本研究通过随机突变对毕赤酵母甲醇诱导型强启动子PCAT1进行改造，以绿色荧光蛋白为表征，经过高通量筛选，获得一个活性范围为25.57%~138.12%的PCAT1启动子库；通过测序发现活性提高了38.12%的PCAT1变体6-39在5’-3’方向上的-118bp处的碱基由A突变为了G。用转录因子结合位点在线预测软件MatInspector分析该PCAT1变体的转录因子结合位点变化，发现该PCAT1变体与野生型的PCAT1相比，F$YMCM结合位点减少，并且F$CSRE和F$YGAL结合位点增多。接着，用MatInspector分析16种毕赤酵母甲醇利用途径基因启动子的转录因子结合位点，发现转录因子F$CSRE结合位点在甲醇诱导型启动子中分布广泛，F$YGAL结合位点仅在组成型的启动子中分布广泛，推测F$CSRE结合位点的增多最有可能提高PCAT1的甲醇诱导活性，为毕赤酵母甲醇利用途径基因启动子关键位点解析提供参考。

In this study, the methanol-inducible strong promoter PCAT1 of Pichia pastoris was modified by random mutation, characterized by green fluorescent protein. After high-throughput screening, a PCAT1 promoter library with an activity range of 25.57%- 138.12% was obtained. It was found that the PCAT1 variant 6-39 whose activity increased by 38.12% was mutated from A to G at the -118 bp in the 5 '-3'direction.The online prediction software for transcription factor binding sites, MatInspector, was used to analyze the change of transcription factor binding sites of the PCAT1 variants. Compared to wild-type PCAT1, the binding site of F$YMCM of the PCAT1 variant was reduced, and the binding sites of F$CSRE and F$YGAL increased. Then, the transcription factor binding sites of 16 promoters of the genes involved in methanol utilization pathway of Pichia pastoris were analyzed by MatInspector. It was found that the binding sites of the transcription factor, F$CSRE, were widely distributed in the methanol-inducible promoters, whilst the binding sites of F$YGAL were extensively distributed only in the constitutive promoter. Thus, the increase of the binding sites of F$CSRE is most likely to increase the methanol-inducing activity of PCAT1, which provides a reference for the analysis of the key sites of the promoters of the genes for the methanol utilization pathway of Pichia pastoris.

广东省重点领域研发计划项目（2019B020210003）