Metagenome mining of pediocin-like bacteriocins and creation of pET-pedABCD, a customizable pediocin expression vector

• Ano de defesa: 2020
• Autor(a) principal: Barrios, Wesley Elias Bhering
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Federal de Viçosa
• Palavras-chave em Português: Pediocinas; Proteínas - Síntese; Expressão gênica; Mineração de dados (Computação); Bioquímica e Biologia Molecular
• Link de acesso: https://locus.ufv.br//handle/123456789/28830

Fermented dairy products, the human intestine, and the bovine rumen are some environments that contain lactic acid bacteria (LAB). In these competitive environments, the synthesis of antimicrobial compounds such as bacteriocins acts to control the population of gram-positive bacteria through various mechanisms, favoring the producer microorganism. The bacteriocins are classified into 3 classes, being class II, subclass A, bacteriocins analogous to pediocin, peptides with ample activity against Gram-positive and negative bacteria. Pediocin PA-1, the precursor of this subclass, acts through the insertion in the plasma membrane of the target bacterium by binding to the mannose carrier widely conserved in gram-positive bacteria or by electrostatic interactions, forming a pore that leads to cell death. The high increase of bacteria with resistance to classical antibiotics highlights the importance of the discovery of new antimicrobials. An alternative to this problem may be in metagenomes, which correspond to the DNA sequencing of microbial communities, providing access to the genetic potential of non- cultivable strains, enabling the discovery of new bacteriocins. In this context, the main objective of this work was the mining of metagenomes of fermented dairy products using the Memi algorithm. To this end, a new standard of amino acids characteristic of class IIa was developed to increase the accuracy of results and decrease false positives. Initially, new characteristic class IIa amino acid standards were proposed from the alignment of deposited sequences. It was possible to define memi-9 as a new standard of amino acids characteristic of class IIa bacteriocins, presenting 92.2% sensitivity and 100% accuracy. The mining of metagenomes resulted in the mapping of 52 already known class IIa bacteriocins and other possible candidates to compose the class. Also, we produced the pET-pedABCD vector by chemical synthesis, for heterologous expression of bacteriocins using all biosynthesis machinery necessary to produce the active peptide in the extracellular culture medium / soluble cell fraction. This is the first modular plasmid theoretically capable of expressing several class IIa bacteriocins by Golden Gate Assembly. pET-pedABCD was transformed into E. coli BL21(DE3) Arctic Express under Kanamicin selection and the presence of the expression cassette was confirmed in two clones by PCR with T7 primers. Analysis of SDS-PAGE corresponding to the insoluble fractionpreliminarily demonstrated the expression of the main protein for processing and transport of the precursor peptide (PedD). Given the importance of antimicrobial resistance in the one health sphere, a class IIa bacteriocin mining method was developed in metagenomes and a low-cost system for the modular production of bacteriocins was created, adding to the arsenal of tools to combat antimicrobial-resistant bacteria. Keywords: Metagenome Mining. Class IIa bacteriocins. Pediocin-like. Amino acid pattern.