Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae

The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose‐containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1) with two strong galactose‐induced promoters (pGAL1 and pGAL10), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC–URA plasmid except that gene expression is mediated by constitutive promoters. Copyright © 2010 John Wiley & Sons, Ltd.     

The role of PaAAC1 encoding a mitochondrial ADP/ATP carrier in the biosynthesis of extracellular glycolipids,mannosylerythritol lipids,in the basidiomycetous yeast Pseudozyma antarctica

Pseudozyma antarctica produces large amounts of the glycolipid biosurfactants known as mannosylerythritol lipids (MEL), which show not only excellent surface‐active properties but also versatile biochemical actions. A gene homologous with a mitochondrial ADP/ATP carrier was dominantly expressed in P. antarctica under MEL‐producing conditions on the basis of previous gene expression analysis. The gene encoding the mitochondrial ADP/ATP carrier of P. antarctica (PaAAC1) contained a putative open reading frame of 954 bp and encodes a polypeptide of 317 amino acids. The deduced translation product shared high identity of 66%, 70%, 69%, 74%, 75% and 52% with the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae (AAC1), S. cerevisiae (AAC2), S. cerevisiae (AAC3), Kluyveromyces lactis (KlAAC), Neurospora crassa (NcAAC) and human (ANT1), respectively, and conserved the consensus sequences of all ADP/ATP carrier proteins. The gene expression by introducing a plasmid pUXV1‐PaAAC1 into the yeast cells increased the MEL production. In addition, the expression of PaAAC1 in which the conserved arginine and leucine required for ATP transport activity were replaced with isoleucine and serine, respectively, failed to increase MEL production. Accordingly, these results suggest that PaAAC1 encoding a mitochondrial ADP/ATP carrier should be involved in MEL biosynthesis in the yeast. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and characterization of a vector set with regulated promoters for systematic metabolic engineering in Saccharomyces cerevisiae

A set of vectors was constructed that enable combined and systematic testing of metabolic pathway genes in Saccharomyces cerevisiae. The vectors are available as CEN/ARS and 2 µ‐based plasmids with a choice of three inducible promoters, P_GAL1, P_CUP1 and P_ADH2. These features offer control over the initiation and level of gene expression. In addition, the vectors can be used as templates to generate PCR fragments for targeted chromosomal integration of gene expression cassettes. Selection markers are flanked by loxP elements to allow efficient CreA‐mediated marker removal and recycling after genomic integration. For each promoter, expression of a bacterial lacZ reporter gene was characterized from plasmid‐based and integrated chromosomal cassettes, and compared to that of the glycolytic P_PGK1 promoter. Plasmid stabilities were also determined. The promoters showed distinct activity profiles useful for modulating expression of metabolic pathway genes. This series of plasmids with inducible promoters extends our previous vector set carrying the constitutive promoters P_PGK1, P_TEF1 and P_HXT7‐391. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface‐active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino‐terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene‐disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low‐temperature tolerance of the yeast. Copyright © 2010 John Wiley & Sons, Ltd.     

Transforming sugars into fat – lipid biosynthesis using different sugars in Yarrowia lipolytica

In an era of ever‐increasing energy demands, a promising technology is being developed: the use of oleaginous microorganisms such as Yarrowia lipolytica to convert waste materials into biofuels. Here, we constructed two Y. lipolytica strains that displayed both increased lipid accumulation and more efficient use of biomass‐derived sugars, including glucose, fructose, galactose and inulin. The first strain, Y. lipolytica YLZ150, was derived from the French wild‐type strain W29. It had inhibited triacylglycerol mobilization (?tgl4 ) and β‐oxidation (?pox1–6 ), and it overexpressed GPD1 , DGA2 , HXK1 , the native Leloir pathway, SUC2 from Saccharomyces cerevisiae and INU1 from Kluyveromyces marxianus . The second strain, Y. lipolytica Y4779, was derived from the Polish A‐101 strain. It had inhibited β‐oxidation (?mfe2 ) and overexpressed GPD1 , DGA1 , HXK1 , YHT3, SUC2 and INU1 . In the first experiment, strain YLZ150 was batch‐cultured in media containing different hexoses; the highest values for lipid concentration and yield of lipids from the substrate were obtained using fructose (20.3 g dm^?3 and 0.14 g g^?1, respectively). In the second experiment, we grew the two strains in fed‐batch cultures to examine lipid biosynthesis from inulin (a fructose polymer). For Y4779, the lipid concentration was 10.3 g dm^?3 and the yield of lipids from substrate was 0.07 g g^?1; in contrast, for YLZ150, these values were 24 g dm^?3 and 0.16 g g^?1, respectively. The YLZ150 strain is thus able to efficiently exploit glucose, fructose, galactose, sucrose and inulin for lipid biosynthesis. Copyright © 2017 John Wiley & Sons, Ltd.     

