Maxim O. Nagornykh

A study of bacterial xylanases from Streptomyces prunicolor, Streptomyces carpinensis, and Streptomyces venezuelae was conducted. The xylanase-encoding genes were cloned from bacterial genomes. Recombinant producers based on E. coli M15(Rep4) and the pQE-30 vector were obtained, and the expression and purification of the recombinant proteins were carried out. Electrophoretically homogeneous preparations of four xylanases from the GH10 and GH11 families were obtained. The xylanases had a near-neutral pH optimum and moderate thermal stability (the enzymes were stable at 70 °C). The xylanases were more stable over a pH range of 5.0 to 8.0. The properties of the xylanases obtained correspond to the properties of most xylanase-producing bacteria of the Streptomyces genus described in the literature. Xylanases from the GH10 family of S. carpinensis form xylose, xylobiose and xylotetraose as main products of xylan hydrolysis. The GH10 xylanase from S. venezuelae hydrolyzes xylan into xylose, xylobiose, xylotriose, xylotetraose and xylopentaose. The main products of xylan hydrolysis by GH11 xylanase from S. prunicolor are xylobiose, xylopentaose and a trace amount of xylose. All four xylanases effectively saccharify cereals, indicating the biotechnological potential of these enzymes.

The widespread use of antibiotics in medicine and agriculture has significantly accelerated the emergence rate of bacterial infections showing multiple antibiotic resistance. Since resistance to conventional antibiotics is developed rather quickly, designing alternative antimicrobial drugs with other mechanisms underlying their effects on bacteria is a promising. The enzymes possessing bactericidal activity may be one option for such antibacterial agents. The study aimed to produce the combination recombinant protein-based products active against bacteria and their biofilms. Soluble forms of five recombinant proteins were produced using the genetic engineering approaches. Two of these have a bacteriolytic effect (endolysins LysK and PM9 from the Staphylococcus aureus bacteriophages), the other are capable of disrupting extracellular DNA matrix in biofilms (two nonspecific nucleases NucA, as well as the DNA-specific deoxyribonuclease I). It has been shown that natural endolysin PM9 with the truncated catalytic domain shows 4 times lower bacteriolytic efficacy compared to the full-size LysK version. Comparative analysis revealed 1.5–2 timed higher efficacy of nonspecific nucleases in terms of bacterial biofilm disruption compared to the DNA-specific deoxyribonuclease I. It has been shown that simultaneous use of endolysins and nucleases has a synergistic antibacterial effect and disrupts biofilms of the pathogenic bacterium Staphylococcus aureus. The findings show the prospects of developing the recombinant protein-based antibacterial drugs.

When developing vaccines and immunotherapeutic and enzymatic drugs, it is usually necessary to obtain a functional protein preparation, which can later be used for immunization or analytical studies in the process of creating a drug. Production in E. coli bacterial cells is the fastest and cheapest way to obtain such proteins. However, recombinant proteins, when produced in E. coli, often form insoluble and functionally inactive aggregates. The purpose of this work is to create a set of expression vectors for rapid screening of optimal conditions for the production of recombinant proteins prone to aggregation in E. coli in soluble form. The work used modern genetic engineering methods, including optimization and de novo synthesis of nucleotide sequences encoding helper polypeptides. For the production and chromatographic purification of proteins, the standard strain BL21(DE3) and the affinity chromatography method using a metal chelate sorbent were used. As a result, a set of nine vectors with helper polypeptides was obtained to increase solubility and increase product yield during the production of recombinant proteins in E. coli cells. The efficiency of obtaining protein drugs prone to molecular aggregation using this set of vectors has been demonstrated by the exampe of three cytokines: interleukin-31 (IL-31), interleukin-33 (IL-33), and transforming growth factor beta (TGF-beta1). The resulting set allows for rapid selection of a suitable helper polypeptide as well as optimization of conditions for obtaining a soluble form of recombinant proteins in E. coli during the pharmaceutical development of therapeutic drugs.

In the genome of Cellulomonas flavigena, two genes that potentially encode endoglucanases — Cfla_2912 and Cfla_2913 were identified. We cloned the genes and created Pichia pastoris-based recombinant producers of two proteins that were expressed from the AOX1 promoter. Each of the endoglucanase molecules contains a GH6 catalytic domain, CBM2 carbohydrate-binding module, and TAT signal peptide. The fermentation of the producers was carried out in a 10 L fermenter; Cfla_2912 and Cfla_2913 were purified using affinity chromatography. The yield comprised 10.3 mg/ml (430 U/ml) for Cfla_2913 and 9 mg/ml (370 U/ml) for Cfla_2912. Cfla_2912 and Cfla_2913 were found to have a high activity against barley β-glucan and lichenan, a weak activity against carboxymethyl cellulose (CMC), phosphoric-acid treated cellulose, and no activity against laminarin, xylan, soluble starch, microcrystalline cellulose, cellobiose, and cellotriose. Thus, the proteins exhibited β-glucanase activity. Both proteins had a neutral pH optimum of about 7.0 and were more stable at neutral and slightly alkaline pH ranging from 7.0 to 9.0. Cfla_2912 and Cfla_2913 showed a moderate thermal stability. The products of barley β-glucan hydrolysis by Cfla_2912 and Cfla_2913 were trisaccharide, tetrasaccharide, and cellobiose. Cfla_2912 and Cfla_2913 efficiently hydrolyzed cereal polysaccharides, which indicate that they may have biotechnological potential.

Gene editing with programmable nucleases opens new perspectives in important practice areas, such as healthcare and agriculture. The most challenging problem for the safe and effective therapeutic use of gene editing technologies is the proper delivery and expression of gene editors in cells and tissues of different organisms. Virus-based and nonviral systems can be used for the successful delivery of gene editors. Here we have reviewed structural elements of nonviral DNA- and RNA-based expression vectors for gene editing and delivery methods in vitro and in vivo.

The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.