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1.
Lager-brewing yeasts in the era of modern genetics.
Gorter de Vries, AR, Pronk, JT, Daran, JG
FEMS yeast research. 2019;(7)
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Abstract
The yeast Saccharomyces pastorianus is responsible for the annual worldwide production of almost 200 billion liters of lager-type beer. S. pastorianus is a hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been studied for well over a century. Scientific interest in S. pastorianus intensified upon the discovery, in 2011, of its S. eubayanus ancestor. Moreover, advances in whole-genome sequencing and genome editing now enable deeper exploration of the complex hybrid and aneuploid genome architectures of S. pastorianus strains. These developments not only provide novel insights into the emergence and domestication of S. pastorianus but also generate new opportunities for its industrial application. This review paper combines historical, technical and socioeconomic perspectives to analyze the evolutionary origin and genetics of S. pastorianus. In addition, it provides an overview of available methods for industrial strain improvement and an outlook on future industrial application of lager-brewing yeasts. Particular attention is given to the ongoing debate on whether current S. pastorianus originates from a single or multiple hybridization events and to the potential role of genome editing in developing industrial brewing yeast strains.
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A CRISPR/Cas9 method to generate heterozygous alleles in Saccharomyces cerevisiae.
EauClaire, SF, Webb, CJ
Yeast (Chichester, England). 2019;(10):607-615
Abstract
Saccharomyces cerevisiae is a genetically facile organism, yet multiple CRISPR/Cas9 techniques are widely used to edit its genome more efficiently and cost effectively than conventional methods. The absence of selective markers makes CRISPR/Cas9 editing particularly useful when making mutations within genes or regulatory sequences. Heterozygous mutations within genes frequently arise in the winners of evolution experiments. The genetic dissection of heterozygous alleles can be important to understanding gene structure and function. Unfortunately, the high efficiency of genome cutting and repair makes the introduction of heterozygous alleles by standard CRISPR/Cas9 technique impossible. To be able to quickly and reliably determine the individual phenotypes of the thousands of heterozygous mutations that can occur during directed evolutions is of particular interest to industrial strain improvement research. In this report, we describe a CRISPR/Cas9 method that introduces specific heterozygous mutations into the S. cerevisiae genome. This method relies upon creating silent point mutations in the protospacer adjacent motif site or removing the protospacer adjacent motif site entirely to stop the multiple rounds of genome editing that prevent heterozygous alleles from being generated. This technique should be able to create heterozygous alleles in other diploid yeasts and different allelic copy numbers in polyploid cells.
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Electron cryomicroscopy observation of acyl carrier protein translocation in type I fungal fatty acid synthase.
Lou, JW, Iyer, KR, Hasan, SMN, Cowen, LE, Mazhab-Jafari, MT
Scientific reports. 2019;(1):12987
Abstract
During fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.
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New insights on yeast and filamentous fungus adhesion in a natural co-immobilization system: proposed advances and applications in wine industry.
Ogawa, M, Bisson, LF, García-Martínez, T, Mauricio, JC, Moreno-García, J
Applied microbiology and biotechnology. 2019;(12):4723-4731
Abstract
Fungi possess extraordinary strength in attachment to biotic and abiotic surfaces. This review focuses on adhesion mechanisms of yeast and filamentous fungi and the proposed combination of the adhesive forces of both organisms in an immobilization system called yeast biocapsules, whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum. The natural adherent properties of each organism, one multicellular and another unicellular, allow yeast to be fixated securely on the filamentous fungi and complete alcoholic fermentation. Following alcoholic fermentation, the hyphae become an inert support for yeast cells while maintaining shape and integrity. Biocapsules have been used successfully in both wine and bioethanol production. Investigation of the potential genes involved in fungal-yeast fusion suggests that natural hydrophobic interactions of both organisms play a major role. Analysis of the possible mechanisms involved in fungus and yeast adhesion, future perspectives on improving yeast immobilization, and proposed applications of the biocapsules are explored.
