Rewiring Methanol-free AOX1 Promoter for Recombinant Proteins Expression in Pichia pastoris | AIChE

Rewiring Methanol-free AOX1 Promoter for Recombinant Proteins Expression in Pichia pastoris

Authors 

Shi, L., State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
Wang, X., East China University of Science and Technology
Wang, J., East China University of Science and Technology

The alcohol oxidase 1 (AOX1) promoter (PAOX1) of Pichia pastoris is frequently used to express recombinant proteins as it leads to high levels of expression in response to methanol. Large amounts of methanol, however, can pose a considerable safety risk in industrial bioprocess. To develop a methanol-free approach, we identified and investigated the roles of three transcriptional suppressors and one activator of PAOX1. Their transcription regulation mechanisms on PAOX1 were first investigated. Then, deletion of the three repressors – Mig1, Mig2, and Nrg1 – and overexpression of the activator – Mit1 – lead to PAOX1 expression in response to glycerol. A novel P. pastoris strain was generated in which Mit1 was overexpressed under the control of PAOX1 in the triple deletion mutant Δmig1Δmig2Δnrg1. When cultured in glycerol, GFP regulated by PAOX1 achieved higher levels of expression to the wide-type strain induced with methanol. The cellular processes of the triple deletion mutant overexpressing Mit1 were studied systematically by RNA-Seq. This novel strain represents a safe and efficient alternative to the traditional methanol-based expression systems.