Heterologous Expression of Bacillus pumilus 3–19 Protease in Pichia pastoris and Its Potential Use as a Feed Additive in Poultry Farming

Proteases are one of the most innovative products used to improve the efficiency of feed additives. Due to their hydrolytic properties, they enhance the absorption of amino acids, which can reduce the protein content and the cost of feeds. The subtilisin-like proteinase of Bacillus pumilus 3–19 is a promising candidate for industrial use as a feed additive. However, in order to obtain a high yield of the enzyme, it is necessary to develop a highly efficient expression system. The aim of the study was to obtain stable expression of the optimized B. pumilus 3–19 protease gene in the Pichia pastoris expression system and evaluate the correlation of enzyme activity with the choice of vector type and signal peptides. The efficient secretion of subtilisin-like protease into the culture fluid of the recombinant yeast strains was confirmed. The study showed that the incubation time affects the synthesis of protease in P. pastoris, and the maximum activity of the enzyme was observed at 72 h of growth of the yeast culture. Yeast strains with constructs based on the low-copy vector pPINK-LC showed higher protease activity (U/mL) in the hydrolysis of azocasein (2.63 ± 0.16 for killer signal peptide (SP), 2.49 ± 0.08 for α-mating factor presequence, 2.19 ± 0.11 for lysozyme SP) than strains with constructs based on the pPINK-HC vector (1.86 ± 0.09 for killer SP, 2.21 ± 0.07 for α-mating factor presequence, 1.31 ± 0.11 for lysozyme SP), regardless of which signal peptide was used. The ability of the recombinant protease to hydrolyze a specific substrate confirms that the enzyme is a member of the subtilisin family. The maximum protease activity was obtained for yeast strains with pPINK-LC-killer-aprBp (5.75 ± 0.08 U/mL) and pPINK-LC-α-mat.factor-aprBp (4.33 ± 0.07 U/mL) constructs. This study demonstrated that the subtilisin-like protease from recombinant P. pastoris strains exhibits proteolytic activity, which depends on the incubation time and the choice of signal peptide and vector. The production of bacillary protease by the heterologous yeast-based expression system makes this system promising for the development of new feed additives for animal husbandry

Identification of Pantoea Phytate-Hydrolyzing Rhizobacteria Based on Their Phenotypic Features and Multilocus Sequence Analysis (MLSA)

Abstract—: Accurate strain identification within the Pantoea genus is difficult due to homologous recombination, which may affect the species boundaries. An integrated approach is presently the most effective one in determining the species of bacteria. Biochemical identification using the API20E system, phylogenetic analysis of the 16S rRNA gene sequences, and MLSA analysis based on partial sequences of the fusA, pyrG, leuS, gyrB, and rpoB genes showed that the soil phytate-hydrolyzing isolates belonged to the genus Pantoea, specifically to the species Pantoea brenneri. It was also established that phytate-hydrolyzing activity of the strains was accompanied by their ability to fix atmospheric nitrogen

Label-free multiple reaction monitoring, a promising method for quantification analyses of specific proteins in bacteria

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity-and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9-and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture

Expression of Pantoea sp. 3.5.1 AgpP Phytase in Three Expression Systems

Nowadays, microbial phytases have been widely used as feed additives that increase the bioavailability of phosphorus for monogastric animals. Still, there is an active search for new phytases, development of effective production systems, and study of their properties. In this study, we compared the biochemical characteristics of bacterial histidine acid phytase of Pantoea sp. 3.5.1 produced in three different expression systems: Escherichia coli, methylotrophic yeast Pichia pastoris, and yeast Yarrowia lipolytica. The maximum activity of the recombinant phytase AgpP-P expressed by P. pastoris occurred at pH 4.0, while the pH optimum of the recombinant AgpP-Y phytase expressed by Y. lipolytica is shifted towards more acidic pH 3.0. The recombinant AgpP-P enzyme is stable at pH values ​​from 2.0 to 5.0, while the AgpP-Y remains active at pH values ​​from 3.0 to 7.0; however, at pH above 8.0, the enzyme becomes inactive. The temperature optimum of recombinant AgpP-P phytase corresponded to 50 °С, while the temperature optimum for AgpP-Y was at 45 °С. The recombinant enzymes AgpP-P and AgpP-Y retained activity at temperatures from 4 to 70 °C and from 4 to 60 °C, respectively. Bivalent metal ions at a concentration of 1 mM had the same effect on the activity of recombinant phytases from E. coli, P. pastoris, and Y. lipolytica: Ca2 +, Mg2 +, and Mn2 + ions more than doubled the activity of enzymes, while Co2 + did not affect the activity of phytases, while ions Zn2 +, Cu2 +, and Fe2 + inhibited the activity of enzymes

