Part:BBa_K5327007:Design

Dihomomethionine N-hydroxylase

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1166
  Illegal SpeI site found at 45
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1166
  Illegal SpeI site found at 45
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1166
  Illegal XhoI site found at 570
  Illegal XhoI site found at 1519
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1166
  Illegal SpeI site found at 45
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1166
  Illegal SpeI site found at 45
  Illegal NgoMIV site found at 262
  Illegal AgeI site found at 1402
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The design of the Dihomomethionine N-hydroxylase gene uses the coding sequence (CDS) from Arabidopsis thaliana, optimized for codon usage in Saccharomyces cerevisiae (S288C) to ensure efficient expression in yeast. This plant cytochrome P-450 enzyme is crucial for converting chain-elongated methionines, such as dihomomethionine, trihomomethionine, and tetrahomomethionine, into their corresponding aldoximes, which are key intermediates in the metabolic pathway. In this experiment, the enzyme specifically catalyzes the conversion of dihomomethionine (DHM). To ensure high-level expression and mRNA stability, the CDC19 promoter (CDC19pBBa_K3772015) and PYK1 terminator (PYK1tBBa_K5327018) were selected. The optimized gene was inserted into a vector and introduced into Saccharomyces cerevisiae S288C via homologous recombination, followed by screening and expression validation using a defective strain. This approach enables Dihomomethionine N-hydroxylase to effectively catalyze the conversion reactions in yeast, enhancing the synthesis of target products and optimizing yeast as a metabolic engineering platform.

Plasmid

Fig 1. The plasmid expression of dihomomethionine N-hydroxylase

Source

Arabidopsis thaliana