FLO gene-dependent phenotypes in industrial wine yeast strains

Most commercial yeast strains are nonflocculent. However, controlled flocculation phenotypes could provide significant benefits to many fermentation-based industries. In nonflocculent laboratory strains, it has been demonstrated that it is possible to adjust flocculation and adhesion phenotypes to desired specifications by altering expression of the otherwise silent but dominant flocculation (FLO) genes. However, FLO genes are characterized by high allele heterogeneity and are subjected to epigenetic regulation. Extrapolation of data obtained in laboratory strains to industrial strains may therefore not always be applicable. Here, we assess the adhesion phenotypes that are associated with the expression of a chromosomal copy of the FLO1, FLO5, or FLO11 open reading frame in two nonflocculent commercial wine yeast strains, BM45 and VIN13. The chromosomal promoters of these genes were replaced with stationary phase-inducible promoters of the HSP30 and ADH2 genes. Under standard laboratory and wine making conditions, the strategy resulted in expected and stable expression patterns of these genes in both strains. However, the specific impact of the expression of individual FLO genes showed significant differences between the two wine strains and with corresponding phenotypes in laboratory strains. The data suggest that optimization of the flocculation pattern of individual commercial strains will have to be based on a strain-by-strain approach.     

Novel wine-mediated FLO11 flocculation phenotype of commercial Saccharomyces cerevisiae wine yeast strains with modified FLO gene expression

Depending on the genetic background of Saccharomyces strains, a wide range of phenotypic adhesion identities can be directly attributed to the FLO11-encoded glycoprotein, which includes asexual flocculation, invasive growth and pseudohyphal formation, flor formation and adhesion to biotic and abiotic surfaces. In a previous study, we reported that HSP30-mediated stationary-phase expression of the native chromosomal FLO11 ORF in two nonflocculent commercial Saccharomyces cerevisiae wine yeast strains, BM45 or VIN13 did not generate a flocculent phenotype under either standard laboratory media or synthetic MS300 must fermentation conditions. In the present study, the BM45- and VIN13-derived HSP30p-FLO11 wine yeast transformants were observed to be exclusively and strongly flocculent under authentic red wine-making conditions, thus suggesting that this specific fermentation environment specifically contributes to the development of a flocculent phenotype, which is insensitive to either glucose or mannose. Furthermore, irrespective of the strain involved this phenotype displayed both Ca(2+)-dependent and Ca(2+)-independent flocculation characteristics. A distinct advantage of this unique FLO11-based phenotype was highlighted in its ability to dramatically promote faster lees settling rates. Moreover, wines produced by BM45-F11H and VIN13-F11H transformants were significantly less turbid than those produced by their wild-type parental strains.     

絮凝基因FLO1及FLO1c高表达提高工业酿酒酵母乙酸耐受性及发酵性能

乙酸是生物质乙醇发酵过程中酵母细胞面临的重要抑制剂之一,对细胞生长及发酵性能有强烈的抑制作用。增强酵母菌对乙酸胁迫的耐受性对提高乙醇产率具有重要意义。用分别带有完整絮凝基因FLO1及其重复序列单元C发生缺失的衍生基因FLO1c的重组表达质粒分别转化非絮凝型工业酿酒酵母CE6,获得絮凝型重组酵母菌株6-AF1和6-AF1c。同时以空载体p YCPGA1转化CE6的菌株CE6-V为对照菌株。与CE6-V相比,絮凝酵母明显提高了对乙酸胁迫的耐受性。在0.6%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.56倍和1.62倍;在1.0%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.21倍和1.78倍。可见絮凝能力改造能明显提高工业酿酒酵母的乙酸胁迫耐受性及发酵性能,而且FLO1内重复序列单元C缺失具有更加明显的效果。     

Possible mechanism for flocculation interactions governed by gene FLO1 in Saccharomyces cerevisiae.

A model is proposed for the mechanism of flocculation interactions in yeasts in which flocculent cells have a recognition factor which attaches to alpha-mannan sites on other cells. This factor may be governed by the expression of the single, dominant gene FLO1. Isogenic strains of Saccharomyces cerevisiae, differing only at FLO1 and the marker genes ade1 and trp1, were developed to examine the components involved in flocculene. Electron microscopy and concanavalin Aferritin labeling of aggregated cells showed that extensive and intense interactions between cell wall mannan layers mediated cell aggregation. The components of the mannan layer essential for flocculence were Ca2+ ions, alpha-mannan carbohydrates, and proteins. By studying the divalent cation dependence at various pH values and in the presence of competing monovalent cations, flocculation was found to be Ca2+ dependent; however, Mg2+ and Mn2+ ions substituted for Ca2+ under certain conditions. Reversible inhibition of flocculation by concanavalin A and succinylated concanavalin A implicated alpha-branched mannan carbohydrates as one essential component which alone did not determine the strain specificity of flocculence, since nonflocculent strains interacted with and competed for binding sites on flocculent cells. FLO1 may govern the expression of a proteinaceous, lectin-like activity, firmly associated with the cell walls of flocculent cells, which bind to the alpha-mannan carbohydrates of adjoining cells. It was selectively and irreversibly inhibited by proteolysis and reduction of disulfide bonds. The potential of this system as a model for the genetic and biochemical control of cell-cell interactions is discussed.     

