Source: AGRICULTURAL RESEARCH SERVICE submitted to NRP
GENOMIC MECHANISMS OF IN SITU DETOXIFICATION OF BIOMASS CONVERSION INHIBITORS FOR ETHANOLOGENIC YEAST SACCHAROMYCES CEREVISIAE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0207531
Grant No.
2006-35504-17359
Cumulative Award Amt.
$380,000.00
Proposal No.
2006-02272
Multistate No.
(N/A)
Project Start Date
Sep 15, 2006
Project End Date
Aug 31, 2011
Grant Year
2006
Program Code
[71.2]- Biobased Products & Bioenergy Production Research
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
PEORIA,IL 61604
Performing Department
NATL CTR FOR AGRIC UTILIZATION RESEARCH
Non Technical Summary
Economic dilute acid hydrolysis of biomass produces inhibitors interfere yeast growth and fermentation. Additional steps to remove the inhibitors add cost and generate extras wastes. This project uses functional genomics to study mechanisms of in situ detoxification of biomass conversion inhibitors for ethanologenic yeast Saccharomyces cerevisiae. Results of this research will provide the guidance for future genetic manipulation and engineering of more robust strains for economic bioethanol production.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51170101040100%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
7010 - Biological Cell Systems;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The overall objective of this project is to elucidate genomic mechanisms of detoxification and tolerance of ethanologenic yeast to biomass conversion inhibitors including furfural and 5-hydroxymethylfurfural (HMF). Specific supporting objectives are: 1) to identify several subsets of genes involved in main pathways relevant to detoxification and tolerance to biomass conversion inhibitors, furfural and HMF, each individually and in combination, using genome-wise transcriptome analysis using microarray and real-time qRT-PCR, and as well as promoter binding motif and protein expression analysis; 2) to identify and characterize novel genes and novel functions specifically responsible for detoxification, biotransformation, and tolerance to furfural and HMF involved in biomass conversion to ethanol; 3) to establish a web-based interactive database to aid microarray data mining and visualization in pathway analysis; and 4) to infer computational models of gene regulatory network of major gene interactions in relevant pathways involved in furfural and HMF detoxification and tolerance verified by fermentation and biological experiments.
Project Methods
This proposal uses functional genomics to study mechanisms of in situ detoxification of biomass conversion inhibitors for ethanologenic yeast Saccharomyces cerevisiae. It addresses significant cost-competitive issues for biomass conversion to affordable fuel by efficient biotransformation to detoxify biomass conversion inhibitors such as furfural and HMF. Finding new technology for converting biomass into biofuel such as ethanol is a key challenge to be overcome for cost-competitive bioenergy. Our study on genomic mechanisms of in situ detoxification of biomass conversion inhibitors involves fundamental research to understand important mechanisms for economic biomass conversion. Understanding the genetic blue print and dissection of the detoxification and tolerance mechanisms will provide more new opportunities to improve the performance of ethanologenic yeast. Such new technology can be expected to accelerate the application and advancement of biofuel and bioenergy development.

Progress 09/15/06 to 08/31/11

Outputs
OUTPUTS: Activities: Developed the first mRNA reference standard for comparable gene expression analyses; Conducted genome expression analyses of tolerant ethanologenic yeast; Created a new qRT-PCR array assay method and developed data processing procedures using a user-friendly interactive computation program; Identified novel genes and candidate genes for stress tolerance involved in lignocellulose-to-ethanol conversion; Discovered and described reprogrammed pathways of tolerant yeast for in situ detoxification of inhibitors derived from biomass pretreatment; Genetically engineered tolerant yeast to enable its xylose utilization capability; Discovered a new yeast producing the highest level of beta-glucosidase activities for lower-cost cellulosic ethanol production; Trained young scientists through teaching graduate level courses of bioinformatics; Mentored postdoctoral research associates, Ph.D. and Master Degree and undergraduate students. Events: Organized and chaired 4 international symposia related to lignocellulose biofuels conversion and microbial stress tolerance; Conducted 2 workshops on unification of gene expression data for comparable analyses for the American Society for Microbiology and the Society for Industrial Microbiology and Biotechnology; Delivered 28 presentations as invited key note speaker, lecturer, panel and research presentations nationally and internationally; Presented 30 oral or poster presentations for professional conferences and meetings. Services: Served as advisor of postdoctoral research associates; Ph.D. student project advisory committee; University professor promotion evaluation panel; Grant