[Improvement of acetic acid tolerance and fermentation performance of industrial Saccharomyces cerevisiae by overexpression of flocculent gene FLO1 and FLO1c].

Flocculent gene FLO1 and its truncated form FLO1c with complete deletion of repeat unit C were expressed in a non-flocculent industrial strain Saccharomyces cerevisiae CE6 to generate recombinant flocculent strains 6-AF1 and 6-AF1c respectively. Both strains of 6-AF1 and 6-AF1c displayed strong flocculation and better cell growth than the control strain CE6-V carrying the empty vector under acetic acid stress. Moreover, the flocculent strains converted glucose to ethanol at much higher rates than the control strain CE6-V under acetic acid stress. In the presence of 0.6% (V/V) acetic acid, the average ethanol production rates of 6-AF1 and 6-AF1c were 1.56 and 1.62 times of that of strain CE6-V, while the ethanol production rates of 6-AF1 and 6-AF1c were 1.21 and 1.78 times of that of strain CE6-V under 1.0% acetic acid stress. Results in this study indicate that acetic acid tolerance and fermentation performance of industrial S. cerevisiae under acetic acid stress can be improved largely by flocculation endowed by expression of flocculent genes, especially FLO1c.

Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker.

With increasing application of Hansenula polymorpha in fundamental research and biotechnology, many more genetic manipulations are required. However, these have been restricted for the finiteness of selectable markers. Here, MazF, a toxin protein from Escherichia coli, was investigated as a counter-selectable marker in H. polymorpha. The lethal effect of MazF on yeast cells suggested that it is a candidate for counter-selection in H. polymorpha. Markerless or scarless gene deletion in H. polymorpha was conducted based on selectable markers cassette mazF-zeoR, in which the zeocin resistance cassette and mazF expression cassette were used as positive and counter-selectable markers, respectively. For markerless deletion, the target region can be replaced by CYC1TT via two-step homologous recombination. For scarless deletion, the innate upstream region (5'UP) of target genes rather than CYC1TT mediates homologous recombination to excise both selectable markers and 5' sequence of target genes. Moreover, scarless deletion can be accomplished by using short homologous arms for the effectiveness of mazF as a counter-selectable marker. The applicability of the strategies in markerless or scarless deletion of PEP4, LEU2, and TRP1 indicates that this study provides easy, time-efficient, and host-independent protocols for single or multiple genetic manipulations in H. polymorpha.

Speciation of chromium in chromium yeast.

High-performance liquid chromatography was used to separate Cr(III) and Cr(VI) in samples with detection by inductively coupled plasma mass spectrometry(ICP-MS). The separation was achieved on a weak anion exchange column. The mobile phase was pH 7.0 ammonium nitrate solution. The redox reaction between Cr(III) and Cr(VI) was avoided during separation and determination. This separation method could be used to separate the samples with large concentration differences between Cr(III) and Cr(VI). The alkaline digestion was used to extract chromium in solid sample, which had no effect on the retention time and the peak area of the Cr(VI). However, the conversion of Cr(VI) from Cr(III) was observed during alkaline digestion, which displayed positive relation with the ratio of Cr(III) and Cr(VI) in samples. Both Cr(III) and Cr(VI) contents of chromium yeasts cultured in media with different chromium additions were determined. The spike recoveries of Cr(VI) for chromium yeasts were in the range of 95-108 %.

Secretion expression of SOD1 and its overlapping function with GSH in brewing yeast strain for better flavor and anti-aging ability.

Superoxide dismutase (SOD) is a significant antioxidant, but unlike glutathione (GSH), SOD cannot be secreted into beer by yeast cells during fermentation, this directly leads to the limited application of SOD in beer anti-aging. In this investigation, we constructed the SOD1 secretion cassette in which strong promoter PGK1p and the sequence of secreting signal factor from Saccharomyces cerevisiae were both harbored to the upstream of coding sequence of SOD1 gene, as a result, the obtained strains carrying this cassette successfully realized the secretion of SOD1. In order to overcome the limitation of previous genetic modification on yeast strains, one new comprehensive strategy was adopted targeting the suitable homologous sites by gene deletion and SOD1 + GSH1 co-overexpression, and the new strain ST31 (Δadh2::SOD1 + Δilv2::GSH1) was constructed. The results of the pilot-scale fermentation showed that the diacetyl content of ST31 was lower by 42 % than that of the host, and the acetaldehyde content decreased by 29 %, the GSH content in the fermenting liquor of ST31 increased by 29 % compared with the host. Both SOD activity test and the positive and negative staining assay after native PAGE indicated that the secreted active SOD in the fermenting liquor of ST31 was mainly a dimer with the size of 32,500 Da. The anti-aging indexes such as the thiobarbituric acid and the resistance staling value further proved that the flavor stability of the beer brewed with strain ST31 was not only better than that of the original strain, but also better than that of the previous engineering strains. The multi-modification and comprehensive improvement of the beer yeast strain would greatly enhance beer quality than ever, and the self-cloning strain would be attractive to the public due to its bio-safety.

