Identification of gene targets eliciting improved alcohol tolerance in Saccharomyces cerevisiae through inverse metabolic engineering

Hong, Min Eui; Lee, Ki Sung; Yu, Byung Jo; Sung, Youngje; Park, Sung Min; Koo, Hyun; Kweon, Dae Hyuk; Park, Jae Chan; Liu, Jing Jing

doi:10.1016/j.jbiotec.2010.06.006

Cited by 74 publications

(46 citation statements)

References 47 publications

(53 reference statements)

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“…All of these observations suggested the need for identifying effective gene targets through methods that directly select resistant yeast strains with traceable genetic perturbations. Successful application of inverse metabolic engineering in the present study and some previous works (34,(61)(62)(63)(64) demonstrated the effectiveness of this approach in discovering novel gene perturbation targets for improving the desirable target phenotypes.…”

Section: Discussionsupporting

confidence: 70%

Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering

Chen

Stabryla

Wei

2016

Appl Environ Microbiol

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bDevelopment of acetic acid-resistant Saccharomyces cerevisiae is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target, WHI2 (encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance in S. cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. The WHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression of WHI2 gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2⌬ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response in S. cerevisiae. Additionally, overexpression of WHI2 and of a cognate phosphatase gene, PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production. Lignocellulosic biomass from nonfood stocks such as agricultural and forestry residues has been identified as the prime source for production of renewable biofuels to substitute for conventional fossil fuels in the face of growing demand for energy and rising concerns about greenhouse gas emissions (1-4). Bioconversion of plant cell wall materials by microbial fermentation is typically preceded by harsh (physico)chemical hydrolysis designed to release sugars; this hydrolysis treatment also generates by-products that are toxic to fermenting microorganisms (5, 6). Since hemicellulose and lignin in the plant cell wall are ubiquitously acetylated (7,8), the typical acidic pretreatment of lignocellulosic biomass generates substantial amounts of acetic acid (with concentrations ranging from 1 g/liter to 15 g/liter) in the resulting hydrolysates (9, 10). Acetic acid severely inhibits cell growth and fermentation activity in Saccharomyces cerevisiae (5, 6, 11-13), the predominant microorganism used in industrial fermentation (14, 15). Therefore, improvement in S. cerevisiae resistance to acetic acid is highly desired and critical for achieving efficient and economically viable bioconversion of cellulosic sugars to biofuels.The toxic effects of acetic acid in S. cerevisiae have been intensively characterized, and toxicity mechanisms have been proposed (10,11,(16)(17)(18)(19)(20). When the external pH is lower than the...

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Section: Discussionsupporting

confidence: 70%

Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering

Chen

Stabryla

Wei

2016

Appl Environ Microbiol

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“…This overlap validates our methodology in identifying gene targets that improve galactose fermentation using a genomic library. Similar approaches have been successfully applied to identify gene targets eliciting many phenotypes of biotechnological importance, such as improved xylose utilization , higher tolerance to ethanol and isobutanol (Hong et al, 2010) and enhanced lycopene production (Jin and Stephanopoulos, 2007;Kang et al, 2005). All of the studies were able to discover novel genetic perturbations and to confirm existing or known genetic perturbations responsible for desired phenotypes.…”

Section: Discussionmentioning

confidence: 99%

Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering

Lee

Hong

Jung

et al. 2010

Biotech & Bioengineering

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102

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Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses.

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“…There have been suggestions that no single gene can endow microbes with tolerance to ethanol and other toxic compounds (14), and until recently little progress had been made in identification of key genetic changes that confer enhanced ethanol tolerance (15). Global transcription machinery engineering has been used to improve tolerance to both glucose and ethanol and to increase productivity in Saccharomyces cerevisiae by altering expression of many genes simultaneously through a single genetic modification (16).…”

Section: Nonrandom Distribution Of Mutations Across the Genome And Theirmentioning

confidence: 99%

“…Global transcription machinery engineering has been used to improve tolerance to both glucose and ethanol and to increase productivity in Saccharomyces cerevisiae by altering expression of many genes simultaneously through a single genetic modification (16). In contrast, Hong et al (15) used an inverse metabolic engineering approach to demonstrate that overexpression of endogenous S. cerevisiae genes (INO1, DOG1, HAL1, or a truncated MSN2) individually can confer improved alcohol tolerance, higher titers, higher volumetric productivities, and increased specific growth rate. However, none of the S. cerevisiae genes that confer improvements in alcohol tolerance are similar to the C. thermocellum adhE gene, and their products perform quite different functions.…”

Section: Nonrandom Distribution Of Mutations Across the Genome And Theirmentioning

confidence: 99%

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Brown

Guss

Karpinets

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

160

145

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Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene ( adhE ), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

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Identification of gene targets eliciting improved alcohol tolerance in Saccharomyces cerevisiae through inverse metabolic engineering

Cited by 74 publications

References 47 publications

Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering

Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering

Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

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