Glutathione levels influence chronological life span of Saccharomyces cerevisiae in a glucose‐dependent manner

Diet plays a key role in determining the longevity of the organisms since it has been demonstrated that glucose restriction increases life span whereas a high‐glucose diet decreases it. However, the molecular basis of how diet leads to the aging process is currently unknown. We propose that the quantity of glucose that fuels respiration influences reactive oxygen species generation and glutathione levels, and both chemical species impact in the aging process. Herein, we provide evidence that mutation of the gene GSH1 in Saccharomyces cerevisiae diminishes glutathione levels. Moreover, glutathione levels were higher with 0.5% than in 10% glucose in the gsh1Δ and wild‐type strains. Interestingly, the chronological life span was lowered in the gsh1Δ strain cultured with 10% glucose but not under dietary restriction. The gsh1Δ strain also showed inhibition of the mitochondrial respiration in 0.5 and 10% glucose but only increased the H₂O₂ levels under dietary restriction. These results correlate well with the GSH/GSSG ratio, which showed a decrease in gsh1Δ strain cultured with 0.5% glucose. Together, these data indicate that glutathione exhaustion impact negatively both the electron transport chain function and the chronological life span of yeast, the latter occurring when a low threshold level of this antioxidant is reached, independently of the H₂O₂ levels.     

Disruption of PMR1 in Kluyveromyces lactis improves secretion of calf prochymosin

BACKGROUND: Chymosin is an important industrial enzyme widely used in cheese manufacturing. Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, only low yields of chymosin (80 U mL^?1 in shake flask culture) have been obtained using K. lactis GG799. The aim of this study was to increase the amount of recombinant calf chymosin secreted by K. lactis GG799 by disrupting the PMR1 gene. RESULTS: Kluyveromyces lactis GG799 harbouring the disrupted PMR1 gene showed reduced growth in ethylene glycol tetraacetic acid‐containing and Ca²⁺‐deficient medium, but Ca²⁺ supplementation eliminated the growth problem. The calf chymosin gene was ligated into the K. lactis GG799 expression vector, generating the plasmid pKLAC1‐N‐prochymosin. The linearised plasmid was homologously integrated into the genome of K. lactis GG799. In shake flask culture, chymosin activity was 496 U mL^?1 in the K. lactis PMR1‐deficient mutant, sixfold higher than that in wild‐type K. lactis GG799. CONCLUSION: Disrupting the PMR1 gene improved chymosin production in K. lactis GG799 sixfold. This knowledge could be applied to industrial chymosin production. Copyright © 2010 Society of Chemical Industry     

The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae

The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19‐1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19‐1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes. The GenBank Accession Nos of the sequences are AF255332 (K.lactis GDI1) and AY007574 (P. pastoris GDI1). Copyright © 2001 John Wiley & Sons, Ltd.     

Low-Affinity glucose carrier gene LGT1 of Saccharomyces cerevisiae,a homologue of the Kluyveromyces lactis RAG1 gene

A low-affinity glucose transporter gene of Saccharomyces cerevisiae was cloned by complementation of the rag1 mutation in a strain of Kluyveromyces lactis defective in low-affinity glucose transport. Gene sequence and effects of null mutation in S. cerevisiae were described. Data indicated that there are multiple genes for low-affinity glucose transport.     

The effects of incorporating wild‐type strains of Lactococcus lactis into Turkish white‐brined cheese (Beyaz peynir) on the fatty acid and volatile content

The development of free fatty acids (FFA) and volatile flavour compounds in the Turkish white‐brined cheese Beyaz peynir made by using three wild strains of Lactococcus lactis subsp. lactis was investigated over 90 days. Results showed that production of both FFA and flavour compounds in the control (PK1) and experimental cheeses (MBLL9, MBLL23 and MBL27) was strain dependent. The hydrolysis of milk fat was more evident in the cheese made using Lc. lactis subsp. lactis MBL27. Considering the production of fat breakdown compounds and acidification activities of the strains MBLL23 and MBL27, the combination of these strains could be proposed for the production of white‐brined cheese.     

Proteolytic properties of Turkish white‐brined cheese (Beyaz peynir) made by using wild‐type Lactococcal strains

The development of proteolysis in white‐brined Turkish cheese made by using wild strains of Lactococcus lactis subsp. lactis (namely MBLL9, MBLL23 and MBL27) was monitored for 90 days. Proteolysis in cheeses was investigated using urea‐PAGE gel electrophoresis of pH 4.6‐insoluble and RP‐HPLC of both 70% ethanol‐insoluble and 70% ethanol‐soluble nitrogen fractions. Results indicated that developments of proteolysis in the experimental cheeses were strain dependent. The degradation of casein fractions was more evident in the cheeses made using strain MBLL23. The lowest levels of proteolysis and development of acidity were obtained in the cheese made using strain MBLL9.     