High-level expression, purification and characterisation of porcine ß-defensin 2 in Pichia pastoris and its potential as a cost-efficient growth promoter in porcine feed.

Porcine ß-defensin 2 (pBD2), a recently discovered porcine defensin that is produced by the intestine, exerts antimicrobial activities and innate immune effects that are linked to intestinal diseases in pigs. Here, we report a codon-optimised protein corresponding to mature pBD2 cDNA that was expressed and purified in Pichia pastoris yeast. The highest amount of secreted protein (3,694.0 mg/L) was reached 144 h into a 150-h induction during high-density cultivation. Precipitation followed by gel exclusion chromatography yielded 383.7 mg/L purified recombinant pBD2 (rpBD2) with a purity of ~93.7 %. Two recombinant proteins of 5,458.5 and 5,258.4 Da were detected in the mass spectrum due to variation in the amino-terminus. The rpBD2 exhibited high antimicrobial activity against a broad range of pig pathogenic bacteria (minimal inhibitory concentration [MIC] 32-128 µg/mL); the highest activity was observed against Salmonella choleraesuis, Staphylococcus aureus and Streptococcus suis (MIC 32-64 µg/mL). However, rpBD2 also inhibited the growth of probiotics such as Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae, but at lower efficacies than the pathogens. Purified or unpurified rpBD2 also maintained high activity over a wide range of pH values (2.0-10.0), a high thermal stability at 100 °C for 40 min and significant resistance to papain, pepsin and trypsin. In addition, the activity of rpBD2 towards S. aureus was unaffected by 10 mM dithiothreitol (DTT) and 20 % dimethyl sulphoxide (DMSO). Our results suggest that pBD2 could be produced efficiently in large quantities in P. pastoris and be a substitute for traditional antibiotics for growth promotion in the porcine industry.

[Diversity and genetic stability of yeast flocculation caused by variation of tandem repeats in yeast flocculin genes].

Yeast flocculation is described as a reversible, asexual and calcium dependent process, in which cells adhere to form flocs by interaction of specific cell surface proteins named flocculins on yeast cells with mannose residues present on the cell wall of adjacent yeast cells. Yeast flocculation provides a very economical and convenient pathway for separation of yeast cells from the fermentation broth or removal of heavy metal ions from effluent. A large number of tandem repeats have been found in genes encoding flocculins, which not only have great regulatory effect on the structure and function of flocculins, generating the diversity of flocculation characteristics, but lead to genetic instability in flocculation as well for driving slippage and recombination reactions within and between FLO genes. Here, the research progress in effect of variation of tandem repeats in FLO genes on flocculation characteristics and genetic stability were reviewed to direct and promote the controllable application of flocculation in industrial fermentation process and environmental remediation.

Deletion of intragenic tandem repeats in unit C of FLO1 of Saccharomyces cerevisiae increases the conformational stability of flocculin under acidic and alkaline conditions.

Flocculation is an attractive property for Saccaromyces cerevisiae, which plays important roles in fermentation industry and environmental remediation. The process of flocculation is mediated by a family of cell surface flocculins. As one member of flocculins, Flo1 is characterized by four families of repeats (designated as repeat units A, B, C and D) in the central domain. It is generally accepted that variation of repeat unit A in length in Flo1 influences the degree of flocculation or specificity for sugar recognization. However, no reports were observed for other repeat units. Here, we compared the flocculation ability and its sensitivity to environmental factors between yeast strain YSF1 carrying the intact FLO1 gene and yeast strains carrying the derived forms of FLO1 with partial or complete deletion of repeats in unit C. No obvious differences in flocculation ability and specificity of carbohydrate recognition were observed among these yeast strains, which indicates the truncated flocculins can stride across the cell wall and cluster the N-terminal domain on the surface of yeast cells as the intact Flo1 thereby improving intercellular binding. However, yeast strains with the truncated flocculins required more mannose to inhibit completely the flocculation, displayed broad tolerance of flocculation to pH fluctuation, and the fewer the repeats in unit C, the stronger adaptability of flocculation to pH change, which was not relevant to the position of deletion. This suggests that more stable active conformation is obtained for flocculin by deletion the repeat unit C in the central domain of Flo1, which was validated further by the higher hydrophobicity on the surface of cells of YSF1c with complete deletion of unit C under neutral and alkaline conditions and the stabilization of GFP conformation by fusion with flocculin with complete deletion of unit C in the central domain.

