Newly identified genes contribute to vanillin tolerance in Saccharomyces cerevisiae

Exploring the mechanisms of tolerance in microorganisms to vanillin, which is derived from lignin, will benefit the design of robust cell factories that produce biofuels and chemicals using lignocellulosic materials. Our objective was to identify the genes related to vanillin tolerance in Saccharomyces cerevisiae. We investigated the effects on vanillin tolerance of several genes that have site mutations in the highly vanillin-tolerant strain EMV-8 compared to its parental line NAN-27. The results showed that overexpression of GCY1, a gene that encodes an aldo-keto reductase that also has mRNA-binding activity, YPR1, a paralog of GCY1 that encodes an aldo-keto reductase, PEX5, a gene that encodes a peroxisomal membrane signal receptor and MBF1, a gene that encodes a multiprotein bridging factor increase the specific growth rates (μ) by 49%, 41%, 44% and 48 %, respectively, in medium containing 6 mmol l⁻¹ vanillin. Among these gene products, Gcy1p and Ypr1p showed NADPH-dependent and NAD(P)H-dependent vanillin reductase activity, respectively. The reductase-inactive mutant Gcy1p^Y56F also increased vanillin tolerance in S. cerevisiae, suggesting that other mechanisms exist. Although TRS85 and PEX5, genes for which the mRNAs are binding targets of Gcy1p, were shown to be related to vanillin tolerance, both the mRNA and protein levels of these genes were not changed by overexpression of GCY1. The relationship between the mRNA-binding activity of Gcy1p and its positive effect on vanillin tolerance is still not clear. Finally, we found that the point mutation D112A in Mbf1p, which disrupts the binding of Mbf1p and the TATA element-binding protein (TBP), did not decrease the positive effect of Mbf1p on vanillin tolerance. This indicates that the binding of Mbf1p and TBP is not necessary for the positive effect on vanillin tolerance mediated by Mbf1p. We have successfully identified new genes related to vanillin tolerance and provided novel targets that can be used to improve the vanillin tolerance of S. cerevisiae. Moreover, we have extended our understanding of the proteins encoded by these genes.     

Centromeric DNA from Saccharomyces uvarum is functional in Saccharomyces cerevisiae

Previous comparisons of centromeric DNA sequences in laboratory strains of Saccharomyces cerevisiae have revealed conserved sequences within 120 base pairs (bp) which appear to be essential for centromere function. We wanted to find out whether centromeric DNA in Saccharomyces strains with different degrees of DNA sequence divergence carry the same conserved sequences or not. Bam HI DNA fragments from two S. cerevisiae strains and one Saccharomyces uvarum strain were cloned into a centromere selection vector and tested for centromere function in a S. cerevisiae laboratory strain. Fragments having centromere function were obtained at approximately equal frequencies from all three strains. Two of the S. uvarum centromeric DNAs and two of the S. cerevisiae centromeric DNAs were sequenced and shown to carry in a 120 bp region sequences essentially like those of centromeric DNA in S. cerevisiae laboratory strains. DNA hybridization to separated chromosomal DNAs revealed that the two newly determined S. cerevisiae centromeric DNA sequences belong to chromosomes V and XIII, respectively. On leave from: Department of Cell and Tumor Biology, Roswell Park Memorial Institute, Buffalo, NY 14263, USA; On leave from: The Biological Laboratories, University of Leiden, The Netherlands     

Molecular cloning and biochemical characterization of a novel erythrose reductase from <Emphasis Type="Italic">Candida magnoliae</Emphasis> JH110

Background  

Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110.     

