Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation

Looser, Verena; Hammes, Frederik; Keller, Markus; Berney, Michael; Kovar, Karin; Egli, Thomas

doi:10.1002/bit.20575

Publication type:	Article in scientific journal
Type of review:	Peer review (publication)
Title:	Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation
Authors:	Looser, Verena Hammes, Frederik Keller, Markus Berney, Michael Kovar, Karin Egli, Thomas
DOI:	10.1002/bit.20575
Published in:	Biotechnology & Bioengineering
Volume(Issue):	92
Issue:	1
Page(s):	69
Pages to:	78
Issue Date:	5-Oct-2005
Publisher / Ed. Institution:	Wiley
ISSN:	1097-0290 0006-3592
Language:	English
Subjects:	Ethidium bromide; Fedbatch; E. coli; Flow cytometry; Physiological state; Propidium iodide; Recombinant membrane protein; Xylene monooxygenase
Subject (DDC):	660.6: Biotechnology
Abstract:	The key to optimizing productivity during industrial fermentations is the ability to rapidly monitor and interpret the physiological state of single microbial cells in a population and to recognize and characterize different sub-populations. Here, a flow cytometry-based method for the reproducible detection of changes in membrane function and/or structure of recombinant E. coli JM101 (pSPZ3) expressing xylene monooxygenase (XMO), was developed. XMO expression led to compromised but not permeabilized cell membranes. This was deduced from the fact that recombinant cells only stained with ethidium bromide (EB) and not with propidium iodide (PI). During the glucose-limited fedbatch cultivation, an increase from 25% to 95% of EB-stained cells was observed, occurring between 2 and 5 h after induction. Control experiments confirmed that this increase was due to the recombinant protein production and not caused by any possible effects of varying substrate availability, high cell density, plasmid replication or the presence of the inducing agent. We hypothesize that the integration of the recombinant protein into the cell membrane physically disrupted the functionality of the efflux pumps, thus resulting in EB-staining of the recombinant cells. This method enabled us to detect changes in the physiological state of single cells 2-4 h before other indications of partial cell damage, such as unbalanced growth, acetate accumulation and an increased CO(2) production rate, were observed. This method therefore shows promise with respect to the further development of an early-warning system to prevent sudden productivity decreases in processes with recombinant E. coli expressing heterologous membrane proteins.
URI:	https://digitalcollection.zhaw.ch/handle/11475/11467
Fulltext version:	Published version
License (according to publishing contract):	Licence according to publishing contract
Departement:	Life Sciences and Facility Management
Organisational Unit:	Institute of Chemistry and Biotechnology (ICBT)
Appears in collections:	Publikationen Life Sciences und Facility Management

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Looser, V., Hammes, F., Keller, M., Berney, M., Kovar, K., & Egli, T. (2005). Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation. Biotechnology & Bioengineering, 92(1), 69–78. https://doi.org/10.1002/bit.20575

Looser, V. et al. (2005) ‘Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation’, Biotechnology & Bioengineering, 92(1), pp. 69–78. Available at: https://doi.org/10.1002/bit.20575.

V. Looser, F. Hammes, M. Keller, M. Berney, K. Kovar, and T. Egli, “Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation,” Biotechnology & Bioengineering, vol. 92, no. 1, pp. 69–78, Oct. 2005, doi: 10.1002/bit.20575.

LOOSER, Verena, Frederik HAMMES, Markus KELLER, Michael BERNEY, Karin KOVAR und Thomas EGLI, 2005. Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation. Biotechnology & Bioengineering. 5 Oktober 2005. Bd. 92, Nr. 1, S. 69–78. DOI 10.1002/bit.20575

Looser, Verena, Frederik Hammes, Markus Keller, Michael Berney, Karin Kovar, and Thomas Egli. 2005. “Flow-Cytometric Detection of Changes in the Physiological State of E. Coli Expressing a Heterologous Membrane Protein during Carbon-Limited Fedbatch Cultivation.” Biotechnology & Bioengineering 92 (1): 69–78. https://doi.org/10.1002/bit.20575.

Looser, Verena, et al. “Flow-Cytometric Detection of Changes in the Physiological State of E. Coli Expressing a Heterologous Membrane Protein during Carbon-Limited Fedbatch Cultivation.” Biotechnology & Bioengineering, vol. 92, no. 1, Oct. 2005, pp. 69–78, https://doi.org/10.1002/bit.20575.