Dataset Information

Improving production of N-glycosylated recombinant proteins by leaky Escherichia coli.

ABSTRACT: Escherichia coli has been considered as a promising host for the production of N-glycosylated therapeutic proteins and glycoconjugate vaccines. In this study, we developed a simple and efficient strategy for improving the production of N-glycosylated recombinant proteins by combining auto-induction with the use of a leaky E. coli strain. A leaky E. coli strain, designated as CLM37-?lpp, was engineered by deleting the Braun's lipoprotein (lpp) gene of E. coli strain CLM37. Three distinct acceptor model N-glycosylated proteins, glyco-tagged human tenth fibronectin type III domain (FN3-Gly), enhanced green fluorescent protein (eGFP-Gly), and scFv of vascular endothelial growth factor receptor 3 (scFv-VEGFR3-Gly) were then expressed in CLM37-?lpp, which carried an N-glycosylation machinery from Campylobacter jejuni for the investigation of glycoprotein production. As much as 75%, 65%, and 60% of the glycosylated FN3-Gly, eGFP-Gly, and scFv-VEGFR3-Gly, respectively, were found in the culture medium. The yields of glycosylated FN3-Gly, eGFP-Gly, and scFv-VEGFR3-Gly were 106?±?7.4 mg/L, 65?±?2.5 mg/L, and 62?±?4.3 mg/L, respectively, which were more than three folds the corresponding yields obtained when these proteins were expressed in CLM37, the unmodified strain. The results suggested that this simplified approach could improve the production of N-glycosylated proteins with E. coli to facilitate large-scale production.

SUBMITTER: Ding N

PROVIDER: S-EPMC6646630 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Improving production of N-glycosylated recombinant proteins by leaky Escherichia coli.

Ding Ning N Ruan Yao Y Fu Xin X Lin Yue Y Yu Hongyou H Han Lichi L Fu Changzhen C Zhang Jianing J Hu Xuejun X

3 Biotech 20190722 8

Escherichia coli has been considered as a promising host for the production of N-glycosylated therapeutic proteins and glycoconjugate vaccines. In this study, we developed a simple and efficient strategy for improving the production of N-glycosylated recombinant proteins by combining auto-induction with the use of a leaky E. coli strain. A leaky E. coli strain, designated as CLM37-Δlpp, was engineered by deleting the Braun's lipoprotein (lpp) gene of E. coli ...[more]

PMID: 31355111

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Project description:UNLABELLED:Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production. IMPORTANCE:The use of microbial host platforms for the production of bulk commodities, such as chemicals and fuels, is now a focus of many biotechnology efforts. Many of these compounds are inherently toxic to the host microbe, which in turn places a limit on production despite efforts to optimize the bioconversion pathways. In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds. We demonstrate that microbial tolerance engineering using transcriptomics data can also identify targets that improve production. Our results include an exporter and a methionine biosynthesis regulator that improve isopentenol production, providing a starting point to further engineer the host for biogasoline production.

Project description:In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

Dataset Information

Improving production of N-glycosylated recombinant proteins by leaky Escherichia coli.

Publications

Improving production of N-glycosylated recombinant proteins by leaky <i>Escherichia coli</i>.

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