Metabolome segregation of four strains of Saccharomyces cerevisiae,Saccharomyces uvarum and Saccharomyces kudriavzevii conducted under low temperature oenological conditions

The monitoring of fermentation at low temperatures (12–15°C) is a current practice in the winery for retention and enhancement of the flavour volatile content of wines. Among Saccharomyces species, Saccharomyces uvarum and Saccharomyces kudriavzevii have revealed interesting industrial properties, including better adaptation at low temperatures. To gather deeper knowledge of the fermentative metabolism at a low temperature of these species together with S. cerevisiae, we performed a comparative metabolomic analysis using four representative strains. We used batch cultures to obtain an exhaustive and dynamic image of the metabolome of strains passing through the sequential stresses related to the winemaking environment. A great variety of intra- and extracellular metabolites (>500 compounds) were quantified across fermentation using distinct chromatographic methods. Besides a global decrease in the lipid composition of the four strains when they entered into the stationary phase, we reported some strain-specific high magnitude changes. Examples of these differences included divergent patterns of production of short-chain fatty acids and erythritol in the S. uvarum strain. Strains also differed in expression for aromatic amino acid biosynthesis and sulphur metabolism, including the glutathione pathway. These data will allow us to refine and obtain the most value of fermentations with this alternative Saccharomyces species.     

In Yarrowia lipolytica erythritol catabolism ends with erythrose phosphate

In response to osmotic stress, the yeast Yarrowia lipolytica produces erythritol, a four‐carbon sugar alcohol, from erythrose‐P, an intermediate of the pentose phosphate pathway. Under non‐stressing conditions (isotonic environment), the produced erythritol is subsequently recycled into erythrose‐P that can feed the pentose phosphate pathway. Herein, gene YALI0F01584g was characterized as involved in the erythritol catabolic pathway. Several experimental evidences suggested that it encodes an erythrulose‐1P isomerase that converts erythrulose‐1P into erythrulose‐4P. On the basis of our previous reports and results gathered in this study with genetically modified strains, including ΔYALI0F01584g and ΔYALI0F01628g disrupted mutants, the entire erythritol catabolic pathway has been characterized.     

Relationship between thermal behaviour,fermentation performance and fatty acid composition in two strains of Saccharomyces cerevisiae

Two Saccharomyces cerevisiae strains, one specifically of S. cerevisiae and another belonging to the physiological race S. uvarum, exhibited associative and dissociate thermal profiles, respectively. The S. cerevisiae subsp. uvarum strain, which displayed the dissociative profile, was characterized by a higher aptitude for fermenting glucose in a superoptimal temperature range as well as by a lower fatty acid unsaturation degree. On the other hand, both strains exhibited a similar fatty acid composition modulation pattern with regard to temperature: the unsaturation level presented two relative maxima at 15 and 40° C. However, on the basis of Central Composite Design results, supplementation with an oleic acid source under semi-anaerobic conditions did not improve the fermentative performances in either strain. the modelling of fermentation rate in relation to certain variables indicated that the fermentative performance at superoptimal temperatures, and particularly the optimal temperature (T _opt) and maximal temperature (T _max) of the strain displaying a dissociative profile, could be increased by acting on medium composition. Correspondence to: M. E. Guerzoni     

Killer toxin producing strains of the yeasts Hanseniaspora uvarum and Pichia kluyveri

By heat treatment killer strains of the type K1 of Saccharomyces cerevisiae that are known to harbour dsRNA plasmids were completely cured, whereas only a small fraction of the clones of the killer type K2 had lost the dsRNA dependent killer character. The K2 killers but not the strains of killer type K1 were easily cured by cycloheximide. Killer strains of Hanseniaspora uvarum were not curable by heat treatment. Curing was successfull with cycloheximide or 5-fluorouracil. Two double-stranded RNA plasmids were detected in the killer strains of H. uvarum. The smaller dsRNA plasmid was absent in the strains that were cured of their killer character by 5-fluorouracil. The killer character of H. uvarum was transferred to S. cerevisiae by spheroplast fusion. The fusion products showing the killer character contained both dsRNA plasmids, obviously the smaller plasmid (M-dsRNA) carries the genes for killer toxin formation. Killer strains of Pichia kluyveri were not curable of their killer character, in these strains no dsRNA plasmids were detected.This paper was kindly supported by a grant from the Deutsche Forschungsgemeinschaft     

