A growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts
| Autor: | Marco J.J. Baur, John van der Oost, Sjoerd C.A. Creutzburg, Teunke van Rossum, Aleksandra Muras, Servé W. M. Kengen |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Isomerase activity Bioengineering Computational biology Isomerase Biosensing Techniques Biology medicine.disease_cause Applied Microbiology and Biotechnology Biochemistry Microbiology 03 medical and health sciences In vivo Microbiologie medicine Escherichia coli Bioluminescence Life Science Mass Screening Selection (genetic algorithm) Research Articles VLAG Sequence Analysis DNA Small molecule Enzymes 030104 developmental biology Luminescent Measurements Bioreporter Biotechnology Research Article |
| Zdroj: | Microbial Biotechnology Microbial Biotechnology 10 (2017) 3 Microbial Biotechnology, 10(3), 625-641 |
| ISSN: | 1751-7915 1751-7907 |
| Popis: | Summary The use of bioreporters in high‐throughput screening for small molecules is generally laborious and/or expensive. The technology can be simplified by coupling the generation of a desired compound to cell survival, causing only positive cells to stay in the pool of generated variants. Here, a dual selection/screening system was developed for the in vivo detection of novel biocatalysts. The sensor part of the system is based on the transcriptional regulator AraC, which controls expression of both a selection reporter (LeuB or KmR; enabling growth) for rapid reduction of the initially large library size and a screening reporter (LuxCDABE; causing bioluminescence) for further quantification of the positive variants. Of four developed systems, the best system was the medium copy system with KmR as selection reporter. As a proof of principle, the system was tested for the selection of cells expressing an l‐arabinose isomerase derived from mesophilic Escherichia coli or thermophilic Geobacillus thermodenitrificans. A more than a millionfold enrichment of cells with l‐arabinose isomerase activity was demonstrated by selection and exclusion of false positives by screening. This dual selection/screening system is an important step towards an improved detection method for small molecules, and thereby for finding novel biocatalysts. |
| Databáze: | OpenAIRE |
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