ZHANG Huijie
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi, ChinaLIAO Simin
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi, ChinaLING Xiaocui
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi, ChinaFENG Jiaxun
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi, ChinaQIN Xiulin
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi, ChinaObjective] Pichia pastoris (syn. Komagataella phaffii) has been extensively used as a versatile cell factory for the production of industrial enzymes and chemicals. However, well-tuned co-expression of multiple genes is a common challenge for P.pastoris in metabolic engineering and synthetic biology. Therefore, in this work, we constructed a set of terminators and paired them with varied promoters to tune the protein levels in P.pastoris.[Methods] We constructed the P.pastoris strains expressing reporter genes (egfp and lacZ) under the control of truncated constitutive 3-phosphoglycerate kinase (PGK1) promoters, and then measured the transcript levels of reporter genes, yEGFP fluorescence intensity and β-galactosidase activity of these strains. Next, we created a total of 27 promoter-terminator pairs to regulate the transcription of egfp, and used 6 promoter- terminator pairs to alter the secretory expression of β-fructofuranosidase (β-Ffase). [Results] The promoter activities of the truncated PPGK1 variants (PPP, PPE, PPG and PPD) relative to that of the native PPGK1 ranged from 70% to 190%. Furthermore, when paired with the weak promoter PPG, moderate promoter PPE, and strong promoter PPD, the terminators had the tuning ranges of 4, 7 and 10 folds (comparing between the strongest and weakest terminator), respectively. Finally, we demonstrated the utility of the promoter-terminator pairs for tuning the expression of the industrial enzyme β-Ffase, which showed an overall tuning range of 6 folds. [Conclusion] The promoter-terminator pairs constructed not only provide valuable information for understanding the modulatory roles of terminator regions in gene expression but also serve as a useful toolbox enabling the metabolic engineering of P.pastoris and the application of P.pastoris in synthetic biology.
ZHANG Huijie, LIAO Simin, LING Xiaocui, FENG Jiaxun, QIN Xiulin. Truncated PGK1 promoter is paired with varied terminators to regulate heterologous gene expression in Pichia pastoris. [J]. Acta Microbiologica Sinica, 2022, 62(7): 2642-2657
Copy
WeChat ID 
Mobile Terminal Acta Microbiologica Sinica ® 2025 All Rights Reserved
