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Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation
BACKGROUND: The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in fav...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2011
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068942/ https://www.ncbi.nlm.nih.gov/pubmed/21414195 http://dx.doi.org/10.1186/1472-6750-11-22 |
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author | Huber, Robert Roth, Simon Rahmen, Natalie Büchs, Jochen |
author_facet | Huber, Robert Roth, Simon Rahmen, Natalie Büchs, Jochen |
author_sort | Huber, Robert |
collection | PubMed |
description | BACKGROUND: The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments. RESULTS: We investigated the effect of different induction times and a concurrent phosphate limitation on the specific productivity of the T7 expression system E.coli BL21(DE3) pRhotHi-2-EcFbFP, which produces the model fluorescence protein EcFbFP upon induction. Therefore, specific online-monitoring tools for small scale cultivations (RAMOS, BioLector) as well as a novel cultivation platform (Robo-Lector) were used for rapid process optimization. The RAMOS system monitored the oxygen transfer rate in shake flasks, whereas the BioLector device allowed to monitor microbial growth and the production of EcFbFP in microtiter plates. The Robo-Lector is a combination of a BioLector and a pipetting robot and can conduct high-throughput experiments fully automated. By using these tools, it was possible to determine the optimal induction time and to increase the specific productivity for EcFbFP from 22% (for unlimited conditions) to 31% of total protein content of the E.coli cells via a phosphate limitation. CONCLUSIONS: The results revealed that a phosphate limitation at the right induction time was suitable to redirect the available cellular resources during cultivation to protein expression rather than in biomass production. To our knowledge, such an effect was shown for the first time for an IPTG-inducible expression system. Finally, this finding and the utilization of the introduced high-throughput experimentation approach could help to find new targets to further enhance the production capacity of recombinant E.coli-strains. |
format | Text |
id | pubmed-3068942 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-30689422011-04-01 Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation Huber, Robert Roth, Simon Rahmen, Natalie Büchs, Jochen BMC Biotechnol Research Article BACKGROUND: The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments. RESULTS: We investigated the effect of different induction times and a concurrent phosphate limitation on the specific productivity of the T7 expression system E.coli BL21(DE3) pRhotHi-2-EcFbFP, which produces the model fluorescence protein EcFbFP upon induction. Therefore, specific online-monitoring tools for small scale cultivations (RAMOS, BioLector) as well as a novel cultivation platform (Robo-Lector) were used for rapid process optimization. The RAMOS system monitored the oxygen transfer rate in shake flasks, whereas the BioLector device allowed to monitor microbial growth and the production of EcFbFP in microtiter plates. The Robo-Lector is a combination of a BioLector and a pipetting robot and can conduct high-throughput experiments fully automated. By using these tools, it was possible to determine the optimal induction time and to increase the specific productivity for EcFbFP from 22% (for unlimited conditions) to 31% of total protein content of the E.coli cells via a phosphate limitation. CONCLUSIONS: The results revealed that a phosphate limitation at the right induction time was suitable to redirect the available cellular resources during cultivation to protein expression rather than in biomass production. To our knowledge, such an effect was shown for the first time for an IPTG-inducible expression system. Finally, this finding and the utilization of the introduced high-throughput experimentation approach could help to find new targets to further enhance the production capacity of recombinant E.coli-strains. BioMed Central 2011-03-17 /pmc/articles/PMC3068942/ /pubmed/21414195 http://dx.doi.org/10.1186/1472-6750-11-22 Text en Copyright ©2011 Huber et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Huber, Robert Roth, Simon Rahmen, Natalie Büchs, Jochen Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation |
title | Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation |
title_full | Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation |
title_fullStr | Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation |
title_full_unstemmed | Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation |
title_short | Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation |
title_sort | utilizing high-throughput experimentation to enhance specific productivity of an e.coli t7 expression system by phosphate limitation |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068942/ https://www.ncbi.nlm.nih.gov/pubmed/21414195 http://dx.doi.org/10.1186/1472-6750-11-22 |
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