A Comparison of the Beneficial Effects of Live and Heat-Inactivated Baker’s Yeast on Nile Tilapia: Suggestions on the Role and Function of the Secretory Metabolites Released from the Yeast

Yeast is frequently used as a probiotic in aquaculture with the potential to substitute for antibiotics. In this study, the involvement and extent to which the viability of yeast cells and thus the secretory metabolites released from the yeast contribute to effects of baker’s yeast was investigated in Nile tilapia. No yeast, live yeast or heat-inactivated baker’s yeast were added to basal diets high in fishmeal and low in soybean (diet A) or low in fishmeal and high in soybean (diet B), which were fed to fish for 8 weeks. Growth, feed utilization, gut microvilli morphology, and expressions of hsp70 and inflammation-related cytokines in the intestine and head kidney were assessed. Intestinal microbiota was investigated using 16S rRNA gene pyrosequencing. Gut alkaline phosphatase (AKP) activity was measured after challenging the fish with Aeromonas hydrophila. Results showed that live yeast significantly improved FBW and WG (P < 0.05), and tended to improve FCR (P = 0.06) of fish compared to the control (no yeast). No significant differences were observed between inactivated yeast and control. Live yeast improved gut microvilli length (P < 0.001) and density (P < 0.05) while inactivated yeast did not. The hsp70 expression level in both the intestine and head kidney of fish was significantly reduced by live yeast (P < 0.05) but not inactivated yeast. Live yeast but not inactivated yeast reduced intestinal expression of tnfα (P < 0.05), tgfβ (P < 0.05 under diet A) and il1β (P = 0.08). Intestinal Lactococcus spp. numbers were enriched by both live and inactivated yeast. Lastly, both live and inactivated yeast reduced the gut AKP activity compared to the control (P < 0.001), indicating protection of the host against infection by A. hydrophila. In conclusion, secretory metabolites did not play major roles in the growth promotion and disease protection effects of yeast. Nevertheless, secretory metabolites were the major contributing factor towards improved gut microvilli morphology, relieved stress status, and reduced intestinal inflammation of Nile tilapia fed diets supplemented with baker’s yeast.     

Functional expression of cloned yeast DNA in Escherichia coli: specific complementation of argininosuccinate lyase (argH) mutations.

Segments of yeast (Saccharomyces cerevisiae) DNA cloned on various plasmid vectors in Escherichia coli can be functionally expressed to produce active enzymes. We have identified several ColE1-DNA(yeast) plasmids capable of complementing argH mutations, including deletions, in E. coli. Variants of the original transformants that grow faster on selective media and contain higher levels of the complementing enzyme activity (argininosuccinate lyase) can be readily isolated. The genetic alterations leading to increased expression of the yeast gene are associated with the cloned yeast DNA segment, rather than the host genome. The yeast DNA segment cloned in these plasmids also specifies a suppressor of the leuB6 mutation in E. coli. The argH and leuB6 complementing activities are expressed from discrete regions of the cloned yeast DNA segment, since the two genetic functions can be separated on individual recloned restriction fragments. The ease with which the bacterial cell can achieve functional high-level gene expression from cloned yeast DNA indicates that there are no significant barriers preventing expression of many yeast genes in E. coli.     

In vitro assembly of minicellulosomes with two scaffoldins on the yeast cell surface for cellulose saccharification and bioethanol production

The present paper reports in vitro strategies for assembly of minicellulosomes with two miniscaffoldins on the Saccharomyces cerevisiae cell surface. It was carried out through incubation of the yeast cells displaying scaffoldins with Escherichia coli lysates containing recombinant cellulases, or using a four-population yeast consortium. The results showed that the display level of miniscaffoldin II was distinctly increased by moving the cellulases production into E. coli or other yeast cells, indicating that the metabolic burden of the yeast host was decreased. The yeast consortium did not show any cellulolytic activity, while the E. coli lysates-treated yeast, whose anchoring miniscaffoldin length was optimized, was able to produce ～1138 mg/L ethanol from microcrystalline cellulose within 4 days. We also confirmed that the yeast-associated minicellulosome moreover showed both higher thermal stability and lower protease accessibility than free minicellulosome. This research promotes the application of S. cerevisiae as a consolidated bioprocessing (CBP) microorganism in cellulosic bioethanol production.     

