Coding

Part:BBa_K5327014

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


Myrosinase 1

Function:[1][2]

Degradation of glucosinolates (glucose residue linked by a thioglucoside bound to an amino acid derivative) to glucose, sulfate and any of the products: thiocyanates, isothiocyanates, nitriles, epithionitriles or oxazolidine-2-thiones. These toxic degradation products can deter insect herbivores. Seems to function in abscisic acid (ABA) and methyl jasmonate (MeJA) signaling in guard cells. Functionally redundant with TGG2. Hydrolyzes sinigrin and, with lower efficiency, p-nitrophenyl beta-D-glucoside.

Usage and Biology

Genome localization:Chromosome: 5; NC_003076.8

Expression diagram:

Fig 1. The expression diagram of myrosinase 1

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of myrosinase 1

The PCR result:

Fig 3. The PCR result of myrosinase 1

Subcellular localization:[3]

Located in the vacuole of cells

Fig 4. The subcellular localization of myrosinase 1

Dynamics data:

Table 1. The dynamics data of myrosinase 1

Design Notes

The design of the Myrosinase 1 gene is based on the coding sequence (CDS) from Arabidopsis thaliana and has been codon-optimized for Saccharomyces cerevisiae (S288C) to ensure its efficient expression. Myrosinase 1 is a key enzyme capable of degrading glucosinolates, and in this experiment, it primarily catalyzes the hydrolysis of Glucoraphanin, producing the target product Sulforaphane, which possesses anticancer, antibacterial, and antioxidant properties. The gene is expressed using the PGI1 promoter (PGI1pBBa_K5327017) and PYK1 terminator (PYK1tBBa_K5327018) to ensure high enzyme expression and mRNA stability in yeast. It is introduced into S. cerevisiae S288C via homologous recombination and screened and validated using auxotrophic selection. This design aims to enhance the activity of Myrosinase 1 in yeast, enabling efficient glucosinolate degradation and optimizing yeast as a metabolic engineering platform.

Plasmid

Fig 1. The plasmid expression of myrosinase 1

Source

Arabidopsis thaliana


References

  1. ↑ STRANGER B E, MITCHELL-OLDS T. Nucleotide variation at the myrosinase-encoding locus, TGG1, and quantitative myrosinase enzyme activity variation in Arabidopsis thaliana [J]. Molecular ecology, 2005, 14(1): 295-309.
  2. ↑ BARTH C, JANDER G. Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense [J]. The Plant journal : for cell and molecular biology, 2006, 46(4): 549-62.
  3. ↑ https://www.uniprot.org/uniprotkb/P37702/entry


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1563
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 421
    Illegal BsaI site found at 1030
    Illegal BsaI site found at 1403
    Illegal BsaI.rc site found at 1493


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