DNA

Part:BBa_K3570000

Designed by: Anton Mykhailiuk   Group: iGEM20_Toulouse_INSA-UPS   (2020-09-28)
Revision as of 16:19, 28 September 2020 by Antonmykhailiuk (Talk | contribs)


GGPP production enhancement in S. cerevisiae

One of the main goals of our project is to make S. Cerevisiae produce β-carotene. This construction is used in tight synergy with BBa_K3570001. Precisely, β-carotene derives from the mevalonate pathway as do limonene and geraniol (figure 1).

Fig. 1: Metabolic pathway of 𝛽-carotene, limonene and geraniol from Acetyl-CoA in engineered yeast.

We, therefore, decided to enhance the mevalonate pathway and the production of GGPP. According to Rabeharindranto et al. 2019, the enhancement of the mevalonate pathway can be achieved by overexpressing the gene HMG1 and CrtE. Our design differs from this publication in the choice of the promoter. The promoter TDH1 has been chosen instead of Gal1/10 because we planned to use this promoter for the regulation of other genes. We thus created the plasmids containing a truncated version of the HMG1 gene from S. Cerevisiae and the CrtE gene from X. Dendrorhous as on the figure 2.

Fig. 2: HMG1-CrtE-pUC19. The integrative locus used is DPP. The selective locus used is HIS. The genes coding for the truncated version of HMG1 comes from S. cerevisiae. The genes coding for CrtE comes from X