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1.  Deciphering the Transcriptional-Regulatory Network of Flocculation in Schizosaccharomyces pombe 
PLoS Genetics  2012;8(12):e1003104.
In the fission yeast Schizosaccharomyces pombe, the transcriptional-regulatory network that governs flocculation remains poorly understood. Here, we systematically screened an array of transcription factor deletion and overexpression strains for flocculation and performed microarray expression profiling and ChIP–chip analysis to identify the flocculin target genes. We identified five transcription factors that displayed novel roles in the activation or inhibition of flocculation (Rfl1, Adn2, Adn3, Sre2, and Yox1), in addition to the previously-known Mbx2, Cbf11, and Cbf12 regulators. Overexpression of mbx2+ and deletion of rfl1+ resulted in strong flocculation and transcriptional upregulation of gsf2+/pfl1+ and several other putative flocculin genes (pfl2+–pfl9+). Overexpression of the pfl+ genes singly was sufficient to trigger flocculation, and enhanced flocculation was observed in several combinations of double pfl+ overexpression. Among the pfl1+ genes, only loss of gsf2+ abrogated the flocculent phenotype of all the transcription factor mutants and prevented flocculation when cells were grown in inducing medium containing glycerol and ethanol as the carbon source, thereby indicating that Gsf2 is the dominant flocculin. In contrast, the mild flocculation of adn2+ or adn3+ overexpression was likely mediated by the transcriptional activation of cell wall–remodeling genes including gas2+, psu1+, and SPAC4H3.03c. We also discovered that Mbx2 and Cbf12 displayed transcriptional autoregulation, and Rfl1 repressed gsf2+ expression in an inhibitory feed-forward loop involving mbx2+. These results reveal that flocculation in S. pombe is regulated by a complex network of multiple transcription factors and target genes encoding flocculins and cell wall–remodeling enzymes. Moreover, comparisons between the flocculation transcriptional-regulatory networks of Saccharomyces cerevisiae and S. pombe indicate substantial rewiring of transcription factors and cis-regulatory sequences.
Author Summary
Flocculation is a process that involves yeast cells adhering to one another to form clumps called flocs. This trait is important for industrial yeast applications as it provides a cost-effective and efficient method to remove yeast cells. The adherence between cells occurs by the binding of glycoproteins known as flocculins and carbohydrate molecules located on the cell surface. To better understand how flocculation works, the genes that encode for flocculins and the transcription factors that regulate their expression need to be identified. In the fission yeast S. pombe, many of the flocculins and transcription factors that function in flocculation are not known. To address this gap in knowledge, we have employed molecular genetics and functional genomic approaches to uncover transcription factors and their target genes that play a role in flocculation. We discover that flocculation in S. pombe is regulated by a complex network of transcription factors that activate and repress themselves, as well as multiple target genes that encode for flocculins and cell wall–remodeling enzymes. The comparison of the flocculation regulatory networks between fission and budding yeasts indicates that they mainly differ in the types of transcription factors and their binding sequences.
doi:10.1371/journal.pgen.1003104
PMCID: PMC3516552  PMID: 23236291
2.  Region of Flo1 Proteins Responsible for Sugar Recognition 
Journal of Bacteriology  1998;180(24):6503-6510.
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.
PMCID: PMC107751  PMID: 9851992
3.  Deletion of Intragenic Tandem Repeats in Unit C of FLO1 of Saccharomyces cerevisiae Increases the Conformational Stability of Flocculin under Acidic and Alkaline Conditions 
PLoS ONE  2013;8(1):e53428.
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.
doi:10.1371/journal.pone.0053428
PMCID: PMC3537727  PMID: 23308221
4.  Alcohol dehydrogenases from Kluyveromyces marxianus: heterologous expression in Escherichia coli and biochemical characterization 
BMC Biotechnology  2014;14:45.
Background
Kluyveromyces marxianus has recently become a species of interest for ethanol production since it can produce ethanol at high temperature and on a wide variety of substrates. However, the reason why this yeast can produce ethanol at high temperature is largely unknown.
