Identification of a novel growth-associated promoter for biphasic expression of heterogenous proteins in Pichia pastoris

Pichia pastoris (P. pastoris) is one of the most popular cell factories for expressing exogenous proteins and producing useful chemicals. The alcohol oxidase 1 promoter (P-AOX1) is the most commonly used strong promoter in P. pastoris and has the characteristic of biphasic expression. However, the inducer for P-AOX1, methanol, has toxicity and poses risks in industrial settings. In the present study, analyzing transcriptomic data of cells collected at different stages of growth found that the formate dehydrogenase (FDH) gene ranked 4960th in relative expression among 5032 genes during the early logarithmic growth phase but rose to the 10th and 1st during the middle and late logarithmic growth phases, respectively, displaying a strict biphasic expression characteristic. The unique transcriptional regulatory profile of the FDH gene prompted us to investigate the properties of its promoter (P-FDH800). Under single-copy conditions, when a green fluorescent protein variant was used as the expression target, the P-FDH800 achieved 119% and 69% of the activity of the glyceraldehyde-3-phosphate dehydrogenase promoter and P-AOX1, respectively. After increasing the copy number of the expression cassette in the strain to approximately four copies, the expression level of GFPuv driven by P-FDH800 increased to approximately 2.5 times that of the strain containing GFPuv driven by a single copy of P-AOX1. Our P-FDH800-based expression system exhibited precise biphasic expression, ease of construction, minimal impact on normal cellular metabolism, and high strength. Therefore, it has the potential to serve as a new expression system to replace the P-AOX1 promoter.