Isolation and characterization of a hexokinase mutant of Schwanniomyces castellii: Consequences on cell production in continuous culture

A mutant of Schwanniomyces castellii with reduced glucose phosphorylation and with practically no phosphorylation of fructose was investigated. Carbon catabolite represion of α-glucosidase and amylases was reduced. Repression of β-galactosidase was normal. We have compared in continuous culture this mutant strain with wild type and another previously described mutant. The relationship between the specific rate of glucose consumption (Qs) and residual glucose concentration (s) in an inverse mode, suggests that there may be two types of transport of glucose. Mutation at the phosphorylation level causes apparent modification of the kinetic parameters of glucose uptake rate. The consequence of mutation at the phosphorylation level on biomass production was discussed.     

Enhancement of nisin production in milk by conjugal transfer of the protease‐lactose plasmid pLP712 to the wild strain Lactococcus lactis UQ2

Lactococcus lactis UQ2 is a wild nisin A producer isolated from a Mexican cheese that grows poorly in milk. Conjugal matings with L. lactis NCDO712 to transfer the Lac+ Prt+ plasmid pLP712 and selection with nisin and lactose yielded L. lactis NCDO712 NisA+. Naturally rifampicin resistant L. lactis UQ2Rif was isolated to provide an additional selective marker. The identity of a transconjugant L. lactis UQ2Rif Lac+ was confirmed by RAPD‐PCR fingerprinting, nisA PCR amplification, nisin production, presence of pLP712 and phospho‐β‐galactosidase activity. This strain performed well in milk and synthesised 200 IU/mL nisin, 40 times more than the original strain.     

The effect of selenium on antioxidant system in erythrocytes and liver of the carp (Cyprinus carpio L.)

The effect of selenium‐supplemented diet (sodium selenate and selenium yeast) on antioxidant in erthrocytes and liver of the carp (Cyprinus carpio L.) fingerlings was studied. With this goal, the activities of glutathione peroxidase (GSH‐Px), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione‐S‐transferase (GST), as well as glutathione (GSH + GSSG) level, were determined. In the group supplemented with sodium selenate, no significant changes in the activity of the above enzymes were recorded in both the erythrocytes and in the liver, with the exception of GST activity that was significantly reduced in the plasma compared with the controls. Glutathione content was at the control level. In the group supplemented with selenium‐yeast, the activities of GSH‐Px, CAT, and SOD were significantly increased in erythrocytes, whereas GST activity and plasma content of GSH + GSSG were reduced compared with the controls. At the same time, the activities of hepatic SOD and GST were increased compared with the controls. These results demonstrate that organically bound selenium (selenium‐yeast) acts more efficiently on antioxidant system of the carp fingerlings than inorganic selenium salts.     

Breeding of high malate‐producing diploid sake yeast with a homozygous mutation in the VID24 gene

Malate is an important taste component of sake (a Japanese alcoholic beverage) that is produced by the yeast Saccharomyces cerevisiae during alcoholic fermentation. A variety of methods for generating high malate‐producing yeast strains have been developed to date. We recently reported that a high malate‐producing strain was isolated as a mutant sensitive to dimethyl succinate (DMS), and that a mutation in the vacuolar import and degradation protein (VID) 24 gene was responsible for high malate productivity and DMS sensitivity. In this work, the relationships between heterozygous and homozygous mutants of VID24 and malate productivity in diploid sake yeast were examined and a method was developed for breeding a higher malate‐producing strain. First a diploid yeast was generated with a homozygous VID24 mutation by genetic engineering. The homozygous integrants produced more malate during sake brewing and grew more slowly in DMS medium than wild‐type and heterozygous integrants. Thus, the genotype of the VID24 mutation influenced the level of malate production and sensitivity to DMS in diploid yeast. Then a homozygous mutant from a heterozygous mutant was obtained without genetic engineering by ultraviolet irradiation and culturing in DMS with nystatin enrichment. The non‐genetically modified sake yeast with a homozygous VID24 mutation exhibited a higher level of malate productivity than the parent heterozygous mutant strain. These findings provide a basis for controlling malate production in yeast, and thereby regulating malate levels in sake. Copyright © 2016 The Institute of Brewing & Distilling     

ERG6 gene deletion modifies Kluyveromyces lactis susceptibility to various growth inhibitors

