Directed evolution of lichenicidin

• Autor(a) principal: Pereira, Marta Daniela Lima Braga Jesus
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/15282
• topic: Biologia molecular; Péptidos - Efeitos terapêuticos; Antibióticos; Bactérias; Mutagénese; Bioengenharia

Lichenicidin is a class II lanthipeptide composed by two peptides, Bliα and Bliβ, that is produced by several strains of Bacillus licheniformis. These peptides act synergistically in order to inhibit the growth of other Gram positive bacteria. Due to their antibacterial activity, they are also designated lantibiotics. Unlike other antimicrobial peptides produced by bacteria, lantibiotics are ribosomally synthesized as an immature peptide and suffer several post-translational modifications to achieve their active form. Thus, it is easier the application of bioengineering strategies aiming the creation of new variants with different bioactivity. The aims of this study were: i) the development a heterologous expression system for the production of lichenicidin allowing a white/blue selection and using E. coli, ii) the generation of two random mutagenesis libraries for each one of the lichenicidin peptides and iii) the selection and analysis of clones with reduced bioactivity when compared with the control. To develop the heterologous system, preliminary tests were conducted that allowed the selection of E. coli Mach1 as the recipient strain. In addition, 37 ºC was established as the best temperature for lichenicidin production. Thus, two random mutagenesis libraries of the structural genes licA1 and licA2, encoding the precursor peptides of Bliα and Bliβ, respectively. Subsequently, the bioactivity of around 4000 clones of each library was analyzed by colony-agar bioassay. In the present study, colonies with improved activity were not possible to detect in any of the libraries. Regarding the Bliα library, about 4,5 % of the clones showed null bioactivity, while 2,6 % and 2,2 % of the clones showed very reduced and activity reduced to half when compared with the control, respectively. In the Bliβ library, the values were very similar since about 5,1 % of the clones showed no bioactivity, 1,4 % showed very reduced activity and 3,8 % had activity reduced to half when compared with the control. Some of these clones were selected for sequencing reaction in order to identify the mutations causing the observed phenotypes. This analysis confirmed the importance of the conserved residues for the bioactivity of both peptides. Moreover, it was found that substitutions involving charged amino acids and prolines result in the production of inactive peptides.