Coding

Part:BBa_K5327005:Design

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


Branched-chain-amino-acid aminotransferase 3, chloroplastic


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


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

Source

Arabidopsis thaliana