The effectiveness of the use of Sinorhizobium meliloti in the cultivation of variable alfalfa (Medicago varia Mart.)

As a result of three-year studies, we have established a growth in the yield and feed advantages of variable alfalfa in the variants with inoculation of seeds with virulent active strains of rhizobia. The total yield augmentation of green mass in the experimental variants over the three years of alfalfa life were 6.8–13.7 % compared to the control ones. The positive effect of inoculation with virulent active rhizobia strains on the total collection of dry matter was expressed in its increase in experimental conditions by 12.6–21.7 %. The highest yield of green mass, as well as dry matter was obtained in the variant with the inoculation of alfalfa seeds with the main production strain 425. The researched factor has a positive effect on the collection of feed units, digestible protein and feed protein units from 1 ha. The collection of feed units per hectare in the experimental versions increases by 1.1–1.3 times, the collection of digestible protein – by 1.2–1.4 times. The maximum substance of feed units and digestible protein per hectare was observed in the version with seed inoculation with strain 425a. The provision of a feed unit with digestible protein increases by 10.44–18.18 g or by 6.1–10.6 %.

Characterization of Methyltransferase from Apramycin Biosynthetic Gene Cluster and Improvement Production of Apramycin in Engineered Streptoalloteichus Tenebrarius

BackgroundApramycin is a structurally unique aminoglycoside, used in veterinary medicine or the treatment of Salmonella, Escherichia coli and Pasteurella multocida infections in farm. Although discovered and used many years ago, many biosynthetic steps of apramycin are still obscure. ResultsIn this study, we identified a HemK family methyltransferase, aprI, involved in apramycin biosynthesis. The function of aprI was studied by using gene disruption and biochemical experiments, and a new aminoglycoside antibiotic demethyl-apramycin was purified from aprI disruption strain. Experiments proved that AprI converted demethyl-aprosamine to aprosamine in vitro. Based on this, the apramycin production strain was improved by overexpression the AprI to decrease the impurity production. ConclusionsWe have identified aprI is a 7’-N-methyltransferase gene in apramycin biosynthesis and confirmed the substrate of methyltransferase. Engineering of aprI resulted in a strain producing a new aminoglycoside demethyl-apramycin and apramycin mono-producing strain with less impurity production. Finally, the yield of demethyl-apramycin in apramycin mono-producing strain decreased from 196±36 mg/L to 51±9 mg/L, and the yield of apramycin increased from 2227±320 mg/L to 2331±210 mg/L.

Preparation and transfer of Burkholderia mallei production strain 5584 in accordance with the biosafety requirements

The Veterinary Service of the Russian Federation takes measures to ensure regular control of livestock health status, to prevent infectious diseases and their introduction into the country; and if such diseases are diagnosed, it takes measures to prevent their spread and contain outbreaks as soon as possible. Success of the taken measures depends on the use of various diagnostic, preventive and therapeutic drugs. In order to produce such medicinal products, biofactories use production and reference strains with stable biological properties, which are stored in national collections of microorganisms. The only keeper of glanders strains is the Laboratory for Collection of Strains of Microorganisms in the FSBSI «FCTRBS-ARRVI», subordinated to the Ministry of Agriculture of the Russian Federation. The following steps were taken due to the official request from FKP Kursk Biofactory – BIOK Company for the transfer of Burkholderia mallei production strain 5584 from the collection of the institution: the strain was passaged in golden hamsters, its viability was determined and biological properties of the culture were studied. The strain was transferred in accordance with the established procedure and in compliance with the biosafety requirements. As the work progressed, Burkholderia mallei strain 5584 culture was isolated and freeze-dried. Before the transfer, biological properties of the freeze-dried Burkholderia mallei strain 5584 were studied for their compliance with the passport data. The obtained results showed that the Laboratory for Collection of Strains of Microorganisms in the FSBSI «FCTRBS-ARRVI» provides optimal conditions to preserve the strain viability and initial biological properties after 5 years of storage. Analysis of the data obtained during the transfer of Burkholderia mallei strain 5584 allowed us to assess the actions taken at all stages of the procedure. It was established that the transfer procedure for the requested glanders production strain complied with the biosafety requirements and regulatory framework regulating the process.

