Disrupting the methionine biosynthetic pathway in Candida guilliermondii: characterization of the MET2 gene as counter‐selectable marker

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine‐O‐acetyltransferase and O‐acetylserine O‐acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead‐containing medium, contrary to the wild‐type strain, which develop as white colonies on this medium. The MET2 wild‐type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene‐expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2‐containing YFP‐expressing plasmid can be easily observed on lead‐containing medium. The MET2 wild‐type allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α‐aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop‐out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of genetic markers for identifying isolates of the species of the genus Fusarium

BACKGROUND: Members of the genus Fusarium are well known as one of the most important plant pathogens causing food spoilage and loss worldwide. Moreover, they are associated with human and animal diseases through contaminated foods because they produce mycotoxins. To control fungal hazards of plants, animals and humans, there is a need for a rapid, easy and accurate identification system of Fusarium isolates with molecular methods. RESULTS: To specify genes appropriate for identifying isolates of various Fusarium species, we sequenced the 18S rRNA gene (rDNA), internal transcribed spacer region 1, 5.8S rDNA, 28S rDNA, β‐tubulin gene (β‐tub), and aminoadipate reductase gene (lys2), and subsequently calculated the nucleotide sequence homology with pair‐wise comparison of all tested strains and inferred the ratio of the nucleotide substitution rates of each gene. Inter‐species nucleotide sequence homology of β‐tub and lys2 ranged from 83.5 to 99.4% and 56.5 to 99.0%, respectively. The result indicated that sequence homologies of these genes against reference sequences in a database have a high possibility of identifying unknown Fusarium isolates when it is more than 99.0%, because these genes had no inter‐species pair‐wise combinations that had 100% homologies. Other markers often showed 100% homology in inter‐species pair‐wise combinations. The nucleotide substitution rate of lys2 was the highest among the six genes. CONCLUSION: The lys2 is the most appropriate genetic marker with high resolution for identifying isolates of the genus Fusarium among the six genes we examined in this study. Copyright © 2011 Society of Chemical Industry     

Metabolism of oligosaccharides and aldehydes and production of organic acids in soymilk by probiotic bifidobacteria

Four strains of Bifidobacterium were assessed for α‐galactosidase activity in MRS broth using p‐nitrophenyl‐α‐d ‐galactopyranoside as substrate. Bifidobacteria were then used to ferment soymilk prepared from soy protein isolate (SPI), with and without d ‐glucose and l ‐cysteine supplementation. Measurements of pH and the quantification of oligosaccharides, organic acids and aldehydes in soymilk were done after 0, 12, 24, 36 and 48 h of incubation. The presence of d ‐raffinose stimulated the α‐galactosidase activity of B. longum BB536 (BB536) and B. pseudolongum 20099 (BP20099). In soymilk, oligosaccharides and aldehydes were effectively metabolized by Bifidobacterium; d ‐glucose and l ‐cysteine supplementation enhanced the hydrolysis of raffinose and stachyose. BB536 and BP20099 degraded a significantly greater level of oligosaccharides and aldehydes than B. animalis Bb‐12 and B. longum 1941 (P < 0.05). Raffinose and stachyose were completely metabolized by BB536 after 48 h. In soymilk without any supplementation, hexanal and pentanal were not detected after 12 h of fermentation with BB536 and BP20099. BB536 was the highest producer of organic acids, with an average acetic acid/l (+)‐lactic acid ratio of 0.7.     

Control of lactic acid bacteria in fermented beverages using lysozyme and nisin: test of traditional beverage boza as a model food system

The objective of this study was to increase quality and limited shelf‐life of boza (3–15 days), a traditional Balkan origin fermented beverage using lysozyme (LYS) and/or nisin (NIS). For this purpose, the effectiveness of NIS, LYS and LYS:NIS combinations was first tested in a broth medium at 4 °C for 3 weeks on Lactobacillus plantarum, one of the frequently isolated lactic acid bacteria in boza. Stability of LYS and NIS in boza, their effects on LAB counts, and chemical and sensory properties of boza were then evaluated during cold storage at 4 °C. Results of LAB counts as well as pH, d ‐ and l ‐lactic acid, and titratable acidity measurements showed that LAB in boza containing NIS (250 μg g^?1) or LYS:NIS (500:250 μg g^?1) could be controlled without reducing LAB counts below 6 log CFU mL^?1 during 2 weeks shelf‐life. In contrast, LYS (500 μg g^?1) alone could not control LAB in boza to delay its acidic spoilage. Positive effects of NIS and LYS:NIS application on quality of boza were also proved with sensory analysis by panelists and e‐nose measurements. This work showed that use of natural GRAS agents in preservation of fermented beverages containing probiotic LAB is possible without affecting their characteristic aroma and flavour.     

