Kurchatov Institute

The experimental data on the study of the taxonomic position and biological properties, including fungicidal and protective activities, of a strain of spore-forming bacteria typified as Bacillus amyloliquefaciens based on the analysis of sequences of variable regions of the 16S rRNA gene are presented. The strain was established to have a wide spectrum of fungicidal activity and to suppress the development of representatives of six genera of phytopathogenic fungi that play a major role in the pathogenesis of agricultural crops, namely, Fusarium (F. solani, F. oxysporum, F. graminearum, F. nivale), Rhizoctonia (R. solani), Alternaria (A. tenuis), Phoma (P. solanicola), Sclerotinia (S. sclerotiorum), Botrytis (B. cinerea), and Magnaporthe (M. grisea). In vitro and in vivo experiments for the culture fluid of the B. amyloliquefaciens 16-1111 strain demonstrated, on the one hand, the absence of phytotoxicity and, on the other hand, protective and stimulating effects on germinating seeds of vegetable crops of the family Cucurbitaceae and plants cultivated in protected ground.

The recombinant strain based on the acidophilic yeast Schizosaccharomyces pombe with a cloned gene of heterologous lactate dehydrogenase (LDH) was used to optimize the biosynthesis of L-lactic acid. For this purpose, the effect of inactivation of pyruvate decarboxylase genes (PDC) on the synthesis of acetate, pyruvate, and ethanol (the main by-product in the synthesis of lactic acid) was studied. Using the wild S. pombe strain, we showed that the Δpdc3 and Δpdc4 deletions did not affect these indicators, while in the Δpdc1 mutant, ethanol biosynthesis was reduced and acetate biosynthesis was increased, and the Δpdc2 mutant accumulated pyruvate. The effect of deletions of the PDC1 and PDC2 genes on lactic acid biosynthesis was tested on a model S. pombe strain containing a heterologous LDH gene from Lactobacillus pentosus. It was shown that in strains with the inactivated PDC2 gene the accumulated pyruvate was poorly consumed for the biosynthesis of lactic acid even in the presence of two different recombinant lactate dehydrogenases; the introduction of the third gene of heterologous lactate dehydrogenase led to loss of viability of the strain. At the same time, in strains with the deleted PDC1 gene, the biosynthesis of lactic acid was enhanced with the introduction of additional LDH genes. The results obtained can be used in the design of industrial lactic acid producing strains.

Previously, it was demonstrated that recombinant spidroins rS1/9 and rS2/12 are able to glue various inorganic and organic materials. It is known that mussel foot proteins have unique adhesive properties associated with a high content of L-3,4-dihydroxyphenylalanine (DOPA), which is formed due to modification of tyrosine residues by the enzyme tyrosinase. We constructed a hybrid protein, which contains recombinant mussel foot protein 3 (Mfp3), as well as the recombinant tyrosinase used for modifying the tyrosine residues in DOPA in all recombinant proteins (rS1/9, rS2/12, and Mfp3 in the composition of the hybrid protein). Such modification led to an enhancement of the adhesive properties of these proteins, while the combination of modified spidroins with Mfp3-DOPA significantly increased their adhesive ability, which was demonstrated in the experiments on gluing various materials. The results obtained indicate the high potential of the hybrid protein containing recombinant modified Mfp3 for use alone or as an additive to recombinant spidroins for medical use.

The production of target proteins in Escherichia coli cells can be greatly simplified if they are synthesized in a biologically active state as part of active inclusion bodies (AIBs), which can easily be isolated from cells by centrifugation. This is a new technology, so the question about the protective properties of AIBs specific for standard inclusion bodies still remains open. This work describes the synthesis of the recombinant protein L6KD-SUMO-[R34-GLP-1(7–37)], which forms AIB, in E. coli BL21(DE3) cells. This protein engineered from a novel, recently developed L6KD-SUMO platform incorporates a modified human glucagon-like peptide-1, R34-GLP-1(7–37), the active substance of Liraglutide-based drugs. It was shown that, the soluble protein His10-SUMO-[R34-GLP-1(7–37)] expressed by E. coli, retained the peptide intact only for 24 h, but the peptide integrity in the AIB composition was maintained over 70 h of cell cultivation. Thus, it is logical to assume that AIB formation has a protective effect on target compounds synthesized by the cell.

A new Escherichia coli–Rhodococcus bireplicon plasmid pRY3-Rho-MCS, which is low-copy in Rhodococcus and multicopy in E. coli and stably inherited in Rhodococcus, was constructed on the basis of the previously used plasmid pRY16. The new plasmid is capable of conjugative transfer from E. coli to R. rhodochrous and R. qingshengii. The resulting R. rhodochrous and R. qingshengii strains with the new plasmid stably overproduced the green fluorescent protein turboGFP under the control of the corynebacterial Ptuf promoter. The efficiency of the target gene expression in the engineered strains was evaluated by flow cytofluorimetry and the possibility of using this technology in the search for highly active promoters for target gene expression in Rhodococcus was demonstrated. The instability of inheritance of the original plasmid pRY16 in R. rhodochrous cells is potentially suitable for the development of CRISPR/Cas-based methods for genome editing of Rhodococcus spp. It is concluded that the obtained tools, the plasmid pRY3-Rho-MCS and the promoter Ptuf, have a significant potential for application in biotechnology, including the development of biocatalysts, as well as producers of bacterial proteins that are difficult to synthesize in E. coli and other model microorganisms.