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.     

The golden root, Rhodiola rosea, prolongs lifespan but decreases oxidative stress resistance in yeast Saccharomyces cerevisiae

The effect of aqueous extract from R. rosea root on lifespan and the activity of antioxidant enzymes in budding yeast Saccharomyces cerevisiae have been studied. The supplementation of the growth medium with R. rosea extract decreased survival of exponentially growing S. cerevisiae cells under H₂O₂-induced oxidative stress, but increased viability and reproduction success of yeast cells in stationary phase. The extract did not significantly affect catalase activity and decreased SOD activity in chronologically aged yeast population. These results suggest that R. rosea acts as a stressor for S. cerevisiae cells, what sensitizes yeast cells to oxidative stress at exponential phase, but induces adaptation in stationary phase cells demonstrating the positive effect on yeast survival without activation of major antioxidant enzymes.     

Expression of a thermostable cellulase gene from a thermophilic anaerobe in Saccharomyces cerevisiae

A cellulase gene from a thermophilic anaerobe was recloned in the yeast Saccharomyces cerevisiae. The maximum level of the gene expression in the recombinant yeast was 4.4 times higher than that in the Escherichia coli transformant harboring the same plasmid. Cellulase activity was observed only within the yeast cells. To compare the enzymatic properties of cellulase produced by the yeast and E. coli transformants, cellulases were purified to homogeneous state by only three purification steps of heat treatment, and cellulose affinity and ion exchange chromatographies. The molecular weights of the enzymes produced by the yeast and E. coli were 3.8 × 10⁴ and 4.0 × 10⁴, respectively by SDS-polyacrylamide gel electrophoresis. Neither of the enzymes was glycosylated. Although the molecular weights were slightly different, enzymatic properties and thermostability were almost indistinguishable between the enzymes produced by the yeast and E. coli transformants.     

N-glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable α-amylase in Saccharomyces cerevisiae

Expression of foreign enzymes in yeast is a traditional genetic engineering approach; however, useful secretory enzymes are not produced in every case. The hyperthermostable α-amylase encoded by the AmyL gene of Bacillus licheniformis was expressed in Saccharomyces cerevisiae; however, it was only weakly produced and was degraded by the proteasome. To determine the cause of low α-amylase production, AmyL was expressed in a panel of yeast mutants harboring knockouts in non-essential genes. Elevated AmyL production was observed in 44 mutants. The knockout genes were classified into six functional categories. Remarkably, all non-essential genes required for N-linked oligosaccharide synthesis and a gene encoding an oligosaccharyl transferase subunit were identified. Immunoblotting demonstrated that differently underglycosylated forms of AmyL were secreted from oligosaccharide synthesis-deficient mutants, while a fully glycosylated form was produced by wild-type yeast, suggesting that N-linked glycosylation of AmyL inhibited its secretion in yeast. Mutational analysis of six potential N-glycosylation sites in AmyL revealed that the N33Q and N309Q mutations remarkably affected AmyL production. To achieve higher AmyL production in yeast, all six N-glycosylation sites of AmyL were mutated. In wild-type yeast, production of the resulting non-glycosylated form of AmyL was threefold higher than that of the glycosylated form.     

Amino acid incorporation on plant ribosomes: The transfer system

Pre-formed Vicia faba phenylalanyl-tRNA was active in a TYMV-RNA-directed Transfer System, whereas a similar tRNA preparation from yeast was not. Thus, lack of charging of yeast tRNA by enzymes from Phaseolus was not the only reason why yeast tRNA would not function in this Transfer System. In the poly U-directed Transfer System; where both types of tRNA were active, the pH and ionic parameters governing the reaction with yeast tRNA were more stringent.     

