R.E., Frazer, S.E. 2019. Factors affecting production of xylitol by the furfural-metabolizing fungus Coniochaeta ligniaria. Current Trends in Microbiology. 12: 109-119.
- Saha, B.C., Kennedy, G.J. 2019. Phosphate limitation alleviates the inhibitory effect of manganese on itaconic acid production by Aspergillus terreus. Biocatalysis and Agricultural Biotechnology. 18:101016.
- Glover, K.D., Kleinjan, J., Jin, Y., Osborne, L., Ingemansen, J., Turnipseed, E., Dykes, L. 2019. Registration of ⿿Focus⿿ hard red spring wheat. Journal of Plant Registrations. 13(1):63-67.
- Qureshi, N., Harry-O'Kuru, R.E., Liu, S., Saha, B. 2018. Yellow top (Physaria fendleri) presscake: a novel substrate for butanol production and reduction in environmental pollution. Biotechnology Progress. 35(3) :e2767.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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.
Impacts
(N/A)
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. doi: 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. 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. doi: 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.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. The overall goal of this project is to develop commercially targeted, integrated bioprocess technologies for production of platform chemicals (triacetic acid lactone, 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 four sub-objectives, all of which fall under National Program 213, Component I: Biochemical Conversion. Under Sub-objective 1.A, initial versions of the construct to be used for identifying new xylose transporters (per Sub-objective 1.B) leaked expression of the marker gene, making it impossible to determine if a transporter improved xylose uptake into the cell. New versions of the marker were developed last fiscal year that show significant growth differences between repressed and induced conditions. Further modifications were made this year to the marker and strain, and growth conditions for testing new xylose transporters using this system have been identified. To facilitate the identification of novel xylose transporters, an alternative test strain is being developed based on the xylose-fermenting haploid yeast strain we developed. During the last fiscal year, genes associated with hexose transport were deleted and this fiscal year genes responsible for uptake were deleted. The final strain is being used as the parent for expressing and testing new transporters. Putative transporters from recently identified xylose-fermenting yeast strains were identified and engineered for expression in the test strain. Two transporters from Spathaspora xylofermentans, when expressed in the parent test strain, resulted in growth on xylose medium. The parent strain was unable to grow using xylose. These new transporters are being evaluated for use to enhance xylose utilization for the production of bioethanol. An ethanol yeast strain which displays good performance under inhibitory conditions was evaluated further to identify a genetic component for ethanol tolerance, acetic acid tolerance, furfural tolerance, and carbon source utilization in this strain. Forty haploid strains derived from ten starting cells are being evaluated for tolerance to the compounds mentioned above. Identification of the best performing strain selects for strains carrying genes with beneficial mutations that enhance the utilization of biomass-derived xylose. Under Sub-objective 2.C, significant progress was made toward itaconic acid (a building block platform chemical with wide applications for the manufacture of various synthetic resins, coatings, polymers and clear plastics) production from lignocellulosic hydrolyzates by a fungal strain. The fungal strain could not grow and produce itaconic acid from dilute acid pretreated and enzymatically saccharified wheat straw hydrolyzate. The hydrolyzate contained significant quantities of a compound at a concentration that was totally inhibitory to itaconic acid production by the fungus. The effects of other compounds present in the hydrolyzate on growth and itaconic acid production by the strain were investigated. Several factors such as pH, temperature and aeration affecting the production of itaconic acid by the fungal strain were investigated. A convenient and reproducible microtiter plate based method for fermentative production of itaconic acid was developed which will greatly aid in screening, developing mutants and optimizing various parameters for itaconic acid production by the fungal strain. One suitable fungal strain was obtained by evaluating twenty fungal strains for production of itaconic acid from mannose, a major sugar component of woody biomass. This strain can be used to produce itaconic acid from the sugars derived from woody biomass especially softwood. Under Sub-objective 3.B, the effects of several factors including temperature and pH on the production of xylitol (a naturally occurring sweetener that has 40% fewer calories than table sugar and has been shown to improve dental health and prevent ear infections) from biomass derived sugar xylose by a fungal mutant strain were investigated. Engineering this strain to carry an extra gene increased the xylitol yield by 11- 22%. The fungus withstands inhibitory compounds that are commonly found in sugars obtained from fibrous biomass and thus has useful properties for biomass utilization. This inhibitor-tolerant strain produced from 0. 