Ethanol Tolerance Research Articles

Screening of Yeast Cells for the Production of Wine from Banana and Pineapple Substrates

Wine, an alcoholic beverage is usually produced from juice of variety of fruits by the fermentative action of microorganisms particularly by yeasts. Several substrates such as pineapple, banana, watermelon, pawpaw and other fruits have been used to produce wine using Saccharomyces species. This study was undertaken using non- Saccharomyces to produce table wine with pineapple and banana fruits as substrates. Standard microbiological procedures were employed for yeasts cell isolation, sugar (sucrose) fermentation test, pH, ethanol, sucrose and glucose tolerance test were carried out respectively. Alcohol production by the yeasts was screened and the isolates were identified by genomic techniques. Twenty-two (22) yeasts isolated from palm wine (YW), banana (YB) and pineapple (YP) were screened for their ability to ferment sugar and fourteen (14) of the yeast isolates were positive while eight (8) were negative. The fourteen (14) isolates were further screened for their ability to tolerate pH, ethanol, sucrose and glucose. Tolerance tests for these fourteen (14) yeast isolates recorded values between a range of 3.0-5.0, 0-10% v/v, 5-20% w/v and 5-25% w/v for pH, ethanol, sucrose and glucose concentrations respectively. Statistically, there was a significant difference in the interaction effect for pH, ethanol, sucrose and glucose tolerance (OD600 nm) for yeast isolates at p value ≤ 0.0001. Five (5) yeast isolates had high tolerance ability to pH, ethanol, sucrose and glucose and were further screened for their ability to produce alcohol. The five (5) yeast isolates were identified as Meyerozyma guilliermondii strain 1621, Pichia guilliermondii strain PX-PAT, Meyerozyma caribbica strain Kw 1S7Y2, Meyerozyma caribbica strain Y-27400, Kodamaea ohmeri strain ww1-1 and they produced alcohol content of 7.6%, 6.5%, 2.9%, 2.5% and 0.3% respectively. Meyerozyma guilliermondii strain 1621 and Pichia guilliermondii strain PX-PAT 18 S isolated from palm wine exhibited good characteristics and produced high quantity of alcohol and are suitable for alcohol fermentation of substrates for wine production.

Improving the enzyme property of ornithine transcarbamylase from Lactobacillus brevis through site-directed mutation

Ornithine transcarbamylase (OTC) is an enzyme that participates in the degradation of arginine by catalyzing the formation of ornithine and carbamoyl phosphate from citrulline, which is a major precursor of ethyl carbamate (EC), a potential carcinogen in fermented foods and beverages. Prior research has not focused on the study of OTC, although it has been previously found to have a negative relationship with EC formation. In this study, nine mutants (H140A, H140W, Q143A, Q143F, Q143W, Q143Y, D236A, D236W, and D236R) were constructed by site-directed mutagenesis using the Discovery Studio software to increase the catalytic properties or thermal stability of the enzyme. Compared with the wild-type enzyme, the catalytic activity of Q143W and H140A mutants increased 2–3 times at the most. The optimal temperature decreased to 35 °C, and the optimal pH decreased to 8.5. Meanwhile Q143W and H140A also exhibited excellent ethanol tolerance, which indicated new possibilities for further application in wine fermentation. The thermostability of D236R was successfully enhanced with a 1.4-fold improvement of t1/2 and T5010 higher by 9.9 °C. It is expected that OTC will be transformed by site-directed mutation to achieve an increase in enzyme activity while also enhancing the thermal stability.

