Coding
MAM1

Part:BBa_K5327002

Designed by: Junbin Li   Group: iGEM24_BUCT   (2024-08-20)


Methylthioalkylmalate synthase 1, chloroplastic

Function:[1][2]

Determines the side chain length of aliphatic glucosinolate structures. Catalyzes exclusively the condensation reactions of both the first and second methionine carbon chain elongation.

Usage and Biology

Genome localization:Chromosome 5 - NC_003076.8

Expression diagram:

Fig 1. The expression diagram of methylthioalkylmalate synthase 1, chloroplastic

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of methylthioalkylmalate synthase 1, chloroplastic

The PCR result:

Fig 3. The PCR result of methylthioalkylmalate synthase 1, chloroplastic

Subcellular localization:[3]

Located in the chloroplast of cells

Fig 4. The subcellular localization of methylthioalkylmalate synthase 1, chloroplastic

Dynamics data:

Table 1. The dynamics data of methylthioalkylmalate synthase 1, chloroplastic

Design Notes

To obtain the key intermediate product dihomomethionine, we optimized the Methylthioalkylmalate synthase 1 (MAM1) gene from Arabidopsis thaliana and codon-optimized it for Saccharomyces cerevisiae (S288C) to ensure its efficient expression in yeast. MAM1 specifically catalyzes the first and second condensation reactions in the methionine carbon chain elongation, determining the side chain length of aliphatic glucosinolates. [4]We selected the CDC19 promoter (CDC19pBBa_K3772015) and ADH1 terminator (ADH1tBBa_K2637012) to drive and stabilize gene expression. After designing the optimized gene, it was inserted into a vector and integrated into yeast through homologous recombination. Defective strains were then used for screening and expression verification. This strategy ensures that MAM1 efficiently catalyzes methionine chain elongation, particularly in producing the key intermediate dihomomethionine, thereby facilitating the synthesis of the desired product.

Plasmid

Fig 5. The plasmid expression of methylthioalkylmalate synthase 1, chloroplastic

Source

Arabidopsis thaliana


References

  1. ↑ TEXTOR S, BARTRAM S, KROYMANN J, et al. Biosynthesis of methionine-derived glucosinolates in Arabidopsis thaliana: recombinant expression and characterization of methylthioalkylmalate synthase, the condensing enzyme of the chain-elongation cycle [J]. Planta, 2004, 218(6): 1026-35.
  2. ↑ BENDEROTH M, TEXTOR S, WINDSOR A J, et al. Positive selection driving diversification in plant secondary metabolism [J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(24): 9118-23.
  3. ↑ https://www.uniprot.org/uniprotkb/Q9FG67/entry
  4. ↑ TEXTOR S, BARTRAM S, KROYMANN J R, et al. Biosynthesis of methionine-derived glucosinolates in Arabidopsis thaliana : recombinant expression and characterization of methylthioalkylmalate synthase, the condensing enzyme of the chain-elongation cycle [J]. Planta, 2004, 218(6): 1026-35.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 907
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 907
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 907
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 907
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 283
    Illegal BsaI.rc site found at 472
    Illegal SapI.rc site found at 591


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