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. 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. 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.

中文翻译：

---

改造过氧化氢酶启动子的调控位点以改善毕赤酵母中的异源蛋白质生产

构建更强的毕赤酵母 PCAT1 启动子，并确定 PCAT1 上影响启动子活性的假定转录因子结合位点 (TFBS)。PCAT1 的合成文库是通过删除或复制启动子序列中推定的 TFBS 基序生成的。CSRE、MIG1、RAP1 和 HAP2/3/4 被发现对 PCAT1 活性有重要影响。与野生型 PCAT1 和 PAOX1 相比，在启动子序列上具有推定 RAP1 结合位点的 PCAT1 变体 P4 显示出更强的活性，PAOX1 是已报道的最强的天然毕赤酵母启动子。这一推论得到了 EGFP（增强型绿色荧光蛋白）和南极念珠菌脂肪酶 B 的证实。转录调节因子 RAP1 的作用可能在 PCAT1 甲醇诱导中很重要。通过在 PCAT1 启动子序列上复制 RAP1 的假定结合位点，可以构建更强的 PCAT1 变体。这种 PCAT1 变体在异源蛋白质生产、代谢工程和合成生物学方面具有潜在价值。