酿酒酵母中双链RNA介导基因沉默技术研究GRE3基因表达抑制

RNA沉默技术作为探索基因功能的实验手段应用于多种生物. 以编码酿酒酵母NADPH依赖型醛糖还原酶的GRE3基因为对象，检测酿酒酵母双链RNA介导的基因沉默效应. 以pESC-LEU为骨架，构建重组质粒psiLENT-GRE3并用于转化酿酒酵母YPH499. 用RT-PCR检测到诱导1 kb RNA双螺旋和136 bp loop结构引起的GRE3基因表达下调. 结果表明，双链RNA介导的基因沉默技术，能够用作降低酿酒酵母某一特定基因表达水平的工具. 并有助于理解芽殖酵母的RNA干扰现象.     

Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes

Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.

     

Redox Interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in Mixed Culture under Enological Conditions

Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.     

Pure and Mixed Genetic Lines of Saccharomyces bayanus and Saccharomyces pastorianus and Their Contribution to the Lager Brewing Strain Genome

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified “pure” strains containing a single type of genome and “hybrid” strains that contained portions of the genomes from the “pure” lines, as well as alleles termed “Lager” that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.     

Deciphering the Hybridisation History Leading to the Lager Lineage Based on the Mosaic Genomes of Saccharomyces bayanus Strains NBRC1948 and CBS380T

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380^T and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380^T harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380^T and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380^T or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus.     

Mitochondrial DNA polymorphism of the yeast <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> var. <Emphasis Type="Italic">uvarum</Emphasis>

Genetic relationships among forty-one strains of Saccharomyces bayanus var. uvarum isolated in different wine regions of Europe and four wild isolates were investigated by restriction analysis (RPLP) of mitochondrial DNA (mtDNA) with four restriction endonucleases, AluI, DdeI, HinfI and RsaI. No clear correlation between origin and source of isolation of S. bayanus var. uvarum strains and their mtDNA restriction profiles was found. On the whole, the mtDNA of S. bayanus var. uvarum is much less polymorphic than that of S. cerevisiae. This observation is in good agreement with results obtained by electrophoretic karyotyping. Unlike wine S. cerevisiae, strains of S. bayanus var. uvarum display a low level of chromosome length polymorphism.     

Rewiring central carbon metabolism for tyrosol and salidroside production in Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for high-level production of aromatic chemicals has received increasing attention in recent years. Tyrosol production from glucose by S. cerevisiae is considered an environmentally sustainable and safe approach. However, the production of tyrosol and salidroside by engineered S. cerevisiae has been reported to be lower than 2 g/L to date. In this study, S. cerevisiae was engineered with a push-pull-restrain strategy to efficiently produce tyrosol and salidroside from glucose. The biosynthetic pathways of ethanol, phenylalanine, and tryptophan were restrained by disrupting PDC1, PHA2, and TRP3. Subsequently, tyrosol biosynthesis was enhanced with a metabolic pull strategy of introducing PcAAS and EcTyrA^M53I/A354V. Moreover, a metabolic push strategy was implemented with the heterologous expression of phosphoketolase (Xfpk), and then erythrose 4-phosphate was synthesized simultaneously by two pathways, the Xfpk-based pathway and the pentose phosphate pathway, in S. cerevisiae. Furthermore, the heterologous expression of Xfpk alone in S. cerevisiae efficiently improved tyrosol production compared with the coexpression of Xfpk and phosphotransacetylase. Finally, the tyrosol yield increased by approximately 135-folds, compared with that of parent strain. The total amount of tyrosol and salidroside with glucose fed-batch fermentation was over 10 g/L and reached levels suitable for large-scale production.     

A possible application of ale brewery strains ofSaccharomyces cerevisiae in lager beer production

The physiological characteristics of two strains of brewery ale yeasts,Saccharomyces cerevisiae, with sedimentation abilities, were investigated to see if the strains were suitable for lager beer production. Compared with typical industrial ale strains ofS. cerevisiae and lager strains ofS. uvarum (nowS. cerevisiae), the investigated strains differ in fermentation dynamics, as well as in biological properties. The differences, however, particularly between the two strains and the lager brewing yeasts, were not significant.     

