Xiaolong Wang1, Menghao Cai2, Lei Shi3, Qi Wang4, Jinxiang Zhu5, Jinjia Wang6, Mian Zhou7, Xiangshan Zhou8, Yuanxing Zhang9,10. 1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. jxwangxl@126.com. 2. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. cmh022199@ecust.edu.cn. 3. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. soshilei@qq.com. 4. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. wangqisam@163.com. 5. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. ywsy_bn@126.com. 6. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. lazeewang@gmail.com. 7. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. mianzhou@ecust.edu.cn. 8. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. xszhou@ecust.edu.cn. 9. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. yxzhang@ecust.edu.cn. 10. Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB), Shanghai, 200237, China. yxzhang@ecust.edu.cn.
Abstract
OBJECTIVE: The regulator in glycerol repression of Pichia pastoris AOX1 promoter (P AOX1 ) is still unclear. RESULTS: A Cys2His2 zinc finger transcriptional repressor PpNrg1 localized to nucleus and participated in the repression of P AOX1 in P. pastoris in glucose and glycerol. Quantitative real-time PCR revealed that PpNrg1 repressed expression of numerous genes involved in methanol utilization and peroxisome biogenesis in 0.02 % glucose and 1 % (v/v) glycerol. Electrophoretic mobility shift assay and DNase I footprinting assay revealed that PpNrg1 bound to five sites of P AOX1 , including two binding sites of PpMxr1, which is an indispensable activator of P AOX1 in P. pastoris. CONCLUSION: Transcriptional repressor PpNrg1 suppresses P AOX1 in glucose and glycerol by directly binding to five sites of P AOX1 , including two binding sites of transcriptional activator PpMxr1.
OBJECTIVE: The regulator in glycerol repression of Pichia pastoris AOX1 promoter (P AOX1 ) is still unclear. RESULTS: A Cys2His2 zinc finger transcriptional repressor PpNrg1 localized to nucleus and participated in the repression of P AOX1 in P. pastoris in glucose and glycerol. Quantitative real-time PCR revealed that PpNrg1 repressed expression of numerous genes involved in methanol utilization and peroxisome biogenesis in 0.02 % glucose and 1 % (v/v) glycerol. Electrophoretic mobility shift assay and DNase I footprinting assay revealed that PpNrg1 bound to five sites of P AOX1 , including two binding sites of PpMxr1, which is an indispensable activator of P AOX1 in P. pastoris. CONCLUSION: Transcriptional repressor PpNrg1 suppresses P AOX1 in glucose and glycerol by directly binding to five sites of P AOX1 , including two binding sites of transcriptional activator PpMxr1.