Prikladnaâ biohimiâ i mikrobiologiâ

The identification of risks associated with novel agricultural products of plant origin obtained via genome editing is an important aspect of genetic engineering. An extensive discussion is currently ongoing worldwide to clarify the similarities and differences between the “old” risks of “classic” GM plants and the “new” ones associated with genome editing, the lack of existing methods for identification and assessment of new risks. We propose here the concept of “safe by design” as applied to protection that is a new interesting tool that introduces good known standards of safety into plant bioengineering. This approach states that design options are identified to minimize or prevent risks and off-target of genome editing at the concept stage. The correlation between experimentally determined and in silico predicted off-target gRNA activity is a major challenge in the CRISPR system application. Today the most studies are focused on efficiency of gRNA design, while we pay attention specifically to the bioinformatics search and study of potential promoters, as the potential risk associates with a possible unplanned change in the transcriptional activity of promoters. We conveyed these strategies in the form of a risk assessment framework for regulation of new genetic technologies.

The review analyzes current information about the anaerobic type of respiration using a non-natural methacrylate compound as an electron acceptor. Both the methacrylate redox systems themselves and the anaerobic bacteria in whose cells they are found are considered. These complexes consist of flavin-containing reductase and multiheme cytochrome(s) c₃. The genes of the components of the methacrylate redox systems of different microorganisms are homologous and are organized into one operon. Methacrylate-reducing activity is determined in the periplasm. The only known bacterial acrylate reductase that reduces the natural compound differs from methacrylate redox systems. The physiological role, origin, and research perspectives for this unique enzyme system are discussed.

The biosynthesis of succinic acid from glucose by the previously engineered E. coli strain SUC1.0 (pMW119-kgd) (MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, P_Lglk, P_tacgalP, ∆aceBAK, ∆glcB, ∆sdhAB, pMW119-kgd) was optimized. The yield of the target substance was increased, upon the activation in the strain of the variant tricarboxylic acid cycle, mediated by the action of heterologous 2-ketoglutarate decarboxylase, due to the intensification of the anaplerotic formation of oxaloacetic acid. Inactivation of the nonspecific thioesterase YciA in the strain did not considerably change the biosynthetic characteristics of the producer. The enhancement of the expression of native phosphoenolpyruvate carboxylase led to an increase in the yield of the target compound by the recombinant synthesizing succinic acid via the reactions of the native tricarboxylic acid cycle from 25 to 42%, and from 67 to 75% upon the induced expression of Mycobacterium tuberculosis 2-ketoglutarate decarboxylase. Expression in the strain of pyruvate carboxylase gene from Bacillus subtilis resulted in an increase in the yield of succinic acid up to 84%. Functioning in whole-cell biocatalyst mode, the engineered strain SUC1.0 P_L-pycA (pMW119-kgd) demonstrated a substrate to target product conversion ratio reaching 93%, approaching the corresponding theoretical maximum.

Strains of Bacillus genus were isolated from soil samples in the permafrost region (Yakutia, Russia). The phenotypic characteristics of the strains are given. The analysis of the obtained data made it possible to assign them to the group Bacillus cereus complex. PCR analysis made it possible to determine the profile of B. cereus toxin synthesis genes in the genomes of the studied strains. Genetic characterization was obtained by RAPD genotyping and using MLVA loci used for genotyping of the anthrax pathogen. The results of genotyping at different levels of resolution made it possible to differentiate the studied strains from the B. anthracis species, to show their intraspecific genetic differences and the degree of relationship. Whole genome sequencing was carried out, based on the data of which MLST genotyping was carried out, which revealed 2 known sequence types and one new one, described for the first time in this work. The results obtained are of practical importance and are extremely interesting from the point of view of the evolution and phylogeography of the B. cereus complex group, since the fact that strains were isolated from permafrost suggests that their age may be much older than expected.

The interest in peroxidases of the basidiomycetes secreted enzyme complex is due to their wide substrate specificity and the ability of these enzymes to participate in the biodegradation of such difficultly degradable biopolymers as lignin. However, due to the difficulty of isolating these enzymes from native sources, their study is difficult. In this work, expression vectors were created that carried the sequence encoding the T. hirsuta LE-BIN072 versatile peroxidase VP2, which was transformed into the genome of the P. canescens strain. Screening of transformants showed the presence of peroxidase activity up to 1 U/mL. Fragments of the target protein in the culture liquid of the selected transformants were identified by mass spectrometric analysis. A new strain of P. canescens pVP2D-6, a producer of the recombinant universal peroxidase VP2 T. hirsuta LE-BIN072, was obtained for the first time, and the ability of the enzyme complex secreted by it to modify alkaline lignin was shown.

Transformation of 11-trifluoroacetate 6α-methylhydrocortisone (11-TFA MHC) by cells of actinobacteria Arthrobacter (Nocardioides) was carried out in the presence of α-cyclodextrin (α-CD). The composition and dynamics of the transformation products accumulation in the culture medium at various pH values and the ratio of α-CD/substrate were studied. It was shown that the addition of α-CD to the transformation medium at pH less than 7 promotes an increase in the rate of 1,2-dehydrogenation with the formation of 6 α-methylprednisolone 11-trifluoroacetate (11-TFA MPL). At pH over 7, the primary process is the hydrolysis of the 11β-trifluoroacetyloxy group. In this case, the participation of α-CD in these processes as an acceptor of the trifluoroacetyl ion is not excluded.