34 to 0.71 g xylitol per g of xylose. Under Sub-objective 4.B, butanol (a superior biofuel than ethanol per gallon basis and has excellent gasoline blending properties) was produced from sweet sorghum bagasse at high solid loading. Sweet sorghum bagasse at 25% solid loading was first pretreated with liquid hot water at a high temperature followed by two stage enzymatic hydrolysis using commercial enzyme preparations. The first stage hydrolyzate (hydrolyzate I) contained about 90 g sugars per liter. The left over residue was then subjected to second stage enzymatic hydrolysis which yielded about half of the sugars obtained in first stage hydrolysis (hydrolyzate II). The fermentation of hydrolyzate II by an acetone butanol ethanol (ABE) producing anaerobic bacterium was started with simultaneous removal of ABE by energy efficient vacuum technique. Then the hydrolyzate I was added in a fed-batch mode to the fermentation vessel, and the fermentation and simultaneous recovery of ABE were continued until all sugars were utilized by the bacterium. Simultaneous removal of ABE by vacuum greatly helped to alleviate the strong inhibitory effects of ABE to the culture. In this process an ABE yield of 0.39 g per g sugar with high productivity was achieved. Accomplishments 01 Improved production of butanol to benefits farmers. Butanol is an advanced biofuel that packs 30% more energy than ethanol on per gallon basis and has excellent blending properties with gasoline. Agricultural Research Service scientists in Peoria, Illinois, developed an integrated fermentation and simultaneous product recovery process that enables 18% more production of butanol from sweet sorghum bagasse that will benefit US farmers, biofuel, and transportation industries. 02 Microtiter plate as microbioreactors for production of itaconic acid. Itaconic acid is a building block platform chemical which is currently produced industrially from corn-derived glucose by fermentation with a fungus. Lignocellulosic biomass has the potential to serve as a low cost source of sugars for production of itaconic acid; however, the fungus could not grow and produce itaconic acid from lignocellulosic biomass hydrolyzates. Agricultural Research Service scientists in Peoria, Illinois, have developed a micro-scale fermentation method for production of itaconic acid in microtiter plate microbioreactors. The new technique is very useful as a convenient, reliable and much cheaper way to investigate the reasons for inhibition of growth and itaconic acid production by the fungus and greatly aid in developing mutant strains, screening and optimization of itaconic acid production. 03 Itaconic acid production from mannose, a sugar component of woody biomass. Itaconic acid has gained importance as a fully sustainable building block platform chemical for wide applications for the manufacture of various synthetic resins, coatings, polymers and clear plastics. It is currently produced industrially from corn-derived glucose by fermentation with a fungus. However, the production cost of itaconic acid must be lowered in order to expand its market. Agricultural Research Service scientists in Peoria, Illinois, found a fungal strain that can be used for production of itaconic acid from mannose in good yield. This fungal strain can be used in woody biomass conversion to itaconic acid. Efficient utilization of mannose together with glucose will lower the production cost of itaconic acid from woody biomass.
Impacts
(N/A)
Publications
- Saha, B.C., Kennedy, G.J., Qureshi, N., Cotta, M.A. 2017. Biological pretreatment of corn stover with Phlebia brevispora NRRL-13108 for enhanced enzymatic hydrolysis and efficient ethanol production. Biotechnology Progress. 33(2):365-374.
- Saha, B.C. 2017. Emerging biotechnologies for production of itaconic acid and its applications as a platform chemical. Journal of Industrial Microbiology and Biotechnology. 44(2): 303-315. doi: 10.1007/s10295-016- 1878-8.
- Hector, R.E., Mertens, J.A. 2017. A synthetic hybrid promoter for xylose- regulated control of gene expression in Saccharomyces yeasts. Molecular Biotechnology. 59(1):24-33. doi: 10.1007/s12033-016-9991-5.
- Solana, M., Qureshi, N., Bertucco, A., Eller, F. 2016. Recovery of butanol by counter-current carbon dioxide fractionation with its potential application to butanol fermentation. Materials. 9(7):530-540.
- Jimenez, D.J., Hector, R.E., Riley, R., Lipzen, A., Kuo, R.C., Amirebrahimi, M., Barry, K.W., Grigoriev, I.V., Dirk van Elsas, J., Nichols, N.N. 2017. Draft genome sequence of Coniochaeta ligniaria NRRL 30616, a lignocellulolytic fungus for bioabatement of inhibitors in plant biomass hydrolysates. Genome Announcements. 5(4):e01476-16. doi: 10.1128/ genomeA.01476-16.
