Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast

Wei, Na; Quarterman, Josh; Kim, Soo Rin; Cate, J.H.D.; Liu, Jing Jing

doi:10.1038/ncomms3580

Cited by 208 publications

(165 citation statements)

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“…The acetic acid concentration in glucose fermentation did not change significantly, most likely due to carbon catabolite repression by glucose, while the acetic acid concentration in xylose fermentation started to decrease after 40 h and was reduced by ϳ1 g/liter at the end of the fermentation experiment. Under anoxic conditions, no acetic acid consumption occurred in glucose or xylose fermentation, as S. cerevisiae does not metabolize acetic acid without oxygen (43). Noticeably, when no acetic acid consumption occurred, there was still significant improvement in fermentation by strain S-WHI2 versus that by the control strain S-C1 ( Fig.…”

Section: Resultsmentioning

confidence: 77%

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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: Resultsmentioning

confidence: 77%

“…For anaerobic fermentation experiments, samples were collected by sterile syringes and 26-gauge BD needles. Yeast cell dry weight was determined using a microwave method as described previously (43). All of the fermentation experiments were performed in duplicate.…”

Section: Methodsmentioning

confidence: 99%

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 redox imbalance is especially problematic during anaerobic fermentation and has led to efforts to either match the cofactor preference of XR and XDH (26) or to use the cofactor-independent xylose isomerase instead (9,27). Wei et al used this redox imbalance to their advantage by redirecting the surplus NADH of xylose consumption to the conversion of acetate to ethanol, showing consumption of 0.031 g acetate (g xylose) Ϫ1 in a Gpd ϩ background (24). No genetic changes were required to satisfy the increased NADPH demand, consistent with findings that the oxidative pentose phosphate pathway flexibly responds to increased NADPH consumption (22).…”

mentioning

confidence: 99%

“…To circumvent this redox constraint, Wei et al elegantly combined the ACS/ADA/ADH pathway with xylose fermentation via xylose reductase (XR) and xylitol dehydrogenase (XDH) (24). Introducing NAD(P)H-specific XR and NADH-specific XDH from Scheffersomyces stipitis into S. cerevisiae is known to confer the ability to ferment xylose (25), but the differing redox cofactor preferences of these enzymes result in increased NADPH demand and overproduction of NADH during growth on xylose.…”

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confidence: 99%

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Henningsen

Hon

Covalla

et al. 2015

Appl Environ Microbiol

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Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter ؊1 acetate during fermentation of 114 g liter ؊1 glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter ؊1 , this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter ؊1and raised the ethanol yield to 7% above the wild-type level. Saccharomyces cerevisiae is the principal microorganism used to produce ethanol, of which 93 billion liters were globally produced in 2014 (1). However, new strains are needed for the production of second-generation biofuels. Pretreatment and monomerization of lignocellulosic substrates produce complex sugar mixtures, including the C 5 sugars xylose and arabinose that are poorly fermented by most wild-type S. cerevisiae strains (2, 3). In addition, a variety of inhibitory compounds are released, such as furfural, hydroxymethylfurfural, methylglyoxal, and acetate (4-6). It is therefore desirable not only to expand the substrate range of S. cerevisiae (3, 7-9) but also to increase its inhibitor tolerance (10-13).Acetate is released during deacylation of hemicellulose and lignin and can be present in concentrations of Ͼ10 g liter Ϫ1 in cellulosic hydrolysates (4,14). At low pH particularly, this weak organic acid is a potent inhibitor of S. cerevisiae metabolism (15-17). Unfortunately, wild-type S. cerevisiae strains are poorly equipped to eliminate acetate from the medium under anoxic conditions. S. cerevisiae can grow on acetate as the sole carbon source under oxic conditions by converting acetate to acetyl coenzyme A (acetyl-CoA) with acetyl-CoA synthetase (ACS) (EC 6.2.1.1) and by either respiring acetyl-CoA in the tricarboxylic acid (TCA) cycle or upgrading acetyl-CoA to four-...

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“…Efficient conversion of carbon substrates, such as arabinose, xylose, and cellobiose, has been materialized through the introduction of hydrolytic enzymes and transporter genes in yeast. Engineered strains of S. cerevisiae capable of utilizing starch and xylose have been reported (Wei et al, 2013). But all these recombinant strains perform well at a fermentation temperature of 30°C but not at the higher temperatures.…”

Section: Substrate Utilization Of Non-conventional Yeastsmentioning

confidence: 99%

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

et al. 2017

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The increasing fossil fuel scarcity has led to an urgent need to develop alternative fuels. Currently microorganisms have been extensively used for the production of first-generation biofuels from lignocellulosic biomass. Yeast is the efficient producer of bioethanol among all existing biofuels option. Tools of synthetic biology have revolutionized the field of microbial cell factories especially in the case of ethanol and fatty acid production. Most of the synthetic biology tools have been developed for the industrial workhorse Saccharomyces cerevisiae. The non-conventional yeast systems have several beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, etc., and synthetic biology have the power to expand these traits. Currently, synthetic biology is slowly widening to the non-conventional yeasts like Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. Herein, we review the basic synthetic biology tools that can apply to non-conventional yeasts. Furthermore, we discuss the recent advances employed to develop efficient biofuel-producing non-conventional yeast strains by metabolic engineering and synthetic biology with recent examples. Looking forward, future synthetic engineering tools' development and application should focus on unexplored non-conventional yeast species.

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Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast

Cited by 208 publications

References 44 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

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

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