Engineering the regulatory site of the catalase promoter for improved heterologous protein production in Pichia pastoris

Abstract

Objectives: To build a stronger Pichia pastoris P_CAT1 promoter and to identify putative transcriptional factor binding sites (TFBSs) on P_CAT1 that affect the activity of the promoter.

Result: A synthetic library of P_CAT1 was generated by deleting or duplicating putative TFBS motifs in the promoter sequence. CSRE, MIG1, RAP1 and HAP2/3/4 were found to have important effects on P_CAT1 activity. The P_CAT1 variant P4 with a putative binding site of RAP1 on the promoter sequence showed a stronger activity compared with that of the wild-type P_CAT1 and P_AOX1, which is the strongest natural P. pastoris promoter that has been reported. This inference was confirmed with EGFP (enhanced green fluorescent protein) and Candida Antarctica lipase B as the reporters.

Conclusion: The role of the transcriptional regulator RAP1 may be important in P_CAT1 methanol induction. A stronger P_CAT1 variant can be constructed by the duplication of the putative binding site of RAP1 on the P_CAT1 promoter sequence. This P_CAT1 variant has potential value for heterologous protein production, metabolic engineering, and synthetic biology.

Keywords: CALB; PCAT1; Pichia pastoris; Promoter; Transcriptional factor binding sites.