Bistability and Formation of the Biofilm Matrix as Adaptive Mechanisms during the Stationary Phase of Bacillus subtilis

Abstract—: Bacilli control behavioral reactions such as motility, biofilm formation, production of enzymes and metabolites, differentiation, and others by integrating a variety of environmental signals through a complex regulatory network. In the natural environment, Bacillus subtilis exists predominantly in the form of biofilms, which has made it an ideal model for studying the molecular strategy of biofilm formation. This paper systematizes information on the main regulatory systems responsible for the loss of mobility and the formation of B. subtilis biofilms, analyzes the behavior of bacteria within the biofilm population, leading to a state of bistability and differentiation into different types of subpopulations. It also evaluates the regulatory relationship between control systems responsible for the synthesis of structural components in the biofilm matrix. Particular emphasis is placed on data concerning signaling mechanisms that trigger the formation of a biofilm and its dispersion. In general, we summarize information about the latest discoveries in this area and their integration into the general idea of these complex microbial communities

The role of various factors in the process of biofilm formation by bacilli

The strategy of existence of microorganisms in the structure of biofilms determines their adaptation to specific environmental conditions. Bacteria of the genus Bacillus are actively used as models for studying the processes of regulation and formation of biofilms. This work aims to assess the effect of proteases secreted by bacilli on the formation of biofilms, as well as on the resistance of recombinant strains to environmental stress factors. We compared the dynamics of biofilm formation by the wild strain B. subtilis 168 and the following strains with altered expression of extracellular proteases: non-protease strains (B. subtilis BG2036, B. subtilis BRB8, and B. subtilis BRB14) and strains with increased protease secretion (B. subtilis (aprBp), B. subtilis (gseBp), and B. subtilis (mprBp)). We also investigated the effect of ethanol and sodium chloride on the ability of the B. subtilis strains to form biofilms in a liquid medium. The bacterial strains were cultured at pH 7.4 and temperature 37 ?C in a synthetic E-medium in U-shaped 96-well plates. Biofilm formation was identified by incubation with crystal violet (CV). We found that the level of biofilm formation is higher (by an average of 10%) in the protease-deficient strains of B. subtilis. The recombinant strains expressing genes of serine proteases form biofilms with a reduced level (by an average of 40%). A strain with the expression of metalloendopeptidase is characterized by an increased level of biofilm formation (up to 10%), which is possibly due to the functional in vivo role of this new enzyme. The wild and protease-deficient strains of B. subtilis respond similarly to ethanol stress: biofilm formation is reduced in both of them. The wild strain exhibits a greater sensitivity to osmotic stress than the protease-deficient one

The effect of Bacillus subtilis GM2 and GM5 probiotics on the growth and fodder digestibility of broiler chickens

The probiotic properties of the Bacillus subtilis GM2 and GM5 strains were studied on 90 Cobb 500 broiler chickens aged one day that were randomly divided into three groups: the control group with the standard diet and two experimental groups (30 chickens per group) with the diet supplemented with B. subtilis GM2 (group 1) and GM5 (group 2) spores. The addition of B. subtilis GM2 and GM5 spores at a concentration of 1·107 CFU/g to the ration of broiler chickens improved their growth rate, as well as increased the digestibility of nutrients and modulated the intestinal microflora in them. The use of probiotics stimulated an increase in the live weight of chickens by 6.30% and 13.78% (p = 0.05) as compared with the control group. The average daily weight gain in experimental groups 1 and 2 amounted to 52.82 ± 0.36 g and 56.54 ± 0.47 g per chicken, which is more than in the control group (49.69 ± 0.40 g) – by 6.30% and 13.79% (p = 0.05), respectively. The administration of probiotics by feeding favored an increase in the population of lactic acid bacteria in the small and large (to a lesser extent) intestine – bacteria belonging to the Enterobacteriaceae, Lactobacillaceae, and Clostridiaceae families were isolated from the intestinal contents and identified. The veterinary-sanitary examination found that the meat of broiler chickens from the experimental groups meets all the GOST requirements for organoleptic, physico-chemical, and bacterioscopic characteristics. Thus, probiotics based on the B. subtilis GM2 and GM5 strains have a positive effect on the growth and fodder digestibility of broiler chickens

