Enhanced growth and recombinant protein production of Escherichia coli by a perfluorinated oxygen carrier in miniaturized fed-batch cultures

Abstract

BackgroundLiquid perfluorochemicals (PFCs) are interesting oxygen carriers in medicine and biotechnology with a high solubility for oxygen. They have been repeatedly used for improving oxygen transfer into prokaryotic and eukaryotic cell cultures, however their application is still limited. Here we show the great benefit of air/oxygen saturated perfluorodecalin (PFD) for high cell density cultivation of Escherichia coli in microwell plates and their positive effect on the soluble production of a correctly folded heterologously expressed alcohol dehydrogenase.ResultsIn EnBase® cultivations the best effect was seen with PFD saturated with oxygen enriched air (appr. 10 μM oxygen per ml) when PFD was added at the time of induction. In contrast the effect of PFD was negligible when it was added already at the time of inoculation. Optimisation of addition time and content of loaded oxygen into the PFD resulted in an increased the cell density by 40% compared to control cultures, and correspondingly also the product yield increased, demonstrated at the example of a recombinant alcohol dehydrogenase.ConclusionsPFCs are a valuable additive in miniaturized cell culture formats. For production of recombinant proteins in low cell density shaken cultures the addition of oxygen-enriched PFD makes the process more robust, i.e. a high product yield is not any more limited to a very narrow cell density window during which the induction has to be done. The positive effect of PFD was even more obvious when it was added during high cell density cultures. The effect of the PFD phase depends on the amount of oxygen which is loaded into the PFD and which thus is a matter of optimisation.

Liquid Perfluorochemicals as Flexible and Efficient Gas Carriers Applied in Bioprocess Engineering: An Updated Overview and Future Prospects

“…A significantly higher yield of genetically modified alcohol dehydrogenase as well as a lower level of inclusion bodies (i.e. insoluble fraction of not-correctly folded protein molecules) achieved in milliliter-scale of dispersed PFD containing cultures of E. coli has been reported by Pilarek et al (2011). The same group has also informed of a 25 % increase in plasmid DNA yield biosynthesised in E. coli cells cultured in PFDsupported microliter-scale system (Grunzel et al, 2014;Pilarek et al, 2013).…”

“…microorganisms, e.g. bacteria (Pilarek et al, 2011;Pilarek et al, 2013a), yeast (Pilarek and Szewczyk, 2008;Pilarek et al, 2006), fungi (Elibol, 2001) or microalgae (Hillig et al, 2013;Hillig et al, 2014), or plant cells (Davey et al 2005;Pilarek and Szewczyk 2008;Sykłowska-Baranek et al 2014) as wells as animal (Douglas et al 2014;Pilarek et al, 2013b;Pilarek et al, 2014;Rappaport, 2003;Shiba et al, 1998) to successfully obtain higher cell densities or to identify positive influence on metabolite excretion due to enhanced the mass transfer effects in culture systems, mainly.…”

Intensification of Organophosphorus Hydrolase Synthesis by Using Substances with Gas-Transport Function

“…Thus, the introduction of PFD at 50 vol. % produces a 40% increase in the biomass output of genetically modified E.coli RB791 pAdh cells, which synthesizes the recombinant alcohol dehydrogenase [17]. In another case, a 20% increase in amount of accumulated biomass for genetically modified E. coli RB791 pQE30adh cells was observed when 50 vol.…”

“…The physical properties of these compounds determine the conditions of their application in biotechnological processes and the possible ways of their recycling for multiple use, and also, most probably, the degree of their influence on the parameters of the biotechnological process. PFH had the lowest boiling temperature and molecular mass, and had the highest solubility in water [11,13,17,30,31].…”

Metabolic engineering and bio-electrochemical synthesis in Pseudomonas putida KT2440 for the production of p-hydroxybenzoate and 2-ketogluconate

“…Therefore, a considerable amount of efforts have been made to provide adequate oxygen during the fermentation, including optimization of the feeding strategy (Salehmin et al, 2013), using oxygen-enriched air, using perfluorocarbon oxygen carrier (Rols et al, 1990), etc. However, these efforts do not fully satisfy the industrial demands as they involve complicated and toilsome work for optimization of specific microbes to produce specific product; using oxygen-enriched air may cause oxidative stress to cell that would lead to impaired cell growth and the decrease productivity (Baez and Shiloach, 2014); the perfluorocarbon emulsion is relatively expensive limited its application in large scale production (Pilarek et al, 2011).…”