Positive and negative selection LYS5MX gene replacement cassettes for use in Saccharomyces cerevisiae

Novel MX cassettes are described that contain the open reading frames (ORFs) of Saccharomyces cerevisiae or Candida albicans LYS5. The LYS5MX and CaLYS5MX cassettes, the targeting efficiencies of which are equivalent to those of other MX cassettes, are positively selected for Lys+ in a lys5 background. Unlike most of the other MX cassettes, the LYS5MX cassettes are also negatively selectable (alpha-aminoadipate-resistant), which will allow the use of the LYS5MX cassettes in plasmid shuffling and will also greatly facilitate marker recycling.     

Formation of 4(5)‐Methylimidazole in Aqueous d‐Glucose‐Amino Acids Model System

The International Agency for Research on Cancer (IRAC) has classified 4(5)‐methylimidazole (4‐MeI) as a group 2B possible human carcinogen. Thus, how 4‐MeI forms in a d ‐glucose (Glu) amino acids (AA) model system is important, as it is how browning is affected. An aqueous solution of Glu was mixed individually in equimolar amounts at 3 concentrations (0.05, 0.1, and 0.15 M) with aqueous solutions of l ‐Alanine (Ala), l ‐Arginine (Arg), Glycine (Gly), l ‐Lysine (Lys), and l ‐Serine (Ser). The Glu‐AA mixtures were reacted at 60, 120, and 160 °C for 1 h. The 4‐MeI levels were measured by gas chromatography‐mass spectrometry after derivatization with isobutylchloroformate. No 4‐MeI was formed at 60 °C for any treatment combination; however, at 120 °C and 0.05 M, Glu‐Arg and Glu‐Lys produced 0.13 and 0.14 mg/kg of 4‐MeI. At 160 °C and 0.05 M all treatment combinations formed 4‐MeI. At 160 °C and 0.15 M, the observed levels of Glu‐Ala, Glu‐Arg, Glu‐Gly, Glu‐Lys, and Glu‐Ser were 0.21, 1.00, 0.15, 0.22, and 0.16 mg/kg. The AA type, reactant concentrations, and temperature significantly affected (P < 0.001) formation of 4‐MeI as well as browning. Glu‐Lys treatment in all combinations produced the most browning, but Glu‐Arg produced the most 4‐MeI. This method showed that foods processed using low temperatures may have reduced levels of 4‐MeI.     

l‐Citrulline Supplementation‐Increased Skeletal Muscle PGC‐1α Expression Is Associated with Exercise Performance and Increased Skeletal Muscle Weight

Scope

l ‐citrulline has recently been reported as a more effective supplement for promoting intracellular nitric oxide (NO) production compared to l ‐arginine. Here, the effect of l ‐citrulline on skeletal muscle and its influence on exercise performance were investigated. The underlying mechanism of its effect, specifically on the expression of skeletal muscle peroxisome proliferator‐activated receptor‐gamma coactivator‐1α (PGC‐1α), was also elucidated.

Methods and results

Six‐week‐old ICR mice were orally supplemented with l ‐citrulline (250 mg kg^?1) daily, and their performance in weight‐loaded swimming exercise every other day for 15 days, was evaluated. In addition, mice muscles were weighed and evaluated for the expression of PGC‐1α and PGC‐1α‐regulated genes. Mice orally supplemented with l ‐citrulline had significantly higher gastrocnemius and biceps femoris muscle mass. Although not statistically significant, l ‐citrulline prolonged the swimming time to exhaustion. PGC‐1α upregulation was associated with vascular endothelial growth factor α (VEGFα) and insulin‐like growth factor 1 (IGF‐1) upregulation. VEGFα and IGF‐1 are important for angiogenesis and muscle growth, respectively, and are regulated by PGC‐1α. Treatment with NG‐nitro‐l ‐arginine methyl ester hydrochloride (l ‐NAME), a nitric oxide synthesis inhibitor, suppressed the l ‐citrulline‐induced PGC‐1α upregulation in vitro.

