Emerging Alternative Production Systems

Sommer, Benjamin; Laux, Holger; Frenzel, André; Jostock, Thomas

doi:10.1002/9783527682423.ch21

Cited by 1 publication

(1 citation statement)

References 292 publications

(186 reference statements)

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“…In the biopharmaceutical sector in 2018, it produced 22% of all therapeutic proteins with active licenses in the USA and the EU [4]. Although this marked a decrease in the share of microbials on the biotherapeutic market compared to the decades before, the emergence of biologics, like antibody fragments (Fab), single domain antibodies (sdAb) and other scaffolds, which can be expressed in prokaryotes, leads to a resurge of E. coli as a production host [5][6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Triggering outer membrane leakiness of a novel E. coli strain for recombinant protein production

Kastenhofer

Rettenbacher

Feuchtenhofer

et al. 2020

Preprint

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BackgroundRecombinant proteins in Escherichia coli are expressed inside the cell. With the growing interest in continuous cultivation, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, we present a novel E. coli production host for growth decoupled recombinant protein production that can leak up to 90% of recombinant protein to the extracellular space. We investigated the effects of the process parameters temperature and specific glucose uptake rate on physiology, productivity, lysis and leakiness. Two model proteins were used, Protein A and a VHH single-domain antibody, and performance was compared to the industrial standard strain BL21(DE3). ResultsWe show that inducible growth repression in the novel E. coli strain enGenes-X-press, the effect of the metabolic burden on host physiology can be greatly reduced compared to BL21(DE3). Furthermore, in both strains, increasing temperature and specific substrate enhanced productivity and leakiness.Using the enGenes-X-press strain, extracellular Protein A and VHH titer reached up to 349 mg/g and 19.6 mg/g, respectively, comprising between 80 and 90% of total soluble product, while keeping cell lysis to a minimum. BL21(DE3) leaked 198 mg/g and 3.9 mg/g of Protein A and VHH to the medium, accounting for only 56% and 34% of total soluble product, respectively. ConclusionsWe confined the parameter space in which outer membrane leakiness can be controlled, while maintaining cell viability. Moreover, our findings demonstrate that the enGenes-X-press strain constitutes a superior host for extracellular production of recombinant protein.

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