Fed-Batch Production of Bacterial Ghosts Using Dielectric Spectroscopy for Dynamic Process Control

Meitz, Andrea; Sagmeister, Patrick; Lubitz, Werner; Herwig, Christoph; Langemann, Timo

doi:10.3390/microorganisms4020018

Cited by 11 publications

(3 citation statements)

References 49 publications

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“…Fed-Batch production of BGs could significantly increase the level of INP per BG and the total yield of INP-BG. 57 The investigations performed were rather concept studies to answer the question whether BGs have the ability to induce ice nucleation at all. BGs from a recombinant Gram-negative bacterium as in our case E. coli C41 carrying INP could have the advantage over live parent bacteria that they are non-living and most important that they are no longer genetically modified organisms (GMOs) but only products thereof.…”

Section: Discussionmentioning

confidence: 99%

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts

Kassmannhuber

Rauscher²,

Schöner³

et al. 2017

Bioengineered

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In a concept study the ability to induce heterogeneous ice formation by Bacterial Ghosts (BGs) from Escherichia coli carrying ice nucleation protein InaZ from Pseudomonas syringae in their outer membrane was investigated by a droplet-freezing assay of ultra-pure water. As determined by the median freezing temperature and cumulative ice nucleation spectra it could be demonstrated that both the living recombinant E. coli and their corresponding BGs functionally display InaZ on their surface. Under the production conditions chosen both samples belong to type II ice-nucleation particles inducing ice formation at a temperature range of between ¡5.6 C and ¡6.7 C, respectively. One advantage for the application of such BGs over their living recombinant mother bacteria is that they are non-living native cell envelopes retaining the biophysical properties of ice nucleation and do no longer represent genetically modified organisms (GMOs).

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Section: Discussionmentioning

confidence: 99%

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts

Kassmannhuber

Rauscher²,

Schöner³

et al. 2017

Bioengineered

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“…2016; Meitz et al . 2016). However, in the batch mode, various factors, such as pressure, aeration rate, agitation rate, nutrient feeding, and so on, will affect the production of BGs, thus further investigations are warranted.…”

Section: Discussionmentioning

confidence: 99%

“…Similar phenomena have been observed among Gram-negative bacteria other than E. coli (Cai et al 2013;Hu et al 2013;Liu et al 2015;Cao et al 2018), which might be related to the evolution of codon usage by phages that increases the effective use of the host translational machinery (Chithambaram et al 2014a(Chithambaram et al , 2014b, while lysis gene E was derived from coliphage phiX174 that infects E. coli and the expression of lysis gene E via codon optimization of S. enteritidis was capable of lysing S. enteritidis more efficiently (data not shown). Other than these profiles, the mutated system described herein, in theory, can be beneficially performed in batch or fed-batch fermentation systems, as there are many reports of the use of temperature-controlled systems for batch production of BGs (Ra et al 2010;Langemann et al 2016;Meitz et al 2016). However, in the batch mode, various factors, such as pressure, aeration rate, agitation rate, nutrient feeding, and so on, will affect the production of BGs, thus further investigations are warranted.…”

Section: Discussionmentioning

confidence: 99%

Controlled expression of lysis gene E by a mutant of the promoter pL of the thermo‐inducible λcI857‐pL system

Gong

Gao

et al. 2020

J Appl Microbiol

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Aims To identify a lambda promoter pL mutant that could extend the thermal stability of the thermo‐inducible λcI857‐pR/pL system and to evaluate the effects of the modified system for the controlled expression of lysis gene E during the production of bacterial ghosts (BGs). Methods and results The promoter pL mutant was identified by random mutagenesis and site‐directed mutagenesis. The results showed that a T → 35C mutation in the pL promoter was responsible for the phenotype alteration. Under the same induction conditions, the lysis rates of the modified lytic system on Escherichia coli and Salmonella enteritidis were significantly lower than that of the control, while the lysis rates of Escherichia coli with the thermo‐inducible lytic system were significantly higher than that of S. enteritidis with the corresponding plasmid (P < 0·05). Conclusions Increasing the heat stability of the thermo‐inducible lytic systems decreased lysis efficiency during the production of BGs. There exist differences in the lysis efficiency of thermo‐inducible lytic systems between different bacterial strains. Significance and Impact of the Study These findings enrich current knowledge about modifications to thermo‐inducible systems and provide a reference for the application of these modified systems for the production of BGs and controlled gene expression in bacteria.

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A novel method to recover inclusion body protein from recombinant E. coli fed-batch processes based on phage ΦX174-derived lysis protein E

Ehgartner

Sagmeister

Langemann

et al. 2017

Appl Microbiol Biotechnol

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Production of recombinant proteins as inclusion bodies is an important strategy in the production of technical enzymes and biopharmaceutical products. So far, protein from inclusion bodies has been recovered from the cell factory through mechanical or chemical disruption methods, requiring additional cost-intensive unit operations. We describe a novel method that is using a bacteriophage-derived lysis protein to directly recover inclusion body protein from Escherichia coli from high cell density fermentation process: The recombinant inclusion body product is expressed by using a mixed feed fed-batch process which allows expression tuning via adjusting the specific uptake rate of the inducing substrate. Then, bacteriophage ΦX174-derived lysis protein E is expressed to induce cell lysis. Inclusion bodies in empty cell envelopes are harvested via centrifugation of the fermentation broth. A subsequent solubilization step reveals the recombinant protein. The process was investigated by analyzing the impact of fermentation conditions on protein E-mediated cell lysis as well as cell lysis kinetics. Optimal cell lysis efficiencies of 99% were obtained with inclusion body titers of >2.0 g/l at specific growth rates higher 0.12 h−1 and inducer uptake rates below 0.125 g/(g × h). Protein E-mediated cell disruption showed a first-order kinetics with a kinetic constant of −0.8 ± 0.3 h−1. This alternative inclusion body protein isolation technique was compared to the one via high-pressure homogenization. SDS gel analysis showed 10% less protein impurities when cells had been disrupted via high-pressure homogenization, than when empty cell envelopes including inclusion bodies were investigated. Within this contribution, an innovative technology, tuning recombinant protein production and substituting cost-intensive mechanical cell disruption, is presented. We anticipate that the presented method will simplify and reduce the production costs of inclusion body processes to produce technical enzymes and biopharmaceutical products.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-017-8281-x) contains supplementary material, which is available to authorized users.

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Fed-Batch Production of Bacterial Ghosts Using Dielectric Spectroscopy for Dynamic Process Control

Cited by 11 publications

References 49 publications

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts

Controlled expression of lysis gene E by a mutant of the promoter pL of the thermo‐inducible λcI857‐pL system

A novel method to recover inclusion body protein from recombinant E. coli fed-batch processes based on phage ΦX174-derived lysis protein E

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