Antibiotic-free selection in E. coli: new considerations for optimal design and improved production

Peubez, Isabelle; Chaudet, Nicolas; Mignon, Charlotte; Hild, Géraldine; Husson, Stéphanie; Courtois, Virginie; Luca, Karelle De; Speck, Denis; Sodoyer, Régis

doi:10.1186/1475-2859-9-65

Cited by 55 publications

(45 citation statements)

References 19 publications

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“…This ineffectiveness of ampicillin, in addition to the deprecation by regulatory authorities of its use in biotherapeutics and biomaterials production due to safety concerns232425, prompted us to investigate other plasmid stabilisation systems. Indeed, use of β-lactam antibiotics such as ampicillin poses safety risks associated with their potential for causing serious hypersensitivity reactions in patients and for leading to the spread of antibiotic resistance traits to the environments2526.…”

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confidence: 99%

Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein

Barroca

Rodrigues

Sobral

et al. 2016

Sci Rep

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Silk-elastin-like proteins (SELPs) are a family of genetically engineered recombinant protein polymers exhibiting mechanical and biological properties suited for a wide range of applications in the biomedicine and materials fields. They are being explored as the next generation of biomaterials but low productivities and use of antibiotics during production undermine their economic viability and safety. We have developed an industrially relevant, scalable, fed-batch process for the high level production of a novel SELP in E. coli in which the commonly used antibiotic selection marker of the expression vector is exchanged for a post segregational suicide system, the separate-component-stabilisation system (SCS). SCS significantly augments SELP productivity but also enhances the product safety profile and reduces process costs by eliminating the use of antibiotics. Plasmid content increased following induction but no significant differences in plasmid levels were discerned when using SCS or the antibiotic selection markers under the controlled fed-batch conditions employed. It is suggested that the absence of competing plasmid-free cells improves host cell viability and enables increased productivity with SCS. With the process developed, 12.8 g L−1 purified SELP was obtained, this is the highest SELP productivity reported to date and clearly demonstrates the commercial viability of these promising polymers.

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confidence: 99%

Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein

Barroca

Rodrigues

Sobral

et al. 2016

Sci Rep

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“…The gene encoding lysis protein E (see Table S1 in the supplemental material) from bacteriophage X174 (obtained from GenBank [NC_001422.1] and synthesized by GeneArt) was, using EcoRI and BamHI, cloned into the rhamnose promoter-based expression vector pLarge (pUC origin, Km r ), which is derived from pRha67 (26), yielding pRL1, and into the tetracycline promoter (tetA)-based expression vector pASK_IBA 3 (pUC origin, Amp r ), yielding pTL1 (27). To be able to clone the gene encoding lysis protein E directly from pRL1 into pASK_IBA 3 , its multiple cloning site was modified by amplifying the entire plasmid and introducing EcoRI and BamHI restriction sites using the Roche Expand long template PCR system (for primer sequences, see Table S1 in the supplemental material) (28). The pHbpD(p15a)-ESAT6 vector (p15A ori, Cm r ) was constructed by substituting the pMB1 origin of replication of pHbpD(⌬d1)-ESAT6 (19) by the PCR-amplified p15A origin of replication of pBAD33 using the EcoRI and SalI restriction sites (for primer sequences, see Table S1 in the supplemental material).…”

Section: Methodsmentioning

confidence: 99%

Autotransporter-Based Antigen Display in Bacterial Ghosts

Hjelm

Söderström

Vikström

et al. 2015

Appl Environ Microbiol

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Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage X174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system. Bacterial ghosts (BGs) are empty, nonliving cell envelopes of Gram-negative bacteria that still possess all surface structures, including immune-stimulating elements like lipopolysaccharides, lipoproteins, and flagella, while not posing any infectious threat (1-3). They are generated by the controlled production of bacteriophage X174 lysis protein E, which leads to the formation of tunnel structures spanning the entire cell envelope (1, 2, 4). How exactly these tunnel structures are formed is not clear (see, e.g., references 1 and 5 to 11). Due to osmotic pressure, the cytoplasmic content of the bacteria is released through these structures, while the cell envelope is mostly preserved (1, 2).BGs have been used as vaccines as well as vehicles for the delivery of antigens, drugs, and DNA (1, 2). For surface display of antigens in bacterial ghosts, the ice nucleation protein (6) and various outer membrane proteins, such as outer membrane protein A, have been used as anchors (12, 13). Recently, it has been shown that antibody responses to heterologous antigens that are secreted by Salmonella cells or exposed at the surface of Salmonella-derived outer membrane vesicles (OMVs) are higher than antibody responses to antigens that are present intracellularly or in the lumen of the OMVs, respectively (e.g., see references 14 to ...

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“…A principal motivação está no fato de este ser um organismo bem caracterizado do ponto de vista genético, fisiológico e de expressão (Choi et al, 2006). Apesar de todas as conhecidas vantagens do uso da E. coli como hospedeiro, a instabilidade plasmidial é uma preocupação significativa para a utilização industrial destes OGMs, uma vez que estas construções utilizam vetores bacterianos que contém um gene de resistência a antibiótico como marcador seletivo (Spirer et al, 2005;Peubez et al, 2010). Os antibióticos (ou seus produtos oriundos da degradação) podem contaminar a biomassa ou produto, tornando-se necessária a sua remoção do produto final e efluente (Spirer et al, 2005).…”

Section: Introductionunclassified

Produção De Enzimas Acessórias Recombinantes Para Suplementação De Coquetel Enzimático De L.THEOBROMAE Para Aplicação Na Hidrólise Do Bagaço

Katayama¹,

Tavares²,

Costa³

et al. 2015

Anais Do XX Congresso Brasileiro De Engenharia Química

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RESUMO -A produção de coquetéis enzimáticos eficientes é imprescindível para a hidrólise de material lignocelulósico visando à obtenção de açúcares fermentescíveis. Uma das abordagens mais promissoras é o enriquecimento de coquetéis fúngicos em termos de atividades que lhes faltam, que podem ser obtidas através de sistemas heterólogos. O objetivo deste trabalho é o desenvolvimento do processo de produção de hidrolases recombinantes em E. coli visando à suplementação do coquetel enzimático do fungo L. theobromae para aplicação eficiente na sacarificação de bagaço de cana-deaçúcar pré-tratado, uma vez que a caracterização do coquetel secretado por este fungo evidenciam a carência de celulases, em especial da classe endoglucanásica, em sua composição. A plataforma de expressão heteróloga está baseada na linhagem comercial E. coli SE1 cujo sistema de estabilização independe de antibiótico, reduzindo custos na produção da enzima, além de facilitar o tratamento dos efluentes. A construção dos clones em SE1 e os ensaios de expressão da endoglucanase demonstram que este sistema de expressão é promissor.

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Antibiotic-free selection in E. coli: new considerations for optimal design and improved production

Cited by 55 publications

References 19 publications

Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein

Antibiotic free selection for the high level biosynthesis of a silk-elastin-like protein

Autotransporter-Based Antigen Display in Bacterial Ghosts

Produção De Enzimas Acessórias Recombinantes Para Suplementação De Coquetel Enzimático De L.THEOBROMAE Para Aplicação Na Hidrólise Do Bagaço

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