Coding

Part:BBa_K5327015

Designed by: Yifan Zhang   Group: iGEM24_BUCT   (2024-08-28)


Cytosolic sulfotransferase 18

Function:[1][2]

Sulfotransferase that utilizes 3'-phospho-5'-adenylyl sulfate (PAPS) as sulfonate donor to catalyze the sulfate conjugation of desulfo-glucosinolates (dsGSs), the final step in the biosynthesis of the glucosinolate core structure. Preferred substrate are the long-chain desulfo-glucosinolates, 7-methylthioheptyl and 8-methylthiooctyl, derived from methionine. Substrate preference is desulfo-benzyl glucosinolate > desulfo-4-methylthiobutyl glucosinolate > desulfo-6-methylthiohexyl glucosinolate > desulfo-3-methylthiopropyl glucosinolate > desulfo-indol-3-yl methyl glucosinolate > desulfo-singrin > desulfo-3-butenyl glucosinolate.

Usage and Biology

Genome localization:Chromosome: 1; NC_003070.9

Expression diagram:

Fig 1. The expression diagram of cytosolic sulfotransferase 18

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of cytosolic sulfotransferase 18

The PCR result

Fig 3. The PCR result of cytosolic sulfotransferase 18

Subcellular localization:[3]

Located in the cytoplasm of cells

Fig 4. The subcellular localization of cytosolic sulfotransferase 18

Dynamics data:

Table 1. The dynamics data of cytosolic sulfotransferase 18

Design Notes

The design of the Cytosolic sulfotransferase 18 (SOT18) gene is based on the coding sequence (CDS) from Arabidopsis thaliana and has been codon-optimized for efficient expression in Saccharomyces cerevisiae (S288C). SOT18 is a sulfotransferase that utilizes 3'-phospho-5'-adenylyl sulfate (PAPS) as a sulfonate donor to catalyze the sulfate conjugation of desulfo-glucosinolates (dsGSs), the final step in the biosynthesis of the glucosinolate core structure.[4]SOT18 prefers long-chain desulfo-glucosinolates derived from methionine, such as 7-methylthioheptyl and 8-methylthiooctyl, as substrates, with varying preferences for other substrates like desulfo-benzyl glucosinolate and desulfo-4-methylthiobutyl glucosinolate. To ensure high-level expression and mRNA stability, the design employs the PGI1 promoter (PGI1pBBa_K5327017) and HXT7 terminator (HXT7tBBa_K5327019). The optimized gene will be cloned into a vector and introduced into yeast S288C through homologous recombination, validated using a knockout strain. This design aims to enhance the efficiency of desulfo-glucosinolate sulfation in yeast, optimizing yeast as a metabolic engineering platform for glucosinolate production.

Plasmid

Fig 1. The plasmid expression of cytosolic sulfotransferase 18

Source

Arabidopsis thaliana


References

  1. ↑ PIOTROWSKI M, SCHEMENEWITZ A, LOPUKHINA A, et al. Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyze the final step in the biosynthesis of the glucosinolate core structure [J]. The Journal of biological chemistry, 2004, 279(49): 50717-25.
  2. ↑ HIRAI M Y, KLEIN M, FUJIKAWA Y, et al. Elucidation of gene-to-gene and metabolite-to-gene networks in arabidopsis by integration of metabolomics and transcriptomics [J]. The Journal of biological chemistry, 2005, 280(27): 25590-5.
  3. ↑ KLEIN M, PAPENBROCK J. The multi-protein family of Arabidopsis sulphotransferases and their relatives in other plant species [J]. Journal of experimental botany, 2004, 55(404): 1809-20.
  4. ↑ KLEIN M, PAPENBROCK J. Kinetics and substrate specificities of desulfo-glucosinolate sulfotransferases in Arabidopsis thaliana [J]. Physiologia plantarum, 2009, 135(2): 140-9.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 64
    Illegal BsaI.rc site found at 150


[edit]
Categories
Parameters
None