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5.
Beer yeast-derived fluorescent carbon dots for photoinduced bactericidal functions and multicolor imaging of bacteria.
Gao, Z, Zhao, CX, Li, YY, Yang, YL
Applied microbiology and biotechnology. 2019;(11):4585-4593
Abstract
Beer yeast-modified fluorescent carbon dots were synthesized via a one-step strategy for photoinduced bactericidal functions and bio-imaging in bacterial viability assessment. The proposed carbon dots (CDs) were used as an visible light-triggered antibacterial material, and the antimicrobial activities of the CDs against Gram-negative model bacterial species (Escherichia coli) were evaluated under conditions of varying other experimental parameters including CDs concentrations and treatment times. The result showed that the CDs have excellent antibacterial performance of bactericidal effect within 120 min of under visible-light irradiation. And the bactericidal efficiency increased with the increasing concentration of CDs and visible-light illumination time. Moreover, the CDs with high quantum yield (21%) possess highly negative zeta potential (- 41.7 mV) and low cytotoxicity, the CDs could serve as an efficient dye for bacterial viability evaluation, they could selectively stain dead E. coli rather than live ones, which make dead E. coli be viewed with multicolor fluorescence under different excitation wavelengths.
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6.
The problem of genetic code misreading during protein synthesis.
Joshi, K, Cao, L, Farabaugh, PJ
Yeast (Chichester, England). 2019;(1):35-42
Abstract
Saccharomyces cerevisiae has been an important model for determining the frequency of translational misreading events, those in which a tRNA pairs incorrectly to the mRNA and inserts an amino acid not specified by the codon in the mRNA. Misreading errors have been quantified in vivo using reporter protein systems or mass spectrometry with both approaches converging on a simple model for most misreading. The available data show that misreading tRNAs must form stereotypical base mismatches that correspond to those that can mimic Watson-Crick base pairs when formed in the ribosomal A site. Errors involving other mismatches occur significantly less frequently. This work debunks the idea of an average misreading frequency of 5 × 10-4 per codon that extends across the genetic code. Instead, errors come in two distinct classes-high frequency and low frequency events-with most errors being of the low frequency type. A comparison of misreading errors in S. cerevisiae and Escherichia coli suggests the existence of a mechanism that reduces misreading frequency in yeast; this mechanism may operate in eukaryotes generally.
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7.
Cross-talk in NAD+ metabolism: insights from Saccharomyces cerevisiae.
James Theoga Raj, C, Lin, SJ
Current genetics. 2019;(5):1113-1119
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Abstract
NAD+ (nicotinamide adenine dinucleotide) is an essential metabolite involved in a myriad of cellular processes. The NAD+ pool is maintained by three biosynthesis pathways, which are largely conserved from bacteria to human with some species-specific differences. Studying the regulation of NAD+ metabolism has been difficult due to the dynamic flexibility of NAD+ intermediates, the redundancy of biosynthesis pathways, and the complex interconnections among them. The budding yeast Saccharomyces cerevisiae provides an efficient genetic model for the isolation and study of factors that regulate specific NAD+ biosynthesis pathways. A recent study has uncovered a putative cross-regulation between the de novo NAD+ biosynthesis and copper homeostasis mediated by a copper-sensing transcription factor Mac1. Mac1 appears to work with the Hst1-Sum1-Rfm1 complex to repress the expression of de novo NAD+ biosynthesis genes. Here, we extend the discussions to include additional nutrient- and stress-sensing pathways that have been associated with the regulation of NAD+ homeostasis. NAD+ metabolism is an emerging therapeutic target for several human diseases. NAD+ preservation also helps ameliorate age-associated metabolic disorders. Recent findings in yeast contribute to the understanding of the molecular basis underlying the cross-regulation of NAD+ metabolism and other signaling pathways.
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8.
The H channel is not a proton transfer path in yeast cytochrome c oxidase.
