Methanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris.
The alcohol oxidase 1 promoter (PAOX1) 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 PAOX1and combinatorially engineering of them, we successfully constructed a methanol-free PAOX1start-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 PAOX1start-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 PAOX1. 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 PAOX1system.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
Jinjia
J
Wang
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
Xiaolong
X
Wang
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
Lei
L
Shi
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
Fei
F
Qi
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
Ping
P
Zhang
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.|Shanghai Collaborative Innovation Center for Biomanufacturing, 130 Meilong Road, Shanghai 200237, China.
Yuanxing
Y
Zhang
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
Xiangshan
X
Zhou
Bioprocessing Technology Institute, 20 Biopolis Way, #06-01 Centros, Singapore.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 21 Lower Kent Ridge Road, Singapore.
Zhiwei
Z
Song
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
Menghao
M
Cai
D042931:Aldehyde Oxidase / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods*; D000432:Methanol / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D020218:Response Elements; D015533:Transcriptional Activation
D005656:Fungal Proteins; D042931:Aldehyde Oxidase; D005947:Glucose; D005990:Glycerol; D000432:Methanol