- Qureshi, N., Liu, S., Hughes, S., Palmquist, D., Dien, B., Saha, B. 2016. Cellulosic butanol (ABE) biofuel production from sweet sorghum bagasse (SSB): Impact of hot water pretreatment and solid loadings on fermentation employing Clostridium beijerinckii P260. BioEnergy Research. 9(4):1167- 1179. doi: 10.1007/s12155-016-9761-z.
- Liu, S., Qureshi, N., Hughes, S.R. 2017. Progress and perspectives on improving butanol tolerance. World Journal of Microbiology and Biotechnology 33(3):51. doi: 10.1007/s11274-017-2220-y.
- Hughes, S.R., Qureshi, N., Lopez-Nunez, J., Jones, M.A., Jarodsky, J.M., Galindo-Leva, L.A., Lindquist, M.R. 2017. Utilization of inulin-containing waste in industrial fermentations to produce biofuels and bio-based chemicals. World Journal of Microbiology and Biotechnology. 33(4):78. doi:10.1007/s11274-017-2241-6.
- Saha, B.C., Kennedy, G.J., Qureshi, N., Bowman, M.J. 2017. Production of itaconic acid from pentose sugars by Aspergillus terreus. Biotechnology Progress. 33(4): 1059-1067. doi: 10.1002/btpr.2485.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Under Sub-objective 1.B, using a strain that was evolutionarily adapted for improved growth on xylose, the complete genome was sequenced and analyzed to determine the genetic changes that result in increased ability to use xylose. A mutation was identified in a gene that is known to play a role in xylose utilization. Experiments were conducted to determine if the newly identified mutation leads to even better xylose utilization than reported literature values. Initial versions of the construct for identifying new xylose transporters leaked expression of the marker gene, making it impossible to determine if a transporter improved xylose uptake into the cell. New versions of the marker were developed that show significant growth differences between repressed and induced conditions. This new version will allow identification of new xylose transporters. Last fiscal year, a new xylose-fermenting yeast strain based on a Brazilian ethanol yeast was generated that performed well. During this fiscal year, haploid versions of the strain were created and screened for xylose metabolism. One strain with enhanced xylose metabolism was identified and its mating type was switched in order to create a diploid strain with the xylose fermentation genes integrated on both copies of chromosome 4. These strains were analyzed for xylose fermentation on pure sugars and were dilute acid pretreated and enzymatically saccharified switchgrass hydrolyzate. To facilitate the identification of novel xylose transporters, a new test strain was developed based on the xylose-fermenting haploid Brazilian yeast strain we developed. This strain will serve as the parent for expressing and testing new transporters. Currently available strains do not metabolize xylose well and make it difficult to evaluate xylose transport. This new strain�s excellent xylose metabolism will allow improved identification of xylose transport. Under Sub-objective 2.B., a mixed sugar (glucose, xylose, arabinose) utilizing itaconic acid producing Aspergillus NREL1972 strain could not grow and produce itaconic acid using dilute acid, liquid hot water, lime, and alkaline peroxide pretreated and enzymatically hydrolyzed wheat straw hydrolyzates at pH 3.1. Detoxification of the hydrolyzate with a fungal strain and by typical overliming did not improve the itaconic acid production. The Aspergillus strain was mutagenized by exposure to UV light and plated on dilute acid pretreated and enzymatically hydrolyzed wheat straw hydrolyzate gradient plates at pH 3.1. A few distinct colonies from the far right of the gradient plate were evaluated for itaconic acid production. The procedure was repeated several times but with no success so far. The strain was adapted in wheat straw hydrolyzate starting with 5% (v/v) at pH 3.1 and after growth transferred to the next concentration level in increments of 5% (v/v). The mutation and adaptation experiments will continue until a suitable mutant strain is obtained within this fiscal year. The effects of inhibitors and commercial enzyme preparations used for saccharification were evaluated at the level present in the hydrolyzate for inhibition of itaconic acid production from mixed sugars. Many of these were found to be strong inhibitors of itaconic acid production. The screening of UV mutants in gradient plates has been expanded to include tolerance to high concentrations of these inhibitors