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 language | English |
|---|---|
| Pages (from-to) | 625-641 |
| Journal | Microbial Biotechnology |
| Volume | 10 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2017 |
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A growth- and bioluminescence-based bioreporter for the in vivo detection of novel biocatalysts. / van Rossum, Teunke; Muras, A.; Baur, Marco J.J.; Creutzburg, Sjoerd C.A.; van der Oost, John; Kengen, Servé W.M.
In: Microbial Biotechnology, Vol. 10, No. 3, 2017, p. 625-641.Research output: Contribution to journal › Article › Academic › peer-review
TY - JOUR
T1 - A growth- and bioluminescence-based bioreporter for the in vivo detection of novel biocatalysts
AU - van Rossum, Teunke
AU - Muras, A.
AU - Baur, Marco J.J.
AU - Creutzburg, Sjoerd C.A.
AU - van der Oost, John
AU - Kengen, Servé W.M.
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
U2 - 10.1111/1751-7915.12612
DO - 10.1111/1751-7915.12612
M3 - Article
VL - 10
SP - 625
EP - 641
JO - Microbial Biotechnology
JF - Microbial Biotechnology
SN - 1751-7907
IS - 3
ER -