Part:BBa_K4879005

Yarrowia lipolytica ScRAD52 gene

RAD52 coding sequence from Saccharomyces cerevisiae, codon optimized for Yarrowia lipolytica. [1]

Usage and Biology

RAD52 is a well-documented protein that helps in facilitating homologous recombination and subsequent DNA repair; found to be well-conserved across many species. It assists in the formation of a nucleoprotein filament on the broken single-stranded DNA (ssDNA) and thus facilitates strand invasion, the first step in homologous DNA repair.

Wild-type Y.lipolytica has a very low probability of homologous recombination (about 0-36%), which was detrimental to the deletion attempts we made to delete faa1 and alk2 genes from our chassis. As ScRAD52 enhances this probability to as high as 95%, we inserted the (ScRAD52 expression construct) into our organism.

The protein structure of ScRAD52, as predicted by AlphaFold.

Design

The transcriptional unit for CvFAP (BBa_K4879011) is designed with the coding sequence flanked by the TEF1 promoter (BBa_K2983053) upstream and the XPR2 terminator (BBa_K3629004) downstream.

References

1. Qingchun Ji, Jie Mai, Ying Ding, Yongjun Wei, Rodrigo Ledesma-Amaro, Xiao-Jun Ji, Improving the homologous recombination efficiency of Yarrowia lipolytica by grafting heterologous component from Saccharomyces cerevisiae, Metabolic Engineering Communications, Volume 11, 2020, e00152, ISSN 2214-0301, https://doi.org/10.1016/j.mec.2020.e00152.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 403
  Illegal BglII site found at 533
  Illegal XhoI site found at 1223
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1207
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Qingchun Ji, Jie Mai, Ying Ding, Yongjun Wei, Rodrigo Ledesma-Amaro, Xiao-Jun Ji. Improving the homologous recombination efficiency of Yarrowia lipolytica by grafting heterologous component from Saccharomyces cerevisiae, Metabolic Engineering Communications, Volume 11, 2020, e00152, ISSN 2214-0301 https://doi.org/10.1016/j.mec.2020.e00152