Influence of Natural Polyphenols on Isolated Yeast Dipodascus magnusii Mitochondria.

The effect of stilbene polyphenols (resveratrol and pinosilvin) and flavonoids (dihydromyricetin, epigallocatechin, and dihydroquercetin) on producing the reactive oxygen species (ROS) due to cell respiration using the isolated mitochondria of the fungus Dipodascus (Endomyces) magnusii was studied. It was shown that the inhibition of the ROS generation with stilbenes is related to both the oxygen consumption inhibition and their antioxidant properties. The degree of manifestation of polyphenol antioxidant properties depended on the number of hydroxyl groups in a polyphenol molecule. Of the flavonoids tested, dihydromyricetin was most active in reducing the ROS generation, but its inhibitory effect on oxygen consumption by mitochondria was significantly lower as compared to that of the stilbenes. The data obtained show that the mechanism of reducing the ROS generation with polyphenols is associated with both the respiratory inhibition and their antioxidant properties.

A New Recombinant Strain of Yarrowia lipolytica Producing Encapsulated Phytase from Obesumbacterium proteus.

A new recombinant strain of Yarrowia lipolytica synthesizing encapsulated highly thermostable phytase of Obesumbacterium proteus, which is recommended for use as a premix component of feed compositions in animal husbandry, was obtained.

Novel efficient promoter of the mitochondrial porin, voltage-dependent anion channel (VDAC), in the genome of the Yarrowia lipolytica yeast.

This article presents the characteristics of the highly inducible promoter of the gene encoding the mitochondrial porin, the voltage-dependent anion channel (VDAC). This promoter is recommended for use in new genetic constructs both in basic research for assessing the adaptive strategy of lower eukaryotes under adverse conditions and in designing new highly competitive transformants producing economically important compounds (proteins, lipids, and organic acids) on its basis.

Modified gene lacZ encoding ß-galactosidase as efficient transcriptional reporter for the Yarrowia lipolytica yeast.

A modified variant of the ß-galactosidase gene from Escherichia coli (lacZ) has been obtained. This was designed for building transcriptional reporter constructs, the product of which possesses a higher proteolytic resistance in the Yarrowia lipolytica yeasts. The study of the activity of mitochondrial voltagedependent porin (voltage-dependent anion channel, VDAC) showed a high efficiency of the reporter system based on the designed lacZ reporter compared to the wild-type lacZ gene.

[Study of the Accumulation of Rec A from Bacillus subtilis in the Mitochondria of a Recombinant Strain of the Yeast Yarrowia lipolytica].

No eukaryotic species has a system for homologous DNA recombination of the mitochondrial genome. We report on an integrative genetic systembased on the pQ-SRUS construct that allows the expression of the RecA recombinase from Bacillus subtilis and its transportation to mitochondria of Yarrowia lipolytica. The targeting of recombinant RecA to mitochondria is provided by leader sequences (5'-UTR and 3-UTR) derived from the SOD2 gene mRNA, which exhibit affinity to the outer mitochondrial membrane and provides cotranslational import of RecA to the inner space of mitochondria. The accumulation of RecA in mitochondria of the Y. lipolytica recombinant strain bearing the pQ-SRUS construct has been shown by immunoblotting of purified mitochondrial preparations.

[Genetic system for maintaining the mitochondrial human genome in yeast Yarrowia lipolytica].

For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.

[Biotechnological Applications of the Extremophilic Yeast Yarrowia lipolytica (Review)].

The review focuses on applications of Y. lipolytica that demonstrate the importance of this organism and the need for its further investigation and application in science and industry. The ability of this yeast species to adapt to various environmental conditions (including extremophilic), to grow on various substrates, and to synthesize useful products makes this strain very promising for biotechnological applications.

[The Engineering of a Yarrowia lipolytica Yeast Strain Capable of Homologous Recombination of the Mitochondrial Genome].

None of the studied eukaryotic species has a natural system for homologous recombination of the mitochondrial genome. We propose an integrated genetic construct pQ-SRUS, which allows introduction of the recA gene from Bacillus subtilis into the nuclear genome of an extremophilic yeast, Yarrowia lipolytica. The targeting of recombinant RecA to the yeast mitochondria is provided by leader sequences (5'-UTR and 3'-UTR) derived from the SOD2 gene mRNA, which exhibits affinity to the outer mitochondrial membrane and thus provides cotranslational transport of RecA to the inner space of the mitochondria. The Y. lipolytica strain bearing the pQ-SRUS construct has the unique ability to integrate DNA constructs into the mitochondrial genome. This fact was confirmed using a tester construct, pQ-NIHN, intended for the introduction of the EYFP gene into the translation initiation region of the Y. lipolytica ND1 mitochondrial gene. The Y. lipolytica strain bearing pQ-SRUS makes it possible to engineer recombinant producers based on Y. lipolytica bearing transgenes in the mitochondrial genome. They are promising for the construction of a genetic system for in vivo replication and modification of the human mitochondrial genome. These strains may be used as a tool for the treatment of human mitochondrial diseases (including genetically inherited ones).