Results
The ethanol fermentation capability of K. marxianus GX-UN120 at 40°С was found to be the same as that of Saccharomyces cerevisiae at 34°С. Zymogram analysis showed that alcohol dehydrogenase 1 (KmAdh1) was largely induced during ethanol production, KmAdh4 was constitutively expressed at a lower level and KmAdh2 and KmAdh3 were almost undetectable. The genes encoding the four alcohol dehydrogenases (ADHs) were cloned from strain GX-UN120. Each KmADH was expressed in Escherichia coli and each recombinant protein was digested with enterokinase to remove the fusion protein. The optimum pH of the purified recombinant KmAdh1 was 8.0 and that of KmAdh2, KmAdh3 and KmAdh4 was 7.0. The optimum temperatures of KmAdh1, KmAdh2, KmAdh3 and KmAdh4 were 50, 45, 55 and 45°C, respectively. The Km values of the recombinant KmAdh1 and KmAdh2 were 4.0 and 1.2 mM for acetaldehyde and 39.7 and 49.5 mM for ethanol, respectively. The Vmax values of the recombinant KmAdh1 and KmAdh2 were 114.9 and 21.6 μmol min-1 mg-1 for acetaldehyde and 57.5 and 1.8 μmol min-1 mg-1 for ethanol, respectively. KmAdh3 and KmAdh4 catalyze the oxidation reaction of ethanol to acetaldehyde but not the reduction reaction of acetaldehyde to ethanol, and the K m values of the recombinant KmAdh3 and KmAdh4 were 26.0 and 17.0 mM for ethanol, respectively. The Vmax values of the recombinant KmAdh3 and KmAdh4 were 12.8 and 56.2 μmol min-1 mg-1 for ethanol, respectively.
Conclusion
These data in this study collectively indicate that KmAdh1 is the primary ADH responsible for the production of ethanol from the reduction of acetaldehyde in K. marxianus. The relatively high optimum temperature of KmAdh1 may partially explain the ability of K. marxianus to produce ethanol at high temperature. Understanding the biochemical characteristics of KmAdhs will enhance our fundamental knowledge of the metabolism of ethanol fermentation in K. marxianus.
doi:10.1186/1472-6750-14-45
PMCID: PMC4062290  PMID: 24885162
Alcohol dehydrogenase; Characterization; Expression; Gene cloning; Kluyveromyces marxianus
5.  FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin. 
Journal of Bacteriology  1996;178(24):7144-7151.
We report the characterization of a gene encoding a novel flocculin related to the STA genes of yeast, which encode secreted glucoamylase. The STA genes comprise sequences that are homologous to the sporulation-specific glucoamylase SGA and to two other sequences, S2 and S1. We find that S2 and S1 are part of a single gene which we have named FLO11. The sequence of FLO11 reveals a 4,104-bp open reading frame on chromosome IX whose predicted product is similar in overall structure to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. An amino-terminal domain containing a signal sequence and a carboxy-terminal domain with homology to GPI (glycosyl-phosphatidyl-inositol) anchor-containing proteins are separated by a central domain containing a highly repeated threonine- and serine-rich sequence. Yeast cells that express FLO11 aggregate in the calcium-dependent process of flocculation. Flocculation is abolished when FLO11 is disrupted. The product of STA1 also is shown to have flocculating activity. When a green fluorescent protein fusion of FLO11 was expressed from the FLO11 promoter on a single-copy plasmid, fluorescence was observed in vivo at the periphery of cells. We propose that FLO11 encodes a flocculin because of its demonstrated role in flocculation, its structural similarity to other members of the FLO gene family, and the cell surface location of its product. FLO11 gene sequences are present in all yeast strains tested, including all standard laboratory strains, unlike the STA genes which are present only in the variant strain Saccharomyces cerevisiae var. diastaticus. FLO11 differs from all other yeast flocculins in that it is located near a centromere rather than a telomere, and its expression is regulated by mating type. Repression of FLO11-dependent flocculation in diploids is conferred by the mating-type repressor al/alpha2.
PMCID: PMC178626  PMID: 8955395
6.  Candida albicans Sfl1 Suppresses Flocculation and Filamentation▿  
Eukaryotic Cell  2007;6(10):1736-1744.
