Applied Biochemistry and Microbiology最新文献

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.

Abstract—Calcium–magnesium silicate ceramic diopside is an effective carrier of BMP-2 and, in combination with a bovine demineralized bone matrix as a scaffold, can induce effective reparative osteogenesis on the model of craniotomy in mice. It was of interest to investigate the efficacy of combined usage of BMP-2-loaded diopside with recombinant spidroins for the induction of osteogenesis. For this purpose, porous implants were prepared based on recombinant spidroin rS1/9, into which BMP-2 was introduced either as BMP-2 loaded diopside particles suspended in hyaluronic acid or by adsorption. Neo-osteogenesis with bone marrow formation occurred in both groups with BMP-2, most pronounced in the group with BMP-2 and diopside on the model of critical size defects in the cranium in mice. Thus, the use of 3D scaffolds based on recombinant spidroin with BMP2-loaded diopside particles in hyaluronic acid can lead to effective induction of osteogenesis. The spidroin scaffolds themselves represent a sufficiently effective carrier for BMP-2.

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 first part of this review is devoted to an analysis of the beneficial effect of flavonoids on the prevention, course, and outcomes of COVID-19. In addition to the well-known antioxidant, anti-inflammatory, immunomodulatory, antithrombotic, and antitumor effects, flavonoids, secondary metabolites of many plants, exhibit antiviral activity and low cytotoxicity. The basic stages of SARS-CoV-2 replication and COVID-19 pathogenesis are considered to identify possible targets for flavonoids. The effect of these polyphenolic compounds on SARS-CoV-2 both in in vitro systems and in animal models that adequately reproduce COVID-19 symptoms have been analyzed. The results of the first clinical trials on the prevention of this disease and the feasibility of using flavonoids, mainly quercetin, as adjuvant therapy are discussed.

Surfactin is a powerful surfactant of biological origin with a wide range of potential applications in industry and medicine. The main factor limiting its active utilization is the high cost of production. Due to the complexity of the structure, the chemical synthesis of surfactin is unprofitable, so the main approach to its production is microbiological synthesis. This work demonstrated for the first time the ability of B. subtilis NCIB 3610 to synthesize surfactin. This strain possesses properties lost by most B. subtilis model laboratory strains. The nucleotide sequences of genomes of B. subtilis NCIB 3610 and B. subtilis PY79 strains have been obtained by nanopore sequencing. А comparative analysis of the obtained genomes with genome of B. subtilis 168, genetically modified derivatives of which are patented surfactin producers, has been performed. No mutations affecting surfactin biosynthesis were found in the NCIB 3610 strain, while in the PY79 strain mutations were identified in genes involved in cellular processes competing with biosynthesis of the surfactin. Thus, the results obtained in this work make it possible to consider B. subtilis NCIB 3610 and PY79 as alternative base strains for the genetic engineering design of surfactin superproducers.

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.

Enhancing cobalt tolerance is crucial for microbial adsorption and recovery of cobalt from wastewater. In this study, metabolic engineering of Saccharomyces cerevisiae was conducted to systematically investigate the effects of modifying cell wall and membrane components and regulating intracellular antioxidant substances glutathione (GSH) and S-adenosylmethionine (SAM) on the improvement of cobalt tolerance. The cobalt tolerance was increased to 157% by overexpression of the erg4 and erg6 genes in the ergosterol metabolic pathways. In the strain where the key cell wall synthesis gene fks2 was knocked out, the cobalt tolerance was increased by 63%. However, this study also found that there was no direct correlation between the intracellular GSH and SAM levels and the cobalt tolerance of the strain. This study provides theoretical support for further improving the tolerance of S. cerevisiae to cobalt ion in the future.