In order to increase the lytic activity of the enzyme, structures of the recombinant modified CHAP domain of the phage K endolysin to Staphylococcus aureus have been developed, including (starting from the N-terminus) a sequence of various cationic peptides HB(X) fused with the sequence of the CHAP domain through the GSG4S linker region. Genetic engineering constructs encoding the new HB(X)-CHAP were obtained, which were cloned and expressed in the recipient strain E. coli BL21(DE3). All variants of HB(X)-CHAP, as well as the control variant of the CHAP domain, were isolated and purified using a single technique involving a combination of cation and anion exchange chromatography. The lytic activity of the obtained enzymes was studied by the turbidimetric method using an autoclavable culture of S. aureus. For the two most promising HB(X)-CHAP variants from the point of view of further use, the main physicochemical characteristics are determined. It was shown that the presence of the GSG4S linker site in the structure of the molecule led to at least a twofold increase in the activity in the lysis of S. aureus cells, while the cationic peptides did not have a positive effect on the lytic activity of endolysin against Staphylococcus aureus. The obtained data may allow a rational approach to the issue of choosing an approach to optimizing the structure in the future and will expand the possibilities for designing endolysins in order to create an effective drug.

Promoters are crucial components of yeast production strains in metabolic engineering. Due to its high activity, the promoter of the RPL25 gene, which encodes the L25 protein of the large ribosomal subunit, is a promising object for investigation and optimization. Here, to investigate the Yarrowia lipolytica pRPL25 promoter structure, a randomized library of its sequences was generated in 100 nucleotide increments. The strength of the obtained promoters was evaluated using the green fluorescent protein as a reporter. The results showed that the minimum functional length of the studied pRPL25 derivatives was 199 bp, and the optimal functional length was 497 bp. When the sequence was shortened to 400 bp, the expression level of the reporter decreased significantly. Based on the above, we propose that the 400–500 bp region of pRPL25 comprises an upstream activating sequence (UAS). The pRPL25 promoter and its derivatives potentially can be used in bioengineering to create metabolic pathways with a precise level of gene expression. Strains constructed on their basis may be more efficient producers of target compounds compared to classical expression systems such as Saccharomyces cerevisiae.

A method for molecular serotyping of Bacillus turingiensis strains has been developed. It is based on comparison of nucleotide sequences of conserved regions of the flaA gene in five B. turingiensis serovars: sotto, kurstaki, israilensis, berliner, and morrisoni. For amplification of the target DNA fragment, a set of primers universal for all studied serovars of B. turingiensis was designed. To assign a strain to a particular serovar, a new algorithm based on the degree of homology of the conserved flaA region was developed. Validation of the method was carried out on 11 strains of B. turingiensis from the collection of the National Bioresource Center All-Russia National Collection of Industrial Microorganisms (VKPM). The results obtained by molecular serotyping fully coincided with the data of serological methods.

The biotechnological production of citric acid (CA) is one of the largest and fastest-growing sectors in the global biotechnology market. It is produced in crystalline form and as citrate salts and is widely employed in the food industry, household chemicals, cosmetics, metallurgy, and the petroleum extraction sector. Global CA production exceeds two million tons per year and is increasing by approximately 4% annually. Traditional biotechnological CA production relies on the utilization of mycelial fungi, specifically Aspergillus niger as the producer. Various alternative CA producers, including yeast and bacteria-based systems, have been described. However, from an economic standpoint, they lag behind industrial Aspergillus niger producers. Among the most promising, environmentally friendly, and high-tech CA producers are strains of the yeast Yarrowia lipolytica. The development of competitive biotechnologies for CA production using Y. lipolytica yeast strains with glucose as a carbon source has become feasible due to advancements in understanding the genetic control of CA transport and metabolism within the cell.

Resistive switching (RS) memory devices incorporated with the capabilities of in situ data sensing, storing and processing are promising for artificial intelligence applications. In this respect, controlling resistance not only...

Influenza infection represents a serious challenge for virological surveillance and healthcare systems in all countries globally. Despite obvious success in control of influenza through vaccination and antiviral drug development, this infection remains poorly controlled due to antigenic drift and fast selection of drug‐resistant viral variants. The design of novel drugs with alternative targets and mechanisms of action is, therefore, an important goal for medical science worldwide. In the current study, we describe the chemical synthesis of novel tetrahydro‐β‐carboline derivatives containing a thiohydantoin fragment, as well as their antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1). In general, the library of compounds was of low toxicity. Of the 23 compounds under investigation, 10 (43.5%) displayed a selectivity index (SI) of 10 or higher, their activity strongly exceeding that of the reference compound rimantadine. The most active compounds have also demonstrated suppressing activity against the phylogenetically distinct influenza virus of type B. These compounds, similar to the reference compound zanamivir, were active at very late stages of the viral cycle (4–6 h postinfection), suggesting interference with processes of virion assembly and budding. However, no direct inhibiting activity against viral neuraminidase has been demonstrated. The results obtained can be considered as a rationale for further structural optimization and study of this group as potential broad‐range antivirals effective against influenza viruses.