Anna Kujumdzieva

Dual targeting of yeast catalase A to peroxisomes and mitochondria

Yeast catalase A (Cta1p) contains two peroxisomal targeting signals (SSNSKF) localized at its C-terminus and within the N-terminal third of the protein, which both can target foreign proteins to peroxisomes. In the present study we demonstrated that Cta1p can also enter mitochondria, although the enzyme lacks a classical mitochondrial import sequence. Cta1p co-targeting was studied in a catalase A null mutant after growth on different carbon sources, and expression of a Cta1p-GFP (green fluorescent protein)-fusion protein or a Cta1p derivative containing either a c-Myc epitope (Cta1p(myc)) or a SKF-extended tag (Cta1p(myc-SKF)). Peroxisomal and mitochondrial co-import of catalase A were tested qualitatively by fluorescence microscopy and functional complementation of a Delta cta1 null mutation, and quantitatively by subcellular fractionation followed by Western blot analysis and enzyme activity assays. Efficient Cta1p import into peroxisomes was observed when cells were cultivated under peroxisome-inducing conditions (i.e. growth on oleate), whereas significant co-import of Cta1p-GFP into mitochondria occurred when cells were grown under respiratory conditions that favour oxygen stress and ROS (reactive oxygen species) accumulation within this organelle. In particular, when cells were grown on the non-fermentable carbon source raffinose, respiration is maximally enhanced, and catalase A was efficiently targeted to the mitochondrial matrix where it presumably functions as scavenger of H2O2 and mitochondrial-derived ROS.

Cu/Zn superoxide dismutase in yeast mitochondria ^ a general phenomenon

Fermentative and respiratory yeast strains of genera Saccharomyces, Kluyveromyces, Pichia, Candida and Hansenula have been investigated for mitochondrial localization of Cu/Zn superoxide dismutase (SOD). Pure mitochondrial fractions were obtained and the specific activities of Cu/Zn and Mn SODs were measured in comparison with those in the corresponding cell-free extracts. The Cu/Zn SOD: Mn SOD ratio in mitochondria and crude extracts was calculated and was considered a specific characteristic of all tested strains. Electrophoretical visualization of SOD patterns provided evidence for possible migration of cytosolic Cu/Zn SOD to mitochondria. The characteristic Cu/Zn SOD profile in mitochondria of all tested strains suggested its ubiquity within the fermentative and respiratory yeasts.

Pigments and Citrinin Biosynthesis by Fungi Belonging to Genus Monascus

Citrinin is a mycotoxin, which is produced by fungi belonging to the genus Monascus, known in biotechnology as producers of azaphilone pigments. The relation between biosynthesis of these secondary metabolites was investigated in different species of the genus Monascus in batch-culture at the following cultivation conditions: T = 28 °C, agitation 220 rpm, and a medium, which induce citrinin production, containing ethanol as a carbon source. The screening was carried out with 16 fungal strains and the biosynthesis of citrinin and pigments was monitored quantitatively at the standard conditions mentioned above. Some kinetic parameters of the process have been determined. The values of the growth yield coefficient YX/C were between 0.32 and 0.57. The amount of the extracellular red and orange pigments at the end of cultivation varied for the different strains between 0.09 and 1.33 OU/ mg dry weight, and 0.15 and 0.96 OU/mg dry weight, respectively. The amount of the total pigments measured was between 0.16 and 3.6 OU/mg dry weight, and between 0.21 and 3.39 OU/mg dry weight. The determined ratio 500 nm/400 nm, characterizing the pigment production, ranged between 0.60 and 1.06. Twelve of the investigated strains produced citrinin and pigments, two of them produced only pigments. Two strains were not able to produce neither pigments nor citrinin. Thus, the biosynthesis of citrinin appeared to be strain-specific and does not correlate with the pigments’ biosynthesis by the fungal strains belonging to the genus Monascus.

