The eisosomes contribute to acid tolerance of yeast by maintaining cell membrane integrity.

Microbes have evolved multiple mechanisms to resist environmental stresses, which are regulated in complex and delicate ways. Though the role of cell membranes in acid resistance from the perspective of physicochemical properties and membrane proteins has been deeply studied, the function of eisosomes is still in its infancy. In this study, we firstly reported the dynamic changes of eisosomes under acid stress and the decreased acid tolerance of yeasts caused by eisosome disruption. Physiological indicators and non-targeted lipid profiling revealed that eisosome disruption caused changes in multiple lipids and imbalances in lipid homeostasis, which are responsible for membrane integrity damage. Thus the increased infiltration of carboxylic acids and the raised ROS levels were detected in strains with disrupted eisosome assembly, resulting in decreased cellular tolerance. The results here provide novel insights into the acid-resistant mechanism of yeasts from the perspective of the cell membrane subdomain, which has practical impacts on green biological manufacturing and food preservation.

Comparative transcriptome analysis reveals the key regulatory genes for higher alcohol formation by yeast at different α-amino nitrogen concentrations.

Higher alcohols are important flavor substance in alcoholic beverages. The content of α-amino nitrogen (α-AN) in the fermentation system affects the formation of higher alcohols by Saccharomyces cerevisiae. In this study, the effect of α-AN concentration on the higher alcohol productivity of yeast was explored, and the mechanism of this effect was investigated through metabolite and transcription sequence analyses. We screened 12 most likely genes and constructed the recombinant strain to evaluate the effect of each gene on high alcohol formation. Results showed that the AGP1, GDH1, and THR6 genes were important regulators of higher alcohol metabolism in S. cerevisiae. This study provided knowledge about the metabolic pathways of higher alcohols and gave an important reference for the breeding of S. cerevisiae with low-yield higher alcohols to deal with the fermentation system with different α-AN concentrations in the brewing industry.

Identification by comparative transcriptomics of core regulatory genes for higher alcohol production in a top-fermenting yeast at different temperatures in beer fermentation.

Undesirable flavor caused by excessive higher alcohols restrains the development of the wheat beer industry. To clarify the regulation mechanism of the metabolism of higher alcohols in wheat beer brewing by the top-fermenting yeast Saccharomyces cerevisiae S17, the effect of temperature on the fermentation performance and transcriptional levels of relevant genes was investigated. The strain S17 produced 297.85 mg/L of higher alcohols at 20 °C, and the production did not increase at 25 °C, reaching about 297.43 mg/L. Metabolite analysis and transcriptome sequencing showed that the metabolic pathways of branched-chain amino acids, pyruvate, phenylalanine, and proline were the decisive factors that affected the formation of higher alcohols. Fourteen most promising genes were selected to evaluate the effects of single-gene deletions on the synthesis of higher alcohols. The total production of higher alcohols by the mutants Δtir1 and Δgap1 was reduced by 23.5 and 19.66% compared with the parent strain S17, respectively. The results confirmed that TIR1 and GAP1 are crucial regulatory genes in the metabolism of higher alcohols in the top-fermenting yeast. This study provides valuable knowledge on the metabolic pathways of higher alcohols and new strategies for reducing the amounts of higher alcohols in wheat beer.

Modulating acetate ester and higher alcohol production in Saccharomyces cerevisiae through the cofactor engineering.

Flavor production by esters or by higher alcohols play a key role in the sensorial quality of fermented alcoholic beverages. In Saccharomyces cerevisiae cells, the syntheses of esters and higher alcohols are considerably influenced by intracellular CoA levels catalyzed by pantothenate kinase. In this work, we examined the effects of cofactor CoA and acetyl-CoA synthesis on the metabolism of esters and higher alcohols. Strains 12α-BAP2 and 12α+ATF1 where generated by deleting and overexpressing BAP2 (encoded branched-chain amino acid permease) and ATF1 (encoded alcohol acetyl transferases), respectively, in the parent 12α strains. Then, 12α-BAP2+CAB1 and 12α-BAP2+CAB3 strains were obtained by overexpressing CAB1 (encoded pantothenate kinase Cab1) and CAB3 (encoded pantothenate kinase Cab3) in the 12α-BAP2 strain, and 12α-BAP2+CAB1+ATF1 and 12α-BAP2+CAB3+ATF1 were generated by overexpressing ATF1 in the pantothenate kinase overexpression strains. The acetate ester level in 12α-BAP2 was slightly changed relative to that in the control strain 12α, whereas the acetate ester levels in 12α-BAP2+CAB1, 12α-BAP2+CAB3, 12α-BAP2+CAB1+ATF1, and 12α-BAP2+CAB3+ATF1 were distinctly increased (44-118% for ethyl acetate and 18-57% for isoamyl acetate). The levels of n-propanol, methyl-1-butanol, isopentanol, isobutanol, and phenethylol levels were changed and varied among the six engineered strains. The levels of acetate esters and higher alcohols can be modulated by changing the CoA and acetyl-CoA levels. The method proposed in this work supplies a practical means of breeding yeast strains by modulating acetate ester and higher alcohol production.