Construction of a Stable α-Galactosidase-Producing Baker's Yeast Strain

Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of α-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel⁻. In this study we integrated the yeast MEL1 gene, which codes for α-galactosidase, into a commercial mel⁰ baker's yeast strain. The Mel⁺ phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel⁺ baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The α-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more α-galactosidase than did a wild-type Mel⁺ strain and may prove useful for commercial production of α-galactosidase.     

Yeast Modulation of Human Dendritic Cell Cytokine Secretion: An In Vitro Study

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current definition of a probiotic.     

Growth Requirements of San Francisco Sour Dough Yeasts and Bakers' Yeast

The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.     

A Wickerhamomyces anomalus Killer Strain in the Malaria Vector Anopheles stephensi

The yeast Wickerhamomyces anomalus has been investigated for several years for its wide biotechnological potential, especially for applications in the food industry. Specifically, the antimicrobial activity of this yeast, associated with the production of Killer Toxins (KTs), has attracted a great deal of attention. The strains of W. anomalus able to produce KTs, called “killer” yeasts, have been shown to be highly competitive in the environment. Different W. anomalus strains have been isolated from diverse habitats and recently even from insects. In the malaria mosquito vector Anopheles stephensi these yeasts have been detected in the midgut and gonads. Here we show that the strain of W. anomalus isolated from An. stephensi, namely WaF17.12, is a killer yeast able to produce a KT in a cell-free medium (in vitro) as well as in the mosquito body (in vivo). We showed a constant production of WaF17.12-KT over time, after stimulation of toxin secretion in yeast cultures and reintroduction of the activated cells into the mosquito through the diet. Furthermore, the antimicrobial activity of WaF17.12-KT has been demonstrated in vitro against sensitive microbes, showing that strain WaF17.12 releases a functional toxin. The mosquito-associated yeast WaF17.12 thus possesses an antimicrobial activity, which makes this yeast worthy of further investigations, in view of its potential as an agent for the symbiotic control of malaria.     

In Situ Analysis of Metabolic Characteristics Reveals the Key Yeast in the Spontaneous and Solid-State Fermentation Process of Chinese Light-Style Liquor

The in situ metabolic characteristics of the yeasts involved in spontaneous fermentation process of Chinese light-style liquor are poorly understood. The covariation between metabolic profiles and yeast communities in Chinese light-style liquor was modeled using the partial least square (PLS) regression method. The diversity of yeast species was evaluated by sequence analysis of the 26S ribosomal DNA (rDNA) D1/D2 domains of cultivable yeasts, and the volatile compounds in fermented grains were analyzed by gas chromatography (GC)-mass spectrometry (MS). Eight yeast species and 58 volatile compounds were identified, respectively. The modulation of 16 of these volatile compounds was associated with variations in the yeast population (goodness of prediction [Q²] > 20%). The results showed that Pichia anomala was responsible for the characteristic aroma of Chinese liquor, through the regulation of several important volatile compounds, such as ethyl lactate, octanoic acid, and ethyl tetradecanoate. Correspondingly, almost all of the compounds associated with P. anomala were detected in a pure culture of this yeast. In contrast to the PLS regression results, however, ethyl lactate and ethyl isobutyrate were not detected in the same pure culture, which indicated that some metabolites could be generated by P. anomala only when it existed in a community with other yeast species. Furthermore, different yeast communities provided different volatile patterns in the fermented grains, which resulted in distinct flavor profiles in the resulting liquors. This study could help identify the key yeast species involved in spontaneous fermentation and provide a deeper understanding of the role of individual yeast species in the community.     