Ethanol production by Klebsiella sp. SWET4 using banana peel as feasible substrate

Ethanol has gained its importance as one of the highly demanded biofuels and is being produced from different food crops or food crop wastes after vigorous pretreatment compelling the production process economically incompatible to some extent. The process also becomes non-ecofriendly as the pretreatment process generally produces acid fumes and toxic effluents as well as consumes high energy. In this current study, the potential of the banana peel as a probable substrate for ethanol production was evaluated with the help of logical prediction. Moreover, a single-step ethanol production process was investigated to eliminate hazardous pretreatment processes. A potential bacterial strain SWET4 was isolated from a sewage treatment pond and was identified as a member of the Klebsiella genus by partial sequencing of the 16S rRNA gene. The isolated strain showed ethanol tolerance up to 12% and exhibited its potential as a cellulolytic strain. The bacterial strain SWET4 was found able to ferment banana peel in MSM broth without any other carbon source with a comparatively prolonged lag phase of cellular growth. Trace of ethanol in banana peel fermentation broth of SWET4 was confirmed by high-pressure liquid chromatography. The optimization study for ethanol production enlightened the fact that high temperature and pH supported higher ethanol production. However, due to the growth restriction of the bacterial cell at higher temperatures and pH, only 40 °C and pH 8 were considered optimum. At the optimum substrate concentration (40 g l−1), ethanol concentration was found to be 5.48 g l−1 after 5 days of incubation which summed as a total yield of 0.137 g of ethanol per g of dried banana peel. This current study evaluates the potential of banana peel as a probable substrate for ethanol production and emphasizes on a new possibility of single-step ethanol production without any pretreatment, although metabolic optimization of the strain SWET4 is required to make the process industrially feasible.

Assessment of ethanol tolerance of Kluyveromyces marxianus CCT 7735 selected by adaptive laboratory evolution.

Kluyveromyces marxianus CCT 7735 shows potential for producing ethanol from lactose; however, its low ethanol tolerance is a drawback for its industrial application. The first aim of this study was to obtain four ethanol-tolerant K. marxianus CCT 7735 strains (ETS1, ETS2, ETS3, and ETS4) by adaptive laboratory evolution. The second aim was to select among them the strain that stood out and to evaluate metabolic changes associated with the improved ethanol tolerance in this strain. The ETS4 was selected for displaying a specific growth rate higher than the parental strain under ethanol stress (122%) and specific ethanol production rate (0.26g/g/h) higher than those presented by the ETS1 (0.22g/g/h), ETS2 (0.17g/g/h), and ETS3 (0.17g/g/h) under non-stress condition. Further analyses were performed with the ETS4 in comparison with its parental strain in order to characterize metabolic changes. Accumulation of valine and metabolites of the citric acid cycle (isocitric acid, citric acid, and cis-aconitic acid) was observed only in the ETS4 subjected to ethanol stress. Their accumulation in this strain may have been important to increase ethanol tolerance. Furthermore, the contents of fatty acid methyl esters and ergosterol were higher in the ETS4 than in the parental strain. These differences likely contributed to enhance ethanol tolerance in the ETS4. KEY POINTS: • K. marxianus ethanol-tolerant strains were selected by adaptive laboratory evolution. • Valine and metabolites of the TCA cycle were accumulated in the ETS4. • High contents of fatty acids and ergosterol contributed to enhance ethanol tolerance.

Factors affecting yeast ethanol tolerance and fermentation efficiency.

Alcohol fermentation is a key process in wine, beer, alcoholic beverage production, bioethanol production by means of carbohydrate sources, and food industry byproducts. There are three key points in these kinds of processes determining their efficiency; enzymatic cellulose lysis into simple sugar molecules, alcohol fermentation rate, and ethanol tolerance of yeast cells. The first process is usually carried out by either the use of pure cellulolytic enzymes, which is a high cost procedure, or by the production of these enzymes from cellulolytic bacteria and filamentous fungi. Lately, Saccharomyces cerevisiae and several other yeasts were genetically modified to express recombinant cellulases in media or display them on the cell surface. Many studies have indicated that the genetic engineering of yeast cells can be a useful approach in increasing the alcoholic fermentation rate as well as their ethanol tolerance. These modifications could be the overexpression of a key protein using a strong promoter or the modification of a specific domain or amino acid which can also lead to the desired outcome. This review focuses on the modifications of a single protein and/or pathways that can lead to the augmentation of ethanol tolerance and alcoholic fermentation efficiency of Saccharomyces cerevisiae.