proposal review nationally and internationally. Products: Published 1 monograph book on Microbial Stress Tolerance for Biofuels: Systems Biology; 12 invited book chapters; 20 peer-reviewed original research articles; 50 conference presentations and abstracts; Synthesized a unique gene used for strain improvement by genetic engineering; Discovered 8 new gene sequences deposited to public database NCBI GenBank; Generated 2 sets of gene expression data and deposited at public database; Created 2 computation models and 1 program that are available for public; Developed the first mRNA standard reference and distributed by MTAs internationally; Created new knowledge and technologies with 5 US patent pending applications; Trained young scientists of 4 postdoctoral research associates in bioenergy and bioinformatics; Taught curriculum courses of Bioinformatics and Special topics in Computational Systems Biology for 4 semesters each with 10 students enrolled; Trained 4 Ph.D. graduate students; 3 Master degree students; 7 undergraduate students; 8 new gene sequences deposited at public database. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Change in knowledge:Our research identified metabolic conversion products of a biomass pretreatment inhibitors and classified the inhibitors by their functional chemical groups. Validation of this structure-function concept clarified literature and led discoveries of new genes and new gene functions related to yeast stress tolerance; More importantly, the approach of structure-function enabled studies on mechanisms of yeast tolerance and the establishment of scientific foundation for in situ detoxification of biomass pretreatment inhibitors while producing ethanol by tolerant yeast; Our development of the first universal mRNA standard references defined valid mRNA quantification ranges for microarray at 1-7000 pg and qRT-PCR at 0.1-1000 pg. It allowed for the first time that quantitative gene expression can be compared within the valid range of 1-1000 pg from different platforms; The creation of robust mRNA reference for qRT-PCR allowed development of a master equation and qRT-PCR array assay for reproducible and comparable analysis of gene expression data; Identified and described reprogrammed pathways for tolerant yeast to in situ detoxify fermentation inhibitors for the first time. The new knowledge filled the gap of detoxification pathways and is a milestone in understanding mechanisms of yeast tolerance to inhibitors liberated by biomass pretreatment; Uncovered major gene regulatory networks of yeast tolerance in response to biomass pretreatment inhibitors and identified tolerance candidate genes. Comprehensive new knowledge at the genome level aids efforts on tolerant yeast development; Developed computational models to interpret and predict gene interactions and regulatory networks in response to biomass pretreatment inhibitors by ethanologenic yeast; Discovered new yeast strain producing high levels of β-glucosidase activities that no additional enzyme is needed for cellulosic ethanol production using simultaneous saccharification and fermentation; Change in actions: The establishment of our in situ detoxification concept and practice provides economic options for traditional inhibitor removal procedures by expensive chemical and physical means. This practice has been popularly adapted and used worldwide; The benefits of universal mRNA standard reference and the robust mRNA reference for gene expression analysis were recognized and adapted in varied fields of applications by academic and industrial sectors. These mRNA standard references were requested and distributed by USDA-ARS through Material Transfer Agreements worldwide including US, Brazil, France, Germany, and New Zealand. Change in conditions: Trained 2 postdoctoral research associates; Trained and graduated 4 Ph.D. students; Graduated 3 Master Degree students; Trained and graduated 7 undergraduate students; Established 8 MTAs (Material Transfer Agreement) in the US and internationally.

Publications

  • Liu, Z.L. 2010. Unification of gene expression data applying standard mRNA quantification reference for comparable analyses. J Microbial Biochem Technol 2:124-126.
  • Ma, M. and Liu, Z.L. 2010. Comparative genome analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4, and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor-stress for Saccharomyces cerevisiae. BMC Genomics 11:660.
  • Ma, M. and Liu, Z.L. 2010. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae. BMC Microbiology 10:169.
  • Ma, M. and Liu, Z.L. 2010. Mechanisms of ethanol tolerance in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 87:829-845.
  • Bowman, M.J., Jordan, D.B., Vermillion, K.E., Braker, J.D., Moon, J. and Liu, Z.L. 2010. Stereochemistry of furfural reduction by an aldehyde reductase from Saccharomyces cerevisiae that contributes to in situ furfural detoxification. Appl. Envir. Microbiol. 76:4926-4932.
  • Liu, Z.L., and Moon, J. 2009. A novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion. Gene 446:1-10.
  • Moon, M. and Liu, Z.L. 2011. Scheffersomyces stipitis xylose transporter (rgt2) mRNA, complete cds. Accession JF343554.