[Effect of tandem repeats adjacent to 3'-terminal of FLO1 on the flocculation function of Saccharomyces cerevisiae].

OBJECTIVE: Many tandem repeats exist in FLO1 gene of Saccharomyces cerevisiae, which might have great regulatory effect on the conformation and function of flocculation protein (flocculin). In this study, we analyzed the effect of 3'-terminal tandem repeats B, C and D complete deletion on the function of flocculin. METHODS: We constructed the derived gene FLO1 bcd with complete deletion of tandem repeats B, C and D of FLO1 by fusion PCR. We then constructed plasmid pYCF1 bcd by insertion of FLO1 bcd into YCp50, and transformed such plasmid, pYCF1 and YCp50 into S. cerevisiae YS58 separately to generate recombinant strains YSF1 bcd, YSF1 and YSP50. We compared the flocculation ability and characteristics of these strains. RESULT: Compared to YSF1, YSF1 bcd displayed only a slight reduction (4%) in flocculation ability in optimal flocculation buffer (50 mmol/L NaAC, pH 4.5). Moreover, the dependence of flocculation on Ca2+, sensitivity to metal ions and ethanol, and the specificity to different sugars showed no obvious difference between strains YSF1 and YSF1 bcd. However, strain YSF1 bcd displayed much higher flocculation levels than strain YSF1 under conditions with extreme pH, high temperature, or high concentration of mannose. CONCLUSION: Combined deletion of tandem repeats B, C and D adjacent to the 3'-terminal of FLO1 increases the conformation stability of flocculin in response to changes of pH, temperature or concentration of mannose in environment, but does not influence the other characteristics of flocculation.

[Improving ergosterol production from molasses by Saccharomyces cerevisiae].

Ergosterol is an economically important metabolite produced by yeast. To improve the production of ergosterol by Saccharomyces cerevisiae YEH56 (pHXA42) from molasses, a cheap and regenerative material, different strategies were applied. First, Plackett-Burman design and central composite design were applied to screen the significant factors in fermentation medium using ergosterol yield (g/L) as the response value. Ergosterol yield reached 371.56 mg/L by using the optimal fermentation medium in shake-flask culture (total sugar in molasses 40 g/L, KH2PO4 1 g/L, K2HPO4 1.86 g/L, CuSO4 x 5H2O 17.5 mg/L, FeSO4 x 7H2O 13.9 mg/L, MgSO4 x 5H2O 12.3 mg/L, corn steep liquor 10 mL/L), which was increased by 29.5% compared with the initial culture. Second, ergosterol yield was increased by 62.1% using a pH-control strategy in a 5-L bioreactor. Third, ergosterol production was improved further by using molasses feeding strategy. After 38 h fermentation, ergosterol yield reached 1 953.85 mg/L, which was 3.2 times of that in batch fermentation. Meanwhile, ergosterol production rate was increased by 42.7% compared with that in the batch culture.

Integrated expression of the α-amylase, dextranase and glutathione gene in an industrial brewer's yeast strain.

Genetic engineering is widely used to meliorate biological characteristics of industrial brewing yeast. But how to solve multiple problems at one time has become the bottle neck in the genetic modifications of industrial yeast strains. In a newly constructed strain TYRL21, dextranase gene was expressed in addition of α-amylase to make up α-amylase's shortcoming which can only hydrolyze α-1,4-glycosidic bond. Meanwhile, 18s rDNA repeated sequence was used as the homologous sequence for an effective and stable expression of LSD1 gene. As a result, TYRL21 consumed about twice much starch than the host strain. Moreover TYRL21 speeded up the fermentation which achieved the maximum cell number only within 3 days during EBC tube fermentation. Besides, flavor evaluation comparing TYRL21 and wild type brewing strain Y31 also confirmed TYRL21's better performances regarding its better saccharides utilization (83% less in residual saccharides), less off-flavor compounds (57% less in diacetyl, 39% less in acetaldehyde, 67% less in pentanedione), and improved stability index (increased by 49%) which correlated with sensory evaluation of final beer product.

