Effect of Berry Extracts on Saccharomyces cerevisiae Yeast

Introduction. Fruit and berry extracts contain biologically active components and acids that can inhibit or activate Saccharomyces cerevisiae. The research objective was to study the effect of berry extracts on the activity of baking yeast S. cerevisiae and the biochemical properties of wheat dough. Study objects and methods. The experiment featured baking yeast Extra and dry berry extracts of raspberries, aronia, sea buckthorn, and rosehip (LLC Wisterra, Altai Region). The study involved standard and industry-specific control methods of raw materials and semi-finished bakery products, as well as som e standard methods of microbiological analysis. Results and discussion. The raspberry extract (3–4%) suppressed the growth and reproduction of the yeast: after 1 h of exposure, the yeast cell count dropped by 1.5–2 times compared to the control sample. The stimulating effect of the sea buckthorn extract increased the growth rate of yeast cells (up to 40% compared to the control). The extracts of aronia and rosehip had practically no effect on the growth rate of yeast cells. However, 2–3% aronia extract increased the fermentation of the dough, as evidenced by a higher dough fermentation property, which was 2 min versus 3 min at the control after 150 min of exposure. Fruit and berry extracts caused a natural increase in the acidity of the dough, which affected the growth rate of yeast cells. Sea buckthorn extracts increased the acidity so much (up to 4.24 pH units) that it could be regarded as acid stress, which increased the growth rate of yeast cells (1.53×106–1.55×106 vs. 1.10×106 in 1 mL of control sample). The lowest growth rate was detected in the samples with the raspberry extract, which is known to have a strong fungistatic effect: the count of yeast cells decreased by 1.5–2 times after an hour of fermentation. Conclusion. Berry extracts can be of practical interest to bakery enterprises as they help to control yeast fermentation and dough maturation time.

eIF3 and Its mRNA-Entry-Channel Arm Contribute to the Recruitment of mRNAs With Long 5′-Untranslated Regions

Translation initiation in eukaryotes is a multi-step pathway and the most regulated phase of translation. Eukaryotic initiation factor 3 (eIF3) is the largest and most complex of the translation initiation factors, and it contributes to events throughout the initiation pathway. In particular, eIF3 appears to play critical roles in mRNA recruitment. More recently, eIF3 has been implicated in driving the selective translation of specific classes of mRNAs. However, unraveling the mechanism of these diverse contributions—and disentangling the roles of the individual subunits of the eIF3 complex—remains challenging. We employed ribosome profiling of budding yeast cells expressing two distinct mutations targeting the eIF3 complex. These mutations either disrupt the entire complex or subunits positioned near the mRNA-entry channel of the ribosome and which appear to relocate during or in response to mRNA binding and start-codon recognition. Disruption of either the entire eIF3 complex or specific targeting of these subunits affects mRNAs with long 5′-untranslated regions and whose translation is more dependent on eIF4A, eIF4B, and Ded1 but less dependent on eIF4G, eIF4E, and PABP. Disruption of the entire eIF3 complex further affects mRNAs involved in mitochondrial processes and with structured 5′-untranslated regions. Comparison of the suite of mRNAs most sensitive to both mutations with those uniquely sensitive to disruption of the entire complex sheds new light on the specific roles of individual subunits of the eIF3 complex.

Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications

Yeast surface display (YSD) is a “whole-cell” platform used for the heterologous expression of proteins immobilized on the yeast’s cell surface. YSD combines the advantages eukaryotic systems offer such as post-translational modifications, correct folding and glycosylation of proteins, with ease of cell culturing and genetic manipulation, and allows of protein immobilization and recovery. Additionally, proteins displayed on the surface of yeast cells may show enhanced stability against changes in temperature, pH, organic solvents, and proteases. This platform has been used to study protein-protein interactions, antibody design and protein engineering. Other applications for YSD include library screening, whole-proteome studies, bioremediation, vaccine and antibiotics development, production of biosensors, ethanol production and biocatalysis. YSD is a promising technology that is not yet optimized for biotechnological applications. This mini review is focused on recent strategies to improve the efficiency and selection of displayed proteins. YSD is presented as a cutting-edge technology for the vectorial expression of proteins and peptides. Finally, recent biotechnological applications are summarized. The different approaches described herein could allow for a better strategy cascade for increasing protein/peptide interaction and production.

Control of β-glucan exposure by the endo-1,3-glucanase Eng1 in Candida albicans modulates virulence

Candida albicans is a major opportunistic pathogen of humans. It can grow as morphologically distinct yeast, pseudohyphae and hyphae, and the ability to switch reversibly among different forms is critical for its virulence. The relationship between morphogenesis and innate immune recognition is not quite clear. Dectin-1 is a major C-type lectin receptor that recognizes β-glucan in the fungal cell wall. C. albicans β-glucan is usually masked by the outer mannan layer of the cell wall. Whether and how β-glucan masking is differentially regulated during hyphal morphogenesis is not fully understood. Here we show that the endo-1,3-glucanase Eng1 is differentially expressed in yeast, and together with Yeast Wall Protein 1 (Ywp1), regulates β-glucan exposure and Dectin-1-dependent immune activation of macrophage by yeast cells. ENG1 deletion results in enhanced Dectin-1 binding at the septa of yeast cells; while eng1 ywp1 yeast cells show strong overall Dectin-1 binding similar to hyphae of wild-type and eng1 mutants. Correlatively, hyphae of wild-type and eng1 induced similar levels of cytokines in macrophage. ENG1 expression and Eng1-mediated β-glucan trimming are also regulated by antifungal drugs, lactate and N-acetylglucosamine. Deletion of ENG1 modulates virulence in the mouse model of hematogenously disseminated candidiasis in a Dectin-1-dependent manner. The eng1 mutant exhibited attenuated lethality in male mice, but enhanced lethality in female mice, which was associated with a stronger renal immune response and lower fungal burden. Thus, Eng1-regulated β-glucan exposure in yeast cells modulates the balance between immune protection and immunopathogenesis during disseminated candidiasis.