  • Moon, M. and Liu, Z.L. 2011. Scheffersomyces stipitis xylose transporter (xut7) mRNA, complete cds. Accession JF343555.
  • Moon, M. and Liu, Z.L. 2011. Scheffersomyces stipitis xylose transporter (xut5) mRNA, complete cds. Accession JF343556.
  • Moon, M. and Liu, Z.L. 2011. Scheffersomyces stipitis xylose transporter (xut6) mRNA, complete cds. Accession JF343557.
  • Moon, M. and Liu, Z.L. 2011. Scheffersomyces stipitis xylose transporter (xut4) mRNA, complete cds. Accession JF343558.
  • Moon, M. and Liu, Z.L. 2011. Scheffersomyces stipitis xylose transporter (sut4) mRNA, complete cds. Accession JF343559.
  • Liu, Z.L. and Moon, J. 2009. Saccharomyces cerevisiae strain NRRL Y-12632 intermediate subclass aldehyde reductase gene, complete cds. Accession No. FJ851468, protein id= ACO72583.
  • Liu, Z.L., Moon, J., and Song, M.Z. 2008. Genomic mechanisms of inhibitor-detoxification for low-cost lignocellulosic bioethanol conversion. 13th International Biotechnology Symposium, Dalian, China.
  • Li,u, Z.L. 2008. Functional genomics studies lead in situ detoxification of fermentation inhibitors for low cost cellulosic ethanol production. World Conference on Industrial Biotechnology. iBIO2008: 41. Hongzhou, China.
  • Slininger, P.J., Liu, Z. 2008. Impact of inoculum production conditions on stress tolerance and fermentation efficiency of natural xylose-fermenting yeasts presented xylose and glucose. 30th Smposium on Biotechnology for Fuels and Chemicals Proceedings. New Orleans, LA. Abstract No. 5734.
  • Liu, Z.L. 2007. Genomic engineering of Saccharomyces cerevisiae for biomass conversion to ethanol. SIM Annual Meeting: 97.
  • Slininger, P.J., Gorsich, S.W. and Liu, Z.L. 2009. Culture nutrition and physiology impact the inhibitor tolerance of the yeast Pichia stipitis NRRL Y-7124. Biotechnol. Bioeng.102:778-790.
  • Liu, Z.L. Palmquist, D.E., Ma, M., Liu, J. and Alexander, N.J. 2009. Application of a master equation for absolute mRNA quantification using qRT-PCR. J. Biotechnol. 143:10-16.
  • Song, M.J., Ouyang, Z, Liu Z.L. 2009. Discrete dynamic system modeling for gene regulatory networks of HMF tolerance for ethanologenic yeast. IET Systems Biology 3:203-218.
  • Liu, Z.L., J. Moon, B.J. Andersh, P.J. Slininger, and S.A. Weber. 2008. Multiple gene mediated NAD(P)H dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 81:743-753.
  • Liu, Z.L., and Slininger, P.J. 2007. Universal external RNA quality controls for mRNA expression analysis using microbial DNA oligo microarray and real time quantitative RT-PCR. J. Microbiol. Methods 68:486-496.
  • Song M.J. and Liu, Z.L. 2007. A Linear Discrete Dynamic System Model for Temporal Gene Interaction and Regulatory Network Influence in Response to Bioethanol Conversion Inhibitor HMF for Ethanologenic Yeast. Lecture Notes Bioinfomatics 4532:77-95.
  • NCBI GenBank: Liu, Z.L. and Moon, M. 2011. Saccharomyces cerevisiae transgenic strain NRRL Y-50463 xylose isomerase (YXI) mRNA, comple cds. Accession JF261697.
  • Slininger, P.J., Liu, Z.L. and Dien, B.S. 2011. Culture nutrition key to inhibitor-tolerant yeast performance. 33rd Smposium on Biotechnology for Fuels and Chemicals. Seattle, WA.
  • Abstracts: Liu, Z.L., Cotta, M.A. and Weber, S.A. 2011. A new yeast producing beta-glucosidase and tolerant to lignocellulose hydrolysate inhibitors for cellulosic ethanol production using SSF. 33rd Symposium on Biotechnology for Fuels and Chemicals. Seattle, WA.
  • Liu, Z.L., Ma, M., and Song, M. 2009. Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways Mol Genet Genomics 282:233-244.
