Transcription coordinates histone amounts and genome content.

Biochemical reactions typically depend on the concentrations of the molecules involved, and cell survival therefore critically depends on the concentration of proteins. To maintain constant protein concentrations during cell growth, global mRNA and protein synthesis rates are tightly linked to cell volume. While such regulation is appropriate for most proteins, certain cellular structures do not scale with cell volume. The most striking example of this is the genomic DNA, which doubles during the cell cycle and increases with ploidy, but is independent of cell volume. Here, we show that the amount of histone proteins is coupled to the DNA content, even though mRNA and protein synthesis globally increase with cell volume. As a consequence, and in contrast to the global trend, histone concentrations decrease with cell volume but increase with ploidy. We find that this distinct coordination of histone homeostasis and genome content is already achieved at the transcript level, and is an intrinsic property of histone promoters that does not require direct feedback mechanisms. Mathematical modeling and histone promoter truncations reveal a simple and generalizable mechanism to control the cell volume- and ploidy-dependence of a given gene through the balance of the initiation and elongation rates.

Omics analysis reveals mechanism underlying metabolic oscillation during continuous very-high-gravity ethanol fermentation by Saccharomyces cerevisiae.

During continuous very-high-gravity (VHG) ethanol fermentation with Saccharomyces cerevisiae, the process exhibits sustained oscillation in residual glucose, ethanol, and biomass, raising a question: how do yeast cells respond to this phenomenon? In this study, the oscillatory behavior of yeast cells was characterized through transcriptome and metabolome analysis for one complete oscillatory period. By analyzing the accumulation of 26 intracellular metabolites and the expression of 90 genes related to central carbon metabolism and stress response, we confirmed that the process oscillation was attributed to intracellular metabolic oscillation with phase difference, and the expression of HXK1, HXT1,2,4, and PFK1 was significantly different from other genes in the Embden-Meyerhof-Parnas pathway, indicating that glucose transport and phosphorylation could be key nodes for regulating the intracellular metabolism under oscillatory conditions. Moreover, the expression of stress response genes was triggered and affected predominately by ethanol inhibition in yeast cells. This progress not only contributes to the understanding of mechanisms underlying the process oscillation observed for continuous VHG ethanol fermentation, but also provides insights for understanding unsteady state that might develop in other continuous fermentation processes operated under VHG conditions to increase product titers for robust production.

Context-specific action of macrolide antibiotics on the eukaryotic ribosome.

Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

Cell-to-cell heterogeneity of phosphate gene expression in yeast is controlled by alternative transcription, 14-3-3 and Spl2.

Dependent on phosphate availability the yeast Saccharomyces cerevisiae expresses either low or high affinity phosphate transporters. In the presence of phosphate yeast cells still express low levels of the high affinity phosphate transporter Pho84. The regulator Spl2 is expressed in approximately 90% of the cells, and is not expressed in the remaining cells. Here we report that deletion of RRP6, encoding an exonuclease degrading non-coding RNA, or BMH1, encoding the major 14-3-3 isoform, resulted in less cells expressing SPL2 and in increased levels of RNA transcribed from sequences upstream of the SPL2 coding region. SPL2 stimulates its own expression and that of PHO84 ensuing a positive feedback. Upon deletion of the region responsible for upstream SPL2 transcription almost all cells express SPL2. These results indicate that the cell-to-cell variation in PHO84 and SPL2 expression is dependent on a specific part of the SPL2 promoter and is controlled by Bmh1 and Spl2.

Global mapping of translation initiation sites by TIS profiling in budding yeast.

Translation initiation site (TIS) profiling allows for the genome-wide identification of TISs in vivo by exclusively capturing mRNA fragments within ribosomes that have just completed translation initiation. It leverages translation inhibitors, such as harringtonine and lactimidomycin (LTM), that preferentially capture ribosomes at start codon positions, protecting TIS-derived mRNA fragments from nuclease digestion. Here, we describe a step-by-step protocol for TIS profiling in LTM-treated budding yeast that we developed to identify TISs and open reading frames in vegetative and meiotic cells. For complete details on the use and execution of this protocol, please refer to Eisenberg et al. (2020).

Lactoferrin perturbs lipid rafts and requires integrity of Pma1p-lipid rafts association to exert its antifungal activity against Saccharomyces cerevisiae.

Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs ergosterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma1-10 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi.

