Enhancement of Copper Uptake of Yeast Through Systematic Optimization of Medium and the Cultivation Process of Saccharomyces cerevisiae.

Copper is an essential trace element for living organisms. Copper enriched by yeast of Saccharomyces cerevisiae is regarded as the biologically available organic copper supplement with great potentiality for application. However, the lower uptake ratio of copper ions makes the production of copper enriched by yeast uneconomically and environmentally unfriendly. In this study, S. cerevisiae Cu-5 with higher copper tolerance and intracellular copper accumulation was obtained by screening of our yeast strains collection. To increase the uptake ratio of copper ions, the medium composition and cultivation conditions for strain Cu-5 were optimized systematically. A medium comprised of glucose, yeast extract, (NH4)2SO4, and inorganic salts was determined, then a novel cultivation strategy including pH control at 5.5 and increasing amounts of yeast extract for a higher concentration of copper ion in the medium was developed. The uptake ratios of copper ions were more than 90% after combining 50 to 100 mg/L copper ions with 3.5 to 5.0 g/L yeast extract, which is the highest until now and is conducive to the cost-effective and environmentally friendly production of bioactive copper in yeast-enriched form.

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 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 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.

[Construction of high sulphite-producing industrial strain of Saccharomyces cerevisiae].

In the process of beer storage and transportation, off-flavor can be produced for oxidation of beer. Sulphite is important for stabilizing the beer flavor because of its antioxidant activity. However, the low level of sulphite synthesized by the brewing yeast is not enough to stabilize beer flavor. Three enzymes involve sulphite biosynthesis in yeast. One of them, APS kinase (encoded by MET14) plays important role in the process of sulphite formation. In order to construct high sulphite-producing brewing yeast strain for beer production, MET14 gene was cloned and overexpressed in industrial strain of Saccharomyces cerevisiae. Primer 1 (5'-TGTGAATTCCTGTACACCAATGGCTACT-3', EcoR I) and primer 2 (5'-TATAAGCTTGATGA GGTGGATGAAGACG-3', HindIII) were designed according to the MET14 sequence in GenBank. A 1.1kb DNA fragment containing the open reading frame and terminator of MET14 gene was amplified from Saccharomyces cerevisiae YSF-5 by PCR, and inserted into YEp352 to generate recombinant plasmid pMET14. To express MET14 gene properly in S. cerevisiae, the recombinant expression plasmids pPM with URA3 gene as the selection marker and pCPM with URA3 gene and copper resistance gene as the selection marker for yeast transformation were constructed. In plasmid pPM, the PGK1 promoter from plasmid pVC727 was fused with the MET14 gene from pMET14, and the expression cassette was inserted into the plasmid YEp352. The dominant selection marker, copper-resistance gene expression cassette CUP1-MTI was inserted in plasmid pPM to result in pCPM. Restriction enzyme analysis showed that plasmids pPM and pCPM were constructed correctly. The laboratory strain of S. cerevisiae YS58 with ura3, trp1, leu2, his4 auxotroph was transformed with plasmid pPM. Yeast transformants were screened on synthetic minimal medium (SD) containing leucine, histidine and tryptophan. The sulphite production of the transformants carrying pPM was 2 fold of that in the control strain YS58, which showed that the MET14 gene on plasmid pPM was expressed functionally in YS58. The industrial brewing yeast strain YSF-38 was transformed with the plasmid pCPM and yeast transformants were selected on YEPD medium containing 4mmol/L copper sulphate. The recombinant strain carrying pCPM showed a 3.2-fold increase in sulphite production when compared to the host strain YSF-38 under laboratory culture conditions. Flask fermentation under brewing-like conditions was performed in Tsingtao Beer Brewery. The sulphite production of the recombinant strain began to be higher than that of the host strain YSF-38 at the fourth day and reached the maximum at the eighth day. At the end of fermentation, the sulphite produced by recombinant strain is 1.4 fold of that in the host strain. The overexpression of MET14 gene in both laboratory and industrial strains of S. cerevisiae increases the sulphite formation. It is the first time to construct high sulphite-producing industrial strain by functional expression of MET14 in S. cerevisiae. Such study provides the foundation for construction of an excellent brewing yeast strain that can produce proper sulphite and can be used in commercial beer production.

[Genetically modified industrial brewing yeast with high-glutathione and low-diacetyl production].

Recombinant plasmid pICG was constructed by replacing the internal fragment of a-acetohydroxyacid synthase (AHAS) gene (ILV2) with a copy of gamma-glutamylcysteine synthetase gene (GSH1) and copper chelatin gene (CUP1) from the industrial brewing yeast strain YSF31. YSF31 was transformed with plasmid pICG linearized by Kpn I and Pst I. A recombinant strain with high-glutathione and low-diacetyl production was selected. The results of fermentation in 100-L bioreactor showed that the lagering time of beer produced for recombinant strain T2 was shortened by 3 days and the shelf life of the beer was prolonged about 50%. It may be more acceptable for the commercial application, as it does not contain foreign DNA.

[The breeding and culture condition optimization of a high-biomass, selenium-enriched yeast strain].

The yeast fusant ZFF-28, which is high in biomass production and rich in selenium, was constructed after mutagenesis and protoplasts fusion between yeast strains. The total selenium content of ZFF-28 is 1.8 and 1.0 times higher than that of the parental strains Saccharomyces cerevisiae ZY-67 and Saccharomyces kluyveri SZY-198 respectively. Using single factor tests and a L16(4(3) x 2(1)) orthogonal design, the cultivation conditions was optimized as: 50mL culture in 250mL shake flasks in molasses containing 6% sugar and 60microg/mL Se at 28 degree C for 25h at 220 r/min, with the initial pH adjusted to 6.0 - 6.5. Under the optimized conditions, the biomass (dry weight) reached 8.2g/L and the Se content of the cells reached 2050microg/g, with organic and inorganic Se contents being 91% and 9% respectively.