Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover
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 de...
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Published in | PloS one Vol. 9; no. 9; p. e107499 |
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Main Authors | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Public Library of Science
15.09.2014
Public Library of Science (PLoS) |
Subjects | |
Online Access | Get full text |
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Summary: | 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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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Undefined-1 ObjectType-Feature-3 content type line 23 AC02-05CH11231; FC02-07ER64494 USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: LSP AJH LBW STW BAS DBH JJC BED VB DHK YZ RL APG TKS. Performed the experiments: LSP RJB RAN AJH ALR MT LQ LBW BDB BLB RCP AJB NU TL CL DT HL ELP JS DHK YZ TKS. Analyzed the data: AJH LBW IMO TKS. Contributed reagents/materials/analysis tools: NU TL CL DT BAS DBH MSW JJC BED VB TKS. Contributed to the writing of the manuscript: LSP RJB AJH LBW CL STW BAS DBH JJC BED VB DHK YZ RL APG TKS. |
ISSN: | 1932-6203 1932-6203 |
DOI: | 10.1371/journal.pone.0107499 |