Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover

Lucas S. Parreiras, Rebecca J. Breuer, Ragothaman Avanasi Narasimhan, Alan J. Higbee, Alex La Reau, Mary Tremaine, Li Qin, Laura B. Willis, Benjamin D. Bice, Brandi L. Bonfert, Rebeca C. Pinhancos, Allison J. Balloon, Nirmal Uppugundla, Tongjun Liu, Chenlin Li, Deepti Tanjore, Irene M. Ong, Haibo Li, Edward L. Pohlmann, Jose Serate & 12 others Sydnor T. Withers, Blake A. Simmons, David B. Hodge, Michael S. Westphall, Joshua J. Coon, Bruce E. Dale, Venkatesh Balan, David H. Keating, Yaoping Zhang, Robert Landick, Audrey P. Gasch, Trey K. Sato

    Research output: Contribution to journalArticle

    • 24 Citations

    Abstract

    The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.

    Original languageEnglish (US)
    Article numbere107499
    JournalPLoS One
    Volume9
    Issue number9
    DOIs
    StatePublished - Sep 15 2014

    Profile

    Xylose
    Ammonia
    Fermentation
    Zea mays
    Saccharomyces cerevisiae
    xylose
    HLA Antigens
    hydrolysates
    Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)
    corn stover
    engineering
    xylose isomerase
    anaerobic conditions
    ammonia
    ethanol
    yeasts
    Isomerases
    Ethanol
    Yeasts
    Abnormal Erythrocytes

    ASJC Scopus subject areas

    • Agricultural and Biological Sciences(all)
    • Biochemistry, Genetics and Molecular Biology(all)
    • Medicine(all)

    Cite this

    Parreiras, L. S., Breuer, R. J., Narasimhan, R. A., Higbee, A. J., La Reau, A., Tremaine, M., ... Sato, T. K. (2014). Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover. PLoS One, 9(9), [e107499]. DOI: 10.1371/journal.pone.0107499

    Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover. / Parreiras, Lucas S.; Breuer, Rebecca J.; Narasimhan, Ragothaman Avanasi; Higbee, Alan J.; La Reau, Alex; Tremaine, Mary; Qin, Li; Willis, Laura B.; Bice, Benjamin D.; Bonfert, Brandi L.; Pinhancos, Rebeca C.; Balloon, Allison J.; Uppugundla, Nirmal; Liu, Tongjun; Li, Chenlin; Tanjore, Deepti; Ong, Irene M.; Li, Haibo; Pohlmann, Edward L.; Serate, Jose; Withers, Sydnor T.; Simmons, Blake A.; Hodge, David B.; Westphall, Michael S.; Coon, Joshua J.; Dale, Bruce E.; Balan, Venkatesh; Keating, David H.; Zhang, Yaoping; Landick, Robert; Gasch, Audrey P.; Sato, Trey K.

    In: PLoS One, Vol. 9, No. 9, e107499, 15.09.2014.

    Research output: Contribution to journalArticle

    Parreiras, LS, Breuer, RJ, Narasimhan, RA, Higbee, AJ, La Reau, A, Tremaine, M, Qin, L, Willis, LB, Bice, BD, Bonfert, BL, Pinhancos, RC, Balloon, AJ, Uppugundla, N, Liu, T, Li, C, Tanjore, D, Ong, IM, Li, H, Pohlmann, EL, Serate, J, Withers, ST, Simmons, BA, Hodge, DB, Westphall, MS, Coon, JJ, Dale, BE, Balan, V, Keating, DH, Zhang, Y, Landick, R, Gasch, AP & Sato, TK 2014, 'Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover' PLoS One, vol 9, no. 9, e107499. DOI: 10.1371/journal.pone.0107499

    Parreiras, Lucas S.; Breuer, Rebecca J.; Narasimhan, Ragothaman Avanasi; Higbee, Alan J.; La Reau, Alex; Tremaine, Mary; Qin, Li; Willis, Laura B.; Bice, Benjamin D.; Bonfert, Brandi L.; Pinhancos, Rebeca C.; Balloon, Allison J.; Uppugundla, Nirmal; Liu, Tongjun; Li, Chenlin; Tanjore, Deepti; Ong, Irene M.; Li, Haibo; Pohlmann, Edward L.; Serate, Jose; Withers, Sydnor T.; Simmons, Blake A.; Hodge, David B.; Westphall, Michael S.; Coon, Joshua J.; Dale, Bruce E.; Balan, Venkatesh; Keating, David H.; Zhang, Yaoping; Landick, Robert; Gasch, Audrey P.; Sato, Trey K. / Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover.

    In: PLoS One, Vol. 9, No. 9, e107499, 15.09.2014.

    Research output: Contribution to journalArticle

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    abstract = "The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.",
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