scholarly journals Engineering of 2,3-Butanediol Dehydrogenase To Reduce Acetoin Formation by Glycerol-Overproducing, Low-Alcohol Saccharomyces cerevisiae

2009 ◽  
Vol 75 (10) ◽  
pp. 3196-3205 ◽  
Author(s):  
Maryam Ehsani ◽  
Maria R. Fern�ndez ◽  
Josep A. Biosca ◽  
Anne Julien ◽  
Sylvie Dequin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Wine Yeast ◽  
Yeast Cells ◽  
Alcoholic Beverages ◽  
Limiting Factor ◽  
Dehydrogenase Reaction ◽  
Potential Factors ◽  
Low Alcohol ◽  
Butanediol Dehydrogenase

ABSTRACT Engineered Saccharomyces cerevisiae strains overexpressing GPD1, which codes for glycerol-3-phosphate dehydrogenase, and lacking the acetaldehyde dehydrogenase Ald6 display large-scale diversion of the carbon flux from ethanol toward glycerol without accumulating acetate. Although GPD1 ald6 strains have great potential for reducing the ethanol contents in wines, one major side effect is the accumulation of acetoin, having a negative sensory impact on wine. Acetoin is reduced to 2,3-butanediol by the NADH-dependent 2,3-butanediol dehydrogenase Bdh1. In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1, coding for native NADH-dependent Bdh1, and the engineered gene BDH1 221,222,223, coding for an NADPH-dependent Bdh1 enzyme with the amino acid changes 221 EIA 223 to 221 SRS 223, in a glycerol-overproducing wine yeast. We have shown that both the amount of Bdh1 and the NADH availability limit the 2,3-butanediol dehydrogenase reaction. During wine fermentation, however, the major limiting factor was the level of synthesis of Bdh1. Consistent with this finding, the overproduction of native or engineered Bdh1 made it possible to redirect 85 to 90% of the accumulated acetoin into 2,3-butanediol, a compound with neutral sensory characteristics. In addition, the production of diacetyl, a compound causing off-flavor in alcoholic beverages, whose production is increased in glycerol-overproducing yeast cells, was decreased by half. The production of higher alcohols and esters, which was slightly decreased or unchanged in GPD1 ald6 cells compared to that in the control cells, was not further modified in BDH1 cells. Overall, rerouting carbons toward glycerol and 2,3-butanediol represents a new milestone in the engineering of a low-alcohol yeast with desirable organoleptic features, permitting the decrease of the ethanol contents in wines by up to 3�.

scholarly journals Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase

2016 ◽  
Vol 82 (6) ◽  
pp. 1706-1721 ◽  
Author(s):  
Eduard Calam ◽  
Eva González-Roca ◽  
M. Rosario Fernández ◽  
Sylvie Dequin ◽  
Xavier Parés ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Formate Dehydrogenase ◽  
Building Blocks ◽  
Yeast Cells ◽  
Content Type ◽  
Permeabilized Cells ◽  
Oxidation Reduction ◽  
New Variant ◽  
Butanediol Dehydrogenase

ABSTRACTButanediol dehydrogenase (Bdh1p) fromSaccharomyces cerevisiaebelongs to the superfamily of the medium-chain dehydrogenases and reductases and converts reversiblyR-acetoin andS-acetoin to (2R,3R)-2,3-butanediol andmeso-2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme, and it is the main enzyme involved in the last metabolic step leading to (2R,3R)-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p in different forms—purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p)—to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of thedelitto perfettomethodology to placeBDH1under the control of theGAL1promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2R,3R)-2,3-butanediol, (2R,3R)-2,3-pentanediol, (2R,3R)-2,3-hexanediol, and (3R,4R)-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells) yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p toward the oxidation/reduction of the hydroxyl/ketone groups from (2R,3R)-2,3-pentanediol/2,3-pentanedione and (2R,3R)-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction.

Information Technology of Color Imaging Assessment of Saccharomyces cerevisiae UCM Y-517 Yeast Volutin Granules

2020 ◽  
Vol 82 (5) ◽  
pp. 30-35
Author(s):  
O.M. Gromozova ◽  
◽  
T.L. Kachur ◽  
V.V. Vishnevsky ◽  
O.S. Sychev ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Information Technology ◽  
Large Scale ◽  
Digital Images ◽  
Yeast Cells ◽  
Qualitative Assessment ◽  
Color Imaging ◽  
Daily Monitoring ◽  
Web Resource ◽  
Software Algorithms

