Yu. I. Deryabina

Properties of 2-C-Methyl-D-Erythritol 2,4-Cyclopyrophosphate, an intermediate in Nonmevalonate Isoprenoid Biosynthesis

Extraction and purification from the biomass of Corynebacterium ammoniagenes of 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate (MEC) was associated with its spontaneous transformation into a number of derivatives (which was due to the pyrophosphate bond lability and the formation of complexes with metals). These derivatives included 1,2-cyclophospho-4-phosphate, 2,4-diphosphate, 2,3-cyclophosphate, 1,4-diphosphate, and 3,5-diphosphate (identified by 1H, 31P, and 13C NMR spectroscopy) and accounted for about 10% of the MEC. When added to a solution of DNA in the presence of the Fenton reagent, MEC prevented DNA decomposition. In addition, MEC slowed down the interaction of the reagent with tempol radicals, which indicates that complexation of ferrous ions by MEC attenuates their ability to catalyze the formation of hydroxyl radicals from hydrogen peroxide. In the presence of 0.23 mM MEC, the rate of respiration of rat liver mitochondria increased by 1.8 times. At 0.1–1.0 mM, MEC activated in vitro proliferation of human Vgamma9 T cells. It is suggested that MEC acts as an endogenous stabilizing agent for bacterial cells subjected to oxidative stress and as an immunomodulator for eukaryotic hosts.

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).

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 μg/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.

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.

New polymer composites based on keratin and polyethylene

New biodegradable composites based on keratin and polyethylene have been produced under shear deformation. It has been demonstrated that the introduction of keratin leads to an increase in elastic modulus and to a decrease in ultimate tensile strength and elongation at break of the compositions. Elongation at break εb depends on the keratin dispersity; the highest εb values are observed for the compositions containing the smallest keratin particles. It has been shown that the compositions are susceptible to mold fungi; i.e., they are biodegradable.

Investigation of mechanical properties, morphology, and biodegradability of compositions based on polylactide and polysaccharides

The blends of polylactide with ethyl cellulose and chitosan are obtained in a Brabender mixer under conditions of high temperature shear deformation at different initial ratios of the reagents. The investigation of physicomechanical properties of the compositions has shown that the systems have high rigidity. Polyethylene glycol (PEG) and high-molecular-weight polyolefin polydecene have been added to the compositions in order to improve their elasticity. It has turned out that polydecene scarcely affects the mechanical characteristics, while PEG leads to a noticeable increase in elongation at break. Biodegradability of the compositions is investigated by the mass loss of samples after holding in soil, fungus resistance tests, and the analysis of the film morphology by scanning electron microscopy after holding in soil. It is found that the introduction of the third component (PEG) leads to an increase in biodegradability of the compositions.

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.

Carbohydrate Spectrum of Extremophilic Yeasts Yarrowia lipolytica under pH Stress

Alterations in the concentrations of cell cytosol carbohydrates of polyextremophilic yeasts Yarrowia lipolytica under stresses of diverse nature were observed. Under pH stress, mannitol was the main storage carbohydrate (up to 89% of the total cytosol carbohydrates), while arabitol, glucose, and inositol were present in insignificant amounts (3 to 6%). Experiments with inhibition of de novo mannitol synthesis by bis(p-nitrophenyl) disulfide revealed that the cytoprotective effect of mannitol was most noticeable in the cells grown under acidic conditions (pH 4.0), while the role of catalase and superoxide dismutase, the enzymes of the first line of antioxidant protection, increased under alkaline conditions (pH 9.0). The constitutively high mannitol content in Y. lipolytica cells was hypothesized to be a part of the core mechanism of stress resistance in this yeast species.

Mitochondrial cytopathies: Their causes and correction pathways

Mitochondrial cytopathies are a heterogeneous group of systemic disorders caused by mutations in mitochondrial or nuclear genome. The review presents some data on pathogenic mutations in mitochondrial DNA leading to the imbalance in the oxidation phosphorylation processes and energy metabolism in the cells and eventually to the development of mitochondrial cytopathy. The pathways of medicated correction are examined, which are aimed at obtaining optimal energy efficiency of mitochondria with impaired functions, increase of the efficiency of energy metabolism in the tissues, as well as prevention of mitochondrial membrane damage by free radicals using antioxidants and membrane protectors. A conclusion is drawn on the inefficiency of currently used therapeutic strategies and the necessity of new approaches, which can be gene therapy of mitochondrial diseases. Some modern methods for gene defects correction, capable of restoring or removing the damaged gene, expressing full gene product, or blocking the mutant or strange genes work are analyzed. It is shown that the described approaches to the gene therapy of human mitochondrial diseases demand the introduction of foreign sequences into nuclear or mitochondrial genome of a living person, which completely excludes their practical application because of the uncertainty of the outcome. A perspective approach in solving this problem may be a creation of a system allowing the correction of defect genes without introducing synthetic nucleotides into the human genome. Phenotypic selection combined with a capacity of homologous recombination, artificially imparted to mitochondria of yeast Yarrowia lipolytica, allows for replication of intact human mitochondrial DNA in yeast mitochondria, supporting a full-size native human mitochondrial DNA in the yeast cells and eliminating pathogenic mutations by means of standard sitedirected PCR mutagenesis. After the correction in the Y. lipolytica cells, copies of mitochondrial DNA of an individual patient may be returned to him using the transfection of mesenchymal stromal cells followed by selection of transfectants grown in minimal culture media, in which the cells with higher respiratory mitochondrial activity will gain the advantage.

New recombinant producer of human ω-amidase based on Escherichia coli

A new artificial gene encoding human ω-amidase (Nit2) adapted for highly efficient expression in E. coli has been established. A pQE-Nit2 plasmid construct controlled by the T5 promoter has been engineered for its expression. The nit2 gene within the pQE-Nit2 construct has optimized codon usage and an artificial 6His-tag sequence inserted directly after the ATG initiation codon. This tag provides the possibility of single-step purification of a product via metal chelate chromatography. The codon-usage optimization involves the inclusion of several codons of extremely rare occurrence in natural E. coli ORFs within a 30 a.a-long N-terminal region. Other codons included in the N-terminus have moderate occurrence in E. coli. The subsequent sequence of the artificial gene has been composed of the most frequently occurring codons in E. coli. The recombinant producer based on the pQE-Nit2 construct allowed purification of the enzyme with an activity of 6.2 ± 0.2 μmol/min/mg protein, which corresponds to or slightly exceeds the specific activity of rat liver Nit2. The omega-amidase preparation is necessary for the screening of potential inhibitors that can be used as candidate drugs to cure hyperammonemia disorders in liver pathologies and oncological diseases.