Genetic engineering to alter carbon flux for various higher alcohol productions by Saccharomyces cerevisiae for Chinese Baijiu fermentation.

Higher alcohols significantly influence the quality and flavor profiles of Chinese Baijiu. ILV1-encoded threonine deaminase, LEU1-encoded α-isopropylmalate dehydrogenase, and LEU2-encoded ß-isopropylmalate dehydrogenase are involved in the production of higher alcohols. In this work, ILV1, LEU1, and LEU2 deletions in α-type haploid, a-type haploid, and diploid Saccharomyces cerevisiae strains and ILV1, LEU1, and LEU2 single-allele deletions in diploid strains were constructed to examine the effects of these alterations on the metabolism of higher alcohols. Results showed that different genetic engineering strategies influence carbon flux and higher alcohol metabolism in different manners. Compared with the parental diploid strain, the ILV1 double-allele-deletion diploid mutant produced lower concentrations of n-propanol, active amyl alcohol, and 2-phenylethanol by 30.33, 35.58, and 11.71%, respectively. Moreover, the production of isobutanol and isoamyl alcohol increased by 326.39 and 57.6%, respectively. The LEU1 double-allele-deletion diploid mutant exhibited 14.09% increased n-propanol, 33.74% decreased isoamyl alcohol, and 13.21% decreased 2-phenylethanol production, which were similar to those of the LEU2 mutant. Furthermore, the LEU1 and LEU2 double-allele-deletion diploid mutants exhibited 41.72 and 52.18% increased isobutanol production, respectively. The effects of ILV1, LEU1, and LEU2 deletions on the production of higher alcohols by α-type and a-type haploid strains were similar to those of double-allele deletion in diploid strains. Moreover, the isobutanol production of the ILV1 single-allele-deletion diploid strain increased by 27.76%. Variations in higher alcohol production by the mutants are due to the carbon flux changes in yeast metabolism. This study could provide a valuable reference for further research on higher alcohol metabolism and future optimization of yeast strains for alcoholic beverages.

Overexpression of SNF4 and deletions of REG1- and REG2-enhanced maltose metabolism and leavening ability of baker's yeast in lean dough.

Maltose metabolism of baker's yeast (Saccharomyces cerevisiae) in lean dough is suppressed by the glucose effect, which negatively affects dough fermentation. In this study, differences and interactions among SNF4 (encoding for the regulatory subunit of Snf1 kinase) overexpression and REG1 and REG2 (which encodes for the regulatory subunits of the type I protein phosphatase) deletions in maltose metabolism of baker's yeast were investigated using various mutants. Results revealed that SNF4 overexpression and REG1 and REG2 deletions effectively alleviated glucose repression at different levels, thereby enhancing maltose metabolism and leavening ability to varying degrees. SNF4 overexpression combined with REG1/REG2 deletions further enhanced the increases in glucose derepression and maltose metabolism. The overexpressed SNF4 with deleted REG1 and REG2 mutant ΔREG1ΔREG2 + SNF4 displayed the highest maltose metabolism and strongest leavening ability under the test conditions. Such baker's yeast strains had excellent potential applications.

Functional analysis of the global repressor Tup1 for maltose metabolism in Saccharomyces cerevisiae: different roles of the functional domains.

BACKGROUND: Tup1 is a general transcriptional repressor of diverse gene families coordinately controlled by glucose repression, mating type, and other mechanisms in Saccharomyces cerevisiae. Several functional domains of Tup1 have been identified, each of which has differing effects on transcriptional repression. In this study, we aim to investigate the role of Tup1 and its domains in maltose metabolism of industrial baker's yeast. To this end, a battery of in-frame truncations in the TUP1 gene coding region were performed in the industrial baker's yeasts with different genetic background, and the maltose metabolism, leavening ability, MAL gene expression levels, and growth characteristics were investigated. RESULTS: The results suggest that the TUP1 gene is essential to maltose metabolism in industrial baker's yeast. Importantly, different domains of Tup1 play different roles in glucose repression and maltose metabolism of industrial baker's yeast cells. The Ssn6 interaction, N-terminal repression and C-terminal repression domains might play roles in the regulation of MAL transcription by Tup1 for maltose metabolism of baker's yeast. The WD region lacking the first repeat could influence the regulation of maltose metabolism directly, rather than indirectly through glucose repression. CONCLUSIONS: These findings lay a foundation for the optimization of industrial baker's yeast strains for accelerated maltose metabolism and facilitate future research on glucose repression in other sugar metabolism.