The regulatable MAL32 promoter in Saccharomyces cerevisiae: characteristics and tools to facilitate its use

Here we describe a set of tools to facilitate the use of maltose and the MAL32 promoter for regulated gene expression in yeast, alone or in combination with the GAL1 promoter. Using fluorescent protein reporters we find that under non‐inducing conditions the MAL32 promoter exhibits a low basal level of expression, similar to the GAL1 promoter, and that both promoters can be induced independently of each other using the respective sugars, maltose and galactose. While their repression upon glucose addition is immediate and complete, we found that the MAL32 and GAL1 promoters each exhibit distinct induction kinetics. A set of plasmids is available to facilitate the application of the MAL32 promoter for chromosomal modifications using PCR targetting and for plasmid based gene expression. Copyright © 2016 John Wiley & Sons, Ltd.     

A new inducible CRISPR-Cas9 system useful for genome editing and study of double-strand break repair in Candida glabrata

In recent years, the CRISPR-Cas9 system has proven extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae and other yeast species such as Candida glabrata. Inducible CRISPR-Cas9 systems have the additional advantage of allowing to separate the transformation step of the organism by the CRISPR-Cas9 system, from the cutting and repair steps. This has indeed been developed in S. cerevisiae, where most inducible expression systems rely on the GAL promoters. Unfortunately, C. glabrata is gal⁻ and lacks the GAL genes, like many other yeast species. We report here the use of a vector expressing cas9 under the control of the MET3 promoter, with the guide RNA cloned into the same plasmid. We show that it can be used efficiently in C. glabrata, for both described outcomes of CRISPR-Cas9-induced chromosome breaks; nonhomologous end joining in the absence of a homologous repair template; and homologous recombination in the presence of such a template. This system therefore allows easy editing of the genome of C. glabrata, and its inducibility may allow identification of essential genes in this asexual yeast, where spore lethality cannot be observed, as well as the study of double-strand break repair.     

Isolation,characterisation and sulphation of soluble polysaccharides isolated from Cucurbita maxima

Using hot water extraction, a large number of polysaccharides were obtained from Cucurbita maxima. A DEAE‐Sepharose CL‐6B chromatography column was used to isolate the major polysaccharides from C. maxima. Two fractions were obtained (LP2‐1 and LP2‐2). LP2‐1 and LP2‐2 consisted of neutral polysaccharides (MW: 1.02 × 10⁴ and 4.32 × 10⁸ g mol^?1, respectively) comprised mainly of galactose units. Analyses by FT‐IR spectrometry, partial acid hydrolysis, periodate oxidation, Smith degradation and GC‐MS indicated that LP2‐1 consisted of 85.3% (1→4) glycosidic linkages and 1.7% (1→3) or (1→6) glycosidic linkages. The LP2‐1 backbone consisted of (1→4)‐linked galactose units, which occasionally branched at O6 or O3. The branches were composed of (1→4)‐linked galactose and terminated with galactose (13%). Two sulphated derivatives (SLP2‐1 and SLP2‐2) with variable degrees of sulphation (DS) were obtained by the sulphur trioxide–pyridine method, without degradation of the polysaccharide. DS of PL2‐1 and PL2‐2 was 0.19 and 0.20, respectively.     