  • Liu, Z.L., Ma, M. and Moon J. 2011. Newly designed ethanologenic yeast Saccharomyces cerevisiae that tolerates lignocellulose hydrolysates and utilizes heterogeneous biomass sugars for cellulosic ethanol conversion. American Society for Microbiology. New Orleans, LA.
  • Slininger, P.J., Liu, Z.L. and Dien, B.S. 2011. Nutrient supplementation key to inhibitor-tolernat yeast development and fermentation performance on switchgrass hydrolysates. 9th Recent Advances in Fermetnation Technology Conference, Marco Island, FL.
  • Zhang, L. Li, S. and Liu, Z.L. 2011. Challenges of cellulosic ethanol production from xylose-extracted corncob residues. 33rd Symposium on Biotechnology for Fuels and Chemicals. Seattle, WA.
  • Liu, Z.L. 2010. Unification of gene expression data from comparative analyses. Yeast Genetics and Molecular Biology Meeting. Vancouver, British Columbia, Canada.
  • Liu, Z.L. and Moon, J. 2010. New aldehyde reductase genes of Saccharomyces cerevisiae contribute in situ detoxification of lignocellulose-to-ethanol conversion inhibitors. The 14th International Biotechnology Symposium and Exhibition. Rimini, Italy.
  • Liu, Z.L., Ma, M., and Cotta, M.A. 2010. Reprogrammed glucose metabolic pathways of inhibitor-tolerant yeast. American Institute of Chemical Engineering Midwest Regional conference. Chicago, IL.
  • Moon, J. and Liu, Z.L. 2010. Protein engineering of GRE2 from Saccharomyces cerevisiae for enhanced detoxification of 5-hydroxymethylfurfural. American Society for Microbiology 110th General Meeting. San Diego, CA.
  • Zhang, Y., Song, J. L and Liu, Z.L. 2010. twzPEA: a topology and working zone based pathway enrichment analysis framework. The International Conference on Intelligent Systems and Molecular Biology. Boston, MA.
  • Liu, Z.L., Ma. M., and Cotta, M.A. 2010. Reprogrammed glucose metabolic pathways of inhibitor-tolerant yeast. American Institute of Chemical Engineering Midwest Regional Conference, Chicago, IL.
  • Slininger, P.J., Moon, J. and Liu, Z.L. 2010. Process design condiserations for optimal production of ethanol from lignocellulose using available yeasts including natural pentose-fermenting yeast and their derivatives. American Institute of Chemical Engineering Midwest Regional Conference, Chicago, IL.
  • Slininger, P.J., Moon, J., Thompson, S.T., Weber, S.A., and Liu, Z.L. 2010. The switch from xylose to glucose stalled by repression of xylose-utilizing enzymes during exposure of Scheffersomyces (Pichia) stipitis to high ethanol concentrations. 32nd Symposium on Biotechnology for Fuels and Chemicals. Clearwater, FL.
  • Liu, Z.L., Ma, M., and Song, M. 2009. Acquired tolerance and in situ detoxification of furfural and HMF through glucose metabolic pathways by Saccharomyces cerevisiae. American Society for Microbiology 109th General Meeting. Philadelphia, PA.
  • Moon, J. and Liu, Z.L. 2010. Protein engineering of GRE2 from Saccharomyces cerevisiae for enhanced detoxification of 5-hydroxymethylfurfural. American Institute of Chemical Engineering Midwest Regional conference. Chicago, IL.
  • Slininger, P.J., Moon J. and Liu, Z.L. 2010. Process design considerations for optimal production of ethanol from lignocellulose using available yeasts, including natural pentose-fermenting yeasts, and their derivatives. American Institute of Chemical Engineering Midwest Regional conference. Chicago, IL.
  • Liu, Z.L. and Weber, S.A. 2010. Cellulosic ethanol production from xylose extracted corncob residue by SSF using inhibitor-and thermal-tolerant yeast Clavispora NRRL Y-50339. American Society for Microbiology 110th General Meeting. San Diego, CA.
  • Ma, M. and Liu, Z.L. 2010. Heat shock protein genes and newly integrated glucose metabolic pathways promote ethanol tolerance of Saccharomyces cerevisiae. American Society for Microbiology 110th General Meeting. San Diego, CA.
  • Liu, Z.L., 2008. Mechanisms of tolerance and in situ detoxification of biomass conversion inhibitors by Saccharomyces cerevisiae. 2nd International Symposium on Bioenergy and Bioprocess Technology, Qingdao, China.
