Zhan Chunjun, Yang Yankun, Zhang Zhenyang, Li Xiang, Liu Xiuxia, Bai Zhonghu
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China.
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
FEMS Yeast Res. 2017 Jun 1;17(4). doi: 10.1093/femsyr/fox015.
In the methylotrophic yeast Pichia pastoris (P. pastoris), the efficient promoter of alcohol oxidase (PAox1) is induced by methanol and repressed by glycerol, but the molecular mechanism is not clear. In this study, the relationship between alcohol oxidase 1 (aox1), methanol expression regulator 1 (mxr1) and glycerol transporter 1 (gt1) was studied. By RT-PCR, it was found that the overexpression of gt1 could increase the glycerol content in cells and repress the expression of mxr1 and aox1, and the deletion of gt1 reduced the glycerol content in cells and promoted the expression of aox1. The overexpression of mxr1 could repress the expression of gt1, and the deletion of mxr1 could promote the expression of gt1 to some extent. By EMSA, Mxr1 binding sites were found in the promoter of gt1 (PGt1) (-141 to -138, CCCC), and Mxr1 could regulate the expression of gt1 by binding to PGt1. The relationships among aox1, mxr1 and gt1 revealed here provide a reference for the understanding of the mechanism of glycerol repression of PAox1.
在甲基营养型酵母巴斯德毕赤酵母（P. pastoris）中，醇氧化酶高效启动子（PAox1）受甲醇诱导且被甘油抑制，但其分子机制尚不清楚。在本研究中，对醇氧化酶1（aox1）、甲醇表达调节因子1（mxr1）和甘油转运蛋白1（gt1）之间的关系进行了研究。通过逆转录聚合酶链反应（RT-PCR）发现，gt1的过表达可增加细胞内甘油含量并抑制mxr1和aox1的表达，而gt1的缺失则降低细胞内甘油含量并促进aox1的表达。mxr1的过表达可抑制gt1的表达，mxr1的缺失在一定程度上可促进gt1的表达。通过电泳迁移率变动分析（EMSA）发现，在gt1启动子（PGt1）（-141至-138，CCCC）中存在Mxr1结合位点，且Mxr1可通过与PGt1结合来调节gt1的表达。此处揭示的aox1、mxr1和gt1之间的关系为理解PAox1的甘油抑制机制提供了参考。
