Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae

Zhu, Lin; Maruyama, Jun‐ichi; Kitamoto, Katsuhiko

doi:10.1007/s00253-013-4795-z

Cited by 22 publications

(16 citation statements)

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“…The NSlDv10 strain contains the deletion of Aovps10 for vacuolar protein sorting receptor to improve the protein secretion by reducing the tra cking pathway from Golgi to vacuoles [32]. In the AUT1-lD-v10-sD strain, beside Aovps10, the tripeptidyl peptidase gene AosedD was deleted in the hyper-producing mutant (AUT1) strain to further increase the protein production [29]. Additionally, the production loss in the culture medium was avoided by the deletion of ten-protease genes in the NSlD-ΔP10 strain [33].…”

Section: Resultsmentioning

confidence: 99%

“…The lamentous fungus Aspergillus oryzae is listed as Generally Regarded As Safe (GRAS) by the U.S. Food and Drug Administration (FDA) due to more than a thousand years of use in Japanese traditional food fermentation [28]. The fungus has been used as a host for heterologous protein production due to the ability to secrete large amounts of proteins into the culture medium [29], and the production of a hetero-oligomeric protein neoculin with the disul de bond was reported [30]. Thus, A. oryzae is expected to be a high potential host for industrial antibody production.…”

Section: Introductionmentioning

confidence: 99%

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Functional production of human antibody by the filamentous fungus Aspergillus oryzae

Huynh

Morita

Sakamoto

et al. 2020

Preprint

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Background: Monoclonal antibodies (mAbs) as biopharmaceuticals take a pivotal role in the current therapeutic applications. Generally mammalian cell lines, such as those derived from Chinese hamster ovaries (CHO), are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, a filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy the human anti-TNFα antibody adalimumab, one of the world’s best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to produce the full length of mAbs by A. oryzae. Generally, N-glycosylation of the antibody affects immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (FcγR) on immune cells. The CRISPR/Cas9 system was used to first delete the Aooch1 gene encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with FcγRIIIa.Results: Adalimumab was expressed in A. oryzae by the fusion protein system with α-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNFα neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of a commercial product Humira®. No apparent binding with the FcγRIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure using the Aooch1 deletion strain, which suggests only a little additional activity of immune effector functions.Conclusion: These results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient’s welfare.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

Huynh

Morita

Sakamoto

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…The NSlDv10 strain contains the deletion of Aovps10 for vacuolar protein sorting receptor to improve the protein secretion by reducing the trafficking pathway from Golgi to vacuoles [32]. In the AUT1-lD-v10-sD strain, beside Aovps10, the tripeptidyl peptidase gene AosedD was deleted in the hyper-producing mutant (AUT1) strain to further increase the protein production [29]. Additionally, the production loss in the culture medium was avoided by the deletion of ten-protease genes in NSlD-ΔP10 strain [33].…”

Section: Resultsmentioning

confidence: 99%

“…Food and Drug Administration (FDA) due to more than a thousand years of use in Japanese traditional food fermentation [28]. The fungus has been used as a favored host for heterologous protein production due to the ability to secrete large amounts of proteins into the culture medium [29], and the production of a hetero-oligomeric protein neoculin with the disulfide bond was reported [30].…”

Section: Introductionmentioning

confidence: 99%

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

Huynh¹,

Morita²,

Sakamoto³

et al. 2020

Preprint

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Background: Monoclonal antibodies (mAbs) as one of the biopharmaceuticals take a pivotal role in the current therapeutic application. Generally, mammalian cell lines such as Chinese hamster ovary (CHO) cell lines are used to produce the recombinant antibody. However, there are still concerns about the high cost and the risk of pathogenic contamination when using mammalian cells. Aspergillus oryzae, the filamentous fungus recognized as a GRAS (Generally Regarded As Safe) organism, has an ability to secrete a large amount of proteins into the culture supernatant, and thus the fungus has been used as one of the cost-effective microbial hosts for heterologous protein production. Pursuing this strategy, human anti-TNFα antibody adalimumab, one of the world’s best-selling antibodies for the treatment of immune-mediated inflammatory diseases including rheumatoid arthritis, was chosen to attempt for producing the full length of mAbs by A. oryzae. Generally, N-glycosylation in antibody affects the immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) via binding to the Fc receptor (FcγR) on immune cells. The CRISPR/Cas9 system was used to firstly delete the Aooch1 encoding a key enzyme for the hyper-mannosylation process in fungi to investigate the binding ability of antibody with FcγRIIIa.Results: The adalimumab was expressed in A. oryzae by the fusion protein system with α-amylase AmyB. The full-length adalimumab consisting of two heavy and two light chains was successfully produced in the culture supernatants. Among the producing strains, the highest amount of antibody was obtained from the ten-protease deletion strain (39.7 mg/L). Two-step purifications by Protein A and size-exclusion chromatography were applied to obtain the high purity sample for further analysis. The antigen-binding and TNFα neutralizing activities of the adalimumab produced by A. oryzae were comparable with those of the commercial product – Humira. No apparent binding with the FcγRIIIa was detected with the recombinant adalimumab even by altering the N-glycan structure under the Aooch1 deletion, which suggests only a little additional activity of immune effector functions.Conclusion: These results demonstrated an alternative low-cost platform for human antibody production by using A. oryzae, possibly offering a reasonable expenditure for patient’s welfare.

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“…A number of industrially useful proteins have been produced using A. oryzae Ito et al 2007 ;Chen et al 2010 ), and hyperproducing mutant strains have also been isolated (Nemoto et al 2009 ). Advances in molecular genetic techniques for generating multiple gene disruptions (Maruyama and Kitamoto 2008 ;Maruyama and Kitamoto 2011 ) have facilitated the molecular breeding of hyperproducing A. oryzae host strains (Yoon et al 2009 ;Yoon et al 2011 ;Zhu et al 2013 ). As recently reported, inhibition of vacuolar degradation machineries, such as vacuolar protein sorting and autophagy, enhanced the ability of heterologous protein production in A. oryzae (Yoon et al 2010 ;Yoon et al 2013 ).…”

Section: Introductionmentioning

confidence: 97%

Stress Biology of Yeasts and Fungi

Kitagaki¹

2015

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Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae

Cited by 22 publications

References 22 publications

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

Functional production of human antibody by the filamentous fungus Aspergillus oryzae

Stress Biology of Yeasts and Fungi

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