Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli
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...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2018-06-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2018.01344/full |
| _version_ | 1811233631792791552 |
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| author | Cauã A. Westmann Luana de Fátima Alves Luana de Fátima Alves Rafael Silva-Rocha María-Eugenia Guazzaroni |
| author_facet | Cauã A. Westmann Luana de Fátima Alves Luana de Fátima Alves Rafael Silva-Rocha María-Eugenia Guazzaroni |
| author_sort | Cauã A. Westmann |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T11:22:57Z |
| format | Article |
| id | doaj.art-1e2362ad443f453d9ade897bc84e9e6d |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-12T11:22:57Z |
| publishDate | 2018-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-1e2362ad443f453d9ade897bc84e9e6d2022-12-22T03:35:17ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2018-06-01910.3389/fmicb.2018.01344365151Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coliCauã A. Westmann0Luana de Fátima Alves1Luana de Fátima Alves2Rafael Silva-Rocha3María-Eugenia Guazzaroni4Department of Cellular and Molecular Biology, FMRP, University of São Paulo, Ribeirão Preto, BrazilDepartment of Biology, FFCLRP, University of São Paulo, Ribeirão Preto, BrazilDepartment of Biochemistry, FMRP, University of São Paulo, Ribeirão Preto, BrazilDepartment of Cellular and Molecular Biology, FMRP, University of São Paulo, Ribeirão Preto, BrazilDepartment of Biology, FFCLRP, University of São Paulo, Ribeirão Preto, BrazilAlthough 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.https://www.frontiersin.org/article/10.3389/fmicb.2018.01344/fullfunctional metagenomicsbi-directional reporterconstitutive promoterssynthetic biologyhigh-throughput screening |
| spellingShingle | Cauã A. Westmann Luana de Fátima Alves Luana de Fátima Alves Rafael Silva-Rocha María-Eugenia Guazzaroni Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli Frontiers in Microbiology functional metagenomics bi-directional reporter constitutive promoters synthetic biology high-throughput screening |
| title | Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli |
| title_full | Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli |
| title_fullStr | Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli |
| title_full_unstemmed | Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli |
| title_short | Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli |
| title_sort | mining novel constitutive promoter elements in soil metagenomic libraries in escherichia coli |
| topic | functional metagenomics bi-directional reporter constitutive promoters synthetic biology high-throughput screening |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2018.01344/full |
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