for production of itaconic acid from mixed sugars. Under Sub-objective 4.A., production of butanol (a superior biofuel than ethanol with energy content close to gasoline on per gallon basis) from agricultural biomass is characterized by two important factors: i) generation of sugar degradation products during biomass pretreatment which inhibit butanol production; and ii) maximum acetone butanol ethanol (ABE) concentration in a batch reactor is limited to 20 g/L because of product (ABE) toxicity to the microbial culture. Increase in ABE production beyond 20 g/L can be achieved by simultaneous removal of ABE as it is produced (process integration). To reduce generation of inhibitory substances during dilute acid pretreatment so that a detoxification step is not required prior to fermentation while maximizing sugar yield, lignocellulosic biomass should be pretreated under optimized conditions of temperature, acid dose and duration of pretreatment. Sweet sorghum bagasse (86 g/L) was pretreated with dilute sulfuric acid (5.0 g/L) at 160 degrees Celsius for zero min holding time followed by enzymatic saccharification at pH 5.0 and 45 degrees Celsius for 72 h using a cocktail of 3 commercial enzyme (cellulase, �- glucosidase, xylanase) preparations. The saccharified hydrolyzate contained 53.6 g total sugars (glucose, xylose, arabinose, mannose, and galactose) suggesting that almost complete saccharification occurred. This was followed by fermentation of the hydrolyzate using an ABE producing anaerobic bacterial strain. The culture (bacterial strain) produced 20.4 g total ABE per L with a productivity of 0.42 g/L.h indicating that the hydrolyzate as such was not inhibitory to the culture. Accomplishments 01 Improved industrial yeast strains for producing bio-ethanol from biomass-derived sugars. Industrial yeasts typically offer improved performance under harsh conditions found in an industrial setting. However, not all industrial yeasts perform well in the presence of additional inhibitors that are generated when producing sugars from biomass feedstocks. A yeast strain from a Brazilian fuel ethanol production facility was found to tolerate these inhibitors. ARS scientists in Peoria, Illinois, engineered the Brazilian yeast strain to express all of the proteins required for converting xylose to ethanol and then identified a strain with excellent performance. When hydrolyzed switchgrass was used as the source of sugars, this strain produced 30% more ethanol than the parent strain. Complete and efficient utilization of all biomass-derived sugars from any feedstock is important to achieve the highest ethanol production. The use of this new strain is expected to decrease the production cost for any process using biomass-derived sugars, thereby increasing profit.
Impacts
(N/A)
Publications
- Mariano, A.P., Ezeji, T.C., Qureshi, N. 2015. Butanol production by fermentation: Efficient bioreactors. In: Snyder, S.W., editor. Commercializing Biobased Products: Opportunities, Challenges, Benefits, and Risks, 2015. RSC Publishing, Cambridge, United Kingdom. p. 48-70. doi: 10.1039/9781782622444-00048.
- Lindquist, M.R., Lopez-Nunez, J.C., Jones, M.A., Cox, E.J., Pinkleman, R.J. , Bang, S.S., Moser, B.R., Jackson, M.A., Iten, L.B., Kurtzman, C.P., Bischoff, K.M., Liu, S., Qureshi, N., Tasaki, K., Rich, J.O., Cotta, M.A., Saha, B.C., Hughes, S.R. 2015. Irradiation of Yarrowia lipolytica NRRL YB- 567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates. Applied Microbiology and Biotechnology. 99(22):9723�9743.
- Okonkwo, C.C., Azam, M.M., Ezeji, T.C., Qureshi, N. 2016. Enhancing ethanol production from cellulosic sugars using Scheffersomyces (Pichia) stipitis. Bioprocess and Biosystems Engineering. 39(7):1023-1032. doi: 10. 1007/s00449-016-1580-2.
- Saha, B.C., Qureshi, N., Kennedy, G.J., Cotta, M.A. 2016. Biological pretreatment of corn stover with white-rot fungus for improved enzymatic hydrolysis. International Biodeterioration and Biodegradation. 109:29-35. doi: 10.1016/j.ibiod.2015.12.020.
- Liu, S., Skory, C., Qureshi, N., Hughes, S. 2016. The yajC gene from Lactobacillus buchneri and Escherichia coli and its role in ethanol tolerance. Journal of Industrial Microbiology and Biotechnology. 43(4):441- 450. doi: 10.1007/s10295-015-1730-6.
- Nichols, N.N., Saha, B.C. 2016. Production of xylitol by a Coniochaeta ligniaria strain tolerant of inhibitors and defective in growth on xylose. Biotechnology Progress. 32(3):606-612. doi: 10.1002/btpr.2259.
