Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation.
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.
Looser
V
V
University of Applied Sciences Zürich (HSW ZFH), Grüental, CH-8820 Wädenswil, Switzerland.
Hammes
F
F
Keller
M
M
Berney
M
M
Kovar
K
K
Egli
Thomas
T
D000085:Acetates / Q000737:chemistry; D019149:Bioreactors; D002244:Carbon / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry; D004996:Ethidium / Q000494:pharmacology; D005285:Fermentation; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D007218:Industrial Microbiology / Q000295:instrumentation / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010105:Oxygenases / Q000378:metabolism; D011419:Propidium / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D000085:Acetates; D003470:Culture Media; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D011419:Propidium; D002244:Carbon; D010105:Oxygenases; C067624:xylene monooxygenase; D004996:Ethidium; D005947:Glucose