Coding

Part:BBa_K5327016

Designed by: Fangxian Chen   Group: iGEM24_BUCT   (2024-08-28)


Flavin-containing monooxygenase FMO GS-OX1

Function:[1][2][3]

Catalyzes the conversion of methylthioalkyl glucosinolates into methylsulfinylalkyl glucosinolates. Able to S-oxygenate both desulfo- and intact 4-methylthiobutyl glucosinolates, but no activity with methionine, dihomomethionine or 5-methylthiopentaldoxime.

Usage and Biology

Genome localization:Chromosome: 1; NC_003070.9

Expression diagram:

Fig 1. The expression diagram of flavin-containing monooxygenase FMO GS-OX1

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of flavin-containing monooxygenase FMO GS-OX1

The PCR result:

Fig 3. The PCR result of flavin-containing monooxygenase FMO GS-OX1

Design Notes

The design of the Flavin-containing monooxygenase FMO GS-OX1 gene is based on the coding sequence (CDS) from Arabidopsis thaliana and has been codon-optimized for efficient expression in Saccharomyces cerevisiae (S288C). FMO GS-OX1 encodes a flavin monooxygenase that catalyzes the conversion of methylthioalkyl glucosinolates into methylsulfinylalkyl glucosinolates.[4]This enzyme is capable of S-oxygenating both desulfo- and intact 4-methylthiobutyl glucosinolates but exhibits no activity with methionine, dihomomethionine, or 5-methylthiopentaldoxime. In this experiment, the GPD promoter (GPDpBBa_K517001) and PYK1 terminator (PYK1tBBa_K5327018) were selected to ensure high-level expression and mRNA stability in yeast. The optimized gene will be cloned into a vector and introduced into S. cerevisiae S288C through homologous recombination, followed by screening and expression validation using a knockout strain. This design aims to enhance the enzymatic activity of FMO GS-OX1 in yeast, thereby increasing the synthesis efficiency of methylsulfinylalkyl glucosinolates and optimizing yeast as a metabolic engineering platform for glucosinolate production.

Plasmid

Fig 1. The plasmid expression of flavin-containing monooxygenase FMO GS-OX1

Source

Arabidopsis thaliana


References

  1. ↑ HANSEN B G, KLIEBENSTEIN D J, HALKIER B A. Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis [J]. The Plant journal : for cell and molecular biology, 2007, 50(5): 902-10.
  2. ↑ KONG W, LI J, YU Q, et al. Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates [J]. Frontiers in plant science, 2016, 7: 1292.
  3. ↑ HANADA K, SAWADA Y, KUROMORI T, et al. Functional compensation of primary and secondary metabolites by duplicate genes in Arabidopsis thaliana [J]. Molecular biology and evolution, 2011, 28(1): 377-82.
  4. ↑ HANSEN B G, KLIEBENSTEIN D J, HALKIER B A. Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis [J]. The Plant journal : for cell and molecular biology, 2007, 50(5): 902-10.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 453
    Illegal EcoRI site found at 1124
    Illegal EcoRI site found at 1223
    Illegal XbaI site found at 700
    Illegal PstI site found at 1034
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 453
    Illegal EcoRI site found at 1124
    Illegal EcoRI site found at 1223
    Illegal NheI site found at 951
    Illegal PstI site found at 1034
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 453
    Illegal EcoRI site found at 1124
    Illegal EcoRI site found at 1223
    Illegal BamHI site found at 793
    Illegal XhoI site found at 300
    Illegal XhoI site found at 309
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 453
    Illegal EcoRI site found at 1124
    Illegal EcoRI site found at 1223
    Illegal XbaI site found at 700
    Illegal PstI site found at 1034
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 453
    Illegal EcoRI site found at 1124
    Illegal EcoRI site found at 1223
    Illegal XbaI site found at 700
    Illegal PstI site found at 1034
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 64
    Illegal BsaI site found at 139
    Illegal BsaI site found at 1257


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