Enhancement of Galactose Uptake from Kappaphycus alvarezii Hydrolysate Using Saccharomyces cerevisiae Through Overexpression of Leloir Pathway Genes.

A total 42.68 g/L monosaccharide with 0.10 g/L HMF was obtained from 10% (w/v) Kappaphycus alvarezii with thermal acid hydrolysis using 350 mM HNO3 at 121 °C for 60 min and enzymatic saccharification with a 1:1 mixture of Viscozyme L and Celluclast 1.5 L for 72 h. To enhance the galactose utilization rate, fermentation was performed with overexpression of GAL1 (galactokinase), GAL7 (galactose-1-phosphate uridyltransferase), GAL10 (UDP-glucose-4-epimerase), and PGM2 (phosphoglucomutase 2) in Saccharomyces cerevisiae CEN.PK2 using CCW12 as a strong promoter. Among the strains, the overexpression of PGM2 showed twofold high galactose utilization rate (URgal) and produced ethanol 1.4-fold more than that of the control. Transcriptional analysis revealed the increase of PGM2 transcription level leading to enhance glucose-6-phosphate and fructose-6-phosphate and plays a key role in ensuring a higher glycolytic flux in the PGM2 strain. This finding shows particular importance in biofuel production from seaweed because galactose is one of the major monosaccharides in seaweeds such as K. alvarezii.

Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase.

Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one of the most common RNA modifications; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome-wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNAMet synthetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (Ψ). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouridine for MetRS binding. Furthermore, polysomal and protein analyses revealed that Ψ1074 mediates translation. Modification of this site occurs presumably by Pus6, a pseudouridine synthetase known to modify MetRS cognate tRNA. Consistently, the deletion of Pus6 leads to a decrease in MetRS association with both tRNAMet and YEF3 mRNA. Furthermore, while global protein synthesis decreases in pus6Δ, translation of YEF3 increases. Together, our data imply that Pus6 'writes' modifications on tRNA and mRNA, and both types of RNAs are 'read' by MetRS for translation regulation purposes. This represents a novel integrated path for writing and reading modifications on both tRNA and mRNA, which may lead to coordination between global and gene-specific translational responses.

Slow Adaptive Response of Budding Yeast Cells to Stable Conditions of Continuous Culture Can Occur without Genome Modifications.

Continuous cultures assure the invariability of environmental conditions and the metabolic state of cultured microorganisms, whereas batch-cultured cells undergo constant changes in nutrients availability. For that reason, continuous culture is sometimes employed in the whole transcriptome, whole proteome, or whole metabolome studies. However, the typical method for establishing uniform growth of a cell population, i.e., by limited chemostat, results in the enrichment of the cell population gene pool with mutations adaptive for starvation conditions. These adaptive changes can skew the results of large-scale studies. It is commonly assumed that these adaptations reflect changes in the genome, and this assumption has been confirmed experimentally in rare cases. Here we show that in a population of budding yeast cells grown for over 200 generations in continuous culture in non-limiting minimal medium and therefore not subject to selection pressure, remodeling of transcriptome occurs, but not as a result of the accumulation of adaptive mutations. The observed changes indicate a shift in the metabolic balance towards catabolism, a decrease in ribosome biogenesis, a decrease in general stress alertness, reorganization of the cell wall, and transactions occurring at the cell periphery. These adaptive changes signify the acquisition of a new lifestyle in a stable nonstressful environment. The absence of underlying adaptive mutations suggests these changes may be regulated by another mechanism.

Cloning, Yeast Expression, and Characterization of a ß-Amyrin C-28 Oxidase (CYP716A249) Involved in Triterpenoid Biosynthesis in Polygala tenuifolia.

Polygala tenuifolia Willd. is a traditional Chinese herbal medicine that is widely used in treating nervous system disorders. Triterpene saponins in P. tenuifolia (polygala saponins) have excellent biological activity. As a precursor for the synthesis of presenegin, oleanolic acid (OA) plays an important role in the biosynthesis of polygala saponins. However, the mechanism behind the biosynthesis of polygala saponins remains to be elucidated. In this study, we found that CYP716A249 (GenBank: ASB17946) oxidized the C-28 position of ß-amyrin to produce OA. Using quantitative real-time PCR, we observed that CYP716A249 had the highest expression in the roots of 2-year-old P. tenuifolia, which provided a basis for the selection of samples for gene cloning. To identify the function of CYP716A249, the strain R-BE-20 was constructed by expressing ß-amyrin synthase in yeast. Then, CYP716A249 was co-expressed with ß-amyrin synthase to construct the strain R-BPE-20 by using the lithium acetate method. Finally, we detected ß-amyrin and OA by ultra-HPLC-Q Exactive hybrid quadrupole-Orbitrap high-resolution accurate mass spectrometry and GC-MS. The results of this study provide insights into the biosynthesis pathway of polygala saponins.

