Analysis of RiPPs biosynthetic clusters of Haloferax mediterranei and Pedobacter lusitanus

• Autor(a) principal: Costa, Thales Viana Labourdette
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/36524
• topic: Secondary metabolites; RiPPs; Lanthipeptides; TOMMs; RT-qPCR; FTIR; Biosensor

Secondary metabolites (SMs) are a diverse group of compounds. The SMs of peptide origin that are synthesized by the ribosome and undergo several post-translational modifications are called RiPPs. Haloferax mediterranei ATCC 33500, a strain of haloarchaea, and Pedobacter lusitanus NL19, a bacterial strain, have biosynthetic genes (clusters) that encode the production of a subtype of RiPPs named lanthipeptides. Additionally, the genome of H. mediterranei also possesses genes involved in the biosynthesis of another group of RiPPs: the haloazolisins. The objectives of this work focused on the analysis of biosynthetic clusters in the two strains aforementioned. As such, for H. mediterranei, specific objectives were to evaluate: i) whether the RiPPs clusters are cryptic and ii) the biomolecular profile of the strain in the absence of the RiPPs´ core enzymes (HaloMs and YcaO). For this, RT-qPCR and FTIR techniques were employed, respectively. For P. lusitanus, the ped15 lanthipeptide gene cluster was selected and the objectives were: i) to evaluate the transcription of pedA15 operon in different culture media, to identify one with active transcription and ii) to initiate the construction of a biosensor that enables the rapid transcriptional analysis of these genes. For this, we used RT-qPCR and cloning techniques, respectively. The results showed that for H. mediterranei all the genes analyzed are transcribed under the conditions evaluated, with a tendency of the number of copies to decrease over time. FTIR analysis revealed that the absence of the enzymes HaloMs or YcaO implied a change in the levels of amides and lipids. Regarding P. lusitanus, SPA medium seems to be the most promising for obtaining a higher number of transcripts of the pedA15 operon. However, a repressor was identified in the ped15 cluster, which is 10X more transcribed than the pedA15 operon. This protein should be taken into consideration for the development of the future biosensor, which was initiated in this work with the cloning of promoter regions upstream of the luciferase operon genes.