Please use this identifier to cite or link to this item: https://doi.org/10.1038/srep41850
Title: Methanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris
Authors: Wang, J
Wang, X
Shi, L
Qi, F
Zhang, P
Zhang, Y
Zhou, X
Song, Z 
Cai, M
Keywords: aldehyde oxidase
fungal protein
glucose
glycerol
methanol
DNA responsive element
gene expression regulation
genetics
growth, development and aging
metabolism
microbiology
Pichia
procedures
transcription initiation
Aldehyde Oxidase
Fungal Proteins
Gene Expression Regulation, Fungal
Glucose
Glycerol
Industrial Microbiology
Methanol
Pichia
Response Elements
Transcriptional Activation
Issue Date: 2017
Publisher: Nature Publishing Group
Citation: Wang, 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
Rights: Attribution 4.0 International
Abstract: The 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.
Source Title: Scientific Reports
URI: https://scholarbank.nus.edu.sg/handle/10635/179757
ISSN: 2045-2322
DOI: 10.1038/srep41850
Rights: Attribution 4.0 International
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