Hyphal morphogenesis in Candida albicans is regulated by multiple pathways which act by either inducing or repressing filamentation. Most notably, Tup1, Nrg1, and Rfg1 are transcriptional repressors, while Efg1, Flo8, Cph1, and Czf1 can induce filamentation. Here, we present the functional analysis of CaSFL1, which encodes the C. albicans homolog of the Saccharomyces cerevisiae SFL1 (suppressor of flocculation) gene. Deletion of CaSFL1 results in flocculation (i.e., the formation of clumps) of yeast cells, which is most pronounced in minimal medium. The flocs contained hyphae already under noninducing conditions, and filamentation could be enhanced with hypha-inducing cues at 37°C. Expression of SFL1 in a heterozygous mutant under the control of the CaMET3 promoter was shown to complement these defects and allowed switching between wild-type and mutant phenotypes. Interestingly, increased expression of SFL1 using a MET3prom-SFL1 construct prior to the induction of filamentation completely blocked germ tube formation. To localize Sfl1 in vivo, we generated a SFL1-GFP fusion. Sfl1-green fluorescent protein was found in the nucleus in both yeast cells and, to a lesser extent, hyphal cells. Using reverse transcription-PCR, we find an increased expression of ALS1, ALS3, HWP1, ECE1, and also FLO8. Our results suggest that Sfl1 functions in the repression of flocculation and filamentation and thus represents a novel negative regulator of C. albicans morphogenesis.
doi:10.1128/EC.00236-07
PMCID: PMC2043394  PMID: 17766464
7.  Effect of oxygenation and temperature on glucose-xylose fermentation in Kluyveromyces marxianus CBS712 strain 
Background
The yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized.
Results
In the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes.
Conclusions
In the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to the impairment of the oxidoreductive pathway being determined by the cofactor imbalance, post-transcriptional and/or post-translational regulation of the pathway enzymes contributes to the efficiency of xylose catabolism in micro-aerobic conditions. Overall, the presented work provides novel information on the fermentation capability of the CBS712 strain that is currently considered as the reference strain of the genus K. marxianus.
doi:10.1186/1475-2859-13-51
PMCID: PMC3991920  PMID: 24712908
Kluyveromyces marxianus; Glucose fermentation; Xylose fermentation; Ethanol production; Oxygen requirement; Xylose reductase; Xylitol dehydrogenase
8.  High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042▿  
Applied and Environmental Microbiology  2008;74(24):7514-7521.
We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura+ transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys− transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus α-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus α-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiae-based molecular genetic tools.
doi:10.1128/AEM.01854-08
PMCID: PMC2607150  PMID: 18931291
9.  Controlled Expression of the Dominant Flocculation Genes FLO1, FLO5, and FLO11 in Saccharomyces cerevisiae▿  
Applied and Environmental Microbiology  2008;74(19):6041-6052.
In many industrial fermentation processes, the Saccharomyces cerevisiae yeast should ideally meet two partially conflicting demands. During fermentation, a high suspended yeast count is required to maintain a satisfactory rate of fermentation, while at completion, efficient settling is desired to enhance product clarification and recovery. In most fermentation industries, currently used starter cultures do not satisfy this ideal, probably because nonflocculent yeast strains were selected to avoid fermentation problems. In this paper, we assess molecular strategies to optimize the flocculation behavior of S. cerevisiae. For this purpose, the chromosomal copies of three dominant flocculation genes, FLO1, FLO5, and FLO11, of the haploid nonflocculent, noninvasive, and non-flor-forming S. cerevisiae FY23 strain were placed under the transcriptional control of the promoters of the ADH2 and HSP30 genes. All six promoter-gene combinations resulted in specific flocculation behaviors in terms of timing and intensity. The strategy resulted in stable expression patterns providing a platform for the direct comparison and assessment of the specific impact of the expression of individual dominant FLO genes with regard to cell wall characteristics, such as hydrophobicity, biofilm formation, and substrate adhesion properties. The data also clearly demonstrate that the flocculation behavior of yeast strains can be tightly controlled and fine-tuned to satisfy specific industrial requirements.
