Coding

Part:BBa_K5327001

Designed by: Junbin Li   Group: iGEM24_BUCT   (2024-08-20)


NADPH--cytochrome P450 reductase 2

Function:[1][2]

Converts 2-oxo acids to branched-chain amino acids. Shows activity with L-Leu, L-Ile and L-Val as amino donors and alpha-keto-glutarate as an amino acceptor, but no activity for D-isomers of Leu, Ile, Val, Asp, Glu or Ala. Acts on methionine and its derivatives and the corresponding 2-oxo acids. Catalyzes the initial deamination of methionine to 4-methylthio-2-oxobutyrate as well as the transamination of other typical intermediates of the methionine chain elongation pathway.

Usage and Biology

Genome localization:Chromosome 3 - NC_003074.8

Expression diagram:

Fig 1. The expression diagram of methionine aminotransferase BCAT4

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of methionine aminotransferase BCAT4

The PCR result :

Fig 3. The PCR result of methionine aminotransferase BCAT4

Subcellular localization:[3]

Located in the cytoplasm of cells

Fig 4. The subcellular localization of methionine aminotransferase BCAT4

Dynamics data:

Table 1. The dynamics data of methionine aminotransferase BCAT4

Design Notes

In this study, BCAT4 plays a crucial role in the chain elongation pathway of aliphatic glucosinolate biosynthesis, particularly exhibiting the highest activity when methionine (Met) is used as a substrate. BCAT4 catalyzes the transamination step in the chain elongation pathway, forming an essential component of Met-derived glucosinolate biosynthesis.[4] When designing the gene, the coding sequence (CDS) from Arabidopsis thaliana (ath) was selected and codon-optimized for Saccharomyces cerevisiae (S288C) to ensure efficient gene expression. BCAT4 was chosen as the target gene due to its crucial role in the biosynthesis of aliphatic glucosinolates. To optimize expression, the ADH1 promoter (ADH1p,BBa_J63005) and the CYC1 terminator (CYC1t,BBa_K2368002) from yeast were selected to effectively control gene transcription and ensure stable expression. After the gene design, the optimized gene was inserted into a vector and introduced into Saccharomyces cerevisiae (S288C) through homologous recombination, followed by screening and expression verification using auxotrophic selection. These steps ensure the stable expression of BCAT4 in yeast, where it functions in transamination within the target metabolic pathway, ultimately achieving efficient synthesis of the desired product.

Plasmid

Fig 5. The plasmid expression of methionine aminotransferase BCAT4

Source

Arabidopsis thaliana


References

  1. ↑ SCHUSTER J, KNILL T, REICHELT M, et al. Branched-chain aminotransferase4 is part of the chain elongation pathway in the biosynthesis of methionine-derived glucosinolates in Arabidopsis [J]. The Plant cell, 2006, 18(10): 2664-79.
  2. ↑ FUNAKOSHI M, SEKINE M, KATANE M, et al. Cloning and functional characterization of Arabidopsis thaliana D-amino acid aminotransferase--D-aspartate behavior during germination [J]. The FEBS journal, 2008, 275(6): 1188-200.
  3. ↑ DIEBOLD R, SCHUSTER J, DäSCHNER K, et al. The branched-chain amino acid transaminase gene family in Arabidopsis encodes plastid and mitochondrial proteins [J]. Plant physiology, 2002, 129(2): 540-50.
  4. ↑ SCHUSTER J, KNILL T, REICHELT M, et al. Branched-chain aminotransferase4 is part of the chain elongation pathway in the biosynthesis of methionine-derived glucosinolates in Arabidopsis [J]. The Plant cell, 2006, 18(10): 2664-79.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 907
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 907
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 907
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 907
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 283
    Illegal BsaI.rc site found at 472
    Illegal SapI.rc site found at 591


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Parameters
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