Algorithms for ribosome traffic engineering and their potential in improving host cells' titer and growth rate.

mRNA translation is a fundamental cellular process consuming most of the intracellular energy; thus, it is under extensive evolutionary selection for optimization, and its efficiency can affect the host's growth rate. We describe a generic approach for improving the growth rate (fitness) of any organism by introducing synonymous mutations based on comprehensive computational models. The algorithms introduce silent mutations that may improve the allocation of ribosomes in the cells via the decreasing of their traffic jams during translation respectively. As a result, resources availability in the cell changes leading to improved growth-rate. We demonstrate experimentally the implementation of the method on Saccharomyces cerevisiae: we show that by introducing a few mutations in two computationally selected genes the mutant's titer increased. Our approach can be employed for improving the growth rate of any organism providing the existence of data for inferring models, and with the relevant genomic engineering tools; thus, it is expected to be extremely useful in biotechnology, medicine, and agriculture.

Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients.

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC's multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.

Cells are surrounded and contained by a plasma membrane consisting of a double layer of fats and proteins. These proteins monitor and facilitate the movement of food, oxygen and messages in and out of the cell, and help neighboring cells communicate. Membrane proteins are manufactured in a cell compartment called the endoplasmic reticulum. Cellular machines called ribosomes visit this compartment's membrane to manufacture proteins that need to be secreted or embedded into the cell's membranes. As these proteins are made, they are pulled into the endoplasmic reticulum so they can be folded correctly and inserted in the membrane. A cellular machine in this compartment's membrane that aids this process is the endoplasmic reticulum membrane protein complex (EMC). Many steps can go wrong during protein assembly, so to control protein quality, the EMC has to accommodate the variety of complex physical features that proteins can have. To explore the activity of the EMC, Miller-Vedam, Bräuning, Popova et al. studied the normal structure of the EMC in both yeast and human cells grown in the lab. These snapshots of the complex in different species had a lot in common, including how the complex was arranged within and around the membrane. Next, Miller-Vedam, Bräuning, Popova et al. generated 50 mutant versions of the EMC in human cells to determine how changing different parts of the complex affected the production of three proteins that rely on the EMC to fold correctly. These proteins were an enzyme called squalene synthase, a signaling protein called the beta adrenergic receptor and sigma intracellular receptor 2, a protein involved in the regulation of cholesterol levels. Mutations in the section of the EMC outside of the endoplasmic reticulum, within the main cellular compartment, negatively impacted the stability of squalene synthase. This section of the EMC provides a platform where proteins can associate before entering the membrane. The part of EMC that spans the membrane contains both a fat-filled cavity and a cavity with a 'door' that is either open or closed. Mutations in this section disrupted the insertion of both squalene synthase and the beta adrenergic receptor into the membrane, a role performed by the cavity with the door. The specific role of the fat-filled cavity is still not fully understood, but a mutation affecting this cavity disrupts the correct production of all three proteins studied. The largest section of the complex, which sits inside the endoplasmic reticulum, protected proteins as they folded, ensuring they were not destroyed for being folded incorrectly before they were fully formed. Mutations in this part of the EMC negatively impacted the stability of sigma intracellular receptor 2 without negatively affecting the other proteins. This molecular dissection of the activity of the EMC provides insights into how membrane proteins are manufactured, stabilized, coordinated, and monitored for quality. These findings could contribute towards the development of new treatments for certain congenital diseases. For example, cystic fibrosis, retinitis pigmentosa, and Charcot-Marie-Tooth disease are all thought to be caused by mutations within membrane proteins that require the EMC during their production.

GCN sensitive protein translation in yeast.

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons-the ramp region-of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

Effect of K225 residue to the catalytic efficiency of Kex2 protease.