Exploring potentials of indigenous yeasts in fermentation of Chambourcin Grapes

Background: Indigenous yeasts present on grape berries have been shown to impact the winemaking process and final wine quality. In this study, we used Chambourcin, a hybrid grape cultivar, to isolate indigenous yeasts for potential use in winemaking. Hybrid grapes are of particularly interest due to higher resistance to cold temperature and fungal diseases. Methods: Yeasts were isolated from spontaneous fermentation of crushed grapes by plating on Dichloran Rose Bengal Chloramphenicol agar and identified by Sanger sequencing. Yeast candidates were selected for their ability to grow in Yeast Extract-Peptone-Dextrose broth supplemented with varying ethanol concentration. Candidate yeasts were chosen for mock fermentation using sterile Chambourcin juice. Volatile and non-volatile compounds that predict wine quality (flavor and aroma) were measured by UPLC and GC-MS. Results: Hanseniaspora uvarum, Starmerella bacillaris, Candida californica, and Zygoascus meyerae were predominantly isolated from Chambourcin. S. bacillaris isolate 180002 and C. californica isolate 180004 were able to tolerate up to 10% ethanol compared to S. cerevisiae (ethanol tolerance up to 12%). Our results demonstrate that isolate 180002 grown in ethanol supplemented medium is comparable to the commercial S. cerevisiae. Various aroma profiles of three main chemical families – esters, higher alcohol and volatile acids in products fermented by S. bacillaris and other isolates were observed. Conclusion: Starmerella bacillaris isolate 180002 demonstrates potential for use in winemaking with hybrid grapes based on ethanol tolerance and production of wine related chemical compounds. This study further supports the application of indigenous grape-associated yeasts in creating flavor and aroma diversity of wine.

Isolation and characterization of yeast strains from Badacsony, Hungary

In modern winery, starter strains are used for wine making to avoid the risk of slow or incomplete fermentation.However, application of commercial starter yeasts sometimes leads to a uniform character of the wines. On the other hand, indigenous (“terroir”) strains are adapted better to local conditions highlighting the specific taste of wine. In this study, we isolated local yeast strains from Badacsony wine region of Hungary and investigated with microbiological and molecular biological tests in order to develop indigenous starter selection method. As many as 480 yeast strains were isolated and grouped using carbohydrate and nitrogen sources. Finally, 80 selected isolates were characterized for important oenological features, including tolerance of glucose, ethanol and acetic acid. Fermentation ability, killer toxin, hydrogen sulfide and acid production of 80 selected isolates were also tested. Isolates were studied by applying two molecular methods based on rRNA gene sequencing and analysis of Ty retrotransposon's delta elements in case of Saccharomyces strains. Our results have shown that the isolated strains belong to 15 yeast species of 8 genera, and the diversity of yeast population was significantly high in the investigated vineyard. We have found that selection for technological properties was a potential way to find suitable strains from the local microbiome, because a high proportion of isolated wild yeast strains show beneficial oenological properties for wine making. Further, we studied 35 available starter yeasts to avoid re-isolation and we identified only 3 starter yeasts from grape and must samples, which can be considered as very low incidence

Modeling the Ethanol Tolerance of the Probiotic Yeast Saccharomyces cerevisiae var. boulardii CNCM I-745 for its Possible Use in a Functional Beer.

Saccharomyces yeasts are able to ferment simple sugars to generate levels of ethanol that are toxic to other yeasts and bacteria. The tolerance to ethanol of different yeasts depends also on the incubation temperature. In this study, the ethanol stress responses of S. cerevisiae and the probiotic yeast S. boulardii CNCM I-745 were evaluated at two temperatures. The growth kinetics parameters were obtained by fitting the Baranyi and Roberts model to the experimental data. The four-parameter logistic Hill equation was used to describe the ethanol tolerance of the yeasts at the temperatures of 28 and 37°C. Adequate determination coefficients were obtained (R2 > 0.91) in all cases. S. boulardii grown at 28°C was selected as the yeast with the best ethanol tolerance (6-8%) for use in the elaboration of functional craft beers.

