Dataset Information

Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant Saccharomyces cerevisiae strains for second-generation bioethanol production.

ABSTRACT:

Background

Robust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance.

Results

Grape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content. Forty newly isolated Saccharomyces cerevisiae strains gave high ethanol yields at 40°C when inoculated in minimal media at high sugar concentrations of up to 200 g/l glucose. In addition, the isolates displayed distinct inhibitor tolerance in defined broth supplemented with increasing levels of single inhibitors or with a cocktail containing several inhibitory compounds. Both the fermentation ability and inhibitor resistance of these strains were greater than those of established industrial and commercial S. cerevisiae yeasts used as control strains in this study. Liquor from steam-pretreated sugarcane bagasse was used as a key selective condition during the isolation of robust yeasts for industrial ethanol production, thus simulating the industrial environment. The isolate Fm17 produced the highest ethanol concentration (43.4 g/l) from the hydrolysate, despite relatively high concentrations of weak acids, furans, and phenolics. This strain also exhibited a significantly greater conversion rate of inhibitory furaldehydes compared with the reference strain S. cerevisiae 27P. To our knowledge, this is the first report describing a strain of S. cerevisiae able to produce an ethanol yield equal to 89% of theoretical maximum yield in the presence of high concentrations of inhibitors from sugarcane bagasse.

Conclusions

This study showed that yeasts with high tolerance to multiple stress factors can be obtained from unconventional ecological niches. Grape marc appeared to be an unexplored and promising substrate for the isolation of S. cerevisiae strains showing enhanced inhibitor, temperature, and osmotic tolerance compared with established industrial strains. This integrated approach of selecting multiple resistant yeasts from a single source demonstrates the potential of obtaining yeasts that are able to withstand a number of fermentation-related stresses. The yeast strains isolated and selected in this study represent strong candidates for bioethanol production from lignocellulosic hydrolysates.

SUBMITTER: Favaro L

PROVIDER: S-EPMC4176503 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Publications

Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant Saccharomyces cerevisiae strains for second-generation bioethanol production.

Favaro Lorenzo L Basaglia Marina M Trento Alberto A Van Rensburg Eugéne E García-Aparicio Maria M Van Zyl Willem H WH Casella Sergio S

Biotechnology for biofuels 20131129 1

<h4>Background</h4>Robust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance.<h4>Results</h4>Grape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content. For ...[more]

PMID: 24286305

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Project description:BackgroundThe cost-effective production of second-generation bioethanol, which is made from lignocellulosic materials, has to face the following two problems: co-fermenting xylose with glucose and enhancing the strain's tolerance to lignocellulosic inhibitors. Based on our previous study, the wild-type diploid Saccharomyces cerevisiae strain BSIF with robustness and good xylose metabolism genetic background was used as a chassis for constructing efficient xylose-fermenting industrial strains. The performance of the resulting strains in the fermentation of media with sugars and hydrolysates was investigated.ResultsThe following two novel heterologous genes were integrated into the genome of the chassis cell: the mutant MGT05196N360F, which encodes a xylose-specific, glucose-insensitive transporter and is derived from the Meyerozyma guilliermondii transporter gene MGT05196, and Ru-xylA (where Ru represents the rumen), which encodes a xylose isomerase (XI) with higher activity in S. cerevisiae. Additionally, endogenous modifications were also performed, including the overproduction of the xylulokinase Xks1p and the non-oxidative PPP (pentose phosphate pathway), and the inactivation of the aldose reductase Gre3p and the alkaline phosphatase Pho13p. These rationally designed genetic modifications, combined with alternating adaptive evolutions in xylose and SECS liquor (the leach liquor of steam-exploding corn stover), resulted in a final strain, LF1, with excellent xylose fermentation and enhanced inhibitor resistance. The specific xylose consumption rate of LF1 reached as high as 1.089 g g-1 h-1 with xylose as the sole carbon source. Moreover, its highly synchronized utilization of xylose and glucose was particularly significant; 77.6% of xylose was consumed along with glucose within 12 h, and the ethanol yield was 0.475 g g-1, which is more than 93% of the theoretical yield. Additionally, LF1 performed well in fermentations with two different lignocellulosic hydrolysates.ConclusionThe strain LF1 co-ferments glucose and xylose efficiently and synchronously. This result highlights the great potential of LF1 for the practical production of second-generation bioethanol.

Project description:BackgroundStrong multiple stress-tolerance is a desirable characteristic for Saccharomyces cerevisiae when different feedstocks are used for economical industrial ethanol production. Random mutagenesis or genome shuffling has been applied for improving multiple stress-tolerance, however, these techniques are generally time-consuming and labor cost-intensive and their molecular mechanisms are unclear. Genetic engineering, as an efficient technology, is poorly applied to construct multiple stress-tolerant industrial S. cerevisiae due to lack of clear genetic targets. Therefore, constructing multiple stress-tolerant industrial S. cerevisiae is challenging. In this study, some target genes were mined by comparative transcriptomics analysis and applied for the construction of multiple stress-tolerant industrial S. cerevisiae strains with prominent bioethanol production.ResultsTwenty-eight shared differentially expressed genes (DEGs) were identified by comparative analysis of the transcriptomes of a multiple stress-tolerant strain E-158 and its original strain KF-7 under five stress conditions (high ethanol, high temperature, high glucose, high salt, etc.). Six of the shared DEGs which may have strong relationship with multiple stresses, including functional genes (ASP3, ENA5), genes of unknown function (YOL162W, YOR012W), and transcription factors (Crz1p, Tos8p), were selected by a comprehensive strategy from multiple aspects. Through genetic editing based on the CRISPR/Case9 technology, it was demonstrated that expression regulation of each of these six DEGs improved the multiple stress-tolerance and ethanol production of strain KF-7. In particular, the overexpression of ENA5 significantly enhanced the multiple stress-tolerance of not only KF-7 but also E-158. The resulting engineered strain, E-158-ENA5, achieved higher accumulation of ethanol. The ethanol concentrations were 101.67% and 27.31% higher than those of the E-158 when YPD media and industrial feedstocks (straw, molasses, cassava) were fermented, respectively, under stress conditions.ConclusionSix genes that could be used as the gene targets to improve multiple stress-tolerance and ethanol production capacities of S. cerevisiae were identified for the first time. Compared to the other five DEGs, ENA5 has a more vital function in regulating the multiple stress-tolerance of S. cerevisiae. These findings provide novel insights into the efficient construction of multiple stress-tolerant industrial S. cerevisiae suitable for the fermentation of different raw materials.

Project description:In the search for renewable sources of energy, bioethanol stands out as a benchmark biofuel because its production is based on a proven technological platform. Bioethanol is produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (~2 SNPs per kilobase), and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, and appear to reflect ectopic homologous recombination between repetitive DNA sequences. Despite the complex karyotype of JAY270, this diploid, when sporulated, had a high frequency of viable spores (~93%). Crosses of haploids derived from JAY270 to a haploid of the unrelated laboratory strain S288c also resulted in diploids that had good spore viability (75-95%). Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis and spore viability, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures that may be associated with a fitness benefit. We also explore features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high cell mass production and fermentation kinetics, high temperature growth and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

Dataset Information

Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant Saccharomyces cerevisiae strains for second-generation bioethanol production.

Background

Results

Conclusions

Publications

Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant Saccharomyces cerevisiae strains for second-generation bioethanol production.

Similar Datasets

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