Regulation of Saccharomyces cerevisiae genetic engineering on the production of acetate esters and higher alcohols during Chinese Baijiu fermentation.

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.

Reduced production of diacetyl by overexpressing BDH2 gene and ILV5 gene in yeast of the lager brewers with one ILV2 allelic gene deleted.

Diacetyl causes an unwanted buttery off-flavor in lager beer. The production of diacetyl is reduced by modifying the metabolic pathway of yeast in the beer fermentation process. In this study, BDH2 and ILV5 genes, coding diacetyl reductase and acetohydroxy acid reductoisomerase, respectively, were expressed using a PGK1 promoter in Saccharomyces cerevisiae, which deleted one ILV2 allelic gene. Diacetyl contents and fermentation performances were examined and compared. Results showed that the diacetyl content in beer was remarkably reduced by 16.52% in QI2-KP (one ILV2 allelic gene deleted), 55.65% in QI2-B2Y (overexpressed BDH2 gene and one ILV2 allelic gene deleted), and 69.13% in QI2-I5Y (overexpressed ILV5 gene and one ILV2 allelic gene deleted) compared with the host strain S2. The fermentation ability of mutant strains was similar to that of S2. Results of the present study can lead to further advances in this technology and its broad application in scientific investigations and industrial beer production.

MAL62 overexpression and NTH1 deletion enhance the freezing tolerance and fermentation capacity of the baker's yeast in lean dough.

BACKGROUND: Trehalose is related to several types of stress responses, especially freezing response in baker's yeast (Saccharomyces cerevisiae). It is desirable to manipulate trehalose-related genes to create yeast strains that better tolerate freezing-thaw stress with improved fermentation capacity, which are in high demand in the baking industry. RESULTS: The strain overexpressing MAL62 gene showed increased trehalose content and cell viability after prefermention-freezing and long-term frozen. Deletion of NTH1 in combination of MAL62 overexpression further strengthens freezing tolerance and improves the leavening ability after freezing-thaw stress. CONCLUSIONS: The mutants of the industrial baker's yeast with enhanced freezing tolerance and leavening ability in lean dough were developed by genetic engineering. These strains had excellent potential industrial applications.

Enhanced leavening properties of baker's yeast by reducing sucrase activity in sweet dough.

Leavening ability in sweet dough is required for the commercial applications of baker's yeast. This property depends on many factors, such as glycolytic activity, sucrase activity, and osmotolerance. This study explored the importance of sucrase level on the leavening ability of baker's yeast in sweet dough. Furthermore, the baker's yeast strains with varying sucrase activities were constructed by deleting SUC2, which encodes sucrase or replacing the SUC2 promoter with the VPS8/TEF1 promoter. The results verify that the sucrase activity negatively affects the leavening ability of baker's yeast strains under high-sucrose conditions. Based on a certain level of osmotolerance, sucrase level plays a significant role in the fermentation performance of baker's yeast, and appropriate sucrase activity is an important determinant for the leavening property of baker's yeast in sweet dough. Therefore, modification on sucrase activity is an effective method for improving the leavening properties of baker's yeast in sweet dough. This finding provides guidance for the breeding of industrial baker's yeast strains for sweet dough leavening. The transformants BS1 with deleted SUC2 genetic background provided decreased sucrase activity (a decrease of 39.3 %) and exhibited enhanced leavening property (an increase of 12.4 %). Such a strain could be useful for industrial applications.

Reduced production of ethyl carbamate for wine fermentation by deleting CAR1 in Saccharomyces cerevisiae.

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.

Enhanced leavening ability of baker's yeast by overexpression of SNR84 with PGM2 deletion.