Transcriptome analysis of Lactobacillus paracasei SMN-LBK under ethanol stress

Lactobacillus paracasei SMN-LBK (serial number: CCTCC M 2017429) is an ethanol-resistant lactic acid bacteria (LAB) in kumiss. However, the anti-ethanol stress mechanism of L. paracasei SMN-LBK remains unclear. Hence, we performed a transcriptome analysis between L. paracasei SX10 (L. paracasei SMN-LBK under 10% ethanol stress strain, abbreviated as SX10) and L. paracasei SMN-LBK (abbreviated as S10) by RNA sequencing. We performed real-time quantitative PCR (RT-qPCR) to verify the accuracy of the transcription data. The transcriptome data revealed that 315 genes exhibited upregulated expression, and 332 genes were downregulated in the SX10 compared with the S10 group. The PFK, LDH, GPDH, and GK genes were upregulated, with a log2-fold change of 1.10, 0.30, 0.56, and 1.512, respectively. A gene ontology enrichment analysis revealed significant enrichment of ribosomes, ribonucleoprotein complex, non-membrane-bounded organelles, and intracellular non-membrane-bound organelles. Analysis using the Kyoto Encyclopedia of Genes and Genomes database revealed differential genes associated with ribosome function, pyruvate metabolism, biosynthesis of amino acids, fatty acid biosynthesis, fatty acid metabolism, ATP-binding cassette (ABC) transporter, glycolysis, and glycerophospholipid metabolism. The RT-qPCR results were consistent with the transcriptome results. Lactococcus lactis NZ9000 is a typical host bacterium. We performed PFK and GK overexpression to verify the function of the L. paracaseiSX10 resistance gene in Lactococcus lactis NZ9000. Using sodium dodecyl sulfate (SDS)-PAGE electrophoresis, these resistance genes were successfully expressed in Lactococcus lactis NZ9000. The survival rate and key enzyme activity of the recombinant strains were determined under ethanol stress. The survival rate of Lactococcus lactis NZ9000-pNZ8148-PFK and Lactococcus lactis NZ9000-pNZ8148-GK under 10% ethanol stress were 3.43- and 3.80-fold higher compared with the Lactococcus lactis NZ9000-pNZ8148 control, respectively. These results indicate that PFK and GK are important for the ethanol tolerance of LAB and can increase the ethanol tolerance of Lactococcus lactis NZ9000. Hence, PFK and GK were identified as key genes of L. paracasei SX10 with a high ethanol tolerance. Our results provide novel insight for further studies to perform a systematic analysis of the differentially expressed genes and to determine their potential functions in the ethanol tolerance mechanism of LAB.

Characterization of enhanced ethanol tolerance in Paramecium caudatum after exposure to low ethanol concentrations

Characterization of enhanced ethanol tolerance in Paramecium caudatum after exposure to low ethanol concentrations

A microbubble-sparged yeast propagation\u2013fermentation process for bioethanol production

BackgroundIndustrial biotechnology will play an increasing role in creating a more sustainable global economy. For conventional aerobic bioprocesses supplying O2 can account for 15% of total production costs. Microbubbles (MBs) are micron-sized bubbles that are widely used in industry and medical imaging. Using a fluidic oscillator to generate energy-efficient MBs has the potential to decrease the costs associated with aeration. However, little is understood about the effect of MBs on microbial physiology. To address this gap, a laboratory-scale MB-based Saccharomyces cerevisiae Ethanol Red propagation–fermentation bioethanol process was developed and analysed.ResultsAeration with MBs increased O2 transfer to the propagation cultures. Titres and yields of bioethanol in subsequent anaerobic fermentations were comparable for MB-propagated and conventional, regular bubble (RB)-propagated yeast. However, transcript profiling showed significant changes in gene expression in the MB-propagated yeast compared to those propagated using RB. These changes included up-regulation of genes required for ergosterol biosynthesis. Ergosterol contributes to ethanol tolerance, and so the performance of MB-propagated yeast in fed-batch fermentations sparged with 1% O2 as either RBs or MBs were tested. The MB-sparged yeast retained higher levels of ergosteryl esters during the fermentation phase, but this did not result in enhanced viability or ethanol production compared to ungassed or RB-sparged fermentations.ConclusionsThe performance of yeast propagated using energy-efficient MB technology in bioethanol fermentations is comparable to that of those propagated conventionally. This should underpin the future development of MB-based commercial yeast propagation.

