Protein Secretion Systems of Corynebacterium glutamicum

Vertès, Alain A.

doi:10.1007/978-3-642-29857-8_13

Cited by 15 publications

(14 citation statements)

References 173 publications

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“…However, those records could be achieved by additional modifications such as addition of Ca 2þ into the media (Teramoto et al, 2011) or sigB disruption in host cell (Watanabe et al, 2013). When we compared the productivity without modification, our system employing a synthetic promoter (P H36 ) was quite competitive to others (Vertes, 2013), and this indicates that synthetic promoters have high potential for the production of heterologous proteins in C. glutamicum.…”

Section: Discussionmentioning

confidence: 99%

“…The usefulness of the synthetic promoters was successfully demonstrated with two recombinant proteins models (antibody fragment and endoxylanase) which were secreted into culture medium. Compared to E. coli as a host for protein production, C. glutamicum has several important features that facilitate the secretion of proteins into the culture medium: (i) significantly lower contamination of endogenous proteins in culture medium, and (ii) the lack of detectable extracellular hydrolytic enzyme activity (Vertes, ). Among two model proteins (XynA and M18 scFv), we recently reported the secretory production of M18 scFv in E. coli (Lee et al accepted for publication in Biotechnol Bioproc Eng ).…”

Section: Discussionmentioning

confidence: 99%

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Isolation of fully synthetic promoters for high‐level gene expression in Corynebacterium glutamicum

Yim

Kang

et al. 2013

Biotech & Bioengineering

166

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Corynebacterium glutamicum is an important industrial organism that is widely used in the production of amino acids, nucleotides and vitamins. To extend its product spectrum and improve productivity, C. glutamicum needs to undergo further engineering, including the development of applicable promoter system. Here, we isolated new promoters from the fully synthetic promoter library consisting of 70-bp random sequences in C. glutamicum. Using green fluorescent protein (GFP) as a reporter, highly fluorescent cells were screened from the library by fluorescent activated cell sorting (FACS). Twenty potential promoters of various strengths were isolated and characterized through extensive analysis of DNA sequences and mRNA transcripts. Among 20 promoters, 6 promoters which have different strengths were selected and their activities were successfully demonstrated using two model proteins (antibody fragment and endoxylanase). Finally, the strongest promoter (P(H36)) was employed for the secretory production of endoxylanase in fed-batch cultivation, achieving production levels of 746 mg/L in culture supernatant. This is the first report of synthetic promoters constructed in C. glutamicum, and our screening strategy together with the use of synthetic promoters of various strengths will contribute to the future engineering of C. glutamicum.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Isolation of fully synthetic promoters for high‐level gene expression in Corynebacterium glutamicum

Yim

Kang

et al. 2013

Biotech & Bioengineering

166

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“…Corynebacterium glutamicum is a gram-positive, non-pathogenic, and non-sporulating bacterium that is considered an industrial workhorse for the production of various L-amino acids, nucleic acids, and vitamins (Diesveld et al, 2009;Lv et al, 2012). Recently, this microorganism has attracted attention as a potential cell factory for the production of recombinant proteins since it exhibits numerous ideal features for protein secretion Vertes, 2013). First, it has a single cellular membrane as a grampositive bacterium, which allows proteins to be secreted into the extracellular medium by simply crossing a single membrane barrier.…”

Section: Introductionmentioning

confidence: 99%

Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum

Yim

Choi

Lee

et al. 2015

Biotech & Bioengineering

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Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum.

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“…C. glutamicum possesses two major protein secretory pathways: the general secretory (Sec) pathway that translocates unfolded proteins to the extracellular spacer, and the twin-arginine translocation (Tat) pathway that catalyzes the export of the folded proteins ( 7 ). A signal peptide is a short peptide in the N-terminal region of the secretory protein precursor, and it plays a key role in determining secretory pathway and efficiency of the secreted protein.…”

Section: Introductionmentioning

confidence: 99%

Development of A Novel Gene Expression System for Secretory Production of Heterologous Proteins via the General Secretory (Sec) Pathway in Corynebacterium glutamicum

Jia

Zhang

et al. 2018

ijbiotech

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BackgroundCorynebacterium glutamicum (C. glutamicum) is a potential host for the secretory production of the heterologous proteins. However, to this date few secretion-type gene expression systems in C. glutamicum have been developed, which limit applications of C. glutamicum in a secretory production of the heterologous proteins.ObjectivesIn this study, a novel and efficient general secretory (Sec) pathway-dependent type gene expression system for the production of heterologous proteins was developed in C. glutamicum.Materials and MethodsThe synthesized cloning/expression cassette C was assembled into the basic E. coli-C. glutamicum shuttle vector pAU2, generating the Sec-dependent type gene expression vector pAU5. Subsequently, the applicability of the C. glutamicum/pAU5 system was tested using the α-amylase AmyE from Bacillus subtilis as a reporter protein.ResultsThe vector pAU5 was successfully constructed. The SDS-PAGE experiment showed the AmyE protein band could be observed in the original culture supernatant of the 14067/pAU5-amyE. The Western blotting experiment showed that the AmyE polypeptide could be detected in the culture supernatant of the 14067/pAU5-amyE, not in the cell lysate of 14067/pAU5-amyE. The α-amylase specific activity of the culture supernatant of 14067/pAU5-amyE was 103.24±7.14 U.mg-1 protein, while no α-amylase activity was detected in the cell homogenate supernatant of 14067/pAU5-amyE. These results demonstrate that the recombinant AmyE was efficiently expressed and completely secreted into the extracellular environmentin an active form in C. glutamicum/pAU5 system.ConclusionsA novel efficient Sec-dependent type gene expression vector pAU5 was constructed in the C. glutamicum. The vector pAU5 employs the strong promoter tac-M for controlling a constitutive transcription of the target gene, the consensus ribosome binding site (RBS) sequence of C. glutamicum to ensure protein translation, and the efficient Sec-type cgR_2070 signal sequence to mediate protein secretion in the C. glutamicum. The C. glutamicum/pAU5 system is an efficient expression system for the secretory production of the heterologous proteins.

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Protein Secretion Systems of Corynebacterium glutamicum

Cited by 15 publications

References 173 publications

Isolation of fully synthetic promoters for high‐level gene expression in Corynebacterium glutamicum

Isolation of fully synthetic promoters for high‐level gene expression in Corynebacterium glutamicum

Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum

Development of A Novel Gene Expression System for Secretory Production of Heterologous Proteins via the General Secretory (Sec) Pathway in Corynebacterium glutamicum

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