  • Liu, Z.L. 2008. Change your conventional practice of qRT-PCR: a simple robust quality control standard for yeast mRNA quantification analysis. 12nd International Congress on Yeast. Kiyv, Ukraine.
  • Liu, Z.L. 2008. Tolerant ethanologenic yeast detoxified biomass conversion inhibitors. USDA MWA Bioenergy Forum, Peoria, IL.
  • Liu, Z.L., and P.J. Slininger. 2007. Absolute mRNA quantification of Pseudomonas fluorescens Pf-5 by qRT-PCR using universal RNA controls. ASM Conference on Pseudomonas. Seattle, WA.
  • Liu, Z.L., 2007. Validation and standardization of gene expression data for microarray and real time quantitative PCR using universal external RNA controls. Cambridge Healthtech Institute Conference on Quantitative PCR, Microarrays, and Biological Validation. Providence, RI.
  • Liu, Z.L. and D. Palmquist. 2007. A robust standard for absolute mRNA quantification of Saccharomyces cerevisiae by qRT-PCR using the universal RNA controls. XXIII International Conference of Yeast Genetics and Molecular Biology. Melbourne, Australia. Yeast 24: S84.
  • Liu, Z.L., J. Moon, B.J. Andersh, P.J. Slininger, and S.A. Weber. 2007. Multiple gene mediated aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae. XXIII International Conference of Yeast Genetics and Molecular Biology. Melbourne, Australia. Yeast 24: S134.
  • Liu, Z.L., B.J. Andersh, and P.J. Slininger, 2007. Mechanisms of in situ detoxification of furfural and HMF by ethanologenic yeast Saccharomyces cerevisiae. 29th Symposium of Biofuels and Chemicals. Abs. 85.
  • Monograph and book chapters: Liu, Z.L. 2011. Microbial Stress Tolerance for Biofuels: Systems Biology. Microbiology Monographs 22, 307 p. Microbiology Monographs vol 22, Springer-Verlag Berlin, Germany.
  • Liu, Z.L. 2011. Unification of gene expression data for comparable analyses under stress conditions. Pages 279-299 in: Microbial Stress Tolerance for Biofuels: Systems Biology, Microbiology Monographs 22. Z.L. Liu ed. Springer-Verlag Berlin, Germany.
  • Liu, Z.L. 2011. Genomics of yeast tolerance and in situ detoxification. Pages 1-28 in: Microbial Stress Tolerance for Biofuels: Systems Biology, Microbiology Monographs 22. Z.L. Liu ed. Springer-Verlag Berlin, Germany.
  • Ma, M. and Liu, Z.L. 2011. Molecular mechanisms of ethanol tolerance in Saccharomyces cerevisiae. Pages 77-115 in: Microbial Stress Tolerance for Biofuels: Systems Biology, Microbiology Monographs 22. Z.L. Liu ed. Springer-Verlag Berlin, Germany.
  • Matsushika, A., Liu, Z.L., Sawayama, S., and Moon, J. 2011. Improving biomass sugar utilization by engineering Saccharomyces cerevisiae. Pages 137-160 in: Microbial Stress Tolerance for Biofuels: Systems Biology, Microbiology Monographs 22. Z.L. Liu ed. Springer-Verlag Berlin, Germany.
  • Liu, Z.L., Ma, M., and Cotta, M.A. 2010. Reprogrammed glucose metabolic pathways of inhibitor-tolerant yeast. Pages 159-185 in: Advances in Medicine and Biology Vol 9, V.B. Leon (ed.) Nova Science Publisher, Hauppauge, NY. USA.
  • Liu, Z.L., and Blaschek, H.P. 2010. Biomass conversion inhibitors and in situ detoxification. Pages 233-258 in: Biomass to Biofuels: Strategies for Global Industries. A. Vertes, N. Qureshi, H. Yukawa, and H. Blaschek (eds.) John Wiley & Sons, Ltd. West Sussex, UK.
  • Liu, Z.L., and Song, M. 2009. Genomic adaptation of Saccharomyces cerevisiae to inhibitors involving biomass conversion to ethanol. Pages 136-155 in: Applied Mycology. M. Rai and Bridge, (eds.) CAB International, UK.