Particulate Mycobacterial Vaccines Induce Protective Immunity against Tuberculosis in Mice

Currently available vaccines fail to provide consistent protection against tuberculosis (TB). New, improved vaccines are urgently needed for controlling the disease. The mycobacterial antigen fusions H4 (Ag85B-TB10.4) and H28 (Ag85B-TB10.4-Rv2660c) have been shown to be very immunogenic and have been considered as potential candidates for TB vaccine development. However, soluble protein vaccines are often poorly immunogenic, but augmented immune responses can be induced when selected antigens are delivered in particulate form. This study investigated whether the mycobacterial antigen fusions H4 and H28 can induce protective immunity when assembled into particulate vaccines (polyester nanoparticle-H4, polyester nanoparticle-H28, H4 nanoparticles and H28 nanoparticles). The particulate mycobacterial vaccines were assembled inside an engineered endotoxin-free production strain of Escherichia coli at high yield. Vaccine nanoparticles were purified and induced long-lasting antigen-specific T cell responses and protective immunity in mice challenged by aerosol with virulent Mycobacterium tuberculosis. A significant reduction of M. tuberculosis CFU, up to 0.7-log10 protection, occurred in the lungs of mice immunized with particulate vaccines in comparison to placebo-vaccinated mice (p < 0.0001). Polyester nanoparticles displaying the mycobacterial antigen fusion H4 induced a similar level of protective immunity in the lung when compared to M. bovis bacillus Calmette-Guérin (BCG), the currently approved TB vaccine. The safe and immunogenic polyester nanoparticle-H4 vaccine is a promising subunit vaccine candidate, as it can be cost-effectively manufactured and efficiently induces protection against TB.

Maximizing PHB content in Synechocystis sp. PCC 6803: development of a new photosynthetic overproduction strain

PHB (poly-hydroxy-butyrate) represents a promising bioplastic variety with good biodegradation properties. Furthermore, PHB can be produced completely carbon-neutral when synthesized in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This model strain has a long history of various attempts to further boost its low amounts of produced intracellular PHB of ~15 % per cell-dry-weight (CDW).We have created a new strain that lacks the regulatory protein PirC (gene product of sll0944), which causes a rapid conversion of the intracellular glycogen pools to PHB under nutrient limiting conditions. To further improve the intracellular PHB content, two genes from the PHB metabolism, phaA and phaB from the known production strain Cupriavidus necator, were introduced under the regime of the strong promotor PpsbA2. The created strain, termed PPT1 (Δsll0944-REphaAB), produced high amounts of PHB under continuous light as well under day-night rhythm. When grown in nitrogen and phosphor depleted medium, the cells produced up to 63 % / CDW. Upon the addition of acetate, the content was further increased to 81 % / CDW. The produced polymer consists of pure PHB, which is highly isotactic.The achieved amounts were the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.

Molecular Genetic Testing of Stability and Identification of Vnukovo-32 Strain Used for Production of the Cultural Concentrated Purified Inactivated Dry Rabies Vaccine

Rabies is an acute viral disease caused by a virus of the Rhabdoviridae family of the Lyssavirus genus, which affects the central nervous system and is characterised by absolute mortality. Vaccination is the only way to prevent the disease in humans. One of the products used for vaccination is a cultural concentrated purified inactivated dry rabies vaccine produced by the Federal State Budgetary Institution of Science “Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences” (hereinafter—Chumakov Center).The aim of the study was to examine the structure of the working virus seed of Vnukovo-32 strain used by the Chumakov Center for rabies vaccine production, to assess its genetic stability during production, to explore the possibility of using molecular genetic methods for identification of the production strain in the finished dosage form, and to study the nucleotide sequence of the CVS strain.Materials and methods: Vnukovo-32 rabies virus production strain, working virus seeds, finished batches of the rabies vaccine, CVS fixed rabies virus strain used in the assessment of specific immunity. The molecular genetic study was performed using RT-PCR followed by restriction and sequencing.Results: the paper presents the results of nucleotide sequence analysis of the G gene fragment obtained from the Vnukovo-32 production strain, batches of the working virus seed, and finished batches of the rabies vaccine produced in 2012, 2018, and 2019, and the CVS fixed rabies virus strain used in the assessment of the vaccine’s specific immunity. The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.Conclusion: Vnukovo-32 and CVS strains used by the Chumakov Center are rabies viruses. Analysis of the nucleotide sequence of the G gene fragment showed that the Vnukovo-32 strain remains stable throughout different production stages. The obtained nucleotide sequence of gene G of the Vnukovo-32 strain was deposited in GenBank (accession number MN116503). The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.