Objectives To build a strongerPichia pastorisP(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 naturalP.pastorispromoter that has been reported. This inference was confirmed with EGFP (enhanced green fluorescent protein) andCandida Antarcticalipase 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.