The research is devoted to the development of color imaging information technology, which is relevant for evaluating the results of cytochemical research in both biology and medicine. The aim of this work was to study the validity of software algorithms and integrated information technology for the qualitative and quantitative analysis of metachromasia reaction of volutin granules of Saccharomyces cerevisiae UCM Y-517 yeast. Methods. The object of this study was Saccharomyces cerevisiae UCM Y-517 yeast from the Ukrainian collection of microorganisms. The yeast was cultivated for 24 hours at 28oC. The metachromatic reaction of volutin granules was detected microscopically by the preparation of yeast cells stained with methylene blue. Digital images of cells were transferred to a specialized web resource for computer processing. Results. The algorithm of computer analysis of images included 3 steps: (1) recognition of single volutin granules, (2) assessment of the magnitude of metachromasia for each granule, and (3) integral estimation of the magnitude of metachromasia for the whole image. Information technology was developed and tested to support the daily monitoring of the metachromasia phenomenon, including obtaining the digital images, their transfer to a remote server, and automatic processing. Information technology is integrated into the web portal of the large-scale biophysical experiment “Heliomed”. Conclusions. The proposed method of computer image processing gave a quantitative and qualitative assessment in the automatic mode of metachromatic staining of volutin granules and confirmed the previously proposed classification of this phenomenon on the basis of visual examination.

scholarly journals Monitoring Stress-Related Genes during the Process of Biomass Propagation of Saccharomyces cerevisiae Strains Used for Wine Making

2005 ◽  
Vol 71 (11) ◽  
pp. 6831-6837 ◽  
Author(s):  
Roberto Pérez-Torrado ◽  
Jose M. Bruno-Bárcena ◽  
Emilia Matallana
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Response ◽  
Biomass Production ◽  
Stress Responses ◽  
Environmental Changes ◽  
Wine Yeast ◽  
Yeast Cells ◽  
Mrna Levels ◽  
Yeast Biomass ◽  
Stress Related Genes

ABSTRACT Physiological capabilities and fermentation performance of Saccharomyces cerevisiae strains to be employed during industrial wine fermentations are critical for the quality of the final product. During the process of biomass propagation, yeast cells are dynamically exposed to a mixed and interrelated group of known stresses such as osmotic, oxidative, thermic, and/or starvation. These stressing conditions can dramatically affect the parameters of the fermentation process and the technological abilities of the yeast, e.g., the biomass yield and its fermentative capacity. Although a good knowledge exists of the behavior of S. cerevisiae under laboratory conditions, insufficient knowledge is available about yeast stress responses under the specific media and growth conditions during industrial processes. We performed growth experiments using bench-top fermentors and employed a molecular marker approach (changes in expression levels of five stress-related genes) to investigate how the cells respond to environmental changes during the process of yeast biomass production. The data show that in addition to the general stress response pathway, using the HSP12 gene as a marker, other specific stress response pathways were induced, as indicated by the changes detected in the mRNA levels of two stress-related genes, GPD1 and TRX2. These results suggest that the cells were affected by osmotic and oxidative stresses, demonstrating that these are the major causes of the stress response throughout the process of wine yeast biomass production.

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

scholarly journals In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1422
Author(s):  
Ousama Al Shanaa ◽  
Andrey Rumyantsev ◽  
Elena Sambuk ◽  
Marina Padkina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Potential Model ◽  
Model Organism ◽  
Rna Aptamers ◽  
Early Stages ◽  
Near Future

RNA aptamers are becoming increasingly attractive due to their superior properties. This review discusses the early stages of aptamer research, the main developments in this area, and the latest technologies being developed. The review also highlights the advantages of RNA aptamers in comparison to antibodies, considering the great potential of RNA aptamers and their applications in the near future. In addition, it is shown how RNA aptamers can form endless 3-D structures, giving rise to various structural and functional possibilities. Special attention is paid to the Mango, Spinach and Broccoli fluorescent RNA aptamers, and the advantages of split RNA aptamers are discussed. The review focuses on the importance of creating a platform for the synthesis of RNA nanoparticles in vivo and examines yeast, namely Saccharomyces cerevisiae, as a potential model organism for the production of RNA nanoparticles on a large scale.

scholarly journals Molecular Basis of Fructose Utilization by the Wine Yeast Saccharomyces cerevisiae: a Mutated HXT3 Allele Enhances Fructose Fermentation

2007 ◽  
Vol 73 (8) ◽  
pp. 2432-2439 ◽  
Author(s):  
Carole Guillaume ◽  
Pierre Delobel ◽  
Jean-Marie Sablayrolles ◽  
Bruno Blondin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Basis ◽  
Alcoholic Fermentation ◽  
Wine Yeast ◽  
Glycolytic Flux ◽  
Hexose Transporter ◽  
Wine Yeasts ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Fructose ◽  
Fructose Fermentation

