Coding

Part:BBa_K5327005

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


Branched-chain-amino-acid aminotransferase 3, chloroplastic

Function:[1][2]

Converts 2-oxo acids to branched-chain amino acids. Acts on leucine, isoleucine and valine. Also involved in methionine chain elongation cycle of aliphatic glucosinolate formation. Catalyzes the conversion of 5-methylthiopentyl-2-oxo and 6-methylthiohexyl-2-oxo acids to their respective Met derivatives, homomethionine and dihomo-methionine, respectively.

Usage and Biology

Genome localization:Chromosome: 3; NC_003074.8

Expression diagram:

Fig 1. The expression diagram of branched-chain-amino-acid aminotransferase 3, chloroplastic

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of branched-chain-amino-acid aminotransferase 3, chloroplastic

The PCR result:

Fig 3. The PCR result of branched-chain-amino-acid aminotransferase 3, chloroplastic

Subcellular localization:[3]

Located in the plastid chloroplast of cells

Fig 4. The subcellular localization of branched-chain-amino-acid aminotransferase 3, chloroplastic

Dynamics data:

Table 1. The dynamics data of branched-chain-amino-acid aminotransferase 3, chloroplastic

Design Notes

The design of the Branched-chain-amino-acid aminotransferase 3 (BCAT3) gene utilizes the coding sequence (CDS) from Arabidopsis thaliana, optimized for codons in Saccharomyces cerevisiae (S288C) to ensure efficient expression in yeast. BCAT3 is crucial for the synthesis of branched-chain amino acids, converting 2-oxo acids into leucine, isoleucine, and valine, and also participates in the methionine chain elongation cycle for aliphatic glucosinolate formation. In this experiment, BCAT3 catalyzes the transamination of Homoketo acid to produce Dihomomethionine. Additionally, it catalyzes the conversion of 5-methylthiopentyl-2-oxo and 6-methylthiohexyl-2-oxo acids to their respective methionine derivatives, homomethionine and dihomo-methionine. To express this enzyme in yeast, the ENO2 promoter (ENO2pBBa_K2765042) and HXT7 terminator (HXT7tBBa_K5327019) were chosen to ensure high-level expression and mRNA stability. After design completion, the optimized gene was inserted into a vector, introduced into Saccharomyces cerevisiae S288C via homologous recombination, and expression was verified through screening. This approach aims to maximize BCAT3’s enzymatic activity in yeast, enhancing the efficiency of branched-chain amino acid synthesis and methionine chain elongation for aliphatic glucosinolates, and optimizing yeast as a metabolic engineering platform.

Plasmid

Fig 5. The plasmid expression of branched-chain-amino-acid aminotransferase 3, chloroplastic

Source

Arabidopsis thaliana


References

  1. ↑ KNILL T, SCHUSTER J, REICHELT M, et al. Arabidopsis branched-chain aminotransferase 3 functions in both amino acid and glucosinolate biosynthesis [J]. Plant physiology, 2008, 146(3): 1028-39.
  2. ↑ LäCHLER K, IMHOF J, REICHELT M, et al. The cytosolic branched-chain aminotransferases of Arabidopsis thaliana influence methionine supply, salvage and glucosinolate metabolism [J]. Plant molecular biology, 2015, 88(1-2): 119-31.
  3. ↑ NIEHAUS T D, NGUYEN T N, GIDDA S K, et al. Arabidopsis and maize RidA proteins preempt reactive enamine/imine damage to branched-chain amino acid biosynthesis in plastids [J]. The Plant cell, 2014, 26(7): 3010-22.


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 site found at 885


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Parameters
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