Identification and functional study of a new FLO10-derivative gene from the industrial flocculating yeast SPSC01

Yeast flocculation is an important property for the brewing industry as well as for ethanol fermentation to facilitate biomass recovery by sedimentation from the fermentation broth, which is cost-effective. In this study, a new flocculating gene FLO10 (spsc) of 4,221 bp homologous to FLO10 was identified in the industrial flocculating yeast SPSC01. Sequence analysis indicated that the N- and C-terminus of the deduced protein of this new FLO gene are 99 % identical to that of FLO10, but more intragenic repeats are included. The study on the function of FLO10 (spsc) by its integrative expression in the non-flocculating industrial yeast indicated severe inhibition in the flocculation of the transformant by mannose and maltose, moderate inhibition by sucrose and glucose and no inhibition by xylose and galactose, and thus the NewFlo type was established. Meanwhile, the flocculation of the transformant was stable when the temperature was below 50 °C and the pH was in the range of 4.0-6.0. Furthermore, the medium containing 250 g/l glucose was completely fermented within 48 h by the transformant, with about 110 g/l ethanol and 5.5 g(DCW)/l biomass produced, and no significant difference in ethanol fermentation performance was observed compared to its wide-type strain. Therefore, the FLO gene and corresponding transformation strategy provide a platform for engineering yeast strains with the flocculation phenotype to facilitate biomass recovery.     

Breeding of flocculent industrial alcohol yeast strains by self-cloning of the flocculation gene FLO1 and repeated-batch fermentation by transformants

A nonflocculent industrial polyploid yeast strain, Saccharomyces cerevisiae 396-9-6V, was converted to a flocculent one by introducing a functional FLO1 gene at the URA3 locus. The flocculent strain FSC27 obtained was a so-called self-cloned strain, having no bacterial DNA. FSC27 cells could be easily recovered for reuse from fermentation mash without any physical energy. The strain produced a concentration of alcohol as high as 396-9-6V, although the fermentation rate of FSC27 was slightly lower than that of 396-9-6V. When uracil was added to the medium or when URA3 was reintroduced into FSC27 (named FSCU-L18), the fermentation rate and the growth rate increased, and the ethanol concentration produced was higher than that produced by the parent strain. The stable flocculation and high ethanol productivity were observed by using FSCU-L18 during 10 cycles of repeated-batch fermentation test.     

Identification of novel activation mechanisms for FLO11 regulation in Saccharomyces cerevisiae

Adhesins play a central role in the cellular response of eukaryotic microorganisms to their host environment. In pathogens such as Candida spp. and other fungi, adhesins are responsible for adherence to mammalian tissues, and in Saccharomyces spp. yeasts also confer adherence to solid surfaces and to other yeast cells. The analysis of FLO11, the main adhesin identified in Saccharomyces cerevisiae, has revealed complex mechanisms, involving both genetic and epigenetic regulation, governing the expression of this critical gene. We designed a genomewide screen to identify new regulators of this pivotal adhesin in budding yeasts. We took advantage of a specific FLO11 allele that confers very high levels of FLO11 expression to wild "flor" strains of S. cerevisiae. We screened for mutants that abrogated the increased FLO11 expression of this allele using the loss of the characteristic fluffy-colony phenotype and a reporter plasmid containing GFP controlled by the same FLO11 promoter. Using this approach, we isolated several genes whose function was essential to maintain the expression of FLO11. In addition to previously characterized activators, we identified a number of novel FLO11 activators, which reveal the pH response pathway and chromatin-remodeling complexes as central elements involved in FLO11 activation.     

Ethanol-induced yeast flocculation directed by the promoter of TPS1 encoding trehalose-6-phosphate synthase 1 for efficient ethanol production

