Part:BBa_I742151

crtE (geranylgeranyl pyrophosphate synthase) coding sequence.

crtE (geranylgeranyl pyrophosphate synthase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis parthway.

Usage and Biology

Team Fudan 2022 used BBa I742151 in retinoid biosynthesis by E. coli.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 715
  
• 1000
  COMPATIBLE WITH RFC[1000]

Contribution by Team 2024 Foshan-GreatBay

To increase the yield of β-carotene in yeast cells, we constructed a new composite part BBa_K5419000 (pX-2-PaCrtE) with crtE (BBa_I742151) gene fragment. This composited part was used together with other composite parts, BBa_K5419005 (pXII-5-BtCrtI), and BBa_K5419009 (pXI-2-XdCrtYB), for the construction of yeast strains with high β-carotene production.

Construction Design

We constructed the plasmid by placing the gene under the regulation of a strong constitutive GAP promoter and a CYC terminator, respectively. Integration sequence was added upstream and downstream of the expression cassette to integrate the target gene into the genome of S. cerevisiae using the CRISPR/Cas9 system (Figure 1).

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Figure 1 Design diagrams of pX-2-PaCrtE.

Experimental Approach

(1) Construction of integration plasmids

Firstly, we obtained the target gene expression frames (GAP promotor-gene-CYC terminator) by PCR amplification, and agarose gel electrophoresis results showed that we succeeded in obtaining these fragments. Next, we double-digested the target fragment and the vector (containing the S. cerevisiae X-2 integration site genes) and obtained the plasmid by enzymatic ligation. Finally, we transformed the enzyme-ligation product into E. coli DH5α competent cells, and the colony PCR and sequencing results showed that we successfully constructed the plasmid (Figure 2).

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Figure 2 The construction results of the pX-2-PaCrtE plasmid.