Construction of self-cloning, indigenous wine strains of Saccharomyces cerevisiae with enhanced glycerol and glutathione production.

To improve wine taste and flavor stability, a novel indigenous strain of Saccharomyces cerevisiae with enhanced glycerol and glutathione (GSH) production for winemaking was constructed. ALD6 encoding an aldehyde dehydrogenases of the indigenous yeast was replaced by a GPD1 and CUP1 gene cassette, which are responsible for NAD-dependent glycerol-3-phosphatase dehydrogenase and copper resistance, respectively. Furthermore, the α-acetohydroxyacid synthase gene ILV2 of the indigenous yeast was disrupted by integration of the GSH1 gene which encodes γ-glutamylcysteine synthetase and the CUP1 gene cassette. The fermentation capacity of the recombinant was similar to that of the wild-type strain, with an increase of 21 and 19 % in glycerol and GSH production. No heterologous DNA was harbored in the recombinant in this study.

[Regulation of tandem repeats on the function of flocculation protein in Saccharomyces cerevisiae].

OBJECTIVE: There are a large numbers of tandem repeats in FLO1, which are highly dynamic components in genome leading to the unstable flocculation profiles in Saccharomyces cerevisiae. The effects of repeated unite B or D deletion on the function of flocculation protein was studied to provide theory basis for constructing genetically stable flocculation gene with minimal size. METHODS: We cloned the intact flocculation gene FLO1 from S. cerevisiae YS59 by PCR, and constructed the derived genes FLO1b and FLO1d with repeated unite B or D deletion respectively by fusion PCR. We analyzed the physiological characteristics of flocculation in yeast strains YSF1, YSF1b and YSF1d containing FLO1, FLO1b and FLO1d respectively. RESULTS: YSF1b and YSF1d displayed almost the same level of Flo1-type flocculation as YSF1. However, flocculation of YSF1b and YSF1d, especially YSF1d was more tolerant to pH change and mannose concentration than strain YSF1. CONCLUSION: Tandem repeats regulate the function of flocculation protein. Deletion of repeated unite B or D, especially D increases the stability of flocculation protein.

[Regulatory effect of FLO1 tandem repeats on the flocculation characteristics and genetic stability in Saccharomyces cerevisiae].

OBJECTIVE: There are a large number of tandem repeats in FLO1, which are highly dynamic components in genome leading to the unstable flocculation profiles in Saccharomyces cerevisiae. The effects of complete or partial deletion of repeated DNA sequence A in FLO1 on the flocculation characteristics and genetic stability in yeast were studied to provide theoretical guide for construction genetically stable flocculation gene with minimal size. METHODS: We constructed the derived gene FLO1a with complete deletion of repeated DNA sequence A in the central domain by fusion PCR, and isolated the derived genes FLO1a1 - FLO1a5 with partial deletion of repeated DNA sequence A at different sites using E. coli DH5alpha carrying the FLO1 gene as selective model. We analyzed the physiological characteristics and genetic stability of flocculation in yeast strains YSF1, YSF1a, and YSF1a1 - YSF1a5 containing FLO1, FLO1a and FLO1a1 - FLO1a5 respectively. RESULTS: No obvious flocculation was observed for yeast strain YSF1a, but various levels of flocculation were observed for strains YSF1a1 - YSF1a5. Flocculation of YSF1a3, YSF1a4 and YSF1a5 were more tolerant to environmental changes than that of strain YSF1, and displayed more genetic stability. CONCLUSION: Repeated DNA sequence A is important for the function of flocculation protein.

Improvement of robustness and ethanol production of ethanologenic Saccharomyces cerevisiae under co-stress of heat and inhibitors.

Bioethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, yeast cells are challenged by various environmental stresses during the industrial process of ethanol production. The robustness under heat, acetic acid, and furfural stresses was improved for ethanologenic S. cerevisiae in this work using genome shuffling. Recombinant yeast strain R32 could grow at 45°C, and resist 0.55% (v/v) acetic acid and 0.3% (v/v) furfural at 40°C. When ethanol fermentation was conducted at temperatures ranging from 30 to 42°C, recombinant strain R32 always gave high ethanol production. After 42 h of fermentation at 42°C, 187.6 ± 1.4 g/l glucose was utilized by recombinant strain R32 to produce 81.4 ± 2.7 g/l ethanol, which were respectively 3.4 and 4.1 times those of CE25. After 36 h of fermentation at 40°C with 0.5% (v/v) acetic acid, 194.4 ± 1.2 g/l glucose in the medium was utilized by recombinant strain R32 to produce 84.2 ± 4.6 g/l of ethanol. The extent of glucose utilization and ethanol concentration of recombinant strain R32 were 6.3 and 7.9 times those of strain CE25. The ethanol concentration produced by recombinant strain R32 was 8.9 times that of strain CE25 after fermentation for 48 h under 0.2% (v/v) furfural stress at 40°C. The strong physiological robustness and fitness of yeast strain R32 support its potential application for industrial production of bioethanol from renewable resources such as lignocelluloses.

[Improvement of thermal adaptability and fermentation of industrial ethanologenic yeast by genomic DNA mutagenesis-based genetic recombination].

Ethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, in the process of industrial production of ethanol, both cell growth and fermentation of ethanologenic S. cerevisiae are dramatically affected by environmental stresses, such as thermal stress. In this study, we improved both the thermotolerance and fermentation performance of industrial ethanologenic S. cerevisiae by combined usage of chemical mutagenesis and genomic DNA mutagenesis-based genetic recombination method. The recombinant S. cerevisiae strain T44-2 could grow at 44 degrees C, 3 degrees C higher than that of the original strain CE6. The survival rate of T44-2 was 1.84 and 1.87-fold of that of CE6 when heat shock at 48 degrees C and 52 degrees C for 1 h respectively. At temperature higher than 37 degrees C, recombinant strain T44-2 always gave higher cell growth and ethanol production than those of strain CE6. Meanwhile, from 30 degrees C to 40 degrees C, recombinant strain T44-2 produces 91.2-83.8 g/L of ethanol from 200 g/L of glucose, which indicated that the recombinant strain T44-2 had both thermotolerance and broad thermal adaptability. The work offers a novel method, called genomic DNA mutagenesis-based genetic recombination, to improve the physiological functions of S. cerevisiae.

Improvement of acetic acid tolerance and fermentation performance of Saccharomyces cerevisiae by disruption of the FPS1 aquaglyceroporin gene.

The FPS1 gene coding for the Fps1p aquaglyceroporin protein of an industrial strain of Saccharomyces cerevisiae was disrupted by inserting CUP1 gene. Wild-type strain, CE25, could only grow on YPD medium containing less than 0.45% (v/v) acetic acid, while recombinant strain T12 with FPS1 disruption could grow on YPD medium with 0.6% (v/v) acetic acid. Under 0.4% (v/v) acetic acid stress (pH 4.26), ethanol production and cell growth rates of T12 were 1.7 ± 0.1 and 0.061 ± 0.003 g/l h, while those of CE25 were 1.2 ± 0.1 and 0.048 ± 0.003 g/l h, respectively. FPS1 gene disruption in an industrial ethanologenic yeast thus increases cell growth and ethanol yield under acetic acid stress, which suggests the potential utility of FPS1 gene disruption for bioethanol production from renewable resources such as lignocelluloses.

Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor.

Glutathione in beer works as the main antioxidant compounds which correlates with beer flavor stability. High residual sugars in beer contribute to major non-volatile components which correlate to high caloric content. In this work, Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and Scharomycopsis fibuligera ALP1 gene encoding alpha-amylase were co-expressed in industrial brewing yeast strain Y31 targeting at alpha-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), and new recombinant strain TY3 was constructed. The glutathione content from the fermentation broth of TY3 increased to 43.83 mg/l compared to 33.34 mg/l from Y31. The recombinant strain showed high alpha-amylase activity and utilized more than 46% of starch after 5 days growing on starch as sole carbon source. European Brewery Convention tube fermentation tests comparing the fermentation broth of TY3 and Y31 showed that the flavor stability index increased to 1.3 fold and residual sugar concentration were reduced by 76.8%, respectively. Due to the interruption of ILV2 gene and ADH2 gene, the amounts of off-flavor compounds diacetyl and acetaldehyde were reduced by 56.93% and 31.25%, comparing with the amounts of these from Y31 fermentation broth. In addition, as no drug-resistance genes were introduced to new recombinant strain, consequently, it should be more suitable for use in beer industry because of its better flavor stability and other beneficial characteristics.