The ERG6 gene encodes an S‐adenosylmethionine dependent sterol C‐24 methyltransferase in the ergosterol biosynthetic pathway. In this work we report the results of functional analysis of the Kluyveromyces lactis ERG6 gene. We cloned the KlERG6 gene, which was able to complement the erg6Δ mutation in both K. lactis and Saccharomyces cerevisiae. The lack of ergosterol in the Klerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthesis pathway as well as the KlPDR5 gene encoding an ABC transporter. The Klerg6Δ mutation resulted in reduced cell susceptibility to amphotericin B, nystatin and pimaricin and increased susceptibility to azole antifungals, fluphenazine, terbinafine, brefeldin A and caffeine. The susceptibility phenotype was suppressed by the KlPDR16 gene encoding one of the phosphatidylinositol transfer proteins belonging to the Sec14 family. Decreased activity of KlPdr5p in Klerg6Δ mutant (measured as the ability to efflux rhodamine 6G) together with increased amount of KlPDR5 mRNA suggest that the zymosterol which accumulates in the Klerg6Δ mutant may not fully compensate for ergosterol in the membrane targeting of efflux pumps. These results point to the fact that defects in sterol transmethylation appear to cause a multitude of physiological effects in K. lactis cells. Copyright © 2016 John Wiley & Sons, Ltd.     

Redirection of Pyruvate Pathway of Lactic Acid Bacteria to Improve Cheese Quality

Metabolic engineering in Lactic acid bacteria (LAB) has focused on changing of pyruvate metabolism to increase production of desired flavor compounds. A constructed mutant strain should contain no foreign DNA and antibiotic resistance genes. Therefore, food grade lactate dehydrogenase (ldh ^d) and diacetyl reductase (dar ^d) mutant strains were created using two plasmid system in this study. Metabolic end products (pyruvate, lactate, formate and acetoin) of these strains in glucose medium and in cheese were determined using HPLC. Created mutant and wild type strains were used as a starter culture in cheese. Compared to the wild type strain, different levels of metabolites were observed in cheese during three weeks of ripening. The ldh ^d strains produced less lactate but high acetoin as a result of gene deletion. Deletion of dar gene decreased the production of acetoin. The dar deficient strains have low diacetyl reductase activity and are able to reduce significant amounts of acetoin but not terminate it completely. Genetic modification made the shift from homolactic to mixed acid fermentation, but the desired compound production hardly improved. The basis of these results and techniques are promising for the further studies.     

Process for improving the quality of direct acidified Cottage cheese

The effects of plain and fermented curd dressing ripened by single (Lactococcus lactis subsp. lactis biovar. diacetylactis) as well as mixed‐strain starter cultures (L. lactis subsp. lactis; L. lactis subsp. cremoris; L. lactis subsp. lactis biovar. diacetylactis:: 1:1:1), different levels of fat (18–24%) in curd dressing and inoculation rate (1–5%) on direct acidified Cottage cheese were observed. Ripened curd dressing containing 22% fat and mixed‐strain starter cultures at 3% imparted a pleasant acidic note, delicate overtones of diacetyl, improved the body and texture, visual appearance and thereby enhanced the overall quality of the product.     

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β‐Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9‐fold higher than that of the wild‐type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild‐type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild‐type strain. The relative value (mnn2 deletion mutant/wild‐type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β‐glucosidase activity using p‐nitrophenyl‐β‐glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high‐molecular‐weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparative proteomic analysis of a Candida albicans DSE1 mutant under filamentous and non‐filamentous conditions

Candida albicans is a common opportunistic pathogen that causes a variety of diseases in immunocompromised hosts. In a pathogen, cell wall proteins are important virulence factors. We previously characterized Dse1 as a cell wall protein necessary for virulence and resistance to cell surface‐disrupting agents, such as Calcofluor white, chitin deposition, proper adhesion and biofilm formation. In the absence of decomplexation, our objectives were to investigate differential proteomic expression of a DSE1 mutant strain compared to the wild‐type strain. The strains were grown under filamentous and non‐filamentous conditions. The extracted cell proteome was subjected to tryptic digest, followed by generation of peptide profiles using MALDI–TOF MS. Generated peptide profiles were analysed and unique peaks for each strain and growth condition mined against a Candida database, allowing protein identification. The DSE1 mutant was shown to lack the chitin biosynthesis protein Chs5, explaining the previously observed decrease in chitin biosynthesis. The wild‐type strain expressed Pra1, involved in pH response and zinc acquisition, Atg15, a lipase involved in virulence, and Sod1, required for oxidative stress tolerance, in addition to proteins involved in protein biosynthesis, explaining the increase in total protein content observed compared to the mutants strain. The mutant, on the other hand, expressed glucoamylase 1, a cell wall glycoprotein involved in carbohydrate metabolism cell wall degradation and biofilm formation. As such, MALDI–TOF MS is a reliable technique in identifying mutant‐specific protein expression in C. albicans. Copyright © 2014 John Wiley & Sons, Ltd.