  • Liu, Z.L., B.C. Saha, and P.J. Slininger. 2008. Lignocellulosic biomass conversion to ethanol by Saccharomyces in: Bioenergy pp17-36. J. Wall, C. Harwood, and A. Demain (eds.) ASM Press. Washington DC.
  • Peer reviewed articles: Liu, Z.L., Weber, S.A., Cotta, M.A., and Li, S. 2011. A new β-glucosidase producing yeast for lower-cost cellulosic ethanol production from xylose-extracted corncob residues by simultaneous saccharification and fermentation. Bioresources Technology doi:10.1016/j.biotech.2011.10.099.
  • Ma, M., Liu, Z.L., and Moon, J. 2011. Genetically engineered inhibitor-tolerant Saccharomyces cerevisiae through a systems biology approach is able to competitively utilize xylose and mixed biomass sugars for anaerobic ethanol fermentation (submitted).
  • Moon, J., Liu, Z.L., Ma, M., Slininger, J.P. 2011. New xylose transporters improve xylose uptake and competitive utilization of inhibitor-tolerant Saccharomyces cerevisiae for ethanol production using divergent sugars (submitted)
  • Moon, J. and Liu, Z.L. 2011. Protein engineering of GRE2 from Saccharomyces cerevisiae for enhanced detoxification of 5-hydroxymethylfurfural. Enzyme and Microbial Technology doi:10.1016/j.enzmictec.2011.10.007
  • Liu, Z.L. 2011. Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysate. Appl Microbiol Biotechnol 90:809-825.
  • Slininger, P.J., Thompson, S.R., Weber, S.C., Liu, Z.L., and Moon, J. 2011. Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag. Biotechnol Bioeng 108:1801-1815.
  • Jordan, D. B., Braker, J.D., Bowman, M.J., Vermillion, K., Moon, J., and Liu, Z.L. 2011. Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural. Biochem Biophy Acta (in press).
  • Zhang, L., Li, S. and Liu, Z.L. 2011. Xylose-extracted corncob residue as a potential substrate for cellulosic ethanol production. Bioresources (in press).


Progress 09/15/07 to 09/14/08

Outputs
OUTPUTS: 1. REPORTED A MECHNISM OF YEAST TOLERANCE TO LIGNOCELLULOSIC ETHANOL CONVERSION INHIBITORS. Using genomic tools and molecular biology methods, we identified genes responsible for tolerance and detoxification of lingocellulosic biomass conversion inhibitors by ethanologenic yeast Saccharomyces cerevisiae. Dissemination of this finding to communities of interest was shared through a peer-reviewed journal article on Applied Microbiology and Biotechnology; and by invited lectures at 1) New Mexico State University, Las Cruces NM, April 23, 2008; 2) Tsinghua University, Beijing, China, May 12, 2008; and 3) American Society for Microbiology 108th General Meeting, June 1-5, 2008. 2. DEVELOPED INHIBITOR TOLERANT ETHANOLOGENIC YEAST. A novel strain of Saccharomyces cerevisiae NRRL Y-50049 was developed with enhanced tolerance that in situ detoxifies common lingocellulosic biomass conversion inhibitors such as furfural and 5-hydroxymethylfurfural (HMF). A USDA utility patent application entitled "Inhibitor tolerant Saccharomyces cerevisiae strain" (USSN60/937,517) was filed on June 23, 2008. 3. DEVELOPED IMPROVED GENE REGULATORY NETWORK MODELING. Based on our previous study, we further developed improved discrete dynamic system modeling for gene regulatory networks of inhibitor tolerance for ethanologenic yeast. Dissemination of this advancement and accomplishment was shared through a peer-reviewed journal article on IET Systems Biology. 4. CHAIRED INTERNATIONAL SYMPOSIUM ON BIOFUELS. An international symposium on "Pathways for Introducing New Biobased Transportation Fuels" was chaired by this Project Director during the World Congress of Industrial Biotechnology, Hongzhou, China, May 18-21, 2008. PARTICIPANTS: Individuals: Dr. Z.L. Liu, Project Director, USDA-ARS-NCAUR, no salary. Dr. M. Song, Co-Project Director, New Mexico State University, summer salary. Partner Organizations: National Center for Agricultural Utilization Research, USDA, Peoria, IL 61604. New Mexico State University, Las Cruces, NM 88003-8001 TARGET AUDIENCES: Biofuel communities including interested groups of academic and industrial sectors PROJECT MODIFICATIONS: Co-PI Dr. S.B. Dolins is under consideration of replacement by another faculty member due to his unavailability.

