Please use this identifier to cite or link to this item: https://doi.org/10.1038/srep41850
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dc.titleMethanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris
dc.contributor.authorWang, J
dc.contributor.authorWang, X
dc.contributor.authorShi, L
dc.contributor.authorQi, F
dc.contributor.authorZhang, P
dc.contributor.authorZhang, Y
dc.contributor.authorZhou, X
dc.contributor.authorSong, Z
dc.contributor.authorCai, M
dc.date.accessioned2020-10-26T02:57:44Z
dc.date.available2020-10-26T02:57:44Z
dc.date.issued2017
dc.identifier.citationWang, J, Wang, X, Shi, L, Qi, F, Zhang, P, Zhang, Y, Zhou, X, Song, Z, Cai, M (2017). Methanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris. Scientific Reports 7 : 41850. ScholarBank@NUS Repository. https://doi.org/10.1038/srep41850
dc.identifier.issn2045-2322
dc.identifier.urihttps://scholarbank.nus.edu.sg/handle/10635/179757
dc.description.abstractThe alcohol oxidase 1 promoter (P AOX1 ) of Pichia pastoris is commonly used for high level expression of recombinant proteins. While the safety risk of methanol and tough process control for methanol induction usually cause problems especially in large-scale fermentation. By testing the functions of trans-acting elements of P AOX1 and combinatorially engineering of them, we successfully constructed a methanol-free P AOX1 start-up strain, in which, three transcription repressors were identified and deleted and, one transcription activator were overexpressed. The strain expressed 77% GFP levels in glycerol compared to the wide-type in methanol. Then, insulin precursor (IP) was expressed, taking which as a model, we developed a novel glucose-glycerol-shift induced P AOX1 start-up for this methanol-free strain. A batch phase with glucose of 40 g/L followed by controlling residual glucose not lower than 20 g/L was compatible for supporting cell growth and suppressing P AOX1 . Then, glycerol induction was started after glucose used up. Accordingly, an optimal bioprocess was further determined, generating a high IP production of 2.46 g/L in a 5-L bioreactor with dramatical decrease of oxygen consumption and heat evolution comparing with the wild-type in methanol. This mutant and bioprocess represent a safe and efficient alternative to the traditional glycerol-repressed/methanol-induced P AOX1 system. © The Author(s) 2017.
dc.publisherNature Publishing Group
dc.rightsAttribution 4.0 International
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.sourceUnpaywall 20201031
dc.subjectaldehyde oxidase
dc.subjectfungal protein
dc.subjectglucose
dc.subjectglycerol
dc.subjectmethanol
dc.subjectDNA responsive element
dc.subjectgene expression regulation
dc.subjectgenetics
dc.subjectgrowth, development and aging
dc.subjectmetabolism
dc.subjectmicrobiology
dc.subjectPichia
dc.subjectprocedures
dc.subjecttranscription initiation
dc.subjectAldehyde Oxidase
dc.subjectFungal Proteins
dc.subjectGene Expression Regulation, Fungal
dc.subjectGlucose
dc.subjectGlycerol
dc.subjectIndustrial Microbiology
dc.subjectMethanol
dc.subjectPichia
dc.subjectResponse Elements
dc.subjectTranscriptional Activation
dc.typeArticle
dc.contributor.departmentDEPT OF BIOCHEMISTRY
dc.description.doi10.1038/srep41850
dc.description.sourcetitleScientific Reports
dc.description.volume7
dc.description.page41850
dc.published.statepublished
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