Assessment of expression of Leloir pathway genes in wild-type galactose-fermenting Streptococcus thermophilus by real-time PCR

In this study, four galactose-positive (Gal⁺) Streptococcus thermophilus strains viz. AJM, JM1, KM3 and AUKD8 and one galactose-negative (Gal^?) S. thermophilus NCDC 218 were used to characterize the organization of Leloir pathway genes using long-range PCR, and expression of these genes were studied using real-time PCR, in presence of different sugars. Long-range PCR results showed that both Gal⁺ and Gal^? isolates, the gal–lac gene order (galRKTEM–lacSZ), are conserved including the size of individual genes. The promoter sequence of the three Gal⁺ isolates (AJM, JM1 and KM3) possessed single base pair deletion at ?28 region of galR and C to T substitution at ?9 box galK region. In contrast, Gal⁺ AUKD8 had A to T substitution at preceding ?25 region of galR. The expression of galK and galM grown in the presence of galactose was significantly higher in case of AJM (30- and 7.6-fold, respectively), followed by KM3 and JM1. In addition, galR, galT and galE showed higher expression in galactose, than in lactose and glucose medium. This study gives a preliminary idea on Leloir pathway gene expression in wild Gal⁺ S. thermophilus, and further studies may throw more light on the role of gal–lac operon in galactose metabolism.     

Yeasts have a four-fold variation in ribosomal DNA copy number

By employing pulsed-field gel electrophoresis we have determined the size of the rDNA cluster in wild-type yeast strains representing genera of Candida, Kluyveromyces, Pachysolen, Schizosaccharomyces and Torulaspora. Although the genome size of the examined species is similar (12·3–13·9 Mb), at least a four-fold variation has been observed between the lowest amount of rDNA repeats in P. tannophilus (28) and the highest in C. glabrata and S. poombe (> 115). In two species the rDNA cluster is represented by two loci, residing either in one (S. pombe) or two chromosomes (C. glabrata).     

Gene expression during activation of Paracoccidioides brasiliensis conidia

This study focuses on gene expression during crucial biological phenomena of the dimorphic fungal human pathogen Paracoccidioides brasiliensis, the conidia‐to‐yeast (C‐Y) transition and the conidia‐to‐mycelia (C‐M) germination. We studied 10 genes involved in different cellular functions: oxidative stress response (alternative oxidase (AOX), superoxide dismutase (SOD), flavodoxin, conserved hypothetical protein (Y20)); cell metabolism (glyceraldehyde‐3‐phosphate dehydrogenase (GADPH), cholestenol Delta‐isomerase (ChDI), glycine dehydrogenase (GDh)) and heat shock response (Heat shock protein 90 (HSP90)), and cell synthesis and wall structure (glucan synthase‐1 (GS‐1), α‐1,3‐glucan synthase (αGS), and mannosyltransferase (MT)). Gene expression was measured during the first 72 h and 96 h of C‐Y and C‐M, respectively, previously shown to be a fundamental time frame for the consolidation of these cellular processes. The gene expression of AOX, GAPDH, HSP90, MT, αGS, and GDh was significantly increased during the C‐Y transition, while SOD, ChDI, GAPDH, MT, GDh, and GS‐1 were increased during C‐M germination. Additionally, some were highly expressed in each process: AOX, HSP90, and αGS during C‐Y; SOD, ChDI, and GS‐1 during C‐M. Altogether, these data add new information regarding gene expression during the C‐Y and C‐M processes. Future research will be targeted to further characterize the true relevance of the studied genes during the morphological transition, either during adaptation to the environment or to the infected host. Copyright © 2011 John Wiley & Sons, Ltd.     

Frequency and Correlation of Some Enteric Indicator Bacteria and Salmonella in Ready‐to‐Eat Raw Vegetable Salads from Mexican Restaurants

Data about Salmonella presence in ready‐to‐eat raw vegetable salads (REVS) consumed in restaurants or sold as REVS in México is not available. The objective of the study was to measure the frequency of coliform bacteria (CB), fecal coliform (FC), Escherichia coli, and Salmonella in REVS from different types of restaurants and determine the correlations of CB, FC, and E. coli versus Salmonella from frequencies and concentration data. The REVS were purchased from 3 types of restaurants: national chain restaurants (A₁, A₂); local restaurants (B₁, B₂); and small restaurants in local markets (C₁, C₂, C₃). Two restaurants for each A and B, and 3 for C, were included. Forty REVS were purchased at each A and B restaurant, and 20 at each C restaurant. CB were tested by plate count using violet red bile agar, FC and E. coli were detected by the most probable number method and E. coli confirmed using IMViC test; conventional method of culture was used for Salmonella. Of 220 analyzed samples, 100% had CB, 95.5% had FC, 83.2% had E. coli, and 6.8% had Salmonella. E. coli frequency was equal to or exceeded 75% in all the cases: 75% (A₁, C₁, C₂), 80% (B₂), 85% (B₁, C₃), and 100% (A₂). Salmonella frequency was equal to or exceeded 2.5% in all cases: 2.5% (A₁), 5% (B₂, C₂), 7.5% (B₁), and 10% (A₂, C₁, C₃). No correlation was observed between FC or E. coli versus Salmonella in the analyzed salads. All the tested salads were of poor quality microbiologically, and microbiological quality did not differ between the restaurants types.     

