Coding

Part:BBa_K5327011

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


UDP-glycosyltransferase 74B1

Function:[1][2]

Involved in the biosynthesis of glucosinolate. In in vitro assay, may use phenylacetothiohydroximate (PATH), but not phenylacetic acid (PAA), indole-3-acetic acid (IAA) or salicylic acid (SA) as substrate. Specific for the thiohydroximate functional group and does not glucosylate the carboxylate group or a hydroxyl group.

Usage and Biology

Genome localization:Chromosome: 1; NC_003070.9

Expression diagram:

Fig 1. The expression diagram of UDP-glycosyltransferase 74B1

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of UDP-glycosyltransferase 74B1

The PCR result:

Fig 3. The PCR result of UDP-glycosyltransferase 74B1

Subcellular localization:[3]

Located in the cytosol and nucleus of cells

Fig 4. The subcellular localization of UDP-glycosyltransferase 74B1

Dynamics data:

Table 1. The dynamics data of UDP-glycosyltransferase 74B1

Design Notes

The design of the UDP-glycosyltransferase 74B1 (UGT74B1) gene is based on the coding sequence (CDS) from Arabidopsis thaliana and has been codon-optimized for efficient expression in Saccharomyces cerevisiae (S288C). UGT74B1 plays a crucial role in glucosinolate biosynthesis, specifically participating in the glycosylation step. It is specific for the thiohydroximate functional group and does not glycosylate the carboxylate group or a hydroxyl group.[4]To ensure high-level expression and mRNA stability, the design employs the PGK1 promoter(BBa_K2637015) and ADH1 terminator(BBa_K2637012). The optimized gene will be cloned into a vector, introduced into yeast S288C through homologous recombination, and validated using a knockout strain. This design aims to enhance the production efficiency of glucosinolates in yeast and further optimize yeast as a platform for metabolic engineering.

Plasmid

Fig 5. The plasmid expression of UDP-glycosyltransferase 74B1

Source

Arabidopsis thaliana


References

  1. ↑ LI Y, BALDAUF S, LIM E K, et al. Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana [J]. The Journal of biological chemistry, 2001, 276(6): 4338-43.
  2. ↑ GRUBB C D, ZIPP B J, LUDWIG-MüLLER J, et al. Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis [J]. The Plant journal : for cell and molecular biology, 2004, 40(6): 893-908.
  3. ↑ https://www.uniprot.org/uniprotkb/O48676/entry
  4. ↑ GRUBB C D, ZIPP B J, LUDWIG-MüLLER J, et al. Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis [J]. The Plant journal : for cell and molecular biology, 2004, 40(6): 893-908.


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 370
    Illegal BamHI site found at 265
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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