Truncated PGK1 promoter is paired with varied terminators to regulate heterologous gene expression in Pichia pastoris

Abstract:

Objective] 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 P_PGK1 variants (P_PP, P_PE, P_PG and P_PD) relative to that of the native P_PGK1 ranged from 70% to 190%. Furthermore, when paired with the weak promoter P_PG, moderate promoter P_PE, and strong promoter P_PD, 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.