Coding

Part:BBa_K5327006

Designed by: Han Cao   Group: iGEM24_BUCT   (2024-08-23)


Gamma-glutamyl peptidase 1

Function:[1][2][3]

Involved in glucosinolate biosynthesis. Hydrolyzes the gamma-glutamyl peptide bond of several glutathione (GSH) conjugates to produce Cys-Gly conjugates related to glucosinolates. The gamma-Glu-Cys-Gly-GSH conjugates are the sulfur-donating molecule in glucosinolate biosynthesis.Converts phenylacetohydroximoyl-GSH to benzylglucosinolate.Can use the GSH conjugate of the camalexin intermediate IAN (GS-IAN) as substrate. Required for the biosynthesis of camalexin, a pathogen-inducible phytoalexin with antibacterial and antifungal properties.


Usage and Biology

Genome localization:Chromosome: 4; NC_003075.7

Expression diagram:

Fig 1. The expression diagram of gamma-glutamyl peptidase 1

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of gamma-glutamyl peptidase 1

The PCR result:

Fig 3. The PCR result of gamma-glutamyl peptidase 1

Subcellular localization:[4]

Located in the cytoplasm and cytosol of cells

Fig 4. The subcellular localization of gamma-glutamyl peptidase 1

Dynamics data:

Table 1. The dynamics data of gamma-glutamyl peptidase 1

Design Notes

The design of the Gamma-glutamyl peptidase 1 (GGP1) gene is based on the coding sequence (CDS) from Arabidopsis thaliana, and it has been optimized for codon usage in Saccharomyces cerevisiae (S288C) to ensure efficient expression in yeast. GGP1 participates in the biosynthesis of glucosinolates by hydrolyzing the γ-glutamyl bond in glutathione (GSH) conjugates, producing Cys-Gly conjugates. [5]It also plays a role in converting phenylacetohydroximoyl-GSH to benzylglucosinolate and is essential in the biosynthesis of camalexin, a phytoalexin with antibacterial and antifungal properties. The gene is expressed in yeast using the ADH1 promoter (ADH1pBBa_J63005) and PYK1 terminator (PYK1tBBa_K5327018) to ensure high expression levels and mRNA stability. The optimized gene is inserted into a vector and introduced into Saccharomyces cerevisiae S288C through homologous recombination, followed by selection and expression validation using a defect type. This design aims to enhance the production of glucosinolates and camalexin-related metabolites in yeast, thereby optimizing yeast as a metabolic engineering platform.

Plasmid

Fig 5. The plasmid expression of methionine aminotransferase BCAT4

Source

Arabidopsis thaliana


References

  1. ↑ GEU-FLORES F, NIELSEN M T, NAFISI M, et al. Glucosinolate engineering identifies a gamma-glutamyl peptidase [J]. Nature chemical biology, 2009, 5(8): 575-7.
  2. ↑ GEU-FLORES F, MøLDRUP M E, BöTTCHER C, et al. Cytosolic γ-glutamyl peptidases process glutathione conjugates in the biosynthesis of glucosinolates and camalexin in Arabidopsis [J]. The Plant cell, 2011, 23(6): 2456-69.
  3. ↑ ITO T, KITAIWA T, NISHIZONO K, et al. Glutathione degradation activity of γ-glutamyl peptidase 1 manifests its dual roles in primary and secondary sulfur metabolism in Arabidopsis [J]. The Plant journal : for cell and molecular biology, 2022, 111(6): 1626-42.
  4. ↑ https://www.uniprot.org/uniprotkb/Q9M0A7/entry
  5. ↑ GEU-FLORES F, MøLDRUP M E, BöTTCHER C, et al. Cytosolic γ-glutamyl peptidases process glutathione conjugates in the biosynthesis of glucosinolates and camalexin in Arabidopsis [J]. The Plant cell, 2011, 23(6): 2456-69.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 175
    Illegal SpeI site found at 571
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 175
    Illegal SpeI site found at 571
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1211
    Illegal BamHI site found at 301
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 175
    Illegal SpeI site found at 571
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 175
    Illegal SpeI site found at 571
    Illegal AgeI site found at 43
  • 1000
    COMPATIBLE WITH RFC[1000]


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