doi:10.1128/AEM.00394-08
PMCID: PMC2565957  PMID: 18708514
10.  Physiological characterization of thermotolerant yeast for cellulosic ethanol production 
The conversion of lignocellulose into fermentable sugars is considered a promising alternative for increasing ethanol production. Higher fermentation yield has been achieved through the process of simultaneous saccharification and fermentation (SSF). In this study, a comparison was performed between the yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus for their potential use in SSF process. Three strains of S. cerevisiae were evaluated: two are widely used in the Brazilian ethanol industry (CAT-1 and PE-2), and one has been isolated based on its capacity to grow and ferment at 42 °C (LBM-1). In addition, we used thermotolerant strains of K. marxianus. Two strains were obtained from biological collections, ATCC 8554 and CCT 4086, and one strain was isolated based on its fermentative capacity (UFV-3). SSF experiments revealed that S. cerevisiae industrial strains (CAT-1 and PE-2) have the potential to produce cellulosic ethanol once ethanol had presented yields similar to yields from thermotolerant strains. The industrial strains are more tolerant to ethanol and had already been adapted to industrial conditions. Moreover, the study shows that although the K. marxianus strains have fermentative capacities similar to strains of S. cerevisiae, they have low tolerance to ethanol. This characteristic is an important target for enhancing the performance of this yeast in ethanol production.
doi:10.1007/s00253-014-5580-3
PMCID: PMC3973951  PMID: 24535257
Simultaneous saccharification and fermentation; Ethanol; Sugarcane bagasse; Thermotolerant yeast
11.  FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast 
Cell  2008;135(4):726-737.
Summary
The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1-expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1+ cells avoid exploitation by non-expressing flo1 cells by self/non-self recognition: FLO1+ cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known “green beard genes”, which direct cooperation towards other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly-evolving social trait.
doi:10.1016/j.cell.2008.09.037
PMCID: PMC2703716  PMID: 19013280
Flocculation; FLO1; drug resistance; evolution; ethanol; selfish gene; Darwin; Dawkins; Hamilton; green beard gene
12.  Cloning and Characterization of CAD1/AAF1, a Gene from Candida albicans That Induces Adherence to Endothelial Cells after Expression in Saccharomyces cerevisiae 
Infection and Immunity  1998;66(5):2078-2084.
Adherence to the endothelial cell lining of the vasculature is probably a critical step in the egress of Candida albicans from the intravascular compartment. To identify potential adhesins that mediate the attachment of this organism to endothelial cells, a genomic library from C. albicans was used to transform a nonadherent strain of Saccharomyces cerevisiae. The population of transformed yeasts was enriched for highly adherent clones by repeated passages over endothelial cells. One clone which exhibited a fivefold increase in endothelial cell adherence, compared with S. cerevisiae transformed with vector alone, was identified. This organism also flocculated. The candidal DNA fragment within this adherent/flocculent organism was found to contain a single 1.8-kb open reading frame, which was designated CAD1. It was found to be identical to AAF1. The predicted protein encoded by CAD1/AAF1 contained features suggestive of a regulatory factor. Consistent with this finding, immunoelectron microscopy revealed that CAD1/AAF1 localized to the cytoplasm and nucleus but not the cell wall or plasma membrane of the transformed yeasts. Because yeasts transformed with CAD1/AAF1 both flocculated and exhibited increased endothelial cell adherence, the relationship between adherence and flocculation was examined. S. cerevisiae expressing either of two flocculation phenotypes, Flo1 or NewFlo, adhered to endothelial cells as avidly as did yeasts expressing CAD1/AAF1. Inhibition studies revealed that the flocculation phenotype induced by CAD1/AAF1 was similar to Flo1. Thus, CAD1/AAF1 probably encodes a regulatory protein that stimulates endothelial cell adherence in S. cerevisiae by inducing a flocculation phenotype. Whether CAD1/AAF1 contributes to the adherence of C. albicans to endothelial cells remains to be determined.
PMCID: PMC108166  PMID: 9573092
13.  Repression and induction of flocculation interactions in Saccharomyces cerevisiae. 
Journal of Bacteriology  1982;150(2):890-899.
The biological control of flocculation interactions by factors related to growth under different conditions of aeration was documented with a new assay for flocculence. The degree of flocculence expressed in a genetically defined Saccharomyces cerevisiae strain (FLO1/FLO1 ade1/ade1) remained constant during aerobic growth but varied with aeration. Flocculence was repressed in anaerobically growing cells but was induced in stationary cells or cells returned to aerobic growth. Repression was correlated with the selective inactivation of cell surface lectin-like components. The changes in flocculence were accompanied by changes in 16 extractable proteins separated by electrophoresis; however, a clear correlation between specific protein bands and flocculence could not be established. The study clearly demonstrated that the phenotypic expression of FLO1 could be reproducibly manipulated for experimental purposes by aeration alone.
