Coding

Part:BBa_K5327007

Designed by: Fangxian Chen   Group: iGEM24_BUCT   (2024-08-26)


Dihomomethionine N-hydroxylase

Function:[1][2]

Catalyzes the conversion of the short chain elongated methionines di-, tri-, and tetrahomomethionine to their respective aldoximes 5-methylthiopentanaldoxime, 6-methylthiohexanaldoxime, and 7-methylheptanaldoxime.

Usage and Biology

Genome localization:Chromosome: 1; NC_003070.9

Expression diagram:

Fig 1. The expression diagram of dihomomethionine N-hydroxylase

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of dihomomethionine N-hydroxylase

The PCR result:

Fig 3. The PCR result of dihomomethionine N-hydroxylase

Subcellular localization:[3]

Located in the endoplasmic reticulum membrane of cells

Fig 4. The subcellular localization of dihomomethionine N-hydroxylase

Dynamics data:

Table 1. The dynamics data of dihomomethionine N-hydroxylase

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. [4] 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 5. The plasmid expression of dihomomethionine N-hydroxylase

Source

Arabidopsis thaliana


References

  1. ↑ HANSEN C H, WITTSTOCK U, OLSEN C E, et al. Cytochrome p450 CYP79F1 from arabidopsis catalyzes the conversion of dihomomethionine and trihomomethionine to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates [J]. The Journal of biological chemistry, 2001, 276(14): 11078-85.
  2. ↑ TANTIKANJANA T, MIKKELSEN M D, HUSSAIN M, et al. Functional analysis of the tandem-duplicated P450 genes SPS/BUS/CYP79F1 and CYP79F2 in glucosinolate biosynthesis and plant development by Ds transposition-generated double mutants [J]. Plant physiology, 2004, 135(2): 840-8.
  3. ↑ REINTANZ B, LEHNEN M, REICHELT M, et al. Bus, a bushy Arabidopsis CYP79F1 knockout mutant with abolished synthesis of short-chain aliphatic glucosinolates [J]. The Plant cell, 2001, 13(2): 351-67.
  4. ↑ HANSEN C H, WITTSTOCK U, OLSEN C E, et al. Cytochrome p450 CYP79F1 from arabidopsis catalyzes the conversion of dihomomethionine and trihomomethionine to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates [J]. The Journal of biological chemistry, 2001, 276(14): 11078-85.


Sequence and Features


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]


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