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Transporter engineering for improved tolerance against alkane biofuels in Saccharomyces cerevisiae.

ABSTRACT: BACKGROUND:Hydrocarbon alkanes, components of major fossil fuels, are considered as next-generation biofuels because their biological production has recently been shown to be possible. However, high-yield alkane production requires robust host cells that are tolerant against alkanes, which exhibit cytotoxicity. In this study, we aimed to improve alkane tolerance in Saccharomyces cerevisiae, a key industrial microbial host, by harnessing heterologous transporters that potentially pump out alkanes. RESULTS:To this end, we attempted to exploit ABC transporters in Yarrowia lipolytica based on the observation that it utilizes alkanes as a carbon source. We confirmed the increased transcription of ABC2 and ABC3 transporters upon exposure to a range of alkanes in Y. lipolytica. We then showed that the heterologous expression of ABC2 and ABC3 transporters significantly increased tolerance against decane and undecane in S. cerevisiae through maintaining lower intracellular alkane level. In particular, ABC2 transporter increased the tolerance limit of S. cerevisiae about 80-fold against decane. Furthermore, through site-directed mutagenesis for glutamate (E988 for ABC2, and E989 for ABC3) and histidine (H1020 for ABC2, and H1021 for ABC3), we provided the evidence that glutamate was essential for the activity of ABC2 and ABC3 transporters, with ATP most likely to be hydrolyzed by a catalytic carboxylate mechanism. CONCLUSIONS:Here, we demonstrated that transporter engineering through expression of heterologous efflux pumps led to significantly improved tolerance against alkane biofuels in S. cerevisiae. We believe that our results laid the groundwork for developing robust alkane-producing yeast cells through transporter engineering, which will greatly aid in next-generation alkane biofuel production and recovery.

SUBMITTER: Chen B

PROVIDER: S-EPMC3598725 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Transporter engineering for improved tolerance against alkane biofuels in Saccharomyces cerevisiae.

Chen Binbin B Ling Hua H Chang Matthew Wook MW

Biotechnology for biofuels 20130213 1

<h4>Background</h4>Hydrocarbon alkanes, components of major fossil fuels, are considered as next-generation biofuels because their biological production has recently been shown to be possible. However, high-yield alkane production requires robust host cells that are tolerant against alkanes, which exhibit cytotoxicity. In this study, we aimed to improve alkane tolerance in Saccharomyces cerevisiae, a key industrial microbial host, by harnessing heterologous transporters that potentially pump out ...[more]

PMID: 23402697

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Project description:BACKGROUND:Hydrocarbon alkanes have been recently considered as important next-generation biofuels because microbial production of alkane biofuels was demonstrated. However, the toxicity of alkanes to microbial hosts can possibly be a bottleneck for high productivity of alkane biofuels. To tackle this toxicity issue, it is essential to understand molecular mechanisms of interactions between alkanes and microbial hosts, and to harness these mechanisms to develop microbial host strains with improved tolerance against alkanes. In this study, we aimed to improve the tolerance of Saccharomyces cerevisiae, a model eukaryotic host of industrial significance, to alkane biofuels by exploiting cellular mechanisms underlying alkane response. RESULTS:To this end, we first confirmed that nonane (C9), decane (C10), and undecane (C11) were significantly toxic and accumulated in S. cerevisiae. Transcriptome analyses suggested that C9 and C10 induced a range of cellular mechanisms such as efflux pumps, membrane modification, radical detoxification, and energy supply. Since efflux pumps could possibly aid in alkane secretion, thereby reducing the cytotoxicity, we formed the hypothesis that those induced efflux pumps could contribute to alkane export and tolerance. In support of this hypothesis, we demonstrated the roles of the efflux pumps Snq2p and Pdr5p in reducing intracellular levels of C10 and C11, as well as enhancing tolerance levels against C10 and C11. This result provided the evidence that Snq2p and Pdr5p were associated with alkane export and tolerance in S. cerevisiae. CONCLUSIONS:Here, we investigated the cellular mechanisms of S. cerevisiae response to alkane biofuels at a systems level through transcriptome analyses. Based on these mechanisms, we identified efflux pumps involved in alkane export and tolerance in S. cerevisiae. We believe that the results here provide valuable insights into designing microbial engineering strategies to improve cellular tolerance for highly efficient alkane biofuel production.