A derived actinomycete, Streptomyces sp. SA0215 was isolated from Saudi Red Sea sediments; the strain was identified by morphology and by phylogenetic analysis of the 16S rRNA gene sequence. The chemical analysis of its culture broth yielded 9 secondary metabolites. The compounds were isolated by a series of chromatographic steps, and their structures established by detailed spectroscopic analysis of the NMR and MS data. Pluramycinone metabolites 1-6 with the structurally related indomycinone analogous as saptomycin-A (1), saptomycin-F (2), α-indomycinone (3), β-indomycinone (4), γ-indomycinone (5), kidamycin (6) and furthermore, resistomycin (7), 1-acetyl-β-carboline (8) and ergosterol peroxide (9) were identified from the culture broth extract. Bioactivity of the indomycinones 1–6 was evaluated in antimicrobial and cytotoxicity assays. β-Indomycinone (4) was found to exhibit potent broad spectrum antibacterial activities against the tested pathogens Klebsiella pneumoniae, Escherichia coli and Staphylococcus aureus with inhibition zones diameters of 18, 20 and 24 mm, respectively, compared to the inhibition zones of the antibiotic reference oxytetracycline. The MIC values against the same pathogens were determined 7.2, 11.5, and 13.7 µg/mL, respectively. While γ-indomycinone (5) displayed high activity against the Gram-positive S. aureus with MIC value 10.3 µg/mL, compared to the potent activity of the crude extract (MIC = 9.6 µg/mL). Moreover, saptomycin-A (1) displayed the highest cytotoxic activity against human HepG2 cells with an IC₅₀ value of 19.3 μM, followed by β-indomycinone with an IC₅₀ value of 23.5 μM, while the extract showed the highest activity against human MCF7 cells with an IC₅₀ value of 30.5 µg/mL.

Tyrosinase-related protein 1 (TRP-1) has been known to play an important role in melanogenesis. While we had studied TRP-1 gene knockout using the CRISPR/Cas9 system, it was found that TRP-1 gene knockout could induce apoptosis of human fibrosarcoma cells (HT1080). The purpose of this study was to investigate the effect of TRP-1 knockout on the induction of apoptosis in HT1080 cells. The TRP-1 KO cells were successfully established using the CRISPR/Cas9 system, and the frameshift mutation was verified by the Sanger DNA sequencing analysis. The modified TRP-1 structure by frameshift mutation caused a decrease in gene and protein expression in TRP-1 KO cells. The TRP-1 KO cells were phenotypically changed and induced apoptosis. The flow cytometry analysis showed the early apoptosis phase. Furthermore, the TRP-1 KO cells decreased the scavenging activity of hydrogen peroxide, compared to normal cells. DNA fragmentation and TUNEL assays confirmed the apoptotic cell death in these cells. The expression levels of proteins such as Caspase 3, Bax, and Bcl-2 were positively modulated in the apoptosis induction of TRP-1 KO cells in a mitochondrial-dependent pathway. The expression levels of Catalase, SOD1, and SOD2 were remarkably decreased in the these cells, compared to normal cells. The expression levels of MAPKs kinase such as p38, ERK1/2, JNK, and NF-κB related to cell proliferation were also reduced in the TRP-1 KO cells. Therefore, our findings suggest that TRP-1 could play an important role in apoptosis in addition to melanin production.

Microbial antioxidative enzymes play a crucial role environmental and biomedical application. The key environmental applications of microbial antioxidative enzymes include addressing challenges related to pollution, waste management, and environmental degradation. The main antioxidant enzymes involved in converting hazardous wastes into nontoxic or biodegradable materials include catalases, superoxide dismutases, xanthine oxidases, and glutathione peroxidases. In living cells, antioxidative enzymes cooperate to shield cells from excess reactive oxygen species derived from endogenous metabolism or the external microenvironment and eliminate oxidative stress. These enzymes are crucial to the pharmaceutical, cosmetic, and food industries as well as in the treatment of a number of diseases. Biotechnological production of these enzymes involves both solid-state and submerged fermentation processes. Solid-state fermentation is gaining popularity over submerged processes because it is economical and environmentally benign. The bioprocessing for the production of antioxidative enzymes involves diverse microorganisms. The enzymes are purified by chromatography methods such as gel filtration, gas chromatography, ion exchange and HPLC. The present study focused on microbial antioxidative enzyme production and its applications. This review comprehensively covers the diversity of antioxidative enzyme-producing microorganisms, the biotechnological production of antioxidative enzymes, the bioprocessing parameters and the applications of antioxidant enzymes in various fields.