Images
PMCID: PMC216442  PMID: 7040344
14.  Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion▿  
Eukaryotic Cell  2007;6(12):2214-2221.
The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.
doi:10.1128/EC.00284-06
PMCID: PMC2168249  PMID: 17921350
15.  Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain 
We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5′ upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.
doi:10.1128/AEM.68.9.4517-4522.2002
PMCID: PMC124111  PMID: 12200308
16.  Polygenic Molecular Architecture Underlying Non-Sexual Cell Aggregation in Budding Yeast 
Cell aggregation in unicellular organisms, induced by either cell non-sexual adhesion to yield flocs and biofilm, or pheromone-driving sexual conjugation is of great significance in cellular stress response, medicine, and brewing industries. Most current literatures have focused on one form of cell aggregation termed flocculation and its major molecular determinants, the flocculation (FLO) family genes. Here, we implemented a map-based approach for dissecting the molecular basis of non-sexual cell aggregation in Saccharomyces cerevisiae. Genome-wide mapping has identified four major quantitative trait loci (QTL) underlying nature variation in the cell aggregation phenotype. High-resolution mapping following up with knockout and allele replacement experiments resolved the QTL into the underlying genes (AMN1, RGA1, FLO1, and FLO8) or even into the causative nucleotide. Genetic variation in the QTL genes can explain up to 46% of phenotypic variation of this trait. Of these genes, AMN1 plays the leading role, differing from the FLO family members, in regulating expression of cell clumping phenotype through inducing cell segregation defect. These findings provide novel insights into the molecular mechanism of how cell aggregation is regulated in budding yeast, and the data will be directly implicated to understand the molecular basis and evolutionary implications of cell aggregation in other fungus species.
doi:10.1093/dnares/dss033
PMCID: PMC3576658  PMID: 23284084
cell aggregation; map-based cloning; QTL analysis; Saccharomyces cerevisiae
17.  High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40°C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills 
A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47°C. At 43°C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43°C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50°C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.
PMCID: PMC239064  PMID: 16347089
18.  Mechanisms of yeast flocculation: comparison of top- and bottom-fermenting strains. 
The flocculation of two brewing yeast strains, top-fermenting strain Saccharomyces cerevisiae MUCL 38485 and bottom-fermenting strain Saccharomyces carlsbergensis MUCL 28285, has been investigated by means of a turbidimetric test. The two strains showed different electrical properties, a different hydrophobicity, and a different surface chemical composition. They flocculated according to completely different mechanisms; however, no correlation between the cell physicochemical properties and the onset of flocculation was found for either strain. Flocculation of the bottom strain was governed by a lectin-mediated mechanism. It was inhibited by mannose and some other sugars, required calcium specifically, occurred in a narrow pH range different from the isoelectric point, and was not influenced by ethanol. The onset of flocculation at the end of the exponential phase was controlled both by the appearance of "active" lectins at the cell surface and by the decrease in sugar concentration in the solution. Flocculation of the top strain was not inhibited by mannose, did not require the addition of calcium, and took place at the cell isoelectric point. Low concentrations of ethanol broadened the pH range in which the cells flocculated, and flocculation was favored by an increase of ionic strength. Adsorbed ethanol may induce flocculation by reducing the electrostatic repulsion between cells, by decreasing steric stabilization, and/or by allowing the protrusion of polymer chains into the liquid phase. The onset of flocculation was controlled by both a change of the cell surface and an increase in ethanol concentration. The only evidence for an adhesin-mediated mechanism was the specific requirement for a small amount of calcium.
