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

OBJECTIVES

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

RESULT

A synthetic library of P 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 activity. The P 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 and P, 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 methanol induction. A stronger P variant can be constructed by the duplication of the putative binding site of RAP1 on the P promoter sequence. This P variant has potential value for heterologous protein production, metabolic engineering, and synthetic biology.