Coding

Part:BBa_K5327009

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


Cytochrome P450 83A1

Function:[1][2]

Involved in the metabolism of aliphatic and aromatic oximes.Involved in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates.


Usage and Biology

Genome localization:Chromosome 4 - NC_003075.7

Expression diagram:

Fig 1. The expression diagram of cytochrome P450 83A1

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of cytochrome P450 83A1

The PCR result:

Fig 3. The PCR result of cytochrome P450 83A1

Subcellular localization:[3]

Located in the endoplasmic reticulum membrane of cells

Fig 4. The subcellular localization of cytochrome P450 83A1

Dynamics data:

Table 1. The dynamics data of cytochrome P450 83A1

Design Notes

The design of the Cytochrome P450 83A1 (CYP83A1) gene utilizes the coding sequence (CDS) from Arabidopsis thaliana, optimized for codon usage in Saccharomyces cerevisiae (S288C) to ensure efficient expression in yeast. CYP83A1 plays a crucial role in the metabolism of aliphatic and aromatic aldoximes, participating in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates.[4]In this study, CYP83A1 metabolizes (E)-aldoximes derived from chain-elongated methionine, such as dihomomethionine. The gene is expressed in yeast using the PGI1 promoter (PGI1pBBa_K5327017) and HXT7 terminator (HXT7tBBa_K5327019) to ensure high-level expression and mRNA stability. The optimized gene was inserted into a vector and introduced into Saccharomyces cerevisiae S288C via homologous recombination, followed by screening and expression validation using a defective strain. This method aims to enhance the synthesis of aliphatic glucosinolates in yeast and optimize yeast as a metabolic engineering platform.

Plasmid

Fig 5. The plasmid expression of cytochrome P450 83A1

Source

Arabidopsis thaliana


References

  1. ↑ NAUR P, PETERSEN B L, MIKKELSEN M D, et al. CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzymes metabolizing oximes in the biosynthesis of glucosinolates in Arabidopsis [J]. Plant physiology, 2003, 133(1): 63-72.
  2. ↑ HEMM M R, RUEGGER M O, CHAPPLE C. The Arabidopsis ref2 mutant is defective in the gene encoding CYP83A1 and shows both phenylpropanoid and glucosinolate phenotypes [J]. The Plant cell, 2003, 15(1): 179-94.
  3. ↑ https://www.uniprot.org/uniprotkb/P48421/entry
  4. ↑ NAUR P, PETERSEN B L, MIKKELSEN M D, et al. CYP83A1 and CYP83B1, Two Nonredundant Cytochrome P450 Enzymes Metabolizing Oximes in the Biosynthesis of Glucosinolates in Arabidopsis [J]. Plant physiology, 2003, 133(1): 63-72.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 911
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 246
    Illegal PstI site found at 911
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 641
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 911
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 911
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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