Construction of hypervesiculation <i>Escherichia coli</i> strains and application for secretory protein production

Ojima, Yoshihiro; Sawabe, T; Konami, Katsuya; Azuma, Masayuki

doi:10.1002/bit.27239

Cited by 32 publications

(37 citation statements)

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“…The cell growth, cell volume, relative OMV production, and average OMV size of each strain of hypervesiculating E. coli , including the single-deletion mutants Δ mlaE and Δ nlpI and the double-deletion mutant Δ mlaE Δ nlpI at the end of a 24 h culture, were measured ( Table 2 ). The OD 660 and relative OMV production based on the SDS-PAGE analysis were taken from our previous study ( Ojima et al, 2020 ).…”

Section: Resultsmentioning

confidence: 99%

“…The WT E. coli K-12 strain BW25113 and its derivatives were obtained from the National BioResource Project [National Institute of Genetics (NIG), Mishima, Japan; Baba et al, 2006 ]. The double-gene knockout mutants were constructed by P1 transduction using the P1kc phage ( Ojima et al, 2020 ). Each strain was transformed with pCA24N- gfp ( Ojima et al, 2018 ) to express the His-tagged GFP in the cytosol.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, green fluorescent protein (GFP) fused with the outer membrane protein W (OmpW) was expressed in OMVs to evaluate their capacity for protein secretion. Western blot analysis showed that OmpW-GFP secretion by the OMVs in the culture medium of Δ mlaE Δ nlpI strain reached 3.3 mg/L; 500 times that of the WT strain ( Ojima et al, 2020 ). Since the OMV production by Δ mlaE Δ nlpI was 30 times that of the WT strain, a 500-fold increase in OmpW-GFP secretion by Δ mlaE Δ nlpI could not be fully explained by an increase in OMV production.…”

Section: Introductionmentioning

confidence: 99%

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Aberrant Membrane Structures in Hypervesiculating Escherichia coli Strain ΔmlaEΔnlpI Visualized by Electron Microscopy

et al. 2021

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Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants ΔnlpI and ΔmlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the ΔmlaEΔnlpI cells was more than 100 times higher than that of WT and ΔnlpI, and about 50 times higher than that of ΔmlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Aberrant Membrane Structures in Hypervesiculating Escherichia coli Strain ΔmlaEΔnlpI Visualized by Electron Microscopy

et al. 2021

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“…The genetic basis of OMV formation is not yet entirely understood but could be connected to a range of genes [37]. Such knowledge has already been successfully applied for the engineering of hypervesiculation to support cell viability under stress conditions [46] or protein secretion in E. coli [47], but has not yet been utilised in the context of optimising Pseudomonas biotechnology and will certainly require fine-tuning to avoid extensive loss of lipids and membrane integrity [48,49]. The above-mentioned urgent response mechanisms enable Pseudomonas species to react quickly to emerging adverse conditions, making them robust candidates for whole-cell biotransformation processes and accessing new-to-nature chemistry.…”

Section: Built-in Cell Envelope Stress Response As Support Of Bioproductionmentioning

confidence: 99%

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways

Bitzenhofer

Kruse

Thies

et al. 2021

Essays in Biochemistry

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Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals.

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“…Although, the manufacturing downstream process required for OMV production is potentially cheap and relatively unsophisticated, current bottlenecks to develop OMV vaccine candidate for Pseudomonas at the preclinical level, are the yields and the detoxification strategies. In fact, low yields are generally obtained from Pseudomonas and an overblebbing genetic strategy, similar to that of E. coli for which extensive knowledge is available in literature [ 137 , 138 , 139 , 140 , 141 ], remains unidentified. Moreover, the purification of P. aeruginosa OMV represent a serious challenge [ 142 , 143 ] due to its tendency to secrete large amounts of extracellular factors such as enzymes, toxins and exopolysaccharide, obviously impairing OMV purification.…”

Section: Outer Membrane Vesicles As Vaccine Platformmentioning

confidence: 99%

Strategies to Tackle Antimicrobial Resistance: The Example of Escherichia coli and Pseudomonas aeruginosa

Antonelli

Cappelli

Cinelli

et al. 2021

IJMS

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Traditional antimicrobial treatments consist of drugs which target different essential functions in pathogens. Nevertheless, bacteria continue to evolve new mechanisms to evade this drug-mediated killing with surprising speed on the deployment of each new drug and antibiotic worldwide, a phenomenon called antimicrobial resistance (AMR). Nowadays, AMR represents a critical health threat, for which new medical interventions are urgently needed. By 2050, it is estimated that the leading cause of death will be through untreatable AMR pathogens. Although antibiotics remain a first-line treatment, non-antibiotic therapies such as prophylactic vaccines and therapeutic monoclonal antibodies (mAbs) are increasingly interesting alternatives to limit the spread of such antibiotic resistant microorganisms. For the discovery of new vaccines and mAbs, the search for effective antigens that are able to raise protective immune responses is a challenging undertaking. In this context, outer membrane vesicles (OMV) represent a promising approach, as they recapitulate the complete antigen repertoire that occurs on the surface of Gram-negative bacteria. In this review, we present Escherichia coli and Pseudomonas aeruginosa as specific examples of key AMR threats caused by Gram-negative bacteria and we discuss the current status of mAbs and vaccine approaches under development as well as how knowledge on OMV could benefit antigen discovery strategies.

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Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Cited by 32 publications

References 25 publications

Aberrant Membrane Structures in Hypervesiculating Escherichia coli Strain ΔmlaEΔnlpI Visualized by Electron Microscopy

Aberrant Membrane Structures in Hypervesiculating Escherichia coli Strain ΔmlaEΔnlpI Visualized by Electron Microscopy

Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways

Strategies to Tackle Antimicrobial Resistance: The Example of Escherichia coli and Pseudomonas aeruginosa

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