Physicochemical characterisation of the exopolysaccharides of Streptococcus thermophilus ST‐143

Exopolysaccharides (EPS) excreted by lactic acid bacteria used in the dairy environment are either liberated into the medium (free EPS) or remain attached to the cells (capsular EPS). After batch fermentation of Streptococcus thermophilus ST‐143 at pH 6.0, three EPS fractions were isolated and purified: free EPS (EPS_f), capsular‐derived EPS (EPS_cd) and the entire total EPS (EPS_total). EPS_f and EPS_cd were uncharged, consisted of rhamnose, galactose, glucose and N‐glucosamine in different amounts and showed a molecular mass of 2.6 × 10⁶ Da (EPS_f) and, in case of EPS_cd, 7.4 × 10³ Da and 1.4 × 10⁵ Da in a ratio of 9: 1. Intrinsic viscosity of the polymers was 1.14 mL mg^?1 (EPS_f), 0.06 mL mg^?1 (EPS_cd) but 1.23 mL mg^?1 for EPS_total. The higher functionality of the entire EPS that was also observed in shear rheology measurements indicates that the presence of small capsular‐derived EPS facilitates the entanglement of the larger polymeric carbohydrates of the EPS_f fraction.     

Anti‐obesity effect of Liupao tea extract by modulating lipid metabolism and oxidative stress in high‐fat‐diet‐induced obese mice

Liupao tea (LPT) is traditional dark Chinese tea. The effect of LPT extract on high‐fat‐diet‐induced obese mice was investigated systematically. The results showed that LPT extract could reduce body weight and significantly alleviate liver damage and fat accumulation. LPT could also decrease the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (AKP), total cholesterol (TC), triglycerides (TG), and low‐density lipoprotein cholesterol (LDL‐C) and increase the level of high‐density lipoprotein cholesterol (HDL‐C) in the liver. It also decreased the serum levels of inflammatory cytokines, including tumor necrosis factor alpha (TNF‐α), interferon gamma (IFN‐γ), interleukin (IL)‐1β, and IL‐6 and increased the serum levels of anti‐inflammatory cytokines, including IL‐10 and IL‐4. Moreover, LPT improved the levels of total superoxide dismutase (T‐SOD), glutathione peroxidase (GSH‐Px), and catalase (CAT) and reduced the level of malondialdehyde (MDA) in the liver. Moreover, LPT could upregulate the mRNA and protein expressions of peroxisome proliferator‐activated receptor alpha (PPAR‐α), lipoprotein lipase (LPL), carnitine palmitoyltransferase 1(CPT1), and cholesterol 7 alpha‐hydroxylase (CYP7A1) and downregulate those of PPAR‐γ and CCAAT/enhancer‐binding protein alpha (C/EBP‐α) in the liver. It also increased the mRNA expression of copper/zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), CAT, gamma‐glutamylcysteine synthetase 1 (GSH1), and GSH‐Px. The components of LPT extract include catechin, rutin, taxifolin, and astragalin, which possibly have a wide range of biological activities. In conclusion, our work verified that LPT extract possessed an anti‐obesity effect and alleviated obesity‐related symptoms, including lipid metabolism disorder, chronic low‐grade inflammation, and liver damage, by modulating lipid metabolism and oxidative stress.     

Monitoring of Yeast Communities and Volatile Flavor Changes During Traditional Korean Soy Sauce Fermentation

Flavor development in soy sauce is significantly related to the diversity of yeast species. Due to its unique fermentation with meju, the process of making Korean soy sauce gives rise to a specific yeast community and, therefore, flavor profile; however, no detailed analysis of the identifying these structure has been performed. Changes in yeast community structure during Korean soy sauce fermentation were examined using both culture‐dependent and culture‐independent methods with simultaneous analysis of the changes in volatile compounds by GC‐MS analysis. During fermentation, Candida, Pichia, and Rhodotorula sp. were the dominant species, whereas Debaryomyces, Torulaspora, and Zygosaccharomyces sp. were detected only at the early stage. In addition, Cryptococcus, Microbotryum, Tetrapisispora, and Wickerhamomyces were detected as minor strains. Among the 62 compounds identified in this study, alcohols, ketones, and pyrazines were present as the major groups during the initial stages, whereas the abundance of acids with aldehydes increased as the fermentation progressed. Finally, the impacts of 10 different yeast strains found to participate in fermentation on the formation of volatile compounds were evaluated under soy‐based conditions. It was revealed that specific species produced different profiles of volatile compounds, some of which were significant flavor contributors, especially volatile alcohols, aldehydes, esters, and ketones.     

