Biochemical Convergence of Mitochondrial Hsp70 System Specialized in Iron-Sulfur Cluster Biogenesis.

Mitochondria play a central role in the biogenesis of iron-sulfur cluster(s) (FeS), protein cofactors needed for many cellular activities. After assembly on scaffold protein Isu, the cluster is transferred onto a recipient apo-protein. Transfer requires Isu interaction with an Hsp70 chaperone system that includes a dedicated J-domain protein co-chaperone (Hsc20). Hsc20 stimulates Hsp70's ATPase activity, thus stabilizing the critical Isu-Hsp70 interaction. While most eukaryotes utilize a multifunctional mitochondrial (mt)Hsp70, yeast employ another Hsp70 (Ssq1), a product of mtHsp70 gene duplication. Ssq1 became specialized in FeS biogenesis, recapitulating the process in bacteria, where specialized Hsp70 HscA cooperates exclusively with an ortholog of Hsc20. While it is well established that Ssq1 and HscA converged functionally for FeS transfer, whether these two Hsp70s possess similar biochemical properties was not known. Here, we show that overall HscA and Ssq1 biochemical properties are very similar, despite subtle differences being apparent - the ATPase activity of HscA is stimulated to a somewhat higher levels by Isu and Hsc20, while Ssq1 has a higher affinity for Isu and for Hsc20. HscA/Ssq1 are a unique example of biochemical convergence of distantly related Hsp70s, with practical implications, crossover experimental results can be combined, facilitating understanding of the FeS transfer process.

Design of orthogonal regulatory systems for modulating gene expression in plants.

Agricultural biotechnology strategies often require the precise regulation of multiple genes to effectively modify complex plant traits. However, most efforts are hindered by a lack of characterized tools that allow for reliable and targeted expression of transgenes. We have successfully engineered a library of synthetic transcriptional regulators that modulate expression strength in planta. By leveraging orthogonal regulatory systems from Saccharomyces spp., we have developed a strategy for the design of synthetic activators, synthetic repressors, and synthetic promoters and have validated their use in Nicotiana benthamiana and Arabidopsis thaliana. This characterization of contributing genetic elements that dictate gene expression represents a foundation for the rational design of refined synthetic regulators. Our findings demonstrate that these tools provide variation in transcriptional output while enabling the concerted expression of multiple genes in a tissue-specific and environmentally responsive manner, providing a basis for generating complex genetic circuits that process endogenous and environmental stimuli.

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24.

Ste24 enzymes, a family of eukaryotic integral membrane proteins, are zinc metalloproteases (ZMPs) originally characterized as "CAAX proteases" targeting prenylated substrates, including a-factor mating pheromone in yeast and prelamin A in humans. Recently, Ste24 was shown to also cleave nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders. Ste24 possesses a unique "α-barrel" structure consisting of seven transmembrane (TM) α-helices encircling a large intramembranous cavity (~14 000 Å3 ). The catalytic zinc, coordinated via a HExxH…E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long-studied class of soluble ZMPs, and as a novel cavity-containing integral membrane protein protease has been minimally explored to date. Informed by homology to well-characterized soluble, gluzincin ZMPs, we develop a model of Ste24 that provides a conceptual framework for this enzyme family, suitable for development and interpretation of structure/function studies. The model consists of an interfacial, zinc-containing "ZMP Core" module surrounded by a "ZMP Accessory" module, both capped by a TM helical "α-barrel" module of as yet unknown function. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 absolutely conserved residues, apportioned unequally among the ZMP Core (18), ZMP Accessory (13), and α-barrel (7) modules. This Tripartite Architecture representation of Ste24 provides a unified image of this enzyme family.

Mitochondrial-encoded endonucleases drive recombination of protein-coding genes in yeast.

