Omix technologies and bioinformatics: an example of use in creating a pharmabiotic based on the Limosilactobacillus fermentum U21 strain

Abstract

Motivation and Aim: Today's world is undergoing revolutionary changes in the development and use of pharmacological preparations based on bacteria and their biologically active ingredients. Such preparations are increasingly referred to as pharmabiotics, as opposed to probiotics, used primarily as dietary supplements and consumed by healthy people. Pharmabiotics are live biotherapeutic drugs and/or their metabolites and components with established pharmacological ingredients, mechanism of action and intended for treating specific diseases. In order to develop pharmabiotics, aside from traditional microbiological and biotechnological approaches, a complex of omics technologies can substantially facilitate the process. Lactobacilli are the important component of the human microbiota and, due to the active synthesis of biologically active compounds and bidirectional communication with the host organism, they can affect the state and antioxidant (AO) status of the macroorganism. The strain of Limosilactobacillus fermentum U-21 showed high AO activity on in vitro and in vivo models [1,2]. In this work, we used a combination of genomic, transcriptomic, and proteomic technologies to identify genes and proteins that potentially determine the unique AO properties of the strain. Methods and Algorithms: The DNA of the L. fermentum U21 strain and the comparison strains L. fermentum 103 and L. fermentum 279 was sequenced and deposited in the NCBI GenBank (WGS PNBB01, PGGI01, PGGE01). The search for genes exhibiting AO properties in the genome of L. fermentum U-21 was carried out using the reference catalog of genes of antioxidant proteins found in various species and strains of lactobacilli [3; https://github.com/Alexey-Kovtun/Catalog] and the developed algorithm for their search [4]. RNA was isolated on a King Fisher automated station with the MagMAX™ mirVana™ Total RNA Isolation Kit (Thermo Fisher Scientific). Ready libraries were sequenced on an Illumina HiSeq 2500. Kallisto v0.46.0 software was used to map reads and evaluate transcript abundance. Differential expression analysis was performed using the edgeR v3.26.8 package integrated into the Degust v4.1.1 web tool. The proteins of the cell-free culture supernatants of L. fermentum strains were separated in SDS-PAGE. For mass spectrometric analysis, the colored protein bands were excised and the peptides were separated using the Ultimate 3000 Nano LC system connected to a Q Exactive HF mass spectrometer through a nanoelectrospray source (Thermo Fischer Scientific) based on the mass spectrometry group of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry. RAS. LC-MS/MS data analysis was performed using PEAKS Studio 8.0 build 2016-0908 software. The primary structures of the generated peptides were analyzed based on the UNIPROT KB protein sequence database (07.2016).