[Redox Status of Extremophilic Yeast Yarrowia Lipolytica During Adaptation to pH-Stress].

In this study we investigated the activities of antioxidant enzymes (superoxide dismutases (SODs) and catalases (CATs)) and the ROS level in cells of Yarrowia lipolytica yeasts grown in a medium with different pH values (4.5, 5.5 and 9.0). It was shown that an increase in the cellular ROS level took place under both acid and alkaline conditions. The growth under extreme conditions was accompanied by a significant increase of SOD activity (by 2.5 times in the acid medium and by 4 times in the alkaline medium), but catalase activity did not change. A study of the electrophoretic profile of catalases showed the presence of three isoforms differing in inhibitor resistance. The electrophoretic profiles of SODs and their inhibitory analysis revealed there are two other isoforms, probably of mitochondrial origin, in addition to Cu and Zn SOD. The role of SOD in pH-adaptation of extremophilic Y. lipolytica yeasts is discussed.

[Comparative analysis of respiratory activity in the wild type strain of Neurospora crassa and its photoreceptor complex mutants].

Cell respiratory activity of protoplasts obtained from the wild type of Neurospora crassa and photoreceptor complex WCC--white collar 1 (wc-1) and white collar 2 (wc-2)--mutants of Neurospora crassa strains was investigated. Respiration inhibition by KCN in the presence of 25 mM succinate was similar in all strains and did not exceed 83-85% against control. The significant induction of KCN-resistant respiratory pathway occurred under 1% glucose oxidation in wc-1 and wc-2 mutants if compared with the wild type strains. The inhibitors of the main (cytochrome) pathway of electron transfer in mitochondria-1 mM KCN and antimycin A (4 microg/ml)--blocked the respiration rate of the protoplasts from N. crassa wild type by 75%, while the cell respiration of wc-1 and wc-2 strains was suppressed by approximately 50%. The specific inhibitor of alternative oxidase--10 mM salicylhydroxamic acid (SHAM)-in combination with the blockers of mitochondrial electron transfer chain caused the total suppression of respiratory activity of protoplasts in all studied strains. It is supposed that an increase of KCN-resistance in WCC mutants under glucose oxidation is connected with alternative oxidase activation as the result of failure in reception and signal transduction of active oxygen species.

[The regulation of peroxisomal matrix enzymes (alcohol oxidase and catalase) formation by the product of the gene Mth1 in methylotrophic yeast Pichia methanolica].

Two independent mutant strains of methylotrophic yeast Pichia methanolica (mth1 arg1 and mth2 arg4) from the initial line 616 (ade1 ade5) were investigated. The mutant strains possessed defects in genes MTH1 and MTH2 which resulted in the inability to assimilate methanol as a sole carbon source and the increased activity of alcohol oxidase (AO). The function of the AUG2 gene encoding one of the subunits of AO and CTA1, a probable homolog of peroxisomal catalase of Saccharomyces cereviseae, was investigated by analyses of the molecular forms of isoenzymes. It was shown that optimal conditions for the expression of the AUG2 gene on a medium supplemented with 3% of methanol leads to an increasing synthesis of peroxisomal catalase. The mutant mth1 possessed a dominant formation of AO isoform with electrophoretic mobility which is typical for isogenic form 9, the product of the AUG2 gene, and a decreased level of peroxisomal catalase. The restoration of growth of four spontaneous revertants of the mutant mth1 (Rmth1) on the methanol containing medium was accompanied by an increase in activity of AO isogenic form 9 and peroxisomal catalase. The obtained results confirmed the functional continuity of the structural gene AUG2 in mutant mth1. The correlation of activity of peroxisomal catalase and AO isogenic form 1 in different conditions evidenced the existence of common regulatory elements for genes AUG2 and CTA1 in methilotrophic yeast Pichia methanolica.

Calcium-dependent nonspecific permeability of the inner mitochondrial membrane is not induced in mitochondria of the yeast Endomyces magnusii.

Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore). For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and P(i), prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria. Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction. E. magnusii mitochondria were highly resistant to the above-mentioned substances. Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters. Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured. It is thus evident that E. magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.