Mitochondrial recombination in yeast is well recognized, yet the underlying genetic mechanisms are not well understood. Recent progress has suggested that mobile introns in mitochondrial genomes (mitogenomes) can facilitate the recombination of their corresponding intron-containing genes through a mechanism known as intron homing. As many mitochondrial genes lack introns, there is a critical need to determine the extent of recombination and underlying mechanism of intron-lacking genes. This study leverages yeast mitogenomes to address these questions. In Saccharomyces cerevisiae, the 3'-end sequences of at least three intron-lacking mitochondrial genes exhibit elevated nucleotide diversity and recombination hotspots. Each of these 3'-end sequences is immediately adjacent to or even fused as overlapping genes with a stand-alone endonuclease. Our findings suggest that SAEs are responsible for recombination and elevated diversity of adjacent intron-lacking genes. SAEs were also evident to drive recombination of intron-lacking genes in Lachancea kluyveri, a yeast species that diverged from S. cerevisiae more than 100 million years ago. These results suggest SAEs as a common driver in recombination of intron-lacking genes during mitogenome evolution. We postulate that the linkage between intron-lacking gene and its adjacent endonuclease gene is the result of co-evolution.

Enhancing Medium-Chain Fatty Acid Ethyl Ester Production During Beer Fermentation Through EEB1 and ETR1 Overexpression in Saccharomyces pastorianus.

Esters are important flavor compounds in alcoholic beverages. Although they are present at trace levels, esters play a key role in the formation of flavors, especially fruity flavors, in beverages. Low ester contents result in bland beer and unpleasant flavor. In this study, three recombinant strains, ethanol O-acyltransferase encoding EEB1 overexpression strain (31194:: EEB1), 2-enoyl thioester reductase encoding ETR1 overexpression strain (31194:: ETR1), and EEB1- ETR1 co-overexpression strain (31194:: EEB1:: ETR1), were constructed. Ethyl hexanoate production by 31194:: EEB1 and 31194:: EEB1:: ETR1 was 106% higher than that by the parental strain. Further, ethyl octanoate production by 31194:: EEB1 and 31194:: EEB1:: ETR1 was enhanced by 47 and 41%, respectively, compared with that of parental strain 31194. However, no difference was observed between 31194:: ETR1 and the parental strain in terms of ethyl hexanoate and ethyl octanoate production. This indicates that although EEB1 overexpression in Saccharomyces pastorianus enhanced ethyl hexanoate and ethyl octanoate production, ETR1 expression levels did not affect the extracellular concentrations of these esters.

Insights into the biotransformation of 2,4,6-trinitrotoluene by the old yellow enzyme family of flavoproteins. A computational study.

Biodegradation is a cost-effective and environmentally friendly alternative to removing 2,4,6-trinitrotoluene (TNT) pollution. However, mechanisms of TNT biodegradation have been elusive. To enhance the understanding of TNT biotransformation by the Old Yellow Enzyme (OYE) family, we investigated the crucial first-step hydrogen-transfer reaction by molecular dynamics simulations, docking technologies and empirical valence bond calculations. We revealed the significance of the π-π stacking conformation between the substrate TNT and the reduced flavin mononucleotide (FMNH2) cofactor, which is a prerequisite for the aromatic ring reduction of TNT. Under the π-π stacking conformation, the barrier of the hydrogen-transfer reaction in the aromatic ring reduction is about 16 kcal mol-1 lower than that of nitro group reduction. Then, we confirmed the mechanism of controlling the π-π stacking, that is, the π-π interaction competition mechanism. It indicates that the π-π stacking of TNT and FMNH2 occurs only when the π-π interaction between FMNH2 and TNT is stronger than that between TNT and several key residues with aromatic rings. Finally, based on the competition mechanism, the formation of π-π stacking of TNT and FMNH2 can be successfully enabled by removing the aromatic ring of those key residues in enzymes that originally only transform TNT through the nitro group reduction. This testified the validity of the π-π interaction competition mechanism. This work theoretically clarifies the molecular mechanism of the first-step hydrogen-transfer reaction for the biotransformation of TNT by the OYE family. It is helpful to obtain the enzymes that can biodegrade TNT through the aromatic ring reduction.

Effect of ILV6 Deletion and Expression of aldB from Lactobacillus plantarum in Saccharomyces uvarum on Diacetyl Production and Wine Flavor.

