Establishment and application of an RNAi system in Pichia pastoris

Shupeng Ruan Aoxue Wang Ying Lin Shuli Liang ^*

• South China University of Technology, Guangzhou, China

Reducing endogenous gene expression is a critical task in the metabolic engineering of microbes. However, current methods for gene knockout or suppression can be cumbersome and time-consuming. Target gene expression can be regulated by introducing plasmids containing corresponding hairpin RNA into the cells, where an RNA interference (RNAi) system has been established. This is the first study to explore the regulation of single-gene and double-gene suppression in Pichia pastoris. Additionally, reporter strains of P. pastoris displaying EGFP on the cell surface were used to screen factors relevant to the heterologous protein secretion pathway.Inhibition of YAP1 and YPS1 reduced EGFP display levels by 83% and 48.8%, respectively. In contrast, inhibiting the expression of the endogenous genes PRB1 and PEP4 increased the level of displayed EGFP by 33.8% and 26.5%, respectively. Furthermore, RNAi was employed to suppress fatty acid synthesis, thereby enhancing the conversion of malonyl-CoA to 3-HP and ultimately improving 3-HP synthesis. This study establishes an RNA interference system in P.2 pastoris, offering a simpler, faster, and more efficient approach for rapid gene inhibition during strain construction. The system holds significant potential for optimizing P. pastoris as a microbial cell factory, providing a valuable tool for industrial applications in microbial cell engineering.