Cargando…
A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis
Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective targe...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2016
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087393/ https://www.ncbi.nlm.nih.gov/pubmed/27690044 http://dx.doi.org/10.3390/s16101604 |
_version_ | 1782463898826309632 |
---|---|
author | Steffen, Victoria Otten, Julia Engelmann, Susann Radek, Andreas Limberg, Michael Koenig, Bernd W. Noack, Stephan Wiechert, Wolfgang Pohl, Martina |
author_facet | Steffen, Victoria Otten, Julia Engelmann, Susann Radek, Andreas Limberg, Michael Koenig, Bernd W. Noack, Stephan Wiechert, Wolfgang Pohl, Martina |
author_sort | Steffen, Victoria |
collection | PubMed |
description | Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum) strain DM1933 in a BioLector(®) microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization. |
format | Online Article Text |
id | pubmed-5087393 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-50873932016-11-07 A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis Steffen, Victoria Otten, Julia Engelmann, Susann Radek, Andreas Limberg, Michael Koenig, Bernd W. Noack, Stephan Wiechert, Wolfgang Pohl, Martina Sensors (Basel) Article Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum) strain DM1933 in a BioLector(®) microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization. MDPI 2016-09-28 /pmc/articles/PMC5087393/ /pubmed/27690044 http://dx.doi.org/10.3390/s16101604 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Steffen, Victoria Otten, Julia Engelmann, Susann Radek, Andreas Limberg, Michael Koenig, Bernd W. Noack, Stephan Wiechert, Wolfgang Pohl, Martina A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis |
title | A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis |
title_full | A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis |
title_fullStr | A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis |
title_full_unstemmed | A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis |
title_short | A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis |
title_sort | toolbox of genetically encoded fret-based biosensors for rapid l-lysine analysis |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087393/ https://www.ncbi.nlm.nih.gov/pubmed/27690044 http://dx.doi.org/10.3390/s16101604 |
work_keys_str_mv | AT steffenvictoria atoolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT ottenjulia atoolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT engelmannsusann atoolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT radekandreas atoolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT limbergmichael atoolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT koenigberndw atoolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT noackstephan atoolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT wiechertwolfgang atoolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT pohlmartina atoolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT steffenvictoria toolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT ottenjulia toolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT engelmannsusann toolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT radekandreas toolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT limbergmichael toolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT koenigberndw toolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT noackstephan toolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT wiechertwolfgang toolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis AT pohlmartina toolboxofgeneticallyencodedfretbasedbiosensorsforrapidllysineanalysis |