Impacts
1. We published the first report on a mechanism of tolerance and in situ detoxification of furfural and HMF by S. cerevisiae that is due to multiple gene mediated NAD(P)H-dependent aldehyde reduction. Our findings explained cofactor preference of individual genes, strain dependent, and multiple gene involvement in the tolerance and the inhibitor detoxification by yeast. Our identification and synthesis of HMF metabolic conversion product allow characterization of fermentation profiles and studies on mechanism of the inhibitor detoxification. Our finding and conclusion of aldehyde reduction involved in the detoxification of furfural and HMF clarified the inconsistency in literature and a misleading concept of furan conversion. This new knowledge aids efficient future inhibitor tolerant strain development. 2. The tolerant NRRL Y-50049 is the first yeast that can be applied as an initial inoculum without preconditioning to in situ detoxifies furfural and HMF while producing ethanol. It provides a necessary material for studies on mechanisms of the tolerance and detoxification. Knowledge obtained from such studies guide future more tolerant commercial strain development. 3. Our recent development of the discrete dynamic system modeling for gene regulatory networks used both simulations and real-world data analysis examples. It received high appraisal by peers, especially on the descent data statistical procedures, the use of the F-test, and stabilization for the modeling development. It provides an example to improve the poor statistical methodology in literature. 4. The international symposium brought front runners of researchers around the world to share their findings and updated information. The successful conference enhanced exchanges and communications among scientists and industrial leaders for advanced research and development in the field of biofuels.

Publications

  • Liu, Z.L., B.C. Saha, and P.J. Slininger. 2008. Lignocellulosic biomass conversion to ethanol by Saccharomyces in: Bioenergy pp17-36. J. Wall, C. Harwood, and A. Demain (eds.) ASM Press. Washington DC.
  • Liu, Z.L., J. Moon, B.J. Andersh, P.J. Slininger, and S.A. Weber. 2008. Multiple gene mediated aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae (in press) DOI 10.1007/s00253-008-1702-0.
  • Liu, Z.L., and Song, M.J. 2008. Genomic adaptation of Saccharomyces cerevisiae to inhibitors involving biomass conversion to ethanol. In: Current Advances in Mycology. M. Rai (eds.) (in press).
  • Song, M.J., Ouyang, Z, Liu Z.L. 2008. Discrete dynamic system modeling for gene regulatory networks of HMF tolerance for ethanologenic yeast. IET Systems Biology (in press).


Progress 09/15/06 to 09/14/07

Outputs
1. We developed the first universal external RNA controls for gene expression analysis that can be applied to different platforms of microarray and real time qRT-PCR. By application of these universal RNA controls, we fabricated high-quality DNA oligo 70-mer microarray for genome Saccharomyces cerevisiae used in this study. This research contributes to objective 1 of this project. Application of the universal RNA control is a basis to guard reliability and reproducibility of microarray data for quality high throughput gene expression analysis. Results of this research were disseminated to research communities and public by an original research article published in Journal of Microbiological Methods (Liu and Slininger, 2007, 68:486-496); an invited lecture by CHI (Chambridge Healthtech Institute) conference (October 2007, Providence, RI); an interview and a following cover story exposure by Genomics and Proteomics (by Senior Editor James Netterwald, Go On Green, G&P); an invited seminar by SuperArray Bioscience Corp. (November 2007, Frederick, MD); two Material Transfer Agreements with two international institutions (Universitatasklinik fur Allgemeine, Tubingen, Germany, June 2007, and INRA (Institut National de la Recherche Agronomique), Le Rheu cedex France, June 2007); an ARS (Agricultural Research Service) prevision patent application (Liu, December 13, 2007); collaboration with other ARS research laboratories (Mycotoxin Research Unit, NCAUR); an invited symposium lecture (May 2006, Jonkoping, Sweden); many responses and distribution of reprint requests (2007); and discussion and exchanges in public events. 2. With unique application of the universal RNA controls, we have completed a set of well-designed microarray experiments to study global gene expression profiling and identified genes responsible for the inhibitor tolerance and detoxification. This research contributes to objectives 1 and 2 of this project. Results and knowledge obtained from this research were disseminated by an original research article submitted for publication (Liu et al., 2008, under review); two invited book chapters (Liu et al., 2007, ASM Press; Liu and Song, 2007, submitted); a presentation at 23rd International Conference on Yeast Molecular Biology and Genetics (June 2007, Melbourne, Australia); an invited lecture at Society for Industrial Microbiology (July 2007, Denver, CO); and an ARS prevision patent application (Liu, June 20, 2007). 3. Using computation modeling, we developed a linear discrete system model to infer gene interactions and regulatory networks for ethanologenic yeast in response to biomass conversion inhibitor 5-hydroxymethylfurfural. This research contributes to objectives 1 and 4 of this project. Results of this research were disseminated to research community by publication of two original research articles (Song and Liu, 2007, Lecture Notes Bioinformatics 4532:77-95; Song, Ouyang, Liu, 2008, submitted); and a conference presentation (RECOMB Satellite Conference on System Biology, November 2007, San Diego).