The gene encoding S. cerevisiae Kex2 protease derivative Kex2-667 (encoding the N-terminal 20th to 667th amino acid residues of Kex2 protease, containing the propeptide, catalytic domain, P domain and Ser/Thr enrichment region) and its 225th amino acid residue mutant K225L were overexpressed in Pichia pastoris. Proteases were purified by dialysis and anion exchange chromatography (Q-FF). Their properties were further investigated. For catalysis efficiency, the value of Kcat/Km of Kex2-667-K225L was 3 folds higher than that of Kex2-667. Both were quite stable at 25 °C and 37 °C after 8 h of incubation at pH5.6, while Kex2-667 remained nearly 90% of the total activity while Kex2-667-K225L remained only 80%. The stability of Kex2-667-K225L was lower than that of Kex2-667 from pH4.0 to pH9.0. Due to the mutation site K225 was located at one of the calcium ion binding sites, it resulted in a tighter calcium ion binding region, which may be the reason why the catalytic efficiency of Kex2-667-K225L was improved while the stability was a little decreased.

TORC1 signaling regulates cytoplasmic pH through Sir2 in yeast.

Glucose controls the phosphorylation of silent information regulator 2 (Sir2), a NAD+ -dependent protein deacetylase, which regulates the expression of the ATP-dependent proton pump Pma1 and replicative lifespan (RLS) in yeast. TORC1 signaling, which is a central regulator of cell growth and lifespan, is regulated by glucose as well as nitrogen sources. In this study, we demonstrate that TORC1 signaling controls Sir2 phosphorylation through casein kinase 2 (CK2) to regulate PMA1 expression and cytoplasmic pH (pHc) in yeast. Inhibition of TORC1 signaling by either TOR1 deletion or rapamycin treatment decreased PMA1 expression, pHc, and vacuolar pH, whereas activation of TORC1 signaling by expressing constitutively active GTR1 (GTR1Q65L) resulted in the opposite phenotypes. Deletion of SIR2 or expression of a phospho-mutant form of SIR2 increased PMA1 expression, pHc, and vacuolar pH in the tor1Δ mutant, suggesting a functional interaction between Sir2 and TORC1 signaling. Furthermore, deletion of TOR1 or KNS1 encoding a LAMMER kinase decreased the phosphorylation level of Sir2, suggesting that TORC1 signaling controls Sir2 phosphorylation. It was also found that Sit4, a protein phosphatase 2A (PP2A)-like phosphatase, and Kns1 are required for TORC1 signaling to regulate PMA1 expression and that TORC1 signaling and the cyclic AMP (cAMP)/protein kinase A (PKA) pathway converge on CK2 to regulate PMA1 expression through Sir2. Taken together, these findings suggest that TORC1 signaling regulates PMA1 expression and pHc through the CK2-Sir2 axis, which is also controlled by cAMP/PKA signaling in yeast.

Efficient Prodrug Activator Gene Therapy by Retroviral Replicating Vectors Prolongs Survival in an Immune-Competent Intracerebral Glioma Model.

Prodrug activator gene therapy mediated by murine leukemia virus (MLV)-based retroviral replicating vectors (RRV) was previously shown to be highly effective in killing glioma cells both in culture and in vivo. To avoid receptor interference and enable dual vector co-infection with MLV-RRV, we have developed another RRV based on gibbon ape leukemia virus (GALV) that also shows robust replicative spread in a wide variety of tumor cells. We evaluated the potential of GALV-based RRV as a cancer therapeutic agent by incorporating yeast cytosine deaminase (CD) and E. coli nitroreductase (NTR) prodrug activator genes into the vector. The expression of CD and NTR genes from GALV-RRV achieved highly efficient delivery of these prodrug activator genes to RG-2 glioma cells, resulting in enhanced cytotoxicity after administering their respective prodrugs 5-fluorocytosine and CB1954 in vitro. In an immune-competent intracerebral RG-2 glioma model, GALV-mediated CD and NTR gene therapy both significantly suppressed tumor growth with CB1954 administration after a single injection of vector supernatant. However, NTR showed greater potency than CD, with control animals receiving GALV-NTR vector alone (i.e., without CB1954 prodrug) showing extensive tumor growth with a median survival time of 17.5 days, while animals receiving GALV-NTR and CB1954 showed significantly prolonged survival with a median survival time of 30 days. In conclusion, GALV-RRV enabled high-efficiency gene transfer and persistent expression of NTR, resulting in efficient cell killing, suppression of tumor growth, and prolonged survival upon CB1954 administration. This validates the use of therapeutic strategies employing this prodrug activator gene to arm GALV-RRV, and opens the door to the possibility of future combination gene therapy with CD-armed MLV-RRV, as the latter vector is currently being evaluated in clinical trials.

