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.     

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.     

Construction of flocculent industrial yeast by the yeast flocculation gene <Emphasis Type="Italic">FLO1</Emphasis>

The structure gene FLO1 from Saccharomyces cerevisiae W303-1A encoding a flocculation protein and the G418 resistance gene kanMX from plasmid pUG6 were amplified by PCR method. The expression vector pYX212 harboring FLO1 gene and kanMX gene was transformed into Angel yeast. The transformant Angel yeast F6 was obtained and showed strong and stable flocculation ability during 20 batches inoculation. And the flocculation ability of the transformant Angel yeast F6 showed no difference in the medium with the initial pH ranging from 3.5 to 6.0. Noteworthily, the flocculation onset of the transformant strain was in the early stationary growth phase, not coincident with the glucose depletion in the cultural medium. And in the experiment the ethanol yield and other properties of the transformant Angel yeast F6 were similar to those of the wild-type strain, although its fermentation time was a little slower comparing with the wild-type strain. Those would be potential application for yeast cells to separate and recycle in the fuel ethanol industry.     

Region of Flo1 Proteins Responsible for Sugar Recognition

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The FLO1 gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of FLO1, named Lg-FLO1, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both FLO1 and Lg-FLO1 are involved in the yeast flocculation phenomenon, the FLO1 gene in the mannose-specific flocculation strain was replaced by the Lg-FLO1 gene. The transformant in which the Lg-FLO1 gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the FLO1 and Lg-FLO1 genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric FLO1 and Lg-FLO1 genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.     

Preparation of high activity whole cell biocatalyst by permeabilization of recombinant flocculent yeast with alcohol

Flocculent yeast Saccharomyces cerevisiae YF234 (MATa ura3–52 trp1Δ2 his ade 2–1 can1–100 sta1 FLO8) cells overexpressing glyoxalase I and having strong flocculation ability were permeabilized with isopropyl alcohol and ethanol under various conditions. The treatment with 40% isopropyl alcohol significantly improves the initial reaction rates of recombinant flocculent yeast cells. Moreover, the reactivity of permeabilized flocculent yeast cells was similar to that of dispersed cells with EDTA. On the other hand, the flocculation ability of yeast cells was not affected by the treatment with alcohol solutions of various concentrations and treatment time length. Therefore, the recombinant flocculent yeast cells permeabilized with alcohol are very effective whole cell biocatalysts.     

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.     

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.     

Co-Flocculation of Yeast Species,a New Mechanism to Govern Population Dynamics in Microbial Ecosystems

Flocculation has primarily been studied as an important technological property of Saccharomyces cerevisiae yeast strains in fermentation processes such as brewing and winemaking. These studies have led to the identification of a group of closely related genes, referred to as the FLO gene family, which controls the flocculation phenotype. All naturally occurring S. cerevisiae strains assessed thus far possess at least four independent copies of structurally similar FLO genes, namely FLO1, FLO5, FLO9 and FLO10. The genes appear to differ primarily by the degree of flocculation induced by their expression. However, the reason for the existence of a large family of very similar genes, all involved in the same phenotype, has remained unclear. In natural ecosystems, and in wine production, S. cerevisiae growth together and competes with a large number of other Saccharomyces and many more non-Saccharomyces yeast species. Our data show that many strains of such wine-related non-Saccharomyces species, some of which have recently attracted significant biotechnological interest as they contribute positively to fermentation and wine character, were able to flocculate efficiently. The data also show that both flocculent and non-flocculent S. cerevisiae strains formed mixed species flocs (a process hereafter referred to as co-flocculation) with some of these non-Saccharomyces yeasts. This ability of yeast strains to impact flocculation behaviour of other species in mixed inocula has not been described previously. Further investigation into the genetic regulation of co-flocculation revealed that different FLO genes impact differently on such adhesion phenotypes, favouring adhesion with some species while excluding other species from such mixed flocs. The data therefore strongly suggest that FLO genes govern the selective association of S. cerevisiae with specific species of non-Saccharomyces yeasts, and may therefore be drivers of ecosystem organisational patterns. Our data provide, for the first time, insights into the role of the FLO gene family beyond intraspecies cellular association, and suggest a wider evolutionary role for the FLO genes. Such a role would explain the evolutionary persistence of a large multigene family of genes with apparently similar function.     

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.     

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.     

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.     

Yeast flocculation: New story in fuel ethanol production

Yeast flocculation has been used in the brewing industry to facilitate biomass recovery for a long time, and thus its mechanism of yeast flocculation has been intensively studied. However, the application of flocculating yeast in ethanol production garnered attention mainly in the 1980s and 1990s. In this article, updated research progress in the molecular mechanism of yeast flocculation and the impact of environmental conditions on yeast flocculation are reviewed. Construction of flocculating yeast strains by genetic approach and utilization of yeast flocculation for ethanol production from various feedstocks were presented. The concept of self-immobilized yeast cells through their flocculation is revisited through a case study of continuous ethanol fermentation with the flocculating yeast SPSC01, and their technical and economic advantages are highlighted by comparing with yeast cells immobilized with supporting materials and regular free yeast cells as well. Taking the flocculating yeast SPSC01 as an example, the ethanol tolerance of the flocculating yeast was also discussed.     

Generation of intra- and interspecific Saccharomyces hybrids with improved oenological and aromatic properties

Non-wine yeasts could enhance the aroma and organoleptic profile of wines. However, compared to wine strains, they have specific intolerances to winemaking conditions. To solve this problem, we generated intra- and interspecific hybrids using a non-GMO technique (rare-mating) in which non-wine strains of S. uvarum, S. kudriavzevii and S. cerevisiae species were crossed with a wine S. cerevisiae yeast. The hybrid that inherited the wine yeast mitochondrial showed better fermentation capacities, whereas hybrids carrying the non-wine strain mitotype reduced ethanol levels and increased glycerol, 2,3-butanediol and organic acid production. Moreover, all the hybrids produced several fruity and floral aromas compared to the wine yeast: β-phenylethyl acetate, isobutyl acetate, γ-octalactone, ethyl cinnamate in both varietal wines. Sc × Sk crosses produced three- to sixfold higher polyfunctional mercaptans, 4-mercapto-4-methylpentan-2-one (4MMP) and 3-mercaptohexanol (3MH). We proposed that the exceptional 3MH release observed in an S. cerevisiae × S. kudriavzevii hybrid was due to the cleavage of the non-volatile glutathione precursor (Glt-3MH) to detoxify the cell from the presence of methylglyoxal, a compound related to the high glycerol yield reached by this hybrid. In conclusion, hybrid generation allows us to obtain aromatically improved yeasts concerning their wine parent. In addition, they reduced ethanol and increased organic acids yields, which counteracts climate change effect on grapes.     

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.     

Construction of a flocculating yeast for fructose production from inulin

Construction of flocculating yeast lacking for fructose utilisation was realised by integration of the FLO1 flocculation gene in the ribosomal DNA of an hexokinase deficient (hxk1, hxk2) Saccharomyces cerevisiae strain (ATCC36859). Simultaneous production of ethanol and fructose was obtained from glucose/fructose mixtures or from hydrolysed Jerusalem artichoke extracts using the transformed yeast in batch fermentations and in a continuous reactor with internal biomass recycle. This allowed the production of 5 g ethanol/L and 48 g sugars/L containing up to 99 % fructose from diluted hydrolysed Jerusalem artichoke extracts containing 60 g sugars/L. © Rapid Science Ltd. 1998