Cocultivation of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> (Jacq.) P. Kumm. with yeasts

Cocultivation of Pleurotus ostreatus with eight yeast species were investigated on water agar. Special mycelial structures contacting with yeast cells were found in such cultures: nipple-like appendages and coralioid hyphae. Three out of eight species, Hanseniaspora uvarum, Rhodotorula minuta, and Saccharomyces cerevisiae were identified as trophic preferendum for P. ostreatus. These three yeast species were used for mushroom cultivation on sunflower seed peel. The biomass of fruiting bodies increased by 52.8–75.7% with the H. uvarum and S. cerevisiae suspension presence in the substrate.     

Formation of l(-)malate by Saccharomyces cerevisiae during fermentation

Summary When grown in a synthetic medium most of the 51 strains of the genera Saccharomyces, Saccharomycodes, Zygosaccharomyces and Schizosaccharomyces investigated formed l-malate during fermentation. The quantity varied between 0.1 and 2.6 g malate per liter. Two strains of Saccharomyces cerevisiae synthesized malate at a rate of about 1.5 g/l. Malate was liberated during the growth phase and not metabolized during the stationary phase. Optimum malate formation was observed at a sugar concentration of about 20% (w/v), at pH 5 and at suboptimal nitrogen concentrations of less than 300 mg N/liter. Of the amino acids aspartate and glutamate were most favourable. If ammonium salts were used as the nitrogen source, significant amounts of malate were formed when the pH was kept constant by buffering. Trace metals had no or only little influence on malate synthesis. Biotin and pantothenate were essential for growth. Added ¹⁴CO₂ led to the formation of approximately equal quantities of labelled malate and succinate by S. cerevisiae strain 52, whereas about ten times more malate than succinate was formed by Saccharomyces uvarum. Avidin strongly inhibited the formation of malate while the inhibiton of succinate synthesis and of growth was comparatively much less. Malate is obviously formed by reduction of oxalacetate, the synthesis of which is catalysed by a biotin-dependent pyruvate carboxylase.     

Total biosynthesis of hydrocortisone from a simple carbon source in yeast

We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.     

The [URE3] prion is not conserved among Saccharomyces species

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating inactive form of the nitrogen catabolism regulator Ure2p. To determine whether the [URE3] prion is conserved in S. cerevisiae-related yeast species, we have developed genetic tools allowing the detection of [URE3] in Saccharomyces paradoxus and Saccharomyces uvarum. We found that [URE3] is conserved in S. uvarum. In contrast, [URE3] was not detected in S. paradoxus. The inability of S. paradoxus Ure2p to switch to a prion isoform results from the primary sequence of the protein and not from the lack of cellular cofactors as heterologous Ure2p can propagate [URE3] in this species. Our data therefore demonstrate that [URE3] is conserved only in a subset of Saccharomyces species. Implications of our finding on the physiological and evolutionary meaning of the yeast [URE3] prion are discussed.     

Comparison of engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> for the production of an optically pure keto alcohol

In this study, the production of enantiomerically pure (1R,4S,6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one ((−)-2) through stereoselective bioreduction was used as a model reaction for the comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli as biocatalysts. For both microorganisms, over-expression of the gene encoding the NADPH-dependent aldo-keto reductase YPR1 resulted in high purity of the keto alcohol (−)-2 (>99% ee, 97–98% de). E. coli had three times higher initial reduction rate but S. cerevisiae continued the reduction reaction for a longer time period, thus reaching a higher conversion of the substrate (95%). S. cerevisiae was also more robust than E. coli, as demonstrated by higher viability during bioreduction. It was also investigated whether the NADPH regeneration rate was sufficient to supply the over-expressed reductase with NADPH. Five strains of each microorganism with varied carbon flux through the NADPH regenerating pentose phosphate pathway were genetically constructed and compared. S. cerevisiae required an increased NADPH regeneration rate to supply YPR1 with co-enzyme while the native NADPH regeneration rate was sufficient for E. coli. Nádia Skorupa Parachin and Magnus Carlquist have contributed equally to the paper.