- Galinda-Leva, L.A., Hughes, S.R., Lopez-Nunez, J.C., Jarodsky, J.M., Erickson, A., Lindquist, M.R., Cox, E.J., Bischoff, K.M., Hoecker, E.C., Liu, S., Qureshi, N., Jones, M.A. 2016. Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799. Journal of Industrial Microbiology and Biotechnology. 43(7):927-939. doi: 10.1007/s10295-016- 1771-5.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Under Sub-objective 1.A., significant progress was made toward improving the ability of industrial yeast strains to use biomass-derived sugars. Several variations of a xylose-inducible promoter were tested to improve the fold-induction of the promoter. A non-radioactive method to analyze xylose uptake into the cell was developed. This new method was used to identify new transporters capable of increasing xylose transport into the cell. Four potential xylose transporters were isolated from a newly identified xylose-utilizing yeast that originated from the Brazilian rain forest. These transporters were evaluated for the ability to improve growth and production of ethanol from xylose. Two yeasts isolated from Brazilian fuel ethanol plants were engineered to ferment xylose and strain development continued using a novel xylose isomerase. Using a strain that was evolutionarily adapted for improved growth on xylose, the complete genome was sequenced and analyzed to determine the genetic changes that result in increased ability to use xylose. Progress toward production of the platform chemical triacetic acid lactone (TAL) continued. It was discovered that high productivity of TAL resulted in modification and degradation of TAL. The modification was identified and several genes were evaluated for their contribution to TAL modification. Under Sub-objective 2.A., one hundred Aspergillus terreus strains obtained from ARS Culture Collection, Peoria, Illinois, were screened for production of itaconic acid from glucose, xylose, and arabinose separately (80 g/L) in shake flasks. Itaconic acid is one of the 12 identified building block chemicals and a platform chemical that can be produced by fermentation. The goal is to identify an A. terreus strain that can effectively produce itaconic acid from all sugars typically present in a lignocellulosic hydrolyzate. A total of 15 strains were found to produce itaconic acid from glucose, xylose and arabinose as substrates. Among these, 10 strains were then subjected for secondary screen to evaluate itaconic acid production from a mixture (80 g/L) of glucose, xylose, and arabinose as the carbon source. The best performing strain with respect to productivity and yield was identified. This strain will be used for adaptation to dilute acid pretreated wheat straw hydrolyzate for inhibitor tolerance and high concentrations of itaconic acid for itaconic acid tolerance. Under Sub-objective 3.A., research to develop a microbe that makes xylitol, a sugar substitute from biomass derived sugar xylose was continued. A micriobial strain was indentified that converts xylose to xylitol. The microbe also has intrinsic tolerance for and grows on the mixture of inhibitory compounds that commonly occur along with the sugars. The growth conditions including aeration, pH, temperature, and glycerol concentration were optimized in controlled small-scale fermentations. The microbe produced up to 0.44 g xylitol/g xylose in a rich medium containing 5% xylose. The activity of the enzyme responsible for xylitol production was also assayed under these conditions. This matters because biomass must be harshly pretreated to release sugars, there are typically inhibitory chemicals present in biomass sugars. Use of a microbe that withstands the inhibitory milieu avoids use of a separate step to remove inhibitors. When confronted with hydrolyzates, the inhibitor-tolerant fungus removed inhibitory chemicals while producing xylitol from biomass sugar xylose. Under Sub-objective 4.A., liquid hot water pretreatment conditions with respect to temperature (160-200�C) and duration of pretreatment (0-15 min) were optimized for generation of fermentable sugars from sweet sorghum bagasse after enzymatic hydrolysis using a mixture of 3 commercial enzyme preparations (cellulase, �-glucosidase, and xylanase). The enzymatic hydrolyzate of sweet sorghum bagasse was then subjected to fermentation to butanol (acetone-butanol-ethanol or ABE with butanol being the major product) by a butanol producing anaerobic bacterium. Butanol is a superior transportation biofuel compared to ethanol with an energy content close to gasoline. It also has several superior fuel properties such as burning clean (less shoot), and transportation in existing pipelines due to being less corrosive. For the fermentation purpose, three parameters were studied: i) inhibition effect due to generated sugar degradation products; ii) accumulation of butanol/aceton-butanol- ethanol (ABE) in the fermentation broth; and iii) rate of production of butanol/ABE (productivity). The hydrolyzate as such was found not to be inhibitory to the culture