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

Fingerprint

Bioluminescence
Biocatalysts
Isomerases
Screening
Arabinose
AraC Transcription Factor
Geobacillus
Enzymes
Growth
Cells
Libraries
Cell Survival
Molecules
Escherichia coli
Technology
Throughput
Sensors

Cite this

@article{f74a32218a34464cab600d17a945f6ff,
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.",
author = "{van Rossum}, Teunke and A. Muras and Baur, {Marco J.J.} and Creutzburg, {Sjoerd C.A.} and {van der Oost}, John and Kengen, {Serv{\'e} W.M.}",
year = "2017",
doi = "10.1111/1751-7915.12612",
language = "English",
volume = "10",
pages = "625--641",
journal = "Microbial Biotechnology",
issn = "1751-7907",
publisher = "Wiley",
number = "3",

}

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 journalArticleAcademicpeer-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 -