Open AccessA growth‐ and bioluminescence‐based bioreporter for the in vivo detection of novel biocatalysts
Author(s) -
Rossum Teunke,
Muras Aleksandra,
Baur Marco J.J.,
Creutzburg Sjoerd C.A.,
Oost John,
Kengen Servé W.M.
Publication year - 2017
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/1751-7915.12612
Subject(s) - bioreporter , computational biology , biology , escherichia coli , bioluminescence , reporter gene , isomerase , high throughput screening , bioluminescence imaging , in vivo , luciferase , biochemistry , microbiology and biotechnology , gene , gene expression , transfection
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 (Lux CDABE ; 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.