Induction and Aggravation of the Endoplasmic-Reticulum Stress by Membrane-Lipid Metabolic Intermediate Phosphatidyl-N-Monomethylethanolamine

Phosphatidylcholine (PC) is produced via two distinct pathways in both hepatocytes and yeast, Saccharomyces cerevisiae. One of these pathways involves the sequential methylation of phosphatidylethanolamine (PE). In yeast cells, the methyltransferase, Cho2, converts PE to phosphatidylmonomethylethanolamine (PMME), which is further modified to PC by another methyltransferase, Opi3. On the other hand, free choline is utilized for PC production via the Kennedy pathway. The blockage of PC production is well known to cause endoplasmic reticulum (ER) stress and activate the ER-stress sensor, Ire1, to induce unfolded protein response (UPR). Here, we demonstrate that even when free choline is sufficiently supplied, the opi3Δ mutation, but not the cho2 Δ mutation, induces the UPR. The UPR was also found to be induced by CHO2 overexpression. Further, monomethylethanolamine, which is converted to PMME probably through the Kennedy pathway, caused or potentiated ER stress in both mammalian and yeast cells. We thus deduce that PMME per se is an ER-stressing molecule. Interestingly, spontaneously accumulated PMME seemed to aggravate ER stress in yeast cells. Collectively, our findings demonstrate the multiple detrimental effects of the low-abundance phospholipid species, PMME.

Molecular Mechanism of Ethanol Fermentation Inhibition via Protein Tyrosine Nitration of Pyruvate Decarboxylase by Reactive Nitrogen Species in Yeast

Protein tyrosine nitration (PTN), in which tyrosine (Tyr) residues on proteins are converted into 3-nitrotyrosine (NT), is one of the post-translational modifications mediated by reactive nitrogen species (RNS). Many recent studies have reported that PTN contributed to signaling systems by altering the structures and/or functions of proteins. This study aimed to investigate connections between PTN and the inhibitory effect of nitrite-derived RNS on fermentation ability using the yeast Saccharomyces cerevisiae. The results indicated that RNS inhibited the ethanol production of yeast cells with increased intracellular pyruvate content. We also found that RNS decreased the activities of pyruvate decarboxylase (PDC) as a critical enzyme involved in ethanol production. Our proteomic analysis revealed that the main PDC isozyme Pdc1 underwent the PTN modification at Tyr38, Tyr157, and Tyr344. The biochemical analysis using the recombinant purified Pdc1 enzyme indicated that PTN at Tyr157 or Tyr344 significantly reduced the Pdc1 activity. Interestingly, the substitution of Tyr157 or Tyr344 to phenylalanine, which is no longer converted into NT, recovered the ethanol production under the RNS treatment conditions. These findings suggest that nitrite impairs the fermentation ability of yeast by inhibiting the Pdc1 activity via its PTN modification at Tyr157 and Tyr344 of Pdc1.

Evaluation of a Yeast–Polypyrrole Biocomposite Used in Microbial Fuel Cells

Electrically conductive polymers are promising materials for charge transfer from living cells to the anodes of electrochemical biosensors and biofuel cells. The modification of living cells by polypyrrole (PPy) causes shortened cell lifespan, burdens the replication process, and diminishes renewability in the long term. In this paper, the viability and morphology non-modified, inactivated, and PPy-modified yeasts were evaluated. The results displayed a reduction in cell size, an incremental increase in roughness parameters, and the formation of small structural clusters of polymers on the yeast cells with the increase in the pyrrole concentration used for modification. Yeast modified with the lowest pyrrole concentration showed minimal change; thus, a microbial fuel cell (MFC) was designed using yeast modified by a solution containing 0.05 M pyrrole and compared with the characteristics of an MFC based on non-modified yeast. The maximal generated power of the modified system was 47.12 mW/m2, which is 8.32 mW/m2 higher than that of the system based on non-modified yeast. The open-circuit potentials of the non-modified and PPy-modified yeast-based cells were 335 mV and 390 mV, respectively. Even though applying a PPy layer to yeast increases the charge-transfer efficiency towards the electrode, the damage done to the cells due to modification with a higher concentration of PPy diminishes the amount of charge transferred, as the current density drops by 846 μA/cm2. This decrease suggests that modification by PPy may have a cytotoxic effect that greatly hinders the metabolic activity of yeast.

Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

Genetic Engineering Technique in Virus-Like Particle Vaccine Construction

Vaccine becomes a very effective strategy to deal with various infectious diseases even to the point of eradication as in the smalpox virus. At present many conventional vaccines such as inactivated and live-attenuated vaccines. However, these vaccine methods have side effects on the population. Viral-like particle (VLP) is an alternative vaccine based on recombinant DNA technology that is safe with the same immunogenicity as conventional viruses. This vaccine has been shown to induce humoral immune responses mediated by antibodies and cellular immune responses mediated by cytotoxic T cells. With these advantages, currently various types of vaccines have only been developed on a VLP basis. VLP can be produced from a variety of recombinant gene expression systems including bacterial cell expression systems, yeast cells, insect cells, mammalian cells, plant cells, and cell-free systems.