High-copy genome integration of 2,3-butanediol biosynthesis pathway in Saccharomyces cerevisiae via in vivo DNA assembly and replicative CRISPR-Cas9 mediated delta integration.

CRISPR Cas9 system is becoming an emerging genome-editing platform and has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. In this study, we developed a novel replicative and integrative CRISPR Cas9 genome-editing platform for large DNA construct in vivo assembly, replication, and high-copy genome integration in Saccharomyces cerevisiae. It harnessed advantages of autonomous replicative sequence in S. cerevisiae, in vivo DNA assembly, CRISPR Cas9, and delta integration. Enhanced green fluorescent protein was used as a marker to confirm large DNA construct in vivo assembly and genome integration. Based on this platform, an efficient 2,3- BDO producing yeast strain was rapidly constructed with up to 25-copy genome integration of 2,3-BDO biosynthesis pathway. Further strain engineering was conducted by multiplex disruption of ADH1, PDC1, PDC5 and MTH1 using a 2µ-based replicative CRISPR Cas9 plasmid containing donor DNAs. As a result, the 2,3-BDO titer was improved by 3.9 folds compared to that obtained by the initially engineered yeast and 50.5 g/L 2,3-BDO was produced by the final engineered yeast strain 36aS5-CFBDO in fed-batch fermentation without strain evolution and process optimization. This study demonstrated that the new replicative and integrative CRISPR Cas9 genome-editing platform was promising in generating an efficient 2,3-BDO-producing S. cerevisiae strain.

High Transcriptional Error Rates Vary as a Function of Gene Expression Level.

Errors in gene transcription can be costly, and organisms have evolved to prevent their occurrence or mitigate their costs. The simplest interpretation of the drift barrier hypothesis suggests that species with larger population sizes would have lower transcriptional error rates. However, Escherichia coli seems to have a higher transcriptional error rate than species with lower effective population sizes, for example Saccharomyces cerevisiae. This could be explained if selection in E. coli were strong enough to maintain adaptations that mitigate the consequences of transcriptional errors through robustness, on a gene by gene basis, obviating the need for low transcriptional error rates and associated costs of global proofreading. Here, we note that if selection is powerful enough to evolve local robustness, selection should also be powerful enough to locally reduce error rates. We therefore predict that transcriptional error rates will be lower in highly abundant proteins on which selection is strongest. However, we only expect this result when error rates are high enough to significantly impact fitness. As expected, we find such a relationship between expression and transcriptional error rate for non-C→U errors in E. coli (especially G→A), but not in S. cerevisiae. We do not find this pattern for C→U changes in E. coli, presumably because most deamination events occurred during sample preparation, but do for C→U changes in S. cerevisiae, supporting the interpretation that C→U error rates estimated with an improved protocol, and which occur at rates comparable with E. coli non-C→U errors, are biological.

Comparative transcriptome analysis of genomic region deletion strain with enhanced L-tyrosine production in Saccharomyces cerevisiae.

OBJECTIVE: To determine the effect of large genomic region deletion in a Saccharomyces cerevisiae strain on tyrosine yield and to identify new genetic modification targets through transcriptome analysis. RESULTS: TAL was used to produce p-coumaric acid (p-CA) from tyrosine to quantity tyrosine yield. S. cerevisiae mutant strain NK14 with deletion of a 23.8 kb genomic region was identified to have p-CA production of 10.3 mg L- 1, while the wild-type strain BY4741 had p-CA production of 1.06 mg L- 1. Analysis of growth patterns and stress tolerance showed that the deletion did not affect the growth phenotype of NK14. Transcriptome analysis suggested that, compared to BY4741, genes related to glycolysis (ENO2, TKL1) and the tyrosine pathway (ARO1, ARO2, ARO4, ARO7, TYR1) were upregulated in NK14 at different levels. Besides genes related to the tyrosine biosynthetic pathway, amino acid transporters (AVT6, VBA5, THI72) and transcription factor (ARO80) also showed changes in transcription levels. CONCLUSIONS: We developed a strain with improved tyrosine yield and identified new genetic modification candidates for tyrosine production.

Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides.

BACKGROUND: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. RESULTS: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with ß-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. CONCLUSIONS: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.