Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli ‘leaky mutant’

Müller, Jakob; Wetzel, David; Flaschel, Erwin; Friehs, Karl; Risse, Joe Max

doi:10.1016/j.jbiotec.2016.01.032

Cited by 12 publications

(14 citation statements)

References 41 publications

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“…Most early publications on the subject usually present qualitative findings on whether periplasmic proteins were found outside the cell or not [183], more recent publications present more detailed information about the extracellular protein concentrations achieved and/or secretion efficiency (see Table 2). Secretion efficiencies of ß95-96% were stated for penicillin G acylase [186] and for streptavidin [187]. Strains with lpp mutation or deletion have recently been described and are gainined attention for protein production.…”

Section: Permanently Improved Permeability By Strain Development Mutmentioning

confidence: 99%

“…Strains with lpp mutation or deletion have recently been described and are gainined attention for protein production. Secretion efficiencies of ß95-96% were stated for penicillin G acylase [186] and for streptavidin [187]. Moreover, an extracellular protein yield of up to 3.5 g/L was achieved, but as indicated in EP 1903105 B1, the secretion efficiency depends on the protein of interest [188].…”

Section: Permanently Improved Permeability By Strain Development Mutmentioning

confidence: 99%

“…Excessive foaming was observed for Triton X-100 supplementation [187,232] and the shorter length variant Triton X-45 was found to be an effective alternative [187]. Additional supplements that enhance extracellular protein production include SDS, Tween 20 and 80, Lactose [233], EDTA, polyethyleneimine (PEI) [234], and sarkosyl [235].…”

Section: Cultivation/fermentationmentioning

confidence: 99%

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Secretion of recombinant proteins from E. coli

Kleiner-Grote

Risse

Friehs

2018

Engineering in Life Sciences

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The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

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Section: Permanently Improved Permeability By Strain Development Mutmentioning

confidence: 99%

Section: Permanently Improved Permeability By Strain Development Mutmentioning

confidence: 99%

Section: Cultivation/fermentationmentioning

confidence: 99%

See 1 more Smart Citation

Secretion of recombinant proteins from E. coli

Kleiner-Grote

Risse

Friehs

2018

Engineering in Life Sciences

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“…In comparison to literature, the maximal concentration for the optimized process surpassed titers for E. coli (typically 1-2.6 mM), but was achieved for a lower productivity, as this parameter is usually in the range of 65 to 340 nM h 21 for this host. [12][13][14]16,17 The productivity was close to the p STY of 67.9 nM h 21 achieved for P. pastoris by Nogueira et al 25 The concentration of SAV was considerably lower than the reference concentrations for the methanol-based production processes for SAV by P. pastoris reported by Casteluber et al (71 mM) 24 and Nogueira et al (11 mM), 25 but was achieved in a shorter cultivation time (72 vs. 167 h and 162 h, respectively). Generally, Casteluber's process is difficult to compare or scale up, because it was performed in spinner flasks aerated through a porous stone at very high volumetric flow rates (4 vvm) and contained a washing step between growth and induction phase, bearing the risk of contamination.…”

Section: Bioreactor Experimentsmentioning

confidence: 99%

“…Since then, multiple studies were based on this host. [13][14][15][16][17] The production of SAV by Bacillus subtilis has also been studied extensively. [18][19][20] Due to limited secretory efficiency and/or cytoplasmic expression, these hosts commonly suffer from intracellular binding of biotin by SAV.…”

Section: Introductionmentioning

confidence: 99%

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Müller

Bruhn

Flaschel

et al. 2016

Biotechnology Progress

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Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin-auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol-based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol-based processes lead to large proportions of biotin-blocked binding sites of streptavidin due to biotin-supplemented media. Targeting these problems, this paper provides strategies for the methanol-free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin-blocked streptavidin. The optimized, easily scalable fed-batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin-free streptavidin and a productivity of 57.8 nM h(-1) based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un-optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h(-1) ), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855-864, 2016.

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Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness

Kastenhofer

Rettenbacher

Feuchtenhofer³

et al. 2020

Biotechnology Journal

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With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X‐press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (qS) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy‐chain single‐domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X‐press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS are used to control productivity and leakiness. In the X‐press strain, extracellular SpA and VHH titer reach up to 349 and 19.6 mg g−1, respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X‐press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.

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Constitutive production and efficient secretion of soluble full-length streptavidin by an Escherichia coli ‘leaky mutant’

Cited by 12 publications

References 41 publications

Secretion of recombinant proteins from E. coli

Secretion of recombinant proteins from E. coli

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness

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