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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Publications at this Location » Publication #264580
Title: Genetic engineering of inhibitor-tolerant Saccharomyces cerevisiae for improved xylose utilization in ethanol production

Author
item MA, MENGGEN - New Mexico State University
item Liu, Zonglin
item Moon, Jaewoong

Submitted to: BioEnergy Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/16/2011
Publication Date: 1/12/2012
Citation: Ma, M., Liu, Z., Moon, J. 2012. Genetic engineering of inhibitor-tolerant Saccharomyces cerevisiae for improved xylose utilization in ethanol production. Bioenergy Research. 5:459-469.

Interpretive Summary: Technical challenges to the economical lignocellulose-to-ethanol production include toxic chemical compounds generated from biomass pretreatment that inhibit microbial growth and fermentation and no desirable microbial strain is available for efficient utilization of the abundant xylose contained in lignocellulosic biomass. Saccharomyces cerevisiae strains genetically engineered to utilize xylose do so at a minimum level and produce xylitol as a byproduct. Using systems biology approaches, we developed a tolerant yeast, analyzed pathway interactions between inhibitor-tolerance and xylose utilization, synthesized a functional yeast xylose isomerase, and genetically engineered a set of functional complementary genes by chromosomal integration and deoxyribonucleic acid recombinant technologies. This research developed a new generation of transgenic ethanologenic yeast that is able to grow on xylose as the sole carbon source and competitively utilize xylose in mixtures of glucose-xylose for anaerobic fermentation in the presence of biomass inhibitors. This research represents a technical breakthrough and contributes new knowledge in basic and applied sciences. This study provides an example of second generation bioethanol yeast strains and will guide efforts of strain design and development for cellulosic ethanol industrial applications.

Technical Abstract: Saccharomyces cerevisiae is the classical ethanologenic yeast but limited in pentose utilization such as xylose harbored in lignocellulosic biomass. For economical lignocellulose-to-ethanol production, a desirable robust biocatalyst needs to be tolerant to lignocellulosic hydrolysates and able to competitively utilize heterogeneous biomass sugars of pentoses and hexsoses. Previously, we developed inhibitor-tolerant ethanologenic yeast NRRL Y-50049 that is able to in situ detoxify common aldehyde inhibitors in biomass hydrolysate such as 2-furaldehyde and 5-(hydroxymethyl)-2-furaldehyde. Here we present a new design enhancing its genetic background of competitive xylose utilization capabilities by a systems biology approach. Using genetic engineering and recombinant deoxyribonucleic acid (DNA) technologies, we introduced an in vitro synthesized yeast xylose isomerase gene (YXIsyn) into a defined chromosomal locus of Y-50049 under a robust ADH1 promoter control. To facilitate efficient xylose uptake and utilization, additional supporting genes of xylose transporter (XUT4 and XUT6), xylolukinase (XKS1), and xylitol dehydrogenase (XYL2) from Scheffersomyces (Pichia) stipitis were transferred into an evolutionarily evolved Y-50049-YXIsyn derivative, resulting in S. cerevisiae NRRL Y-50463 (Y-50049-YXIsyn-XUT4-XUT6-XKS1-XYL2). The newly engineered strain was able to grow on xylose as sole carbon source and ferment mixed sugars (glucose-xylose, 50:50 g l-1) anaerobically in the presence of 15 mM each of furfural and hydroxymethyl furfural (HMF). Its tolerance and competitive utilization of xylose in the presence of glucose outperformed existing recombinant S. cerevisiae strains using xylose isomerase pathway to date with an ethanol yield of 38.6 g l**-1 at a volumetric ethanol production rate of 0.40 and 0.54 g l**-1 h**-1 for inhibitor containing and non-inhibitor fermentations, respectively.