Engineering the regulatory site of the catalase promoter for improved heterologous protein production in Pichia pastoris
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ORIGINAL RESEARCH PAPER
Engineering the regulatory site of the catalase promoter for improved heterologous protein production in Pichia pastoris Luyuan Nong . Yiming Zhang . Yehong Duan . Shulin Hu . Ying Lin . Shuli Liang
Received: 23 December 2019 / Accepted: 29 July 2020 Ó Springer Nature B.V. 2020
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. Keywords Pichia pastoris Promoter Transcriptional factor binding sites PCAT1 CALB
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-020-02979-x) contains supplementary material, which is available to authorized users. L. Nong Y. Zhang Y. Duan S. Hu Y. Lin S. Liang (&) Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China e-mail: [email protected] L. Nong Y. Zhang Y. Duan S. Hu Y. Lin S. Liang Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China
The methylotrophic yeast Pichia pastoris (Komagataella phaffii) has many advantages for protein expression (Gellissen 2000). P. pastoris has the ability to grow quickly on minimal media and is well suited for high-density fermentation, which makes the process of fermentation economical and efficient (Zhu et al. 2019). Increasing the transcription level is one of the critical steps for heterologous protein production, and the selection of an effective promoter is a key step for increasing the transcription level. Several promoters of P. pastoris, either constitutive or inducible, have been engineered and used (Juturu and Wu 2018).
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Biotechnol Lett
Catalase is an important enzyme in the methanol utilization pathway in P. pastoris. The promoter of the catalase (PCAT1) show th
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