Construction of an industrial brewing yeast strain to manufacture beer with low caloric content and improved flavor.

In this study, the problems of high caloric content, increased maturation time and off-flavors in commercial beer manufacture arising from residual sugar, diacetyl, and acetaldehyde levels were addressed. A recombinant industrial brewing yeast strain (TQ1) was generated from T1 [Lipomyces starkeyi dextranase gene (LSD1) introduced, alpha-acetohydroxyacid synthase gene (ILV2) disrupted] by introducing Saccharomyces cerevisiae glucoamylase (SGA1) and a strong promoter PGK1 while disrupting the genes coding alcohol dehydrogenase (ADH2). The highest glucoamylase activity for TQ1 was 93.26 U/ml compared with host strain T1 (12.36 U/ml) and wild-type industrial yeast strain YSF5 (10.39 U/ml), respectively. European Brewery Convention (EBC) tube fermentation tests comparing the fermentation broths of TQ1 with T1 and YSF5 showed that the real extract were reduced by 15.79% and 22.47%; the main residual maltotriose concentration were reduced by 13.75% and 18.82%; the caloric content were reduced by 27.18 and 35.39 calories per 12 oz. Due to the disruption of ADH2 gene in TQ1, the off-flavor acetaldehyde concentration in the fermentation broth were 9.43% and 13.28% respectively lower than that of T1 and YSF5. No heterologous DNA sequences or drug-resistance genes were introduced into TQ1. So, the gene manipulations in this work properly solved the addressed problems in commercial beer manufacture.

[Regulation role of sterol C-24 methyltransferase and sterol C-8 isomerase in the ergosterol biosynthesis of Saccharomyces cerevisiae].

OBJECTIVE: Ergosterol is a fungal metabolite with economic importance. For ergosterol biosynthesis, to identify the bottleneck enzymes in the metabolic pathway is of crucial importance. METHODS: Sterol C-8 isomerase encoding gene ERG2 was cloned from Saccharomyces cerevisiae by PCR. To evaluate the effect of ERG2 overexpression on the sterol content in the yeast, the expression plasmid pPERG2 was constructed and transformed into S. cerevisiae strain YS58 to generate recombinant strain YS58 (pPERG2). In addition,the regulation role of sterol C-24 methyltransferase, encoded by ERG6, in ergosterol biosynthesis was further verified by analysis of sterol components and levels in yeast strains overexpressing ERG6, ERG2 respectively, or overexpressing ERG6 and ERG2 simultaneously. RESULTS: Ergosterol content and all sterol intermediates increased largely by overexpressing ERG6 in S. cerevisiae. Although the overexpression of sterol C-8 isomerase encoded by ERG2 alone had negative effect on ergosterol biosynthesis, overexpression of ERG6 and ERG2 simultaneously led to an increased ergosterol level, which was 1.41-fold of that in empty vector strain, 1.92-fold of that in ERG2 only overexpressing strain and 1.12-fold of that in ERG6 only overexpressing strain. CONCLUSION: These results demonstrated that sterol C-24 methyltransferase is an important bottleneck enzyme for ergosterol biosynthesis in S. cerevisiae.

Recombinant industrial brewing yeast strains with ADH2 interruption using self-cloning GSH1+CUP1 cassette.

A self-cloning module for gene knock-out and knock-in in industrial brewing yeast strain was constructed that contains copper resistance and gamma-glutamylcysteine synthetase gene cassette, flanked by alcohol dehydrogenase II gene (ADH2) of Saccharomyces cerevisiae. The module was used to obtain recombined strains RY1 and RY2 by targeting the ADH2 locus of host Y1. RY1 and RY2 were genetically stable. PCR and enzyme activity analysis of RY1 and RY2 cells showed that one copy of ADH2 was deleted by GSH1+CUP1 insertion, and an additional copy of wild type was still present. The fermentation ability of the recombinants was not changed after genetic modification, and a high level of glutathione (GSH) was secreted, resulting from GSH1 overexpression, which codes for gamma-glutamylcysteine synthetase. A pilot-scale brewing test for RY1 and RY2 indicated that acetaldehyde content in fermenting liquor decreased by 21-22%, GSH content increased by 20-22% compared with the host, the antioxidizability of the recombinants was improved, and the sensorial evaluation was also better than that of the host. No heterologous DNA was harbored in the recombinants; therefore, they could be applied in the beer industry in terms of their biosafety.