Applied Biochemistry and Microbiology最新文献

It is known that sorbitol metabolism in the yeast K. kurtzmanii and K. phaffii is limited by the level of sorbitol dehydrogenase expression. In the present work, we obtained transformants of K. kurtzmanii and K. phaffii that synthesized the thermostable endoglucanase Egh12 of Thielavia terrestris, xyloglucanase Xgh12b of Aspergillus cervinus, and cathepsin-L CathL of Tribolium castaneum under the control of the methanol-inducible promoter P_AOX1 and had different rates of sorbitol metabolism due to additional expression of the sorbitol dehydrogenase gene. Synthesis and secretion of the target enzymes were investigated on media containing methanol and sorbitol in different ratios. The results obtained showed that an increase in the rate of sorbitol metabolism in the producers had a negative effect on the synthesis of target proteins up to complete inhibition of their production. At the same time, low-efficiency sorbitol assimilation by these strains, limited by the expression of sorbitol dehydrogenase or by the concentration of sorbitol in the medium, promoted an increase in the secretion of Egh12 and increased enzyme production up to 30% by stimulating the growth of the cell biomass.

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 P_tuf 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 P_tuf, 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.

A new recipient strain, Komagataella phaffii VKPM Y-5229 (Δhis4Δopi1), has been developed. Its use allows constructing strains with increased production of recombinant proteins. The OPI1 gene encoding the Opi1 transcription regulator, a functional analogue of the negative regulator of lipid biosynthesis Opi1p from Saccharomyces cerevisiae, was identified. The OPI1 gene was knocked out in the K. phaffii VKPM Y-5013 (Δhis4) strain using the highly efficient CRISPR/Cas9 genome editing system. The effect of OPI1 gene inactivation on the production of recombinant heterologous enzymes, Citrobacter gillenii phytase and Priestia flexa β-amylase, was studied. The average productivity of transformants based on the VKPM Y-5229 (Δhis4Δopi1) strain secreting phytase or β-amylase increased by 18 and 25%, respectively, compared to the productivity of transformants derived from the parent strain. Furthermore, it was demonstrated that inactivation of the OPI1 gene in a multicopy phytase-producing strain led to an increase in productivity by more than 26%. The results obtained can be employed for developing industrial strains producing heterologous proteins based on K. phaffii, as well as for increasing the production of targeted proteins in existing producing strains.

Abstract—In the first part of the review presented, the role of L-methionine in the vital activity of various organisms and its application are considered. The main methods of obtaining it are presented briefly. The pathways of biosynthesis of L-methionine and its precursors, as well as their regulation in industrially significant bacterial species Escherichia coli and Corynebacterium glutamicum, are described. The current information on the uptake and assimilation reduction of sulfate and thiosulfate in these bacteria as the most important elements of metabolism limiting the possibility of oversynthesis of L-methionine is presented. At the same time, some approaches used in the creation of effective producer strains for the production of L-methionine using biotechnological methods are mentioned.

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.

This review is devoted to the main approaches of systems metabolic engineering used in the development of Corynebacterium glutamicum–based producers of L-valine, an essential proteinogenic amino acid that is widely used as a feed additive. This direction of engineering is based on the concepts of systems biology, considering the living organism as a holistic system with complex interaction between elements. This review considers the following approaches: laboratory evolution as a way to obtain new mutations that increase L-valine production; the use of biosensors to select clones with such mutations; “omic” data obtained for C. glutamicum strains capable of producing L-valine and related amino acids; modification of genes encoding global regulatory factors; and modeling of metabolic pathways relevant to amino acid production in C. glutamicum using full genomic data. Each of the approaches is evaluated in terms of its effectiveness in increasing the productivity of strains and improving their technological characteristics. In addition, information is presented on modern methods of genome editing of C. glutamicum, without which it is impossible to carry out a large number of targeted modifications necessary for the realization of systems metabolic engineering approaches.

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 recombinant strain of the vaccinia virus, VV-mNIS-dGF, has been constructed. The genome of this strain contains an insertion of the mouse sodium/iodide symporter transgene (mNIS). This insertion is located in the deleted region of the viral thymidine kinase gene (tk). In addition, the viral growth factor gene (vgf) was deleted, ensuring that the virus had an attenuated effect on normal cells without affecting its replication in cancer ones. The antitumor activity of the VV-mNIS-dGF strain in two mouse cancer models, melanoma B16-F10 and colorectal carcinoma CT-26, was studied. In both models, the strain exhibited high oncolytic activity, efficiently expressed the mNIS and accumulated iodide in tumor cells in both in vitro experiments and in vivo on mouse allografts. Quantification of the iodide content in tumors was performed by elemental analysis using X-ray Fluorescence Analysis with Synchrotron Radiation (XRF-SR). The results indicate the possibility of using the VV-mNIS-dGF strain for both therapy and diagnosis (theranostics) of malignant tumors.

Abstract—Heavy metal (HM) pollution has become one of the important threats to biological processes occurring in soil and to soil microbiota. This threat also extends to the active component of the soil microbial biota—fungi. In soils polluted with HMs, fungal communities undergo significant structural changes, leading, in particular, to an increase in the proportion and diversity of HM-resistant fungi, among which opportunistic human pathogens and facultative plant pathogens are found. The strains of fungi opportunistic for humans isolated from polluted soils have pathogenic properties to a greater extent than the strains isolated from clean soil. The implementation of pathogenic properties is determined by a number of molecular factors (virulence factors), among which are the activity of certain groups of enzymes, the action of antioxidant defense mechanisms and effector proteins, the synthesis of melanin and toxins, and modification of the lipid and carbohydrate composition of the cell. Heavy metals and soil pollution with them can influence the implementation of the pathogenic properties of fungi and fungus-like organisms that are toxic for humans, animals, and plants. This review provides data from the literature on molecular virulence factors and their modifications under the influence of HMs. Considering that at the moment there is no clear understanding of the direction of the action of HMs on the implementation of pathogenic properties by fungi, the literature data analyzed can still contribute to understanding the consequences of soil pollution with HMs for the development of opportunistic human pathogens and facultative phytopathogens.

Abstract—The Brevibacillus laterosporus strain with a high level of bactericidal and fungicidal activity was used for the first time for the biosynthesis of silver-containing nanoparticles of various compositions. Nanomaterial samples were obtained by reduction of Ag⁺ from AgNO₃ solution or by a chemical reaction between Na₂S₂O₃ and AgNO₃ in the presence of B. laterosporus cells. When assessing the chemical composition and crystal structure of the nanomaterial, it was found that the shape of the nanocrystals was close to spherical, and the particle sizes varied from 2 to 15 nm, with an average value of 5–7 nm. The values of the ζ-potentials of the samples were close and were –23…–25 mV. The dimensions of the hydrodynamic diameter indicated the presence of a bio-layer on the surface of nanocrystals of various types. The presence of proteins on the surface of nanoparticles was established by electrophoresis, and the selectivity of sorption of protein molecules of various molecular weights from the culture liquid of B. laterosporus on their surface was shown. Several proteins have been identified by mass spectroscopy. A difference in the level of biocidal activity of silver and silver sulfide nanoparticles formed in the presence of B. laterosporus cells was found in relation to gram(+) and gram(–) bacteria and yeast.