Coding

Part:BBa_K5327003

Designed by: Han Cao   Group: iGEM24_BUCT   (2024-08-20)


3-isopropylmalate dehydratase large subunit, chloroplastic

Function:[1][2]

Catalyzes the isomerization between 2-isopropylmalate and 3-isopropylmalate, via the formation of 2-isopropylmaleate.Functions in both the biosynthesis of leucine and in the methionine chain elongation pathway of aliphatic glucosinolate formation.

Usage and Biology

Genome localization:Chromosome 4 - NC_003075.7

Expression diagram:

Fig 1. The expression diagram of 3-isopropylmalate dehydratase large subunit, chloroplastic

Corresponding enzyme structure:

Fig 2. The corresponding enzyme structure of 3-isopropylmalate dehydratase large subunit, chloroplastic

The PCR result:

Fig 3. The PCR result of 3-isopropylmalate dehydratase large subunit, chloroplastic

Subcellular localization:[3]

Located in the plastid and chloroplast stroma of cells

Fig 4. The subcellular localization of 3-isopropylmalate dehydratase large subunit, chloroplastic

Design Notes

IPMI (isopropylmalate isomerase) is a heterodimer consisting of a large subunit (LSU) and a small subunit (SSU), and it catalyzes the isomerization between 2-isopropylmalate and 3-isopropylmalate. In Arabidopsis thaliana, there is one LSU gene and three SSU genes. [4] Among them, SSU2 and SSU3 have the capability to catalyze methionine chain elongation, while SSU1 is an important component of leucine biosynthesis.[5] We chose LSU1 and SSU2 to construct our pathway. To ensure efficient expression in Saccharomyces cerevisiae (S288C), we codon-optimized the 3-isopropylmalate dehydratase large subunit （LSU1） gene sourced from Arabidopsis thaliana. LSU1 plays a crucial role in catalyzing the isomerization between 2-isopropylmalate and 3-isopropylmalate, which is essential not only for leucine biosynthesis but also for the methionine chain elongation pathway in aliphatic glucosinolate formation. We selected the TEF1 promoter (TEF1pBBa_K319003) and ADH1 terminator (ADH1tBBa_K2637012) to drive and stabilize gene expression. After designing the optimized gene, it was inserted into a vector and integrated into yeast via homologous recombination, followed by screening and expression verification using defective strains. This approach ensures that LSU1 effectively catalyzes the key isomerization reactions in the methionine chain elongation pathway, thereby contributing to the efficient synthesis of aliphatic glucosinolates.

Plasmid

Fig 5. The plasmid expression of 3-isopropylmalate dehydratase large subunit, chloroplastic

Source

Arabidopsis thaliana


References

  1. ↑ SURESHKUMAR S, TODESCO M, SCHNEEBERGER K, et al. A genetic defect caused by a triplet repeat expansion in Arabidopsis thaliana [J]. Science (New York, NY), 2009, 323(5917): 1060-3.
  2. ↑ HE Y, CHEN B, PANG Q, et al. Functional specification of Arabidopsis isopropylmalate isomerases in glucosinolate and leucine biosynthesis [J]. Plant & cell physiology, 2010, 51(9): 1480-7.
  3. ↑ ZYBAILOV B, RUTSCHOW H, FRISO G, et al. Sorting signals, N-terminal modifications and abundance of the chloroplast proteome [J]. PloS one, 2008, 3(4): e1994.
  4. ↑ HE Y, CHEN B, PANG Q, et al. Functional specification of Arabidopsis isopropylmalate isomerases in glucosinolate and leucine biosynthesis [J]. Plant & cell physiology, 2010, 51(9): 1480-7.
  5. ↑ KNILL T, REICHELT M, PAETZ C, et al. Arabidopsis thaliana encodes a bacterial-type heterodimeric isopropylmalate isomerase involved in both Leu biosynthesis and the Met chain elongation pathway of glucosinolate formation [J]. Plant molecular biology, 2009, 71(3): 227-39.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1289
    Illegal BamHI site found at 363
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 164


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