Impacts
1. Our development of universal external RNA controls was initiated prior to this NRI award. With its completion, we successfully applied these quality controls to this project. Our universal RNA control is the first of its kind since the high throughput expression technology appeared. It provides consistent and reliable reference to guard reliability and reproducibility of gene expression data. Applications of such quality control made it possible to compare expression data obtained from different experimental sets, different laboratories, and different platforms of microarray and real time qRT-PCR. Application of the universal RNA control in this project using S. cerevisiae genome microarray provided the first example to demonstrate enhanced gene expression technology and thereafter significant outcomes of reliable data. Such obtained microarray data significantly reduced misleading false signals for data interpretation. The development and application of such quality controls impact expression community around the world for improved quality research using high throughput technology. Soon after our publication of this research, a highly reputable Japanese research institute developed a similar control system for human genome research citing our published work. 2. We identified over 300 genes that are differentially expressed significantly in response to biomass conversion inhibitor 5-hydroxymethylfurfural (HMF) by genomic expression analysis using microarray. We further identified functional enzyme encoding genes contributing detoxification of inhibitor furfural and HMF. Using the first tolerant strain S. cerevisiae NRRL Y-50049 developed through the extension of this project, we demonstrated multiple gene mediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and HMF by the ethanologenic yeast. Development of tolerant strain is a key for low-cost biomass conversion to ethanol and such a need is well recognized by biofuels communities internationally. The detoxification mechanism illustrated by our research will significantly enhance more tolerant strain development. New knowledge of inhibitor conversion pathways relevant to glycolysis reported in our original research article will stimulate interests of research in this area, advance our understanding of tolerant mechanisms, and reduce the time needed to develop more tolerant yeast for cost-efficient bioethanol conversion. As an example, results of our basic research on detoxification mechanism in this project have showed significant impact to guide more tolerant strain development for potential industrial applications. 3. By a close collaboration between NCAUR and New Mexico State University through this project, we established discrete dynamic system modeling for gene networks of HMF tolerance for ethanologenic yeast. This accomplishment impacts new computation modeling system development, aid gene interaction analysis and inferring global regulatory networks, and benefit for more tolerant strain development with continued refinement of our system modeling.

Publications

  • 1. Liu, Z.L., and Slininger, P.J. 2007. Universal external RNA quality controls for mRNA expression analysis using microbial DNA oligo microarray and real time quantitative RT-PCR. J. Microbiol. Methods 68:486-496.
  • 2. Liu, Z.L., B.C. Saha, and P.J. Slininger. 2007. Lignocellulosic biomass conversion to ethanol by Saccharomyces. In: A.L. Demain, C. Harwood, and J. Wall (eds.) Bioenergy. ASM Press. Washington, DC.
  • 3. Liu, Z.L., J. Moon, B.J. Andersh, P.J. Slininger, and S.A. Weber. 2008. Multiple gene mediated aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae (submitted).
  • 4. Liu, Z.L., and Song, M.J. 2008. Genomic adaptation of Saccharomyces cerevisiae to inhibitors involving biomass conversion to ethanol. In: Current Advances in Mycology. M. Rai (eds.) (submitted).
  • 5. Song M.J. and Liu, Z.L. 2007. A Linear Discrete Dynamic System Model for Temporal Gene Interaction and Regulatory Network Influence in Response to Bioethanol Conversion Inhibitor HMF for Ethanologenic Yeast. Lecture Notes Bioinfomatics 4532:77-95.
  • 6. Song, M.J., Ouyang, Z, Liu Z.L. 2008. Discrete dynamic system modeling for gene regulatory networks of HMF tolerance for ethanologenic yeast. IET Systems Biology (submitted).