Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

Matsuda, Yoshihiko; Itaya, Hiroshi; Kitahara, Yuki; Theresia, Natalia Maria; Kutukova, Ekaterina A.; Yomantas, Yurgis Antanas Vladovich; Date, Masayo; Kikuchi, Yoshimi; Wachi, Masaaki

doi:10.1186/1475-2859-13-56

Cited by 49 publications

(40 citation statements)

References 51 publications

(57 reference statements)

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“…Efforts to improve the protein secretion ability of C. glutamicum have previously focused on: overexpression of genes related to protein secretion channels (Kikuchi et al, 2009), isolation of factors for enhanced protein secretion (Watanabe et al, 2013), deletion of cell wall protein encoding genes (Matsuda et al, 2014), and optimization of culture conditions (Teramoto et al, 2011). glutamicum has ideal features that facilitate efficient protein secretion and has been widely used for production of various recombinant proteins.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Besides, C. glutamicum strain ATCC13869 was commercialized as protein expression system under the trademark “CORYNEX” by the Japanese company Ajinomoto. This system was recently improved for secretory antibody Fab fragment production by deletion of cspB and pbp1a [11]. …”

Section: Introductionmentioning

confidence: 99%

Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum

et al. 2016

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BackgroundTechnical bulk enzymes represent a huge market, and the extracellular production of such enzymes is favorable due to lowered cost for product recovery. Protein secretion can be achieved via general secretion (Sec) pathway. Specific sequences, signal peptides (SPs), are necessary to direct the target protein into the translocation machinery. For example, >150 Sec-specific SPs have been identified for Bacillus subtilis alone. As the best SP for a target protein of choice cannot be predicted a priori, screening of homologous SPs has been shown to be a powerful tool for different expression organisms. While SP libraries between closely related species were successfully applied to optimize recombinant protein secretion, this was not investigated for distantly related species. Therefore, in this study a Sec SP library from low-GC firmicutes B. subtilis is investigated to optimize protein secretion in high-GC actinobacterium Corynebacterium glutamicum using cutinase from Fusarium solani pisi as model protein.ResultsA homologous SP library (~150 SP) for recombinant cutinase secretion in B. subtilis was successfully transferred to C. glutamicum as alternative secretion host. Cutinase secretion in C. glutamicum was quantified using an automated micro scale cultivation system for online growth monitoring, cell separation and cutinase activity determination. Secretion phenotyping results were correlated to those from a previous study, in which the same SP library was used to optimize secretion of the same cutinase but using B. subtilis as host. Strikingly, behavior of specific SP-cutinase combinations was changed dramatically between B. subtilis and C. glutamicum. Some SPs showed comparable cutinase secretion performances in both hosts, whereas other SPs caused diametrical extracellular cutinase activities.ConclusionThe optimal production strain for a specific target protein of choice still cannot be designed in silico. Not only the best SP for a target protein has to be evaluated each time from scratch, the expression host also affects which SP is best. Thus, (heterologous) SP library screening using high-throughput methods is considered to be crucial to construct an optimal production strain for a target protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0604-6) contains supplementary material, which is available to authorized users.

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“…Furthermore, a variety of other commercially interesting metabolites can be produced with C. glutamicum (Becker and Wittmann, ), such as organic acids (Wendisch et al ., ; Okino et al ., ; Litsanov et al ., 2012a,b; Wieschalka et al ., ), diamines (Mimitsuka et al ., ; Kind and Wittmann, ; Schneider and Wendisch, ) or alcohols (Inui et al ., ; Smith et al ., ; Blombach et al ., ; Yamamoto et al ., ). Despite its complex cell envelope (Bansal‐Mutalik and Nikaido, ; Marchand et al ., ; Laneelle et al ., ), C. glutamicum is also an efficient host for the secretory production of heterologous proteins (see Kikuchi et al ., ; Scheele et al ., ; Matsuda et al ., ; and references therein). Based on the broad spectrum of products and its robustness in large‐scale production processes, C. glutamicum has become a platform and model organism in industrial biotechnology (Eggeling and Bott, ; Burkovski, ; Yukawa and Inui, ).…”

Section: Introductionmentioning

confidence: 99%

A chromosomally encoded T7 RNA polymerase‐dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single‐cell level

Kortmann

Kuhl

Klaffl

et al. 2014

Microbial Biotechnology

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Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-d-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg−1. It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum.

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Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

Cited by 49 publications

References 51 publications

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

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

Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum

A chromosomally encoded T7 RNA polymerase‐dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single‐cell level

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