A growth- and bioluminescence-based bioreporter for the in vivo detection of novel biocatalysts

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.

Bibliographic Details

Main Authors:	van Rossum, Teunke, Muras, A., Baur, Marco J.J., Creutzburg, Sjoerd C.A., van der Oost, John, Kengen, Servé W.M.
Format:	Article/Letter to editor biblioteca
Language:	English
Subjects:	Life Science,
Online Access:	https://research.wur.nl/en/publications/a-growth-and-bioluminescence-based-bioreporter-for-the-in-vivo-de
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