Dough-leavening ability is one of the main aspects considered when selecting a baker's yeast strain for baking industry. Generally, modification of maltose metabolic pathway and known regulatory networks of maltose metabolism were used to increase maltose metabolism to improve leavening ability in lean dough. In this study, we focus on the effects of PGM2 (encoding for the phosphoglucomutase) and SNR84 (encoding for the H/ACA snoRNA) that are not directly related to both the maltose metabolic pathway and known regulatory networks of maltose metabolism on the leavening ability of baker's yeast in lean dough. The results show that the modifications on PGM2 and/or SNR84 are effective ways in improving leavening ability of baker's yeast in lean dough. Deletion of PGM2 decreased cellular glucose-1-phosphate and overexpression of SNR84 increased the maltose permease activity. These changes resulted in 11, 19 and 21% increases of the leavening ability for PGM2 deletion, SNR84 overexpression and SNR84 overexpression combining deleted PGM2, respectively.

Decreased production of higher alcohols by Saccharomyces cerevisiae for Chinese rice wine fermentation by deletion of Bat aminotransferases.

An appropriate level of higher alcohols produced by yeast during the fermentation is one of the most important factors influencing Chinese rice wine quality. In this study, BAT1 and BAT2 single- and double-gene-deletion mutant strains were constructed from an industrial yeast strain RY1 to decrease higher alcohols during Chinese rice wine fermentation. The results showed that the BAT2 single-gene-deletion mutant strain produced best improvement in the production of higher alcohols while remaining showed normal growth and fermentation characteristics. Furthermore, a BAT2 single-gene-deletion diploid engineered strain RY1-Δbat2 was constructed and produced low levels of isobutanol and isoamylol (isoamyl alcohol and active amyl alcohol) in simulated fermentation of Chinese rice wine, 92.40 and 303.31 mg/L, respectively, which were 33.00 and 14.20 % lower than those of the parental strain RY1. The differences in fermentation performance between RY1-Δbat2 and RY1 were minor. Therefore, construction of this yeast strain is important in future development in Chinese wine industry and provides insights on generating yeast strains for other fermented alcoholic beverages.

Effects of MAL61 and MAL62 overexpression on maltose fermentation of baker's yeast in lean dough.

The predominant fermentable sugar in lean dough is maltose. To improve the leavening ability of baker's yeast in lean dough, maltose metabolism should be improved. Maltase (alpha-glucosidase, encoded by MAL62) and maltose permease (encoded by MAL61) are the major factors involved in maltose metabolism. The major rate-limiting factor in maltose metabolism and leavening ability of baker's yeast remains unclear. In this work, MAL61 and/or MAL62 overexpression strains were constructed to investigate the decisive factor for maltose metabolism of industrial baker's yeast in lean dough. Our results show that elevated maltose permease activity by MAL61 overexpression yielded less improvement in maltose fermentation compared to elevated maltase activity by MAL62 overexpression. Significant increase in maltase activity by MAL62 overexpression could result in a 44% increase in leavening ability of industrial baker's yeast in lean dough and a 39% increase in maltose metabolism in a medium containing glucose and maltose. Thus, maltase was the rate-limiting factor in maltose fermentation of industrial baker's yeast in lean dough. This study lays a foundation for breeding of industrial baker's yeast for quick dough leavening.

Effects of MIG1, TUP1 and SSN6 deletion on maltose metabolism and leavening ability of baker's yeast in lean dough.

BACKGROUND: Glucose repression is a global regulatory system in baker's yeast. Maltose metabolism in baker's yeast strains is negatively influenced by glucose, thereby affecting metabolite productivity (leavening ability in lean dough). Even if the general repression system constituted by MIG1, TUP1 and SSN6 factors has already been reported, the functions of these three genes in maltose metabolism remain unclear. In this work, we explored the effects of MIG1 and/or TUP1 and/or SSN6 deletion on the alleviation of glucose-repression to promote maltose metabolism and leavening ability of baker's yeast. RESULTS: Results strongly suggest that the deletion of MIG1 and/or TUP1 and/or SSN6 can exert various effects on glucose repression for maltose metabolism. The deletion of TUP1 was negative for glucose derepression to facilitate the maltose metabolism. By contrast, the deletion of MIG1 and/or SSN6, rather than other double-gene or triple-gene mutations could partly relieve glucose repression, thereby promoting maltose metabolism and the leavening ability of baker's yeast in lean dough. CONCLUSIONS: The mutants of industrial baker's yeast with enhanced maltose metabolism and leavening ability in lean dough were developed by genetic engineering. These baker's yeast strains had excellent potential industrial applications.

Optimization of 2,3-butanediol production by Enterobacter cloacae in simultaneous saccharification and fermentation of corncob residue.