Project description:BackgroundThe fermentation of lignocellulose hydrolysates to ethanol requires robust xylose-capable Saccharomyces cerevisiae strains able to operate in the presence of microbial inhibitory stresses. This study aimed at developing industrial S. cerevisiae strains with enhanced tolerance towards pretreatment-derived microbial inhibitors, by identifying novel gene combinations that confer resistance to multiple inhibitors (thus cumulative inhibitor resistance phenotype) with minimum impact on the xylose fermentation ability. The strategy consisted of multiple sequential delta-integrations of double-gene cassettes containing one gene conferring broad inhibitor tolerance (ARI1, PAD1 or TAL1) coupled with an inhibitor-specific gene (ADH6, FDH1 or ICT1). The performances of the transformants were compared with the parental strain in terms of biomass growth, ethanol yields and productivity, as well as detoxification capacities in a synthetic inhibitor cocktail, sugarcane bagasse hydrolysate as well as hardwood spent sulphite liquor.ResultsThe first and second round of delta-integrated transformants exhibited a trade-off between biomass and ethanol yield. Transformants showed increased inhibitor resistance phenotypes relative to parental controls specifically in fermentations with concentrated spent sulphite liquors at 40% and 80% v/v concentrations in 2% SC media. Unexpectedly, the xylose fermentation capacity of the transformants was reduced compared to the parental control, but certain combinations of genes had a minor impact (e.g. TAL1 + FDH1). The TAL1 + ICT1 combination negatively impacted on both biomass growth and ethanol yield, which could be linked to the ICT1 protein increasing transformant susceptibility to weak acids and temperature due to cell membrane changes.ConclusionsThe integration of the selected genes was proven to increase tolerance to pretreatment inhibitors in synthetic or industrial hydrolysates, but they were limited to the fermentation of glucose. However, some gene combination sequences had a reduced impact on xylose conversion.

Project description:BackgroundIn the yeast Saccharomyces cerevisiae, which is widely applied for industrial bioethanol production, uptake of hexoses is mediated by transporters with a facilitated diffusion mechanism. In anaerobic cultures, a higher ethanol yield can be achieved when transport of hexoses is proton-coupled, because of the lower net ATP yield of sugar dissimilation. In this study, the facilitated diffusion transport system for hexose sugars of S. cerevisiae was replaced by hexose-proton symport.ResultsIntroduction of heterologous glucose- or fructose-proton symporters in an hxt0 yeast background strain (derived from CEN.PK2-1C) restored growth on the corresponding sugar under aerobic conditions. After applying an evolutionary engineering strategy to enable anaerobic growth, the hexose-proton symporter-expressing strains were grown in anaerobic, hexose-limited chemostats on synthetic defined medium, which showed that the biomass yield of the resulting strains was decreased by 44.0-47.6%, whereas the ethanol yield had increased by up to 17.2% (from 1.51 to 1.77 mol mol hexose-1) compared to an isogenic strain expressing the hexose uniporter HXT5. To apply this strategy to increase the ethanol yield on sucrose, we constructed a platform strain in which all genes encoding hexose transporters, disaccharide transporters and disaccharide hydrolases were deleted, after which a combination of a glucose-proton symporter, fructose-proton symporter and extracellular invertase (SUC2) were introduced. After evolution, the resulting strain exhibited a 16.6% increased anaerobic ethanol yield (from 1.51 to 1.76 mol mol hexose equivalent-1) and 46.6% decreased biomass yield on sucrose.ConclusionsThis study provides a proof-of-concept for the replacement of the endogenous hexose transporters of S. cerevisiae by hexose-proton symport, and the concomitant decrease in ATP yield, to greatly improve the anaerobic yield of ethanol on sugar. Moreover, the sugar-negative platform strain constructed in this study acts as a valuable starting point for future studies on sugar transport or development of cell factories requiring specific sugar transport mechanisms.

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Transporter engineering for improved tolerance against alkane biofuels in Saccharomyces cerevisiae.

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Transporter engineering for improved tolerance against alkane biofuels in Saccharomyces cerevisiae.

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