[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.

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.

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.

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.

[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.

[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.

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

Porcine β-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.

[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.

[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.

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.

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.

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.

[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.

[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.

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.

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.

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.

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.

[Optimized expression of the L1 protein of human papillomavirus in Hansenula polymorpha].

The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha.

[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.

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.

[Construction of a new brewing yeast strain with secretive alpha-amylase activity and reduced diacetyl production].

Saccharomycopsis fibuligera possesses high alpha-amylase and glucoamylase activities that enable it to utilize raw starch as a carbon source. A expression cassette containing the promoter sequence of 3-phosphogylycerate kinase gene (PGK1p), the alpha factor signal sequence from Saccharomyces cerevisiae and the alpha-amylase coding sequence of S. fibuligera was constructed.

Genetic modification of industrial yeast strains to obtain controllable NewFlo flocculation property and lower diacetyl production.

The expression cassette I10 containing the new-found flocculation gene, FLONS, was transformed into an industrial strain Saccharomyces cerevisiae YSF5. Upstream activating sequences of the S. cerevisiae alcohol dehydrogenase II (ADH2) gene promoter (P(U-ADH2)) were used to regulate the expression of FLONS; alpha-acetolactate synthase gene ILV2 was chosen for homologous recombination of I10 to the YSF5 chromosome; copper binding metallothionein (encoded by CUP1) was used for selection of transformants.

Uricase production by a recombinant Hansenula polymorpha strain harboring Candida utilis uricase gene.

Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae alpha-factor signal peptide as the secretory sequence. Recombinant H.

New industrial brewing yeast strains with ILV2 disruption and LSD1 expression.

New industrial brewing yeast strains, free of vector sequences and drug-resistance genes, were constructed by disrupting alpha-acetohydroxyacid synthase (AHAS) gene (ILV2) and introducing Lipomyces starkeyi dextranase (DEX) gene (LSD1) as a selective marker. The resulting recombinant strains can survive on YNB minimal medium plate with dextran T-70 as sole carbon source and showed lower AHAS activity. Fermentation test with recombinant strains in 500 ml conical flask confirmed DEX activity and lower AHAS activity compared with their host strain.