Conclusion

Supplementation with l ‐citrulline upregulates skeletal muscle PGC‐1α levels resulting in higher skeletal muscle weight that improves time to exhaustion during exercise.

     

Cloning and sequence of the LYS2 homologue gene from the osmotolerant yeast Pichia sorbitophila

We have isolated the Pichia sorbitophila LYS2 (PsLYS2) gene by complementation of a lys2 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 4197 bp and the deduced translation product shares a global identity of 66% and 58% to the Lys2 protein homologues of Candida albicans and S. cerevisiae, respectively. Analysis of PsLYS2 sequence suggests that, similarly to S. cerevisiae LYS2, it codes for a polypeptide having two separate enzymatic activities which reside in different domains of the protein, including an adenylate domain, an acyl-carrier site and a short-chain reductase domain. Several GCN4- and NIT2-binding motifs have been matched in the promotor sequence of PsLYS2. In addition, upstream of the sequenced PsLYS2 sequence, we have found the 3'-terminal half of a gene of same orientation encoding a RAD16-like protein, a genomic organization similar to that of C. albicans.     

Isolation and characterization of the carbon catabolite‐derepressing protein kinase Snf1 from the stress tolerant yeast Torulaspora delbrueckii

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae snf1Δ mutant strain. DNA sequence analysis revealed that the fragment contained a complete open reading frame (ORF), which shares a high similarity with the S. cerevisiae energy sensor protein kinase Snf1. The cloned TdSNF1 gene was able to restore growth of the S. cerevisiae snf1Δ mutant strain on media containing nonfermentable carbon sources. Furthermore, cells of the Tdsnf1Δ mutant were unable to proliferate under nonfermenting conditions. Finally, protein domain analysis showed that TdSnf1p contains a typical catalytic protein kinase domain (positions 41–293), which is also present in other Snf1p homologues. Within this region we identified a protein kinase ATP‐binding region (positions 48–71) and a consensus Ser/Thr protein kinase active site (positions 160–172). The GenBank Accession No. for the sequenced DNA fragment is HM131845. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non‐homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR‐amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour‐intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ‐mediated integrative transformation with PCR‐amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.     

The Pichia pastoris dihydroxyacetone kinase is a PTS1-containing,but cytosolic,protein that is essential for growth on methanol

Dihydroxyacetone kinase (DAK) is essential for methanol assimilation in methylotrophic yeasts. We have cloned the DAK gene from Pichia pastoris by functional complementation of a mutant that was unable to grow on methanol. An open reading frame of 1824 bp was identified that encodes a 65·3 kDa protein with high homology to DAK from Saccharomyces cerevisiae. Although DAK from P. pastoris contained a C-terminal tripeptide, TKL, which we showed can act as a peroxisomal targeting signal when fused to the green fluorescent protein, the enzyme was primarily cytosolic. The TKL tripeptide was not required for the biochemical function of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P. pastoris dakΔ mutant. Peroxisomes, which are essential for growth of P. pastoris on methanol, were present in the dakΔ mutant and the import of peroxisomal proteins was not disturbed. The dakΔ mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the dakΔ mutant was unable to grow on methanol as the sole carbon source but was able to grow on dihydroxyacetone at a much slower rate. The metabolic pathway explaining the reduced growth rate of the dakΔ mutant on dihydroxyacetone is discussed. The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number AF019198. © 1998 John Wiley & Sons, Ltd.     

Combining mutations in the incoming and outgoing pheromone signal pathways causes a synergistic mating defect in Saccharomyces cerevisiae

Mating pheromones stimulate Saccharomyces cerevisiae yeast cells to form a pointed projection that becomes the site of cell fusion during conjugation. To investigate the role of mating projections, we screened for mutations that enhanced the weak mating defect of MAT a ste2‐T326 cells that are defective in forming pointed projections. These cells are also 10‐fold more sensitive to α‐factor pheromone because ste2‐T326 encodes truncated α‐factor receptors that are not regulated properly. Mutations in AXL1, STE6 and FUS3 were identified in the screen. AXL1 was studied further because it is required for efficient a ‐factor pheromone production and for selecting the site for bud morphogenesis. Mutation of AXL1 did not enhance the morphogenesis or pheromone sensitivity defects of ste2‐T326. Instead, the synergistic mating defect was apparently due to decreased a ‐factor production because the axl1Δ ste2‐T326 cells mated well with a sst2 α mating partner that is supersensitive to a ‐factor. When combined with a wild‐type mating partner, the ste2‐T326 axl1Δ cells failed to mate because they did not lock cell walls, one of the earliest steps in conjugation. Analysis of axl1Δ in combination with other mutations that cause defects in morphogenesis or pheromone sensitivity (e.g. bar1, sst2, afr1) indicated that both phenotypes of ste2‐T326 cells, supersensitivity to α‐factor and the defect in forming pointed projections, contributed to the synergistic mating defect. We suggest a model that the synergistic mating defect is caused by the combined effects of ste2‐T326 and axl1Δ on the presentation of a ‐factor to partner cells. Altogether, these results demonstrate an important linkage between the incoming and outgoing pheromone signals during the intercellular communication that promotes yeast mating. Copyright © 1999 John Wiley & Sons, Ltd.     