Improving Industrially Relevant Phenotypic Traits by Engineering Chromosome Copy Number in Saccharomyces pastorianus.

The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S. pastorianus remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple non-targeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same set-up yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid S. pastorianus genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement.

Stiffness of brewers’ yeast under ethanol stress investigated by atomic force microscopy

Yeast cell exposed under ethanol environment changes the mechanical properties, in addition to physiological properties including the ability of growth and ethanol tolerance. However, it is unclear how the temporal variation in ethanol concentration, CEtOH, occurring during brewing, influences the mechanical properties of yeast cells. Here, we investigated the stiffness of brewers’ yeast cells in response to the change in CEtOH using atomic force microscopy (AFM). We found that as CEtOH was abruptly changed into a critical value, the survival rate of cells was drastically decreased while cells rather survived as CEtOH was gradually increased toward the critical value for a few days. Furthermore, our AFM measurements showed that cells that acquired the ethanol tolerance significantly decreased their stiffness. The result indicated an intimate relationship between cell stiffness and ethanol tolerance that depends on the pathway of changing ethanol environments.

Effect of exogenous proline on the ethanolic tolerance and malolactic performance of Oenococcus oeni.

The use of malolactic starter cultures, often offer no guarantee of microbiological success due to the chemical and physical factors (pH, ethanol, SO2, nutrient availability) that occur during the winemaking process. This study was born with the aim of improving the performance of the lactic acid bacteria used as a starter culture in the de-acidification of wines. Two commercial strains of Oenococcus oeni, were used. Was evaluated the effect of exogenous l-proline added during the bacterial growth, on the improvement of their survival in the presence of different ethanol concentrations and their ability to degrade l-malic acid in synthetic wine with the presence of 12% (v/v) and 13% (v/v) of ethanol. The results showed that l-proline improve ethanol tolerance and so the malolactic performances of O. oeni. This work represents an important strategy to ensure good vitality and improve the performance of the malolactic starter.

Screening and Fermentation Characteristics of Excellent Boza Strain

Boza is a special fermented dairy product in Xinjiang Uyghur Autonomous Region (XUAR). Boza has the characteristics of CO2 mouthfeel, low sugar content, and dense texture, and is produced from barley, millet, or corn as raw materials using the traditional natural fermentation technology. In this study, six strains each of yeast and lactic acid bacteria isolated from XUAR Boza were used as test strains for single strain selection and composite starter screening. The analytical indicators mainly included ethanol production ability, acid production, tolerance, fermentation force, and growth. Saccharomyces cerevisiae Y8 and Lactobacillus plantarum L9 were selected as the fermentation strains suitable for Boza fermentation. The results will provide scientific inputs for utilization by the Boza fermentation industry.

Enhanced ethanol tolerance in Lysinibacillus sp.

Enhanced ethanol tolerance in Lysinibacillus sp.

Expression of acetaldehyde dehydrogenase (aldB) improved ethanol production from xylose by the ethanologenic Escherichia coli RM10.

An endogenous homoethanol pathway (glucose/1.2 xylose => 2 pyruvate => 2 ethanol) was previously engineered in Escherichia coli SZ410 via eliminating acid-producing pathways and anaerobic expression of the pyruvate dehydrogenase complex (aceEF-lpd operon). This ethanologenic derivative was subsequently engineered through adaptive evolution and partial deletion of the RNase G, resulting in an improved strain of E. coli RM10 for ethanol production using C6 and C5 sugars. Nevertheless, compared to the ethanol tolerance and/or ethanol titer achieved by industrial yeast, further incremental improvement of RM10 was needed for ethanol production using cellulosic biomass derived C6 and C5 sugars. In this study, the role of aldB gene (encoding for acetaldehyde dehydrogenase, AldB, which oxidizes acetaldehyde to acetic acid) was evaluated for ethanol/acetaldehyde tolerance and xylose fermentation by RM10. Deletion of aldB gene decreased ethanol tolerance, fermentative cell growth and ethanol production from xylose; while overexpression of aldB gene improved fermentative cell growth, and increased ethanol production from xylose. The improvement is likely attributed to preventing acetaldehyde accumulation (a toxic intermediate of homoethanol pathway) via AldB catalyzed oxidation.