Yeast flocculation is an important trait in the brewing industry as well as in ethanol production, through which biomass can be recovered by cost-effective sedimentation. However, mass transfer limitation may affect yeast growth and ethanol fermentation if the flocculation occurs earlier before fermentation is completed. In this article, a novel type of cell-cell flocculation induced by trehalose-6-phosphate synthase 1 (TPS1) promoter was presented. The linear cassette HO-P(TPS1)-FLO1(SPSC01)-KanMX4-HO was constructed to transform the non-flocculating industrial yeast S. cerevisiae 4126 by chromosome integration to obtain a new flocculating yeast strain, ZLH01, whose flocculation was induced by ethanol produced during fermentation. The experimental results illustrated that flocculation of ZLH01 was triggered by 3% (v/v) ethanol and enhanced as ethanol concentration increased till complete flocculation was achieved at ethanol concentration of 8% (v/v). Real time PCR analysis confirmed that the expression of FLO1(SPSC01) was dependent on ethanol concentration. The growth and ethanol fermentation of ZLH01 were improved significantly, compared with the constitutive flocculating yeast BHL01 engineered with the same FLO gene but directed by the constitutive 3-phosphoglycerate kinase promoter PGK1, particularly under high temperature conditions. These characteristics make the engineered yeast more suitable for ethanol production from industrial substrates under high gravity and temperature conditions. In addition, this strategy offers advantage in inducing differential expression of other genes for metabolic engineering applications of S. cerevisiae.     

絮凝选择载体的构建及β葡萄糖苷酶基因在酿酒酵母中的表达

从强絮凝酿酒酵母(Saccharomyces cerevisiae)ABXL-1D菌株中用PCRA-法扩增到絮凝基因(Flocculation gene,FLO1),构建以絮凝基因作选择标记的酿酒酵母表达栽体：用该栽体表达Bacillus polymyxa的β-葡萄糖苷酶基因,转化子可直接从沉淀中筛选。摇瓶培养细胞得到的β-葡萄糖苷酶比活力为3．91u／mg蛋白。在发酵葡萄糖和纤维二糖混合底物时,转化子的葡萄糖残存量明显低于受体菌。这将有利于利用纤维素发酵生产酒精。     

Exploration of a natural reservoir of flocculating genes from various Saccharomyces cerevisiae strains and improved ethanol fermentation using stable genetically engineered flocculating yeast strains

Flocculating yeast strains with good fermentation ability are desirable for brewing industry as well as for fuel ethanol production, however, the genetic diversity of the flocculating genes from natural yeast strains is largely unexplored. In this study, FLO1, FLO5, FLO9, FLO10 and FLO11 PCR products were obtained from 16 yeast strains from various sources, and the PCR product amplified from FLO1 of the self-flocculating yeast strain SPSC01 was used for the construction of expression cassette flanked by homologous fragments of the endonuclease gene HO for chromosome integration. A genetically engineered flocculating yeast BHL01 with good fermentation performance was obtained by transforming an industrial strain Saccharomyces cerevisiae 4126 with the expression cassette. The fermentation performances of SPSC01 and BHL01 in flask fermentation were compared using 208 g/L glucose. BHL01 completed the fermentation 8 h earlier than SPSC01, while no significant difference between BHL01 and S. cerevisiae 4126 was observed. In very high gravity repeated batch ethanol fermentation using 255 g/L glucose, BHL01 maintained stable flocculation for at least over 24 batches, while SPSC01 displayed severe deflocculation under the same conditions. The natural reservoir of flocculating genes from yeast strains may represent an unexplored gene source for the construction of new flocculating yeast strains for improved ethanol production.     

Saccharomyces Cerevisiae S288c Has a Mutation in Flo8, a Gene Required for Filamentous Growth

Diploid strains of baker's yeast Saccharomyces cerevisiae can grow in a cellular yeast form or in filaments called pseudohyphae. This dimorphic transition from yeast to pseudohyphae is induced by starvation for nitrogen. Not all laboratory strains are capable of this dimorphic switch; many grow only in the yeast form and fail to form pseudohyphae when starved for nitrogen. Analysis of the standard laboratory strain S288C shows that this defect in dimorphism results from a nonsense mutation in the FLO8 gene. This defect in FLO8 blocks pseudohyphal growth in diploids, haploid invasive growth, and flocculation. Since feral strains of S. cerevisiae are dimorphic and have a functional FLO8 gene, we suggest that the flo8 mutation was selected during laboratory cultivation.     

Toward a global database for the molecular typing of Saccharomyces cerevisiae strains

Most of the yeast strains used in fermented beverages and foods are classified as Saccharomyces cerevisiae. However, different strains are suitable for different fermentation processes. The purpose of this work is the proposal of a standardized methodology for the molecular genotyping of S. cerevisiae strains based on polymorphisms at microsatellite loci and/or single nucleotide polymorphisms (SNPs). Single nucleotide variants in the coding region of FLO8, a key regulator of flocculation and pseudohyphae formation, were analyzed in a subset of Uruguayan wine strains. Polymorphism analysis at nine microsatellite loci (selected from 33 loci tested) was performed in a collection of 120 strains, mostly wine strains, from different origins. From a total of 184 different alleles scored, 50 were exclusive alleles that could identify 29 strains. Four selected microsatellite loci are located within or near genes of putative enological interest. The Uruguayan strains are highly diverse and evenly distributed in the phylogenetic reconstructions, suggesting an evolutionary history previous to human use. The Saccharomyces cerevisiae Microsatellites and SNPs Genotyping Database is presented (www.pasteur.edu.uy/yeast). Comparison of standardized results from strains coming from different settings (industrial, clinical, environmental) will provide a reliable and growing source of information on the molecular biodiversity of S. cerevisiae strains.     

