Part:BBa_K4879004

Yarrowia lipolytica guaB marker

Mycophenolic acid resistance marker, codon optimized for Yarrowia lipolytica

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Usage and Biology

Due to the limited number of auxotrophic markers available for Yarrowia lipolytica, we had to use antibiotic-resistance markers for the selection of possible attempted deletions in our chassis. In order to use the selection marker, we had to use 1000-2000µg/ml of mycophenolic acid (MPA) in our selection plates.

MPA tends to kill cells by inhibiting inosine monophosphate dehydrogenase (IMPDH), an important enzyme in the de novo biosynthesis of GMP. The selection marker gene, which codes for an IMPDH variant, confers resistance by bypassing this inhibition.

The protein structure of IMPDH variant, as predicted by AlphaFold

Design

The transcriptional unit for the guaB marker (BBa_K4879010) is designed with the coding sequence flanked by the TEF1 promoter (BBa_K2983053) upstream and the XPR2 terminator (BBa_K3629004) downstream.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 565
  Illegal EcoRI site found at 1019
  Illegal PstI site found at 878
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 565
  Illegal EcoRI site found at 1019
  Illegal PstI site found at 878
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 565
  Illegal EcoRI site found at 1019
  Illegal BamHI site found at 1081
  Illegal XhoI site found at 851
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 565
  Illegal EcoRI site found at 1019
  Illegal PstI site found at 878
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 565
  Illegal EcoRI site found at 1019
  Illegal PstI site found at 878
  Illegal NgoMIV site found at 868
  Illegal NgoMIV site found at 1093
  
• 1000
  COMPATIBLE WITH RFC[1000]