Diacetyl generates an aromatic off-flavor in wine at a high level. The present study expressed α-acetolactate decarboxylase (ALDB) from Lactobacillus plantarum and/or inactivated acetohydroxyacid synthase (Ilv6) in Saccharomyces uvarum, and the effects on diacetyl production and wine flavor in mutants were investigated through sequential fermentation and cofermentation in mixed cultures of S. uvarum and L. plantarum. The diacetyl content of WYDΔ6 (disrupted one ILV6 allele), WYSΔ6 ( ILV6 complete deletion), WYADΔ6 (disrupted one ILV6 allele with aldB expression), and WYASΔ6 ( ILV6 complete deletion with aldB expression) decreased by 25.71%, 41.30%, 47.77%, and 50.00%, respectively, after sequential fermentation and decreased by 15.15%, 26.72%, 35.26%, and 43.80%, respectively, after cofermentation, compared with that of the parental strain. In addition, Ilv6 inactivation not only decreased the acetic acid content but also balanced the flavor profile in wine effectively. This work provided a valuable insight into the metabolic pathway of diacetyl and wine flavor in S. uvarum.

Induced expression of the alcohol acetyltransferase gene ATF1 in industrial yeast Saccharomyces pastorianus TUM 34/70.

Targeted induced gene expression for industrial fermentation processes in food and beverage production could fulfill future demands. To avoid metabolic burden and disturbances owing to the fermentation procedure, induced gene expression is necessary for combating stress, such as that caused by temperature shifts that occur during the transition from fermentation to maturation in the brewing process. The aim of this study was to target gene expression in industrial yeast using stress-responsive promoters and homologues of the selection marker SMR1. Self-cloning strains of the industrial brewing yeast Saccharomyces pastorianus TUM 34/70 were constructed to overexpress the alcohol acetyltransferase (ATF1) gene under the control of inducible promoters P SSA3, P HSP104 and P UBI4. Transcription analysis shows the highest induction after 72 h of shock situation for P HSP104 with 1.3-fold and P UBI4 with 2.2-fold. Further, at the end of shock situation the concentrations of ethyl acetate were 1.2- and 1.3-fold higher than the wild type for P HSP104 and P UBI4, respectively. In addition, the influence of the final temperature and temporal sequence of temperature shock to 4°C had a major impact on expression patterns. Therefore, these data show that temperature-induced gene expression of self-cloning industrial yeast could be an option for optimization of the beverage fermentation.

Chemoenzymatic synthesis of sucuronic acid using d-glucurono-6,3-lactone and sucrose as raw materials, and properties of the product.

Using d-glucurono-6,3-lactone (GlcL) and sucrose (Suc) as raw materials, we synthesized sucuronic acid (SucA), in which the d-glucose (Glc) residue of Suc was replaced with d-glucuronic acid, by a three-step chemoenzymatic method. In the 1st chemical step, methyl d-glucuronate (GlcAM) was synthesized by treating GlcL with a strong base anion exchange resin, Amberlite IRA402BL OH AG, in anhydrous methanol. In the 2nd step, which included an enzyme reaction, methyl sucuronate (SucAM) was synthesized from GlcAM and fructose by exploiting the transfructosylation activity of the Microbacterium saccharophilum K-1 ß-fructofuranosidase, a reaction that is suppressed in the presence of high-concentration Glc. In this reaction, the addition of a Suc-non-assimilating yeast, Saccharomyces bisporus NBRC1131, to the reaction mixture increased the amount of SucAM generated, because Glc was removed from the mixture by this yeast. In the 3rd chemical step for producing sodium sucuronate (SucA·Na), SucAM was treated with Amberlite IRA402BL OH AG in water to hydrolyze SucAM's ester bond, and product was then treated with NaOH. The molar yield of SucA·Na from GlcL was 34.2%. SucA was stable at 37 °C in buffer solutions at pH 3, 5, 7, or 9. However, at temperatures exceeding 75 °C, the glycosidic bond of this disaccharide was hydrolyzed not only in acidic buffers (pH 3 and 5) but also in alkaline buffer (pH 9). SucA was not a suitable substrate for the ß-fructofuranosidases of M. saccharopilum K-1 and Saccharomyces cerevisiae.

Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase.