Bioactivities of essential oil and extract of Thymus argaeus,Turkish endemic wild thyme

BACKGROUND: Thymus argaeus Boiss. & Bal. (Lamiaceae), an endemic plant species of Turkey known as wild thyme, is traditionally used as a spice and a wild tea in the Inner Anatolia region of Turkey. In this study the composition of the essential oil and the antimicrobial and antioxidant effects of the methanolic extract and essential oil of T. argaeus were determined. RESULTS: The main components of the essential oil were linalool (499 g kg^?1), α‐terpineol (150 g kg^?1), linalyl acetate (97 g kg^?1) and thymol (94 g kg^?1). The total phenolic, flavanol and flavonol contents of the extract were 83.31 ± 0.59 mg gallic acid equivalent g^?1, 6.26 ± 0.00 mg catechin equivalent g^?1 and 28.81 ± 0.21 mg rutin equivalent g^?1 respectively. The antioxidant activities of the extract and essential oil determined by the 2,2‐diphenyl‐1‐picrylhydrazyl radical‐scavenging method were 830.18 ± 0.42 and 20.47 ± 2.3 mg g^?1 respectively. The antimicrobial activities of the extract and essential oil against 13 bacteria and two yeasts were studied by the agar diffusion method. The micro‐organisms most sensitive to the essential oil were Aeromonas hydrophila and Pseudomonas aeruginosa, while the micro‐organism most sensitive to the extract was P. aeruginosa. CONCLUSION: Only the extract of T. argaeus could be used as a natural antioxidant, while both the extract and the essential oil could be useful as natural antimicrobial agents in food preservation. Copyright © 2009 Society of Chemical Industry     

<Emphasis Type="Italic">α</Emphasis>-Galactosidases production by <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> UFV-1

Extracellular and intracellular α-galactosidases were produced by yeast Debaryomyces hansenii UFV-1 grown on different media with several carbon sources. D. hansenii grown in YP-medium (1% yeast extract and 2% peptone) presented maximum cell mass (8.45 mg/mL) after 36 h of cultivation, with lactose as carbon source, followed by sucrose, glucose, raffinose, and galactose. Higher extracellular and intracellular α-galactosidases activities were observed at 48 h of D. hansenii cultivation in YPmedium containing galactose (0.97 and 5.27 U/mL) and lactose (1.28 and 4.88 U/mL), supporting the evidence for the model of induction for the yeast GAL/MEL regulon, such as described in Sacharomyces cereviseae.     

Proteomic characterisation of subclover seed storage proteins during germination

BACKGROUND: Early seedling development is a critical step in the establishment of subclover (Trifolium subterraneum), an economically important and widespread pasture legume. In this study the seed storage proteome of this non‐model species was characterised in mature dry seeds and during imbibition by using two‐dimensional electrophoresis coupled with tandem mass spectrometry. RESULTS: The phenol‐extracted proteome of subclover dry seeds consisted of 97 polypeptide spots displayed within a window of pI 3–10 and molecular mass 10–150 kDa. De novo sequencing coupled with MS BLAST search enabled the confident identification of 61 proteins, which corresponded to 59 7S vicilin‐ and two 11S legumin‐type globulins. The experimental mobility of vicilin isoforms along with peptide mapping indicated that low‐molecular‐mass polypeptides might account for the post‐translational proteolysis of small vicilin subunits according to the model described for those of pea. Analysis of quantitative changes in the seed storage proteome upon imbibition showed that vicilin catabolism according to a site‐specific process was favoured during early seedling growth in T. subterraneum. CONCLUSION: The establishment of a seed proteome map for T. subterraneum pointed to vicilins as dominant proteins in mature seeds whose catabolism features during early seedling growth may be of relevance under environmental conditions. Copyright © 2009 Society of Chemical Industry