Malkamäki, A, Meunier, B, Reidelbach, M, Rich, PR, Sharma, V
Biochimica et biophysica acta. Bioenergetics. 2019;(9):717-723
Abstract
Cytochrome c oxidases (CcOs) in the respiratory chains of mitochondria and bacteria are primary consumers of molecular oxygen, converting it to water with the concomitant pumping of protons across the membrane to establish a proton electrochemical gradient. Despite a relatively well understood proton pumping mechanism of bacterial CcOs, the role of the H channel in mitochondrial forms of CcO remains debated. Here, we used site-directed mutagenesis to modify a central residue of the lower span of the H channel, Q413, in the genetically tractable yeast Saccharomyces cerevisiae. Exchange of Q413 to several different amino acids showed no effect on rates and efficiencies of respiratory cell growth, and redox potential measurements indicated minimal electrostatic interaction between the 413 locus and the nearest redox active component heme a. These findings clearly exclude a primary role of this section of the H channel in proton pumping in yeast CcO. In agreement with the experimental data, atomistic molecular dynamics simulations and continuum electrostatic calculations on wildtype and mutant yeast CcOs highlight potential bottlenecks in proton transfer through this route. Our data highlight the preference for neutral residues in the 413 locus, precluding sufficient hydration for formation of a proton conducting wire.
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9.
Ergosterol Turnover in Yeast: An Interplay between Biosynthesis and Transport.
Sokolov, SS, Trushina, NI, Severin, FF, Knorre, DA
Biochemistry. Biokhimiia. 2019;(4):346-357
Abstract
Sterols are important components of biological membranes that determine the physicochemical properties of lipid bilayer and regulate the functioning of membrane proteins. Being insoluble in water, sterols cannot diffuse between the membrane compartments separated by an aqueous phase. For this reason, distribution of sterols across cellular membranes is rather uneven. Membrane-to-membrane transport of sterols occurs mainly in a non-vesicular fashion and is provided by Lam and Osh proteins. In this review, we discuss the consequences of impairments in sterol biosynthesis and transport mostly relying on the studies performed on the model organism Saccharomyces cerevisiae. Despite the fact that molecular mechanisms underlying the functioning of Lam and Osh proteins are well established, the biological roles of these proteins are still unclear, because deletions of corresponding genes do not affect yeast phenotype. At the same time, disruptions in the biosynthesis of ergosterol, the major sterol of S. cerevisiae, lead to either cell death or reduced stress resistance. However, under certain conditions (e.g., mild salt or thermal stresses), a decrease in the ergosterol levels causes an increase in cell resistance. This suggests that the cells possess a mechanism facilitating rapid adjustment of the plasma membrane sterol content. We argue that the biological role of Lam proteins is, in particular, fast optimization of sterol composition of cell membranes.
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10.
Utilization of prickly pear waste for baker's yeast production.
Diboune, N, Nancib, A, Nancib, N, Aníbal, J, Boudrant, J
Biotechnology and applied biochemistry. 2019;(5):744-754
Abstract
The feasibility of baker's yeast production using fruits and peels of Opuntia ficus indica (OFI) as carbohydrate feedstock was investigated. Two response surface methodologies involving central composite face centered design (CCFD) were successfully applied. The effects of four independent variables on baker's yeast production from OFI fruit juice was evaluated using the first CCFD. The best results were obtained with 24 H of inoculum age, 30 °C temperature, 200 rpm of agitation, and 10% inoculum size. At the maximum point, the biomass concentration reached 9.29 g/L. A second CCFD was performed to optimize the sugar extraction from OFI fruit peels. The potential of these latter as a fermentation substrate was determined. From the experimental results, the OFI fruit peel is an appropriate carbon source for the production of baker's yeast. The maximum biomass concentration was 12.51 g/L. Different nitrogen supplements were added to promote the yields of baker's yeast. Corn steep liquor was found to be the best alternative nutrient source of casein hydrolysate and yeast extract for baker's yeast production.