文献类型：期刊文献

英文题名：Methanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris

作者：Wang, Jinjia[1];Wang, Xiaolong[1];Shi, Lei[1];Qi, Fei[1];Zhang, Ping[1];Zhang, Yuanxing[1,2];Zhou, Xiangshan[1];Song, Zhiwei[3,4];Cai, Menghao[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg, 130 Meilong Rd, Shanghai 200237, Peoples R China;[3]Bioproc Technol Inst, 20 Biopolis Way,06-01 Ctr, Singapore, Singapore;[4]Natl Univ Singapore, Yong Loo Lin Sch Med, Dept Biochem, 21 Lower Kent Ridge Rd, Singapore, Singapore

年份：2017

卷号：7

外文期刊名：SCIENTIFIC REPORTS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000393174400001)】；

基金：This work was supported by Chinese National High Technology Research and Development Program (2014AA093501), National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204), Fundamental Research Funds for the Central Universities (22A201514040).

语种：英文

摘要：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.