Luyuan Nong1,2, Yiming Zhang1,2, Yehong Duan1,2, Shulin Hu1,2, Ying Lin1,2, Shuli Liang3,4. 1. Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China. 2. Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China. 3. Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China. shuli@scut.edu.cn. 4. Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China. shuli@scut.edu.cn.
Abstract
OBJECTIVES: To build a stronger Pichia pastoris PCAT1 promoter and to identify putative transcriptional factor binding sites (TFBSs) on PCAT1 that affect the activity of the promoter. RESULT: A synthetic library of PCAT1 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 PCAT1 activity. The PCAT1 variant P4 with a putative binding site of RAP1 on the promoter sequence showed a stronger activity compared with that of the wild-type PCAT1 and PAOX1, 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 PCAT1 methanol induction. A stronger PCAT1 variant can be constructed by the duplication of the putative binding site of RAP1 on the PCAT1 promoter sequence. This PCAT1 variant has potential value for heterologous protein production, metabolic engineering, and synthetic biology.
OBJECTIVES: To build a stronger Pichia pastoris PCAT1 promoter and to identify putative transcriptional factor binding sites (TFBSs) on PCAT1 that affect the activity of the promoter. RESULT: A synthetic library of PCAT1 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 PCAT1 activity. The PCAT1 variant P4 with a putative binding site of RAP1 on the promoter sequence showed a stronger activity compared with that of the wild-type PCAT1 and PAOX1, 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 PCAT1 methanol induction. A stronger PCAT1 variant can be constructed by the duplication of the putative binding site of RAP1 on the PCAT1 promoter sequence. This PCAT1 variant has potential value for heterologous protein production, metabolic engineering, and synthetic biology.