Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli

Autor(es)

Westmann, Caua A. ^[1] ; Alves, Luana de Fatima ^{[2, 3]} ; Silva-Rocha, Rafael ^[1] ; Guazzaroni, Maria-Eugenia ^[2]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Dept Cellular & Mol Biol, FMRP, Ribeirao Preto - Brazil

^[2] Univ Sao Paulo, Dept Biol, FFCLRP, Ribeirao Preto - Brazil

^[3] Univ Sao Paulo, Dept Biochem, FMRP, Ribeirao Preto - Brazil

• Tipo de documento: Artigo Científico
• Fonte: FRONTIERS IN MICROBIOLOGY; v. 9, JUN 20 2018.
• Citações Web of Science: 5

Resumo

Although functional metagenomics has been widely employed for the discovery of genes relevant to biotechnology and biomedicine, its potential for assessing the diversity of transcriptional regulatory elements of microbial communities has remained poorly explored. Here, we experimentally mined novel constitutive promoter sequences in metagenomic libraries by combining a bi-directional reporter vector, high-throughput fluorescence assays and predictive computational methods. Through the expression profiling of fluorescent clones from two independent soil sample libraries, we have analyzed the regulatory dynamics of 260 clones with candidate promoters as a set of active metagenomic promoters in the host Escherichia coli. Through an in-depth analysis of selected clones, we were able to further explore the architecture of metagenomic fragments and to report the presence of multiple promoters per fragment with a dominant promoter driving the expression profile. These approaches resulted in the identification of 33 novel active promoters from metagenomic DNA originated from very diverse phylogenetic groups. The in silico and in vivo analysis of these individual promoters allowed the generation of a constitutive promoter consensus for exogenous sequences recognizable by E. coli in metagenomic studies. The results presented here demonstrates the potential of functional metagenomics for exploring environmental bacterial communities as a source of novel regulatory genetic parts to expand the toolbox for microbial engineering. (AU)

• Processo FAPESP: 16/06323-4 - Novas ferramentas para abordagens metagenômicas na prospecção de celulases
• Beneficiário: Luana de Fátima Alves
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 15/04309-1 - Novas abordagens para melhorar a prospecção funcional de biocatalizadores em bibliotecas metagenômicas
• Beneficiário: María Eugenia Guazzaroni
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 16/05472-6 - Engenharia e prospecção de novos elementos regulatórios em bactéria através de abordagens de biologia sintética
• Beneficiário: Cauã Antunes Westmann
• Modalidade de apoio: Bolsas no Brasil - Mestrado