Mitochondrial calcium transport systems: properties, regulation, and taxonomic features.

Currently available information on properties and regulation of mitochondrial Ca2+ transporting systems in eukaryotic cells is summarized. We describe in detail kinetic properties and effects of inhibitors and modulators on the energy-dependent Ca2+ uptake through the Ca2+ uniporter, as well as on Na+-dependent and Na+-independent pathways for Ca2+ release in mammalian mitochondria. Special emphasis is placed on Ca2+ transport systems (for ion uptake and release) in mitochondria of higher plants, algae, and yeasts. Potential physiological implications of mitochondrial Ca2+ fluxes (influx and efflux), e.g., regulation of activity of Ca2+-dependent enzymes of the Krebs cycle, maintaining of cellular Ca2+ homeostasis, and engagement in pathophysiological processes, are discussed.

Isolation and characterization of tightly coupled mitochondria from wild type and nap mutant Neurospora crassa.

A fast and reproducible procedure was elaborated for isolation of tightly coupled mitochondria from wild type and nap-mutant Neurospora crassa cells harvested at different growth stages. The isolated mitochondrial preparations had controlled metabolic states and were tightly coupled, i.e., displayed good respiratory control and had close to the theoretically expected maximal ADP/O ratios upon oxidation of Krebs cycle intermediates and exogenous NADH. They contained the fully competent respiratory chain with all three points of energy conservation. Oxidation of all examined substrates by mitochondria from both wild type and mutant cells was mediated by two alternative terminal oxidative systems, albeit to varying extent, with the more pronounced engagement of the alternative oxidase in the stationary growth phase and with a minor contribution of this non-phosphorylating pathway in the substrate oxidation by mutant mitochondria. Oxidation of NAD-dependent substrates by mitochondria from the two cell types was accommodated via both rotenone-sensitive and rotenone-insensitive pathways, while the level of rotenone-insensitive pathway in mutant cells was lower than in wild type cells. It is suggested that a more limited contribution of alternative non-phosphorylating oxidative pathways to the total respiration in mutant cells, as compared with wild type cells, could, at least partially, explain an elevated ATP level in these cells. However, the absence of principal differences in the arrangement of the respiratory chain in mitochondria of wild type and mutant cells implies that the elevated ATP level in the nap mutant is largely related to reduced ATP expenses for transport processes in these cells.

[Conditions for forming ethanol during bioconversion of cellulose-containing raw material]. / Usloviia obrazovaniia tanola pri biokonversii tselliulozosoderzhashchego syr'ia.

Several natural associations composed by thermophilic anaerobic bacteria capable of utilizing various cellulose materials at 60 +/- 2 degrees C and pH 6.0-7.0 were isolated from the sludge of Kamchatka geothermal springs. The rate of ethanol production (up to 1.7 g/l per day) and the concentration of ethanol in the medium (up to 1.2%), as well as the fermentation period (10-15 days) were determined under anaerobic conditions in the presence of cellulose, coniferous sawdust, newsprint, or paper pulp as a carbon source. Microorganisms were found that inhibited the production of ethanol. The initial pH value was found to influence both the ethanol production rate and ethanol/acetate ratio. A pH decrease from 7.0 to 5.0 led to 6.7-fold increased the ethanol production and caused a 23.8-fold increase in the ethanol/acetate ratio.

[Selection of a mixed culture of cellulosolytic thermophilic anaerobes from various natural sources]. / Selektsiia smeshannoi kul'tury tselliulozoliticheskikh termofil'nykh anaérobov iz raznykh prirodnykh istochnikov.

Microbial associations capable of converting cellulose-containing substrates to ethanol and organic acids were isolated from natural sources. The resulting mixed cultures utilized cellulose, cellobiose, glucose, maize residue, cotton, and flax boon producing ethanol (up to 0.9 g/l) and acetic acid (up to 0.8 g/l). The most complete conversion of cellulose-containing substrates occurred at 60 degrees C, pH 7.0. The selected association of thermophilic anaerobic bacteria produced 0.64 g ethanol per g substrate utilized at the ethanol/acetate ratio 4.7:1.

Adaptation to salt stress in a salt-tolerant strain of the yeast Yarrowia lipolytica.

We have studied the cellular mechanisms underlying adaptation to salt stress in a newly isolated osmo- and salt-tolerant strain of the yeast Yarrowia lipolytica. When cells are incubated in the presence of 9% NaCl, a rapid change in their size and shape is observed. Salt stress is accompanied by an increase in the intracellular level of glycerol, free amino acids (notably proline and aliphatic amino acids), and Na+, as well as by changes in lipid and fatty acid composition.