PMCID: PMC167332  PMID: 7574609
19.  Potential Probiotic Kluyveromyces marxianus B0399 Modulates the Immune Response in Caco-2 Cells and Peripheral Blood Mononuclear Cells and Impacts the Human Gut Microbiota in an In Vitro Colonic Model System 
Considering the increase in the consumption of yeasts as human probiotics, the aim of this study was to broadly investigate the beneficial properties of the lactic yeast Kluyveromyces marxianus (formerly Kluyveromyces fragilis) B0399. Several potential probiotic traits of K. marxianus B0399 were investigated by using in vitro assays, including adhesion and immune modulation, and the effect of the administration of 107 CFU/day of K. marxianus B0399 on the composition and metabolic activity of the human intestinal microbiota was investigated in a 3-stage continuous-culture system simulating the human colon. We demonstrated that this strain was highly adhesive to human enterocyte-like Caco-2 cells and modulated the immune response, inducing proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs). In the presence of inflammatory stimulation with lipopolysaccharide (LPS), K. marxianus B0399 provoked decreases in the levels of production of proinflammatory cytokines in PBMCs and Caco-2 cells, thus ameliorating the inflammatory response. Furthermore, K. marxianus B0399 impacted the colonic microbiota, increasing the bifidobacterial concentration in the stages of the colonic model system simulating the proximal and transverse colon. The amounts of the short-chain fatty acids acetate and propionate also increased following yeast supplementation. Finally, K. marxianus B0399 was found to induce a decrease of the cytotoxic potential of the culture supernatant from the first stage of the colonic model system. The effects of K. marxianus B0399 on adhesion, immune function, and colonic microbiota demonstrate that this strain possesses a number of beneficial and strain-specific properties desirable for a microorganism considered for application as a probiotic.
doi:10.1128/AEM.06385-11
PMCID: PMC3272993  PMID: 22156412
20.  Hydrolysis of Agave fourcroydes Lemaire (henequen) leaf juice and fermentation with Kluyveromyces marxianus for ethanol production 
BMC Biotechnology  2014;14:14.
Background
Carbon sources for biofuel production are wide-ranging and their availability depends on the climate and soil conditions of the land where the production chain is located. Henequen (Agave fourcroydes Lem.) is cultivated in Yucatán, Mexico to produce natural fibers from the leaves, and a juice containing fructans is produced during this process. Fructans can be hydrolyzed to fructose and glucose and metabolized into ethanol by appropriate yeasts. In Mexico, different Agave species provide the carbon source for (distilled and non-distilled) alcoholic beverage production using the stem of the plant, whilst the leaves are discarded. In this work, we investigated the effect of thermal acid and enzymatic hydrolysis of the juice on the amount of reducing sugars released. Growth curves were generated with the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus and fermentations were then carried out with Kluyveromyces marxianus to determine alcohol yields.
Results
With thermal acid hydrolysis, the greatest increase in reducing sugars (82.6%) was obtained using 5% H2SO4 at 100°C with a 30 min reaction time. Statistically similar results can be obtained using the same acid concentration at a lower temperature and with a shorter reaction time (60°C, 15 min), or by using 1% H2SO4 at 100°C with a 30 min reaction time. In the case of enzymatic hydrolysis, the use of 5.75, 11.47 and 22.82 U of enzyme did not produce significant differences in the increase in reducing sugars. Although both hydrolysis processes obtained similar results, the difference was observed after fermentation. Ethanol yields were 50.3 ± 4 and 80.04 ± 5.29% of the theoretical yield respectively.
Conclusions
Final reducing sugars concentrations obtained with both thermal acid and enzymatic hydrolysis were similar. Saccharomyces cerevisiae, a good ethanol producer, did not grow in the hydrolysates. Only Kluyveromyces marxianus was able to grow in them, giving a higher ethanol yield with the enzymatic hydrolysate. The leaves account for a non-negligible weight of the total agave plant biomass, so this work complements the knowledge already developed on agave fermentations by making it possible to produce ethanol from almost the entire plant (stem and leaves).
doi:10.1186/1472-6750-14-14
PMCID: PMC3932041  PMID: 24529165
Biofuel; Sugars; Oligofructans; Hydrolysis; Pretreatments
21.  Adaptive Evolution of a Lactose-Consuming Saccharomyces cerevisiae Recombinant▿  
The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (β-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media.
doi:10.1128/AEM.00186-08
PMCID: PMC2268319  PMID: 18245248
22.  Thermal adaptability of Kluyveromyces marxianus in recombinant protein production 
Background
Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins.
Results
The recombinant strains were able to grow up to 40°C and, along the tested temperature interval (5-40°C), the specific growth rates (μ) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15°C and 30°C. The secretion of the endogenous β-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous β-fructofuranosidase production was temperature dependent, with the highest yield at 30°C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5°C and 40°C, albeit each one presented a different optimal production temperature (15, 40, 5-30°C for HSA, GAA and Sod1p, respectively).