Bacterial enzymes as potential feed additives in poultry farming

The problem of obtaining economic benefits in poultry breeding from complete absorption of feeds by domestic fowls through increasing the digestibility of nutrients remains urgent. The addition of exogenous enzymes to broiler rations enhances the energy and protein intake by improving the substrate bioavailability. The presence of antinutritional dietary factors in poultry feeds (non-starch polysaccharides, protease inhibitors, lectins, and phytates) indicates the possibility of using exogenous enzymes. We assessed the potential of histidine acid phytase Pantoea sp. 3.5.1 and subtilisin-like proteinase B. pumilus as feed additives. When the enzymes pass through the digestive tract, they are exposed to elevated body temperatures of the fowl (40 ?C), pH changes in different parts of the digestive tract, and the influence of bile. Thus, it was important to study the stability of the enzymes under these conditions. It was shown that proteinase maintains its activity throughout the gastrointestinal tract of the fowl: in a weakly acidic environment (pH 5.5, goiter), the enzyme activity remained full (100%); at pH 2.9 (stomach), its decreased by 40%; and under the alkaline conditions of pH 6.5–8.0 (small and large intestine), its values were restored and increased by 13% relative to the control group. When exposed to bile at the concentrations from 0.01% to 0.25% for 1 h, phytase retained more than 50% of its activity (p < 0.05). At the concentrations of bile from 0.01% to 0.05% (1-h exposure), the activity of proteinase remained at the control level. The data obtained show that microbial enzymes in the digestive tract of domestic fowls retain catalytic activity. In the course of the balance experiment, the group of chickens treated with proteinase at the concentration of 10 U/kg demonstrated the highest nitrogen and calcium digestibility coefficients (56.4% and 59.5%, respectively). The use of phytase (1000 U/kg) had a significant impact on the absorption of phosphorus (the digestibility coefficient was 40.5% (0.30 g)). Thus, B. pumilus proteinase and Pantoea sp. 3.5.1 phytase have the potential to be used as feed additives for chickens

Data on the genome analysis of the probiotic strain Bacillus subtilis GM5

In the present study, we report data on the draft genome sequence of a lipopeptide producing rhizospheric Bacillus subtilis GM5 isolate. The genome consists of 4,271,280 bp with a GC-pair content of 43.3%. A total of 4518 genes including 75 tRNA genes, 3 operons coding for rRNA genes and 56 pseudogenes were annotated. Gene clusters responsible for the biosynthesis of secondary metabolites were validated. Six of the thirty-three clusters identified in the genome code for antimicrobial non-ribosomal peptides synthesis. The Whole Genome Shotgun project of B. subtilis GM5 has been deposited in the NCBI database under the accession number NZ_NKJH00000000 (https://www.ncbi.nlm.nih.gov/nuccore/NZ_NKJH00000000.1). Keywords: Bacillus subtilis, Analysis and assembly of the genome, Antimicrobial lipopeptide

Genomic and phenotypic analysis of siderophore-producing Rhodococcus qingshengii strain S10 isolated from an arid weathered serpentine rock environment

© 2020, Springer-Verlag GmbH Germany, part of Springer Nature. The success of members of the genus Rhodococcus in colonizing arid rocky environments is owed in part to desiccation tolerance and an ability to extract iron through the secretion and uptake of siderophores. Here, we report a comprehensive genomic and taxonomic analysis of Rhodococcus qingshengii strain S10 isolated from eathered serpentine rock at the arid Khalilovsky massif, Russia. Sequence comparisons of whole genomes and of selected marker genes clearly showed strain S10 to belong to the R. qingshengii species. Four prophage sequences within the R. qingshengii S10 genome were identified, one of which encodes for a putative siderophore-interacting protein. Among the ten non-ribosomal peptides synthase (NRPS) clusters identified in the strain S10 genome, two show high homology to those responsible for siderophore synthesis. Phenotypic analyses demonstrated that R. qingshengii S10 secretes siderophores and possesses adaptive features (tolerance of up to 8% NaCl and pH 9) that should enable survival in its native habitat within dry serpentine rock