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

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

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

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