Lipid production by Monascus purpureus albino strain

Monascus purpureus DSM 1379 strain was used to study pigment and lipid synthesis and it was shown that both processes are concurrent. In order to obtain only lipids a white mutant has been selected from the red pigment producing strain. This stable mutant lost its ability to produce pigments and revealed morphological and biochemical differences compared to the parent strain. Kinetics of the lipid biosynthesis of the albino mutant in submerged cultures have been studied at C/N ratios of 10:1 and 80:1. The estimation of the specific rates of glucose and nitrogen consumption along with the lipid production indicated more efficient lipid synthesis at C/N ratio 80:1, i.e. 72% (w/w) against 43% (w/w) for C/N ratio 10:1. The neutral, phospho- and glycolipid composition was not influenced by the C/N ratio and nitrogen source used. The investigation of the fatty acid content showed that the major fatty acids were C18:1 (45.5%), and C16 (22.1%). The selected mutant is able to produce high amount of lipids consisting of 88% triglycerides with a percentage of saturated fatty acids 51%.

Characterization of a non-pigment producing Monascus purpureus mutant strain

A characterization of a non-pigment producing mutant Monascus purpureus M12 compared with its parental strain Monascus purpureus Went CBS 109.07 has been performed aiming to investigate the relation between pigment biosynthesis and other characteristics of these fungi. A comparison has been made of morphological features, some physiological properties and biochemical activities of both strains. The albino mutant exhibits an anamorph life cycle, high conidia forming capability, slower radial growth rate and temperature sensitivity. The assimilation capacity of both strains for mono-, disaccharides and some alcohols is in the same range (YX/C 0.2 – 0.35), while the red strain has a higher fermentation capacity. In a selected albino mutant, the growth rate, metabolic activity and capacity for production of typical for Monascus fungi secondary metabolites were reduced considerably. Hydrolytic activity towards natural substrates expressed through glucoamylase and protease was approximately 10 fold lower in the non pigment producing strain (0.05 – 0.08 U/mg protein and 0.01 – 0.07 U/mg protein respectively) compared with the red one. Important qualitative differences between both strains was found in fatty acid composition and in the production of citrinin and monacolin. The mutant strain possessed C17, C20 and C22 fatty acids and did not produce citrinin.

The composition of the microflora of boza, an original bulgarian beverage

ABSTRACT A total of 293 strains of lactic acid bacteria and 78 yeast strains from original Bulgarian boza were isolated. The rod-shaped strains were identified as Lactobacillus salivarius, Lactobacillus sakei, Lactobacillus maltaromicus, Lactobacillus fermentum, Lactobacillus parabuchneri, Lactobacillus paracasei subsp. paracasei and Weissella confusa. The strains of genus Leuconostoc were identified as Leuconostoc lactis, Leuconostoc amelibiosum, Leuconostoc mesenteroides subsp. dextranicum and Leuconostoc pseuclomesenteroides. The yeasts isolated belonged to the species Saccharomyces cerevisiae, Pichia membranifaciens, Dekkera bruxellensis and Nadsonici commutata. During the storage of boza the total number of the lactic acid bacteria increased slightly up to 48 h, while the n umber of yeasts increased considerably. The changes in pH and total acidity during the storage of boza were in accordance with the changes in viable count of lactic acid bacteria and their activity.