The cofactor engineering strategy can relieve the metabolic stress induced by expression of recombinant protein in cellular metabolism related to cofactor and energy reactions. To study the effect of cofactor regeneration on recombinant protein expression, NADH oxidase (noxE) was engineered in P. pastoris expressing lipase B (GSCALB). Expression of noxE in P. pastoris (GSCALBNOX) increased NAD+ levels by 85% with a concomitant reduction in NADH/NAD+ ratio of 67% compared to GSCALB. The change in the redox level positively influenced the methanol uptake rate and made 34% augment in CALB activity. The decline in NADH level (44%) by noxE expression had lowered the adenylate energy charge (AEC) and ATP level in GSCALBNOX. In order to regenerate ATP in GSCALBNOX, adenylate kinase (ADK1) gene from S. cerevisiae S288c was co-expressed. Expression of ADK1 showed a remarkable increase in AEC and co-expression of both the genes synergistically improved CALB activity. This study shows the importance of maintenance of cellular redox homeostasis and adenylate energy charge during recombinant CALB expression in P. pastoris.

Enantioselective Reduction of Citral Isomers in NCR Ene Reductase: Analysis of an Active-Site Mutant Library.

A deeper understanding of the >99 % S-selective reduction of both isomers of citral catalyzed by NCR ene reductase was achieved by active-site mutational studies and docking simulation. Though structurally similar, the E/Z isomers of citral showed a significantly varying selectivity response to introduced mutations. Although it was possible to invert (E)-citral reduction enantioselectivity to ee 46 % (R) by introducing mutation W66A, for (Z)-citral it remained ≥88 % (S) for all single-residue variants. Residue 66 seems to act as a lever for opposite binding modes. This was underlined by a W66A-based double-mutant library that enhanced the (E)-citral derived enantioselectivity to 63 % (R) and significantly lowered the S selectivity for (Z)-citral to 44 % (S). Formation of (R)-citronellal from an (E/Z)-citral mixture is a desire in industrial (-)-menthol synthesis. Our findings pave the way for a rational enzyme engineering solution.

The Diversification of Evolutionarily Conserved MAPK Cascades Correlates with the Evolution of Fungal Species and Development of Lifestyles.

The fungal kingdom displays an extraordinary diversity of lifestyles, developmental processes, and ecological niches. The MAPK (mitogen-activated protein kinase) cascade consists of interlinked MAPKKK, MAPKK, and MAPK, and collectively such cascades play pivotal roles in cellular regulation in fungi. However, the mechanism by which evolutionarily conserved MAPK cascades regulate diverse output responses in fungi remains unknown. Here we identified the full complement of MAPK cascade components from 231 fungal species encompassing 9 fungal phyla. Using the largest data set to date, we found that MAPK family members could have two ancestors, while MAPKK and MAPKKK family members could have only one ancestor. The current MAPK, MAPKK, and MAPKKK subfamilies resulted from duplications and subsequent subfunctionalization during the emergence of the fungal kingdom. However, the gene structure diversification and gene expansion and loss have resulted in significant diversity in fungal MAPK cascades, correlating with the evolution of fungal species and lifestyles. In particular, a distinct evolutionary trajectory of MAPK cascades was identified in single-celled fungi in the Saccharomycetes. All MAPK, MAPKK, and MAPKKK subfamilies expanded in the Saccharomycetes; genes encoding MAPK cascade components have a similar exon-intron structure in this class that differs from those in other fungi.

New family of pectinase genes PGU1b-PGU3b of the pectinolytic yeast Saccharomyces bayanus var. uvarum.

Using yeast genome databases and literature data, we have conducted a phylogenetic analysis of pectinase PGU genes from Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and hybrid taxon S. pastorianus (syn. S. carlsbergensis). Single PGU genes were observed in all Saccharomyces species, except S. bayanus. The superfamily of divergent PGU genes has been documented in S. bayanus var. uvarum for the first time. Chromosomal localization of new PGU1b, PGU2b, and PGU3b genes in the yeast S. bayanus var. uvarum has been determined by molecular karyotyping and Southern hybridization.

Characterisation of the broad substrate specificity 2-keto acid decarboxylase Aro10p of Saccharomyces kudriavzevii and its implication in aroma development.