Flocculation gene variability in industrial brewer’s yeast strains

The brewer’s yeast genome encodes a ‘Flo’ flocculin family responsible for flocculation. Controlled floc formation or flocculation at the end of fermentation is of great importance in the brewing industry since it is a cost-effective and environmental-friendly technique to separate yeast cells from the final beer. FLO genes have the notable capacity to evolve and diverge many times faster than other genes. In actual practice, this genetic variability may directly alter the flocculin structure, which in turn may affect the flocculation onset and/or strength in an uncontrolled manner. Here, 16 ale and lager yeast strains from different breweries, one laboratory Saccharomyces cerevisiae and one reference Saccharomyces pastorianus strain, with divergent flocculation strengths, were selected and screened for characteristic FLO gene sequences. Most of the strains could be distinguished by a typical pattern of these FLO gene markers. The FLO1 and FLO10 markers were only present in five out of the 18 yeast strains, while the FLO9 marker was ubiquitous in all the tested strains. Surprisingly, three strongly flocculating ale yeast strains in this screening also share a typical ‘lager’ yeast FLO gene marker. Further analysis revealed that a complete Lg-FLO1 allele was present in these ale yeasts. Taken together, this explicit genetic variation between flocculation genes hampers attempts to understand and control the flocculation behavior in industrial brewer’s yeasts.     

Chromosomal location of Lg-FLO1 in bottom-fermenting yeast and the FLO5 locus of industrial yeast

Aims: To determine the chromosomal location and entire sequence of Lg-FLO1, the expression of which causes the flocculation of bottom-fermenting yeast. Methods and Results: Two cosmid clones carrying DNA from a bottom-fermenting yeast chromosome VIII right-arm end were selected by colony hybridization. Sequencing revealed that the clones contained DNA derived from a Saccharomyces cerevisiae type chromosome VIII and a Saccharomyces bayanus type chromosome VIII, both from bottom-fermenting yeast. Conclusions: Lg-FLO1 is located on the S. cerevisiae type chromosome VIII at the same position as the FLO5 gene of the laboratory yeast S. cerevisiae S288c. The unique chromosome VIII structure of bottom-fermenting yeast is conserved among other related strains. FLO5 and Lg-FLO1 promoter sequences are identical except for the presence of three 42 bp repeats in the latter, which are associated with gene activity. Flocculin genes might have been generated by chromosomal recombination at these repeats. Significance and Impact of the Study: This is the first report of the exact chromosomal location and entire sequence of Lg-FLO1. This information will be useful in the brewing industry for the identification of normal bottom-fermenting yeast. Moreover, variations in the FLO5 locus among strains are thought to reflect yeast evolution.     

Purification and partial characterization of a flocculin from brewer's yeast.

Analysis of a shear supernatant from flocculent, "fimbriated" Saccharomyces cerevisiae brewer's yeast cells revealed the presence of a protein involved in flocculation of the yeast cells and therefore designated a flocculin. The molecular mass of the flocculin was estimated to be over 300 kDa, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography of the flocculin yielded an aggregate with an apparent molecular weight of > 2,000. The flocculin was found to be protease sensitive, and the sequence of its 16 N-terminal amino acids revealed at least 69% identity with the predicted N terminus of the putative protein encoded by the flocculation gene FLO1. The flocculin was isolated from flocculent S. cerevisiae cells, whereas only a low amount of flocculin, if any, could be isolated from nonflocculent cells. The flocculin was found to stimulate the flocculation ability of flocculent yeast cells without displaying lectinlike activity (that is, the ability to agglutinate yeast cells).     

絮凝基因内衔接重复序列与酵母菌絮凝特性多样性及遗传稳定性

存在于酵母菌细胞表面的絮凝蛋白与邻近细胞表面寡聚甘露糖链相互作用,从而使细胞相互聚集形成细胞团的生理过程称为酵母菌絮凝。编码絮凝蛋白的基因中存在大量衔接重复序列,这些重复序列的变化不但使酵母菌呈现出絮凝特性的多样性,而且由重复序列驱动的基因内或基因间重组使酵母菌的絮凝特性具有非常明显的遗传不稳定性。文中综述了基因内重复序列对酵母菌絮凝特性和遗传稳定性的影响,将为基于序列调控策略改进酵母菌絮凝特性及遗传稳定性奠定理论基础,为絮凝特性在发酵工业或环境修复过程中的可控应用提供新的解决策略。