Multiple Small RNAs Interact to Co-regulate Ethanol Tolerance in Zymomonas mobilis.

sRNAs represent a powerful class of regulators that influences multiple mRNA targets in response to environmental changes. However, very few direct sRNA–sRNA interactions have been deeply studied in any organism. Zymomonas mobilis is a bacterium with unique ethanol-producing metabolic pathways in which multiple small RNAs (sRNAs) have recently been identified, some of which show differential expression in ethanol stress. In this study, we show that two sRNAs (Zms4 and Zms6) are upregulated under ethanol stress and have significant impacts on ethanol tolerance and production in Z. mobilis. We conducted multi-omics analysis (combining transcriptomics and sRNA-immunoprecipitation) to map gene networks under the influence of their regulation. We confirmed that Zms4 and Zms6 bind multiple RNA targets and regulate their expressions, influencing many downstream pathways important to ethanol tolerance and production. In particular, Zms4 and Zms6 interact with each other as well as many other sRNAs, forming a novel sRNA–sRNA direct interaction network. This study thus uncovers a sRNA network that co-orchestrates multiple ethanol related pathways through a diverse set of mRNA targets and a large number of sRNAs. To our knowledge, this study represents one of the largest sRNA–sRNA direct interactions uncovered so far.

Differential Contribution of the Parental Genomes to a S. cerevisiae × S. uvarum Hybrid, Inferred by Phenomic, Genomic, and Transcriptomic Analyses, at Different Industrial Stress Conditions.

In European regions of cold climate, S. uvarum can replace S. cerevisiae in wine fermentations performed at low temperatures. S. uvarum is a cryotolerant yeast that produces more glycerol, less acetic acid and exhibits a better aroma profile. However, this species exhibits a poor ethanol tolerance compared with S. cerevisiae. In the present study, we obtained by rare mating (non-GMO strategy), and a subsequent sporulation, an interspecific S. cerevisiae × S. uvarum spore-derivative hybrid that improves or maintains a combination of parental traits of interest for the wine industry, such as good fermentation performance, increased ethanol tolerance, and high glycerol and aroma productions. Genomic sequencing analysis showed that the artificial spore-derivative hybrid is an allotriploid, which is very common among natural hybrids. Its genome contains one genome copy from the S. uvarum parental genome and two heterozygous copies of the S. cerevisiae parental genome, with the exception of a monosomic S. cerevisiae chromosome III, where the sex-determining MAT locus is located. This genome constitution supports that the original hybrid from which the spore was obtained likely originated by a rare-mating event between a mating-competent S. cerevisiae diploid cell and either a diploid or a haploid S. uvarum cell of the opposite mating type. Moreover, a comparative transcriptomic analysis reveals that each spore-derivative hybrid subgenome is regulating different processes during the fermentation, in which each parental species has demonstrated to be more efficient. Therefore, interactions between the two subgenomes in the spore-derivative hybrid improve those differential species-specific adaptations to the wine fermentation environments, already present in the parental species.

Improvement of Ethanol Tolerance by Inactive Protoplast Fusion in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a typical fermentation yeast in beer production. Improving ethanol tolerance of S. cerevisiae will increase fermentation efficiency, thereby reducing capital costs. Here, we found that S. cerevisiae strain L exhibited a higher ethanol tolerance (14%, v/v) than the fermentative strain Q (10%, v/v). In order to enhance the strain Q ethanol tolerance but preserve its fermentation property, protoplast fusion was performed with haploids from strain Q and L. The fusant Q/L-f2 with 14% ethanol tolerance was obtained. Meanwhile, the fermentation properties (flocculability, SO2 production, α-N assimilation rate, GSH production, etc.) of Q/L-f2 were similar to those of strain Q. Therefore, our works established a series of high ethanol-tolerant strains in beer production. Moreover, this demonstration of inactivated protoplast fusion in industrial S. cerevisiae strain opens many doors for yeast-based biotechnological applications.