Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
Biotechnol Lett. 2020 Dec;42(12):2703-2709. doi: 10.1007/s10529-020-02979-x. Epub 2020 Aug 17.
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.
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.
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.
构建更强的毕赤酵母 P 启动子，并鉴定影响启动子活性的 P 上的假定转录因子结合位点 (TFBS)。
通过删除或重复启动子序列中的假定 TFBS 基序，生成了一个 P 的合成文库。CSRE、MIG1、RAP1 和 HAP2/3/4 被发现对 P 活性有重要影响。与野生型 P 和 P（报道的最强天然 P. pastoris 启动子）相比，在启动子序列上具有假定 RAP1 结合位点的 P 变体 P4 显示出更强的活性。这一推断通过 EGFP（增强型绿色荧光蛋白）和 Candida Antarctica lipase B 作为报告基因得到了证实。
转录调节因子 RAP1 的作用可能在 P 甲醇诱导中很重要。通过在 P 启动子序列上重复假定的 RAP1 结合位点，可以构建更强的 P 变体。该 P 变体在异源蛋白生产、代谢工程和合成生物学方面具有潜在价值。