A COMBINATION OF GLUTAMINASE AND pH CONTROL PREVENTS THE NONENZYMATIC CONVERSION OF l-GLUTAMINE INTO l-2-PYRROLIDINE-5-CARBOXYLIC ACID IN FOOD PROCESSING

A strategy using Alicyclobacillus F‐62 glutaminase was established to minimize the nonenzymatic production of l ‐2‐pyrrolidine‐5‐carboxylic acid (l ‐pyroglutamic acid, savorless amino acid) and maximize the enzymatic production of l ‐glutamic acid (savorous [umami] amino acid) from l ‐glutamine in food‐protein processing. The nonenzymatic production rate of l ‐pyroglutamic acid was influenced by pH and temperature, and decreased over a pH range of 4–6 at 30C. Under optimum conditions (30C and pH 5.0), the l ‐glutamic and l ‐pyroglutamic acids obtained from 1 mM l ‐glutamine in the presence of glutaminase from Alicyclobacillus F‐62 were 0.96 and 0.03 mM, respectively. This novel method is useful for the efficient production of l ‐glutamic acid during food‐protein processing.     

PER1, GUP1 and CWH43 of methylotrophic yeast Ogataea minuta are involved in cell wall integrity

In eukaryotes, the glycosylphosphatidylinositol (GPI) modification of many glycoproteins on the cell surface is highly conserved. The lipid moieties of GPI‐anchored proteins undergo remodelling processes during their maturation. To date, the products of the PER1, GUP1 and CWH43 genes of the yeast Saccharomyces cerevisiae have been shown to be involved in the lipid remodelling. Here, we focus on the putative GPI remodelling pathway in the methylotrophic yeast Ogataea minuta. We found that the O. minuta homologues of PER1, GUP1 and CWH43 are functionally compatible with those of S. cerevisiae. Disruption of GUP1 or CWH43 in O. minuta caused a growth defect under non‐permissive conditions. The O. minuta per1Δ mutant exhibited a more fragile phenotype than the gup1Δ or cwh43Δ mutants. To address the role of GPI modification in O. minuta, we assessed the effect of these mutations on the processing and localization of the O. minuta homologues of the Gas1 protein; in S. cerevisiae, Gas1p is an abundant and well‐characterized GPI‐anchored protein. We found that O. minuta possesses two copies of the GAS1 gene, which we designate GAS1A and GAS1B. Microscopy and western blotting analysis showed mislocalization and/or lower retention of Gas1Ap and Gas1Bp within the membrane fraction in per1Δ or gup1Δ mutant cells, suggesting the significance of lipid remodelling for GPI‐anchored proteins in O. minuta. Localization behaviour of Gas1Bp differed from that of Gas1Ap. Our data reveals, for the first time (to our knowledge), the existence of genes related to GPI anchor remodelling in O. minuta cells.     

The Yarrowia lipolytica PAH1 homologue contributes but is not required for triacylglycerol biosynthesis during growth on glucose

The PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the Mg²⁺-dependent dephosphorylation of phosphatidate to produce diacylglycerol, which can be acylated to form triacylglycerol (TAG). In the model oleaginous yeast Yarrowia lipolytica, TAG is the major lipid produced, and its biosynthesis requires a continuous supply of diacylglycerol, which can be provided by the PAP reaction. However, the regulation of Pah1 has not been studied in detail in Y. lipolytica, and thus its contribution to the biosynthesis of TAG in this yeast is not well understood. In this work, we examined the regulation of the PAH1-mediated PAP activity and Pah1 abundance and localization in cells growing on glucose. We found that Pah1 abundance and localization were regulated in a growth-dependent manner, yet the loss of Pah1 did not have a major effect on PAP activity. We also examined the effects of the Y. lipolytica pah1Δ mutation on cell physiology and lipid biosynthesis. The lack of Pah1 in the pah1Δ mutant resulted in a moderate decrease in TAG levels and an increase in phospholipid levels. These results showed that Pah1 contributed to TAG biosynthesis in Y. lipolytica but also suggested the presence of other activities in the pah1Δ mutant that compensate for the loss of Pah1. Also, the levels of linoleic acid were elevated in pah1Δ cells with a concomitant decrease in the oleic acid levels suggesting that the pah1Δ mutation affected the biosynthesis of fatty acids.     

Yeast mutants affected in viability upon starvation have a modified phospholipid composition

Selection of mutations, based on the suppression of rvs161Δ defects, was performed. Ten mutants were obtained, ranged amongst four complementation groups, named SUR1, SUR2, SUR3 and SUR4. All sur mutations also suppress a mutation in another gene, RVS167, indicating that all six genes are involved in the same biological pathway. The sur mutant cells have abnormal morphologies in stationary phase, i.e. dumbbell-like in sur1, sur2 or sur3 strains and multibudded in sur4 strains. Several phenotypic characteristics of the physiological suppressors as well as the rvs161Δ strain itself led us to analyse the phospholipid composition of the mutants. The assays show an overall decrease of the phospholipid amounts and modifications in the relative contents of some phospholipid classes in sur mutant cells.     

Relationship between lunasin's sequence and its inhibitory activity of histones H3 and H4 acetylation

Scope: Dysfunction of histone acetyltransferases (HATs) or histone deacetylases (HDACs) involved in histones acetylation has been associated with cancer. Inhibitors of these enzymes are becoming potential targets for new therapies. Methods and Results: This study reports by Western‐Blot analysis, that peptide lunasin is mainly an in vitro inhibitor of histone H4 acetylation by P300/cAMP‐response element‐binding protein (CBP)‐associated factor (PCAF), with IC₅₀ values dependent on the lysine position sensitive to be acetylated (0.83 μM (H4‐Lys 8), 1.27 μM (H4‐Lys 12) and 0.40 μM (H4‐Lys 5, 8, 12, 16)). Lunasin is also capable of inhibiting H3 acetylation (IC50 of 5.91 μM (H3‐Lys 9) and 7.81 μM (H3‐Lys 9, 14)). Studies on structure‐activity relationship establish that lunasin's sequence are essential for inhibiting H4 acetylation whereas poly‐D sequence is the main active sequence responsible for H3 acetylation inhibition. Lunasin also inhibits H3 and H4 acetylation and cell proliferation (IC₅₀ of 181μM) in breast cancer MDA‐MB‐231 cells. Moreover, this peptide decreases expression of cyclins and cyclin dependent kinases‐4 and ‐6, implicated in cell cycle pathways. Conclusion: Results from this study demonstrates lunasin's role as modulator of histone acetylation and protein expression that might contribute on its chemopreventive properties against breast cancer.     

Identification of a gene encoding a homocitrate synthase isoenzyme of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, most of the LYS structural genes have been identified except the genes encoding homocitrate synthase and α-aminoadipate aminotransferase. Expression of several LYS genes responds to an induction mechanism mediated by the product of LYS14 and an intermediate of the pathway, α-aminoadipate semialdehyde (αAASA) as an inducer. This activation is modulated by the presence of lysine in the growth medium leading to an apparent repression. Since the first enzyme of the pathway, homocitrate synthase, is feedback inhibited by lysine, it could be a major element in the control of αAASA supply. During the sequencing of chromosome IV of S. cerevisiae, the sequence of ORF D1298 showing a significant similarity with the nifV gene of Azotobacter vinelandii was reported. Disruption and overexpression of ORF D1298 demonstrate that this gene, named LYS20, encodes a homocitrate synthase. The disrupted segregants are able to grow on minimal medium and exhibit reduced but significant homocitrate synthase indicating that this activity is catalysed by at least two isoenzymes. We have also shown that the product of LYS20 is responsible for the greater part of the lysine production. The different isoforms are sensitive to inhibition by lysine but only the expression of LYS20 is strongly repressed by lysine. The N-terminal end of homocitrate synthase isoform coded by LYS20 contains no typical mitochondrial targeting sequence, suggesting that this enzyme is not located in the mitochondria.