Corncob residue, a waste in xylose or xylitol production, was utilized to produce 2,3-butanediol (2,3-BD) via simultaneous saccharification and fermentation (SSF). This study developed the optimal conditions for production of 2,3-BD by using a heat-resistant strain, Enterobacter cloacae UV4, to perform SSF of the corncob residue. Urea, lactic acid, sodium citrate, and MgSO4 , selected by the Plackett-Burman experiment, were determined to be significant independent variables to conduct the response surface experiment. With the optimized medium, a total production of 28.923 g/L for 2,3-BD and acetoin (BA) was obtained at 60 H. Furthermore, 43.162 g/L of BA production and 0.553 g/L/H of productivity were obtained by fed-batch SSF, which was 0.424 g diol/g consumed corncob residue. The results suggest that the waste corncob residue could be used as an available substrate for the production of 2,3-BD by E. cloacae UV4, as well as a potential resource to improve the economics of microbial compound production.

Enhanced production of 2,3-butanediol by overexpressing acetolactate synthase and acetoin reductase in Klebsiella pneumoniae.

Mutants with overexpression of α-acetolactate synthase (ALS), α-acetolactate decarboxylase, and acetoin reductase (AR), either individually or in combination, were constructed to improve 2,3-butanediol (2,3-BD) production in Klebsiella pneumoniae. The recombinant strains were characterized in terms of the enzyme activity, 2,3-BD yield, and expression levels. The recombinant K. pneumoniae strain (KG-rs) that overexpressed both ALS and AR showed an improved 2,3-BD yield. When cultured in the media with five different carbon sources (glucose, galactose, fructose, sucrose, and lactose), the mutant exhibited higher 2,3-BD productivity and production than the parental strain in all the tested carbon sources except for lactose. The 2,3-BD production of KG-rs in a batch fermentation with glucose as the carbon source was 12% higher than that of the parental strain.

[Optimization of synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid].

OBJECTIVE: To optimize the synthetic pathway and fermentation process of yeast cell factories for production of oleanoic acid. METHOD: Using the DNA assembler method, one copy of Glycyrrhiza glabra beta-amyrin synthase (GgbAS), Medicago truncatula oleanolic acid synthase (MtOAS) and Arabidopsis thaliana cytochrome P450 reductase 1 (AtCPR1) genes were introduced into Saccharomyces cerevisiae strain BY-OA, resulting in strain BY-20A. YPD medium with different glucose concentration were then used to cultivate strain BY-2OA. RESULT: Increasing gene copies of GgbAS, MtOAS and AtCPR1 resulted in increased beta-amyrin and oleanolic acid production. The strain BY-2OA produced 136.5 mg x L(-1) beta-amyrin and 92.5 mg x L(-1) oleanolic acid, which were 54% and 30% higher than the parent strain BY-OA. Finally, the titer of oleanolic acid increased to 165.7 mg x L(-1) when cultivated in YPD medium with 40 mg x L(-1) glucose. CONCLUSION: Production of oleanoic acid increased significantly in the yeast strain BY-2OA, which can provide the basis for creating an alternative way for production of oleanoic acid in place of extraction from plant sources.

Simultaneous determination of furfural, acetic acid, and 5-hydroxymethylfurfural in corncob hydrolysates using liquid chromatography with ultraviolet detection.

A single-laboratory validation study was conducted using HPLC for detecting and quantifying acetic acid, furfural, and 5-hydroxymethylfurfural (HMF) in corncob hydrolysates. A pretreatment procedure using dilute sulfuric acid was optimized for corncob hydrolysis. The final hydrolysates were analyzed by HPLC using a C18 RP column with aqueous 0.01% (v/v) H2SO4-CH3OH (95 + 5) as the mobile phase at a flow rate of 1 mL/min. The wavelengths for detecting the three compounds were changed to their optimal UV detection wavelengths at the time of elution. The wavelength detection adjustments were as follow: 205 nm (0 to 4 min); 284 nm (4 to 7 min); and 276 nm (7 to 10 min). Separation was achieved with a chromatographic run time of 10 min. The calibration curves for the three compounds had correlation coefficients (r2) > or = 99.8%. The analytical range, as defined by the calibration curves, was 0.5-10 mg/L for acetic acid, 0.4-22 mg/L for furfural, and 0.1-18 mg/L for HMF. The LODs for acetic acid, furfural, and HMF were estimated to be 0.05, 0.03, and 0.02 mg/L, respectively; the LOQs were 0.196, 0.135, and 0.074 mg/L, respectively. The RSD values for the intraday precision study ranged from 0.31 to 2.22%, and from 0.57 to 2.43% for the interday study. The mean recovery rates in all compounds were between 100.08 and 101.49%.