Part:BBa_K3239005

Prm1 transcription factor in the P.pastoris GS115 strain

Transcription factor in the P. pastoris GS115 strain. Self-upregulates Prm1 expression and upregulates Mit1 and AOX1 expression.

Usage and Biology

The AOX1 promoter is homogenous to the P. pastoris GS115 strain and is critical in the regulation of AOX1 (alcohol oxidase) expression in the methylotrophic yeast. Upon methanol induction, the Mxr1 protein is expressed and the PRM1 promoter is activated. The Mxr1 protein derepresses AOX1 promoter activation, whereas the Prm1 protein not only self-upregulates but also upregulates the expression of the Mit1 protein. Prm1 and Mit1 both upregulate AOX1 expression, allowing the yeast to instantly produce enough alcohol oxidase to metabolize methanol. Mit1 however, also downregulates the expression of Prm1, inhibiting the over-activation of Prm1 expression and subsequent Mit1 and alcohol oxidase expression.

Functional Parameters

A single-base mutation is introduced for compatibility with Type IIS. This single mutation does not affect the protein sequence.

Reference

Liang, S., Wang, B., Pan, L., Ye, Y., He, M., Han, S., … Lin, Y. (2012). Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing. BMC Genomics, 13(1). https://doi.org/10.1186/1471-2164-13-738

Shi, L., Wang, J., Wang, X., Zhang, Y., Song, Z., Cai, M., & Zhou, X. (2019). Transcriptome and metabolome analyses reveal global behaviour of a genetically engineered methanol-independent Pichia pastoris strain. Process Biochemistry, 76(August 2018), 46–54. https://doi.org/10.1016/j.procbio.2018.10.014

Wang, X., Wang, Q., Wang, J., Zhou, M., Shi, L., Zhou, X., … Shen, W. (2016). Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 ( AOX1 ) Promoter in Pichia pastoris. Journal of Biological Chemistry, 291(12), 6245–6261. https://doi.org/10.1074/jbc.m115.692053

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1034
  Illegal PstI site found at 1556
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1034
  Illegal NheI site found at 2815
  Illegal PstI site found at 1556
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1034
  Illegal BglII site found at 387
  Illegal BamHI site found at 1917
  Illegal BamHI site found at 2836
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1034
  Illegal PstI site found at 1556
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1034
  Illegal PstI site found at 1556
  
• 1000
  COMPATIBLE WITH RFC[1000]