ABSTRACT Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

Current State of (Dis)Integration: Public Health and Fusion Centers

2020 ◽  
Vol 17 (2) ◽  
Author(s):  
Cody Minks ◽  
Anke Richter
Keyword(s):  
Public Health ◽  
Law Enforcement ◽  
Information Sharing ◽  
Homeland Security ◽  
Large Scale ◽  
Ad Hoc ◽  
Limiting Factor ◽  
Fusion Centers ◽  
Public Health Emergencies ◽  
Public Health Information

AbstractObjectiveResponding to large-scale public health emergencies relies heavily on planning and collaboration between law enforcement and public health officials. This study examines the current level of information sharing and integration between these domains by measuring the inclusion of public health in the law enforcement functions of fusion centers.MethodsSurvey of all fusion centers, with a 29.9% response rate.ResultsOnly one of the 23 responding fusion centers had true public health inclusion, a decrease from research conducted in 2007. Information sharing is primarily limited to information flowing out of the fusion center, with little public health information coming in. Most of the collaboration is done on a personal, informal, ad-hoc basis. There remains a large misunderstanding of roles, capabilities, and regulations by all parties (fusion centers and public health). The majority of the parties appear to be willing to work together, but there but there is no forward momentum to make these desires a reality. Funding and staffing issues seem to be the limiting factor for integration.ConclusionThese problems need to be urgently addressed to increase public health preparedness and enable a decisive and beneficial response to public health emergencies involving a homeland security response.

scholarly journals Eukaryotic translation factor eIF5A contributes to acetic acid tolerance in Saccharomyces cerevisiae via transcriptional factor Ume6p

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yanfei Cheng ◽  
Hui Zhu ◽  
Zhengda Du ◽  
Xuena Guo ◽  
Chenyao Zhou ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Adaptive Response ◽  
Acid Tolerance ◽  
Peptide Bond ◽  
Transcriptional Factor ◽  
Yeast Cells ◽  
Acetic Acid Tolerance ◽  
Translation Factor ◽  
Industrial Strains

Abstract Background Saccharomyces cerevisiae is well-known as an ideal model system for basic research and important industrial microorganism for biotechnological applications. Acetic acid is an important growth inhibitor that has deleterious effects on both the growth and fermentation performance of yeast cells. Comprehensive understanding of the mechanisms underlying S. cerevisiae adaptive response to acetic acid is always a focus and indispensable for development of robust industrial strains. eIF5A is a specific translation factor that is especially required for the formation of peptide bond between certain residues including proline regarded as poor substrates for slow peptide bond formation. Decrease of eIF5A activity resulted in temperature-sensitive phenotype of yeast, while up-regulation of eIF5A protected transgenic Arabidopsis against high temperature, oxidative or osmotic stress. However, the exact roles and functional mechanisms of eIF5A in stress response are as yet largely unknown. Results In this research, we compared cell growth between the eIF5A overexpressing and the control S. cerevisiae strains under various stressed conditions. Improvement of acetic acid tolerance by enhanced eIF5A activity was observed all in spot assay, growth profiles and survival assay. eIF5A prompts the synthesis of Ume6p, a pleiotropic transcriptional factor containing polyproline motifs, mainly in a translational related way. As a consequence, BEM4, BUD21 and IME4, the direct targets of Ume6p, were up-regulated in eIF5A overexpressing strain, especially under acetic acid stress. Overexpression of UME6 results in similar profiles of cell growth and target genes transcription to eIF5A overexpression, confirming the role of Ume6p and its association between eIF5A and acetic acid tolerance. Conclusion Translation factor eIF5A protects yeast cells against acetic acid challenge by the eIF5A-Ume6p-Bud21p/Ime4p/Bem4p axles, which provides new insights into the molecular mechanisms underlying the adaptive response and tolerance to acetic acid in S. cerevisiae and novel targets for construction of robust industrial strains.

scholarly journals Screening and Genetic Network Analysis of Genes Involved in Freezing and Thawing Resistance in DaMDHAR—Expressing Saccharomyces cerevisiae Using Gene Expression Profiling

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 219
Author(s):  
Il-Sup Kim ◽  
Woong Choi ◽  
Jonghyeon Son ◽  
Jun Hyuck Lee ◽  
Hyoungseok Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Transcription Factors ◽  
Stress Tolerance ◽  
Genetic Network ◽  
Yeast Cells ◽  
Enzymatic Antioxidants ◽  
Freezing And Thawing ◽  
Metal Ion Homeostasis ◽  
Transgenic Yeast

The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3′-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, β-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress.

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