Conclusions
K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures.
doi:10.1186/1475-2859-12-34
PMCID: PMC3655038  PMID: 23587421
23.  Harvesting of microalgae by bio-flocculation 
Journal of Applied Phycology  2010;23(5):849-855.
The high-energy input for harvesting biomass makes current commercial microalgal biodiesel production economically unfeasible. A novel harvesting method is presented as a cost and energy efficient alternative: the bio-flocculation by using one flocculating microalga to concentrate the non-flocculating microalga of interest. Three flocculating microalgae, tested for harvesting of microalgae from different habitats, improved the sedimentation rate of the accompanying microalga and increased the recovery of biomass. The advantages of this method are that no addition of chemical flocculants is required and that similar cultivation conditions can be used for the flocculating microalgae as for the microalgae of interest that accumulate lipids. This method is as easy and effective as chemical flocculation which is applied at industrial scale, however in contrast it is sustainable and cost-effective as no costs are involved for pre-treatment of the biomass for oil extraction and for pre-treatment of the medium before it can be re-used.
doi:10.1007/s10811-010-9591-x
PMCID: PMC3172406  PMID: 21957329
Harvesting; Microalgae; Bio-flocculation
24.  Ethanol-Induced Leakage in Saccharomyces cerevisiae: Kinetics and Relationship to Yeast Ethanol Tolerance and Alcohol Fermentation Productivity 
Ethanol stimulated the leakage of amino acids and 260-nm-light-absorbing compounds from cells of Saccharomyces cerevisiae. The efflux followed first-order kinetics over an initial period. In the presence of lethal concentrations of ethanol, the efflux rates at 30 and 36°C were an exponential function of ethanol concentration: keX = keXmeE (X-Xm), where keX and keXm are the efflux rate constants, respectively, in the presence of a concentration X of ethanol or the minimal concentration of ethanol, Xm, above which the equation was applicable, coincident with the minimal lethal concentration of ethanol. E is the enhancement constant. At 36°C, as compared with the corresponding values at 30°C, the efflux rates were higher and the minimal concentration of ethanol (Xm) was lower. The exponential constants for the enhancement of the rate of leakage (E) had similar values at 30 or 36°C and were of the same order of magnitude as the corresponding exponential constants for ethanol-induced death. Under isothermic conditions (30°C) and up to 22% (vol/vol) ethanol, the resistance to ethanol-induced leakage of 260-nm-light-absorbing compounds was found to be closely related with the ethanol tolerance of three strains of yeasts, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Saccharomyces bayanus. The resistance to ethanol-induced leakage indicates the possible adoption of the present method for the rapid screening of ethanol-tolerant strains. The addition to a fermentation medium of the intracellular material obtained by ethanol permeabilization of yeast cells led to improvements in alcohol fermentation by S. cerevisiae and S. bayanus. The action of the intracellular material, by improving yeast ethanol tolerance, and the advantages of partially recycling the fermented medium after distillation were discussed.
PMCID: PMC202571  PMID: 16347612
25.  Comparison of ethanol production from cheese whey permeate by two yeast strains 
The objective of the present laboratory scale experiment was to compare ethanol production by Kluyveromyces marxianus strain ATCC8554 and Candida kefyr ATCC 14245 from unconcentrated and concentrated cheese whey permeate. The results indicated that ethanol production was greater when using concentrated whey permeate (9.8% lactose) compared to unconcentrated whey permeate (4.9% lactose) by both the yeasts, especially in presence of growth supplements. The rate and extent of ethanol formation increased noticeably and partly linearly for both the yeasts with sharp and partly linear decrease in both lactose and Chemical Oxygen Demand (COD), especially after the first 10 h of fermentation; total time of fermentation was 60 h. The optimum pH and temperature conditions for ethanol production were 4.8 and 30º C respectively. Klu. marxianus strain had greater ethanol producing ability from cheese permeate whey than Can. kefyr.
doi:10.1007/s13197-011-0309-0
PMCID: PMC3550851  PMID: 24082274
Whey permeate; Ethanol; Lactose; COD; Kluyveromyces marxianus; Candida kefyr

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