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

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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.

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

Profile

corn stover
Xylose
xylose
hydrolysates
Ammonia
Yeast
Fermentation
Zea mays
Saccharomyces cerevisiae
engineering
ammonia
fermentation
Fibers
xylose isomerase
anaerobic conditions
Ethanol
Yeasts
ethanol
yeasts
Xylitol

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. 2014 ; Vol. 9, No. 9.
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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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AU - Keating,David H.

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