Sequence-specific binding of Taz1p dimers to fission yeast telomeric DNA

The fission yeast (Schizosaccharomyces pombe) taz1 gene encodes a telomere-associated protein. It contains a single copy of a Myb-like motif termed the telobox that is also found in the human telomere binding proteins TRF1 and TRF2, and Tbf1p, a protein that binds to sequences found within the sub-telomeric regions of budding yeast (Saccharomyces cerevisiae) chromosomes. Taz1p was synthesised in vitro and shown to bind to a fission yeast telomeric DNA fragment in a sequence specific manner that required the telobox motif. Like the mammalian TRF proteins, Taz1p bound to DNA as a preformed homodimer. The isolated Myb-like domain was also capable of sequence specific DNA binding, although with less specificity than the full-length dimer. Surprisingly, a protein extract produced from a taz1–fission yeast strain still contained the major telomere binding activity (complex I) we have characterised previously, suggesting that there could be other abundant telomere binding proteins in fission yeast. One candidate, SpX, was also synthesised in vitro, but despite the presence of two telobox domains, no sequence specific binding to telomeric DNA was detected.     

The Yeast Deletion Collection: A Decade of Functional Genomics

The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, and haploids of both MATa and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general.     

Molecular characterization of the human COQ5 C-methyltransferase in coenzyme Q10 biosynthesis

Coq5 catalyzes the only C-methylation involved in the biosynthesis of coenzyme Q (Q or ubiquinone) in humans and yeast Saccharomyces cerevisiae. As one of eleven polypeptides required for Q production in yeast, Coq5 has also been shown to assemble with the multi-subunit complex termed the CoQ-synthome. In humans, mutations in several COQ genes cause primary Q deficiency, and a decrease in Q biosynthesis is associated with mitochondrial, cardiovascular, kidney and neurodegenerative diseases. In this study, we characterize the human COQ5 polypeptide and examine its complementation of yeast coq5 point and null mutants. We show that human COQ5 RNA is expressed in all tissues and that the COQ5 polypeptide is associated with the mitochondrial inner membrane on the matrix side. Previous work in yeast has shown that point mutations within or adjacent to conserved COQ5 methyltransferase motifs result in a loss of Coq5 function but not Coq5 steady state levels. Here, we show that stabilization of the CoQ-synthome within coq5 point mutants or by over-expression of COQ8 in coq5 null mutants permits the human COQ5 homolog to partially restore coq5 mutant growth on respiratory media and Q₆ content. Immunoblotting against the human COQ5 polypeptide in isolated yeast mitochondria shows that the human Coq5 polypeptide migrates in two-dimensional blue-native/SDS-PAGE at the same high molecular mass as other yeast Coq proteins. The results presented suggest that human and Escherichia coli Coq5 homologs expressed in yeast retain C-methyltransferase activity but are capable of rescuing the coq5 yeast mutants only when the CoQ-synthome is assembled.     

Patterns of Human Oral Yeast Species Distribution on Hainan Island in China

Infections by yeast strains of the genus Candida are among the most prevalent fungal infections of humans. These yeasts are common residents of the oral mucosa and other body surfaces. Since most yeast infections are due to endogenous strains and that species of Candida differ in virulence properties and in intrinsic susceptibilities to antifungal drugs, understanding the human commensal yeast flora can help designing effective treatment and prevention strategies against yeast infections. Here, we report the patterns of yeast species distributions in the oral cavities of 1,799 people from Hainan Island in southern China. Based on sequence information at the fungal barcode locus ITS regions, 368 of the 415 obtained oral yeast strains were identified as belonging to 26 yeast species, while the remaining 47 strains all showed significant sequence divergence to the currently described species. The four most common yeast species were C. albicans (42 %), C. tropicalis (20 %), C. glabrata (5.5 %), and C. parapsilosis (4.1 %) and 10 of the 26 yeast species were represented by only one strain each. Our analyses identified that the gender of hosts and ethnical background showed no contribution to oral yeast species distributions. However, the health status, place of birth, current residency, and the age of hosts all showed significant contributions to the distributions of the four dominant yeast species. We compared our results with those reported previously and discussed the potential mechanisms for the observed differences in oral yeast species distributions.