INFLUENCE OF CARBON AND NITROGEN SOURCES ON GROWTH AND PIGMENT PRODUCTION BY MONASCUS PILOSUS C1 STRAIN

ABSTRACT Pigments produced by Monascus fungi are widely used in food and biotechnology as natural colorants, flavors and preservatives. There are various factors influencing the pigments productions, among which are the nutrient media composition, pH, ambient temperature, mode of cultivation. In the present study the influence of different carbon and nitrogen sources on the pigments biosynthesis by Monascus pilosus C1 strain was investigated during batch cultivation at the following conditions: T = 30°C, pH 6.0, agitation 300 rpm and modified Chapec—Dox medium with different carbon (a range of sugars and alcohols) or nitrogen sources for 7 days. Some process kinetic parameters—YX/C, YP/C, Vav, were calculated and analized. It was found that the micelial growth, expressed as dry weight (DW) and the specific pigment production (SPP) were strongly stimulated by glucose as a carbon source. The DW and SPP reached values of 10.89 g DW l−1, and 1.97 OU mg DW−1 and 1.01 OU mg DW−1 for the total red and yellow pigments, respectively. Sodium glutamate combined with glucose increased effectively the pigments production up to 2.29 OU mg DW−1 and 1.67 OU mg DW−1 respectively for the total red and yellow pigments. The obtained yield coefficient of the studied processes indicated the feasibility of Monascus pilosus C1 strain for pigments production. None of the investigated carbon or nitrogen sources provoked citrinin biosynthesis.

Over-expression of DAAO and catalase in Kluyveromyces marxianus through media optimization, permeabilization and GA stabilization techniques.

The selected thermotolerant, lactose-utilizing yeast strain Kluyveromyces marxianus NBIMCC 8362 possesses high specific d-amino acid oxidase activity (60Ug(-1)), which was increased nine-fold (545Ug(-1)) by design of the growth medium and conditions for d-amino oxidase induction. Applying an optimized simple and rapid procedure for chemical permeabilization of K. marxianus cells with the cationic detergent cetyltrimethylammonium bromide, the enzyme activities (d-amino acid oxidase and catalase) of the cells have been further increased for up to 43- and 58-fold, respectively. However, the enzyme activities of the permeabilized cells decreased rapidly due to the leakage of the enzymes. Treating the permeabilized cells with 0.1% glutaraldehyde at 4°C for 10min stabilized the enzyme in the cells and prevented their outflow. The process is stable for 10 cycles and the productivity measured was 16.6mmmoll(-1)h(-1). The d-alanine transformation efficiency of K. marxianus permeabilized and GA entrapted cells was 98%.

Role of Glutathione S-Transferases and Glutathione in Arsenic and Peroxide Resistance in Saccharomyces Cerevisiae: A Reverse Genetic Analysis Approach

ABSTRACT Glutathione S-transferases (GSTs) define an enzyme family of multifunctional proteins with important roles in cellular detoxification of exogenous and endogenous compounds. The availability of the complete genome sequence and of disruption mutants for all genes/ORFs of Saccharomyces cerevisiae facilitates genome-wide experimental approaches for the detailed understanding of eukaryotic detoxification pathways. In this work, the role of glutathione metabolism and in particular the specific requirement for GSTs for adaptation to oxidative and arsenic stresses in S. cerevisiae was evaluated by screening of a wide selection of gene-specific disruption mutants from the EUROSCARF collection.

Non-enzymatic roles for the URE2 glutathione S-transferase in the response of Saccharomyces cerevisiae to arsenic.

The response of Saccharomyces cerevisiae to arsenic involves a large ensemble of genes, many of which are associated with glutathione-related metabolism. The role of the glutathione S-transferase (GST) product of the URE2 gene involved in resistance of S. cerevisiae to a broad range of heavy metals was investigated. Glutathione peroxidase activity, previously reported for the Ure2p protein, was unaffected in cell-free extracts of an ure2Δ mutant of S. cerevisiae. Glutathione levels in the ure2Δ mutant were lowered about threefold compared to the isogenic wild-type strain but, as in the wild-type strain, increased 2–2.5-fold upon addition of either arsenate (AsV) or arsenite (AsIII). However, lack of URE2 specifically caused sensitivity to arsenite but not to arsenate. The protective role of URE2 against arsenite depended solely on the GST-encoding 3′-end portion of the gene. The nitrogen source used for growth was suggested to be an important determinant of arsenite toxicity, in keeping with non-enzymatic roles of the URE2 gene product in GATA-type regulation.