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

Teunke van Rossum*, A. Muras, Marco J.J. Baur, Sjoerd C.A. Creutzburg, John van der Oost, Servé W.M. Kengen

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)625-641
JournalMicrobial Biotechnology
Volume10
Issue number3
DOIs
Publication statusPublished - 2017

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