BACKGROUND: The yeast amino acid catabolism plays an important role in flavour generation since higher alcohols and acetate esters, amino acid catabolism end products, are key components of overall flavour and aroma in fermented products. Comparative studies have shown that other Saccharomyces species, such as S. kudriavzevii, differ during the production of aroma-active higher alcohols and their esters compared to S. cerevisiae. RESULTS: In this study, we performed a comparative analysis of the enzymes involved in the amino acid catabolism of S. kudriavzevii with their potential to improve the flavour production capacity of S. cerevisiae. In silico screening, based on the severity of amino acid substitutions evaluated by Grantham matrix, revealed four candidates, of which S. kudriavzevii Aro10p (SkAro10p) had the highest score. The analysis of higher alcohols and esters produced by S. cerevisiae then revealed enhanced formation of isobutanol, isoamyl alcohol and their esters when endogenous ARO10 was replaced with ARO10 from S. kudriavzevii. Also, significant differences in the aroma profile were found in fermentations of synthetic wine must. Substrate specificities of SkAro10p were compared with those of S. cerevisiae Aro10p (ScAro10p) by their expression in a 2-keto acid decarboxylase-null S. cerevisiae strain. Unlike the cell extracts with expressed ScAro10p which showed greater activity for phenylpyruvate, which suggests this phenylalanine-derivative to be the preferred substrate, the decarboxylation activities measured in the cell extracts with SkAro10p ranged with all the tested substrates at the same level. The activities of SkAro10p towards substrates (except phenylpyruvate) were higher than of those for ScAro10p. CONCLUSIONS: The results indicate that the amino acid variations observed between the orthologues decarboxylases encoded by SkARO10 and ScARO10 could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds. The knowledge on the important enzyme involved in higher alcohols biosynthesis by S. kudriavzevii could be of scientific as well as of applied interest.

[Identification and polymorphism of pectinase genes PGU in the Saccharomyces bayanus complex].

Pectinase (endo-polygalacturonase) is the key enzyme splitting plant pectin. The corresponding single gene PGU1 is documented for the yeast S. cerevisiae. On the basis of phylogenetic analysis of the PGU nucleotide sequence available in the GenBank, a family of divergent PGU genes is found in the species complex S. bayanus: S. bayanus var. uvarum, S. eubayanus, and hybrid taxon S. pastorianus. The PGU genes have different chromosome localization.

Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as ß-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. ß-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of ß-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.

Variation in α-acetolactate production within the hybrid lager yeast group Saccharomyces pastorianus and affirmation of the central role of the ILV6 gene.

A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer's wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid (S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling α-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form.

RApid Parallel Protein EvaluatoR (RAPPER), from gene to enzyme function in one day.

Cell-free transcription-translation systems offer an effective and versatile platform to explore the impact of genetic variations on protein function. We have developed a protocol for preparing linear, mutagenic DNA templates for direct use in the PURE system, enabling the fast and semi-quantitative evaluation of amino acid variations on catalytic activity and stereo-selectivity in native and engineered variants of Old Yellow Enzyme.

High-efficiency genome editing and allele replacement in prototrophic and wild strains of Saccharomyces.

Current genome editing techniques available for Saccharomyces yeast species rely on auxotrophic markers, limiting their use in wild and industrial strains and species. Taking advantage of the ancient loss of thymidine kinase in the fungal kingdom, we have developed the herpes simplex virus thymidine kinase gene as a selectable and counterselectable marker that forms the core of novel genome engineering tools called the H: aploid E: ngineering and R: eplacement P: rotocol (HERP) cassettes. Here we show that these cassettes allow a researcher to rapidly generate heterogeneous populations of cells with thousands of independent chromosomal allele replacements using mixed PCR products. We further show that the high efficiency of this approach enables the simultaneous replacement of both alleles in diploid cells. Using these new techniques, many of the most powerful yeast genetic manipulation strategies are now available in wild, industrial, and other prototrophic strains from across the diverse Saccharomyces genus.

The genetics of a putative social trait in natural populations of yeast.

The sharing of secreted invertase by yeast cells is a well-established laboratory model for cooperation, but the only evidence that such cooperation occurs in nature is that the SUC loci, which encode invertase, vary in number and functionality. Genotypes that do not produce invertase can act as 'cheats' in laboratory experiments, growing on the glucose that is released when invertase producers, or 'cooperators', digest sucrose. However, genetic variation for invertase production might instead be explained by adaptation of different populations to different local availabilities of sucrose, the substrate for invertase. Here we find that 110 wild yeast strains isolated from natural habitats, and all contained a single SUC locus and produced invertase; none were 'cheats'. The only genetic variants we found were three strains isolated instead from sucrose-rich nectar, which produced higher levels of invertase from three additional SUC loci at their subtelomeres. We argue that the pattern of SUC gene variation is better explained by local adaptation than by social conflict.