at all and the fermentation resulted in accumulation of significant amount of ABE (13.27-15.50 g/L). The butanol productivity was higher (0.43-0.65 g/L.h) than when using glucose or corn (0.35 g/L.h) as a feedstock. Accomplishments 01 New industrial yeast strains for producing bio-ethanol. Brewer�s yeasts are the preferred organism for industrial ethanol production, but not all yeasts are tolerant to industrial processes. Two yeasts originally isolated from Brazilian fuel ethanol production facilities have been shown to perform well under harsh industrial conditions. These yeasts are extremely efficient at converting glucose to ethanol but they are not able to use xylose, the second most abundant sugar in lignocellulosic biomass. Agricultural Research Service scientists in Peoria, Illinois, have engineered these strains to express all of the proteins required for conversion of xylose to ethanol. One of these two strains was capable of rapid utilization of xylose when it was the only sugar available. The engineered strains consumed all of the xylose and made 70% more ethanol compared to other engineered industrial strains. Complete utilization of all biomass-derived sugars from the feedstock is important to achieve the highest productivity. This new technology is expected to promote the economics of cellulosic ethanol, furthering rural development. 02 Developed a novel pretreatment process for conversion of sweet sorghum bagasse to butanol. Sweet sorghum is potentially a high energy and biomass crop that is regarded as one of the most promising crops for biofuel production. Agricultural Research Service scientists in Peoria, Illinois, have developed a novel process to pretreat sweet sorghum bagasse with liquid hot water to generate fermentable sugars after enzymatic hydrolysis. The pretreated sweet sorghum bagasse after enzymatic hydrolysis was easily fermented to butanol (acetone-butanol- ethanol/ABE with butanol as major product) by ABE producing anaerobic bacterium. Additionally, butanol/ABE productivity was found to be 23- 86% higher than when using glucose or corn as a feedstock. Butanol is an advanced biofuel that packs 30% more energy than ethanol on a per gallon basis. The developed process would benefit the biofuel companies and the United States transportation industry and reduce dependency on imported oil. 03 A microbe that produces xylitol. Xylitol, a natural sugar substitute used in foods and pharmaceuticals, can be produced from biomass sugars. However, due to necessarily harsh pretreatment used to break down biomass fibers, the biomass sugars typically contain inhibitors that are detrimental to the process. Agricultural Research Service scientists in Peoria, Illinois, developed a microbe that produces xylitol while also withstanding the mixture of inhibitory compounds that commonly occur along with the sugars. Culture conditions were optimized for maximum xylitol production. A microbe that withstands inhibitors would avoid use of a separate processing step to remove inhibitors and allow more economical production of xylitol. This approach will elevate lower-value biomass to a higher-value sugar substitute. 04 Itaconic acid production from lignocellulosic biomass. Itaconic acid is one of the 12 identified building block chemicals and a platform chemical that can be produced by fermentation. Agricultural Research Service scientists in Peoria, Illinois, that can produce itaconic acid from lignocellulosic biomass derived sugars such as glucose, xylose and arabinose with good yields. This strain has potential to be used for itaconic acid production from lignocellulosic hydrolyzates by fermentation. Itaconic acid is a promising product that can be manufactured from cellulosic biomass that will promote rural development.
Impacts
(N/A)
Publications
- Hughes, S.R., Cox, E.J., Bang, S.S., Pinkelman, R.J., Lopez-Nunez, J.C., Saha, B.C., Qureshi, N., Gibbons, W.R., Fry, M.R., Moser, B.R., Bischoff, K.M., Liu, S., Sterner, D.E., Butt, T.R., Reidmuller, S.B., Jones, M.A., Riano-Herrera, N.M. 2015. Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated platform. Journal of Laboratory Automation. DOI: 10.1177/2211068215573188.
- Qureshi, N. 2014. Consolidated processes for product recovery. In: Qureshi, N., Hodge, D., Vertes, A., editors. Biorefineries: Integrated Biochemical Processes for Liquid Biofuels. Amsterdam, The Netherlands: Elsevier. p. 141-160.
- Qureshi, N. 2014. Integrated bioprocessing and simultaneous product recovery for butanol production. In: Qureshi, N., Hodge, D., Vertes, A., editors. Biorefineries: Integrated Biochemical Processes for Liquid Biofuels. Amsterdam, The Netherlands: Elsevier. p. 205-224.
- Ezeji, T.C., Liu, S., Qureshi, N. 2014. Mixed sugar fermentation by Clostridia and metabolic engineering for butanol production. In: Qureshi, N., Hodge, D., Vertes, A., editors. Biorefineries: Integrated Biochemical Processes for Liquid Biofuels. Amsterdam, The Netherlands: Elsevier. p. 191-204.
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