title: A growth- and bioluminescence-based bioreporter for the in vivo detection of novel biocatalysts.
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.
authors.affiliation: Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708, WE Wageningen, The Netherlands.
authors.forename: Teunke
authors.initials: T
authors.lastname: van Rossum
authors.affiliation: Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708, WE Wageningen, The Netherlands.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Muras
authors.affiliation: Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708, WE Wageningen, The Netherlands.
authors.forename: Marco J J
authors.initials: MJJ
authors.lastname: Baur
authors.affiliation: Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708, WE Wageningen, The Netherlands.
authors.forename: Sjoerd C A
authors.initials: SCA
authors.lastname: Creutzburg
authors.affiliation: Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708, WE Wageningen, The Netherlands.
authors.forename: John
authors.initials: J
authors.lastname: van der Oost
authors.affiliation: Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708, WE Wageningen, The Netherlands.
authors.forename: Servé W M
authors.initials: SWM
authors.lastname: Kengen
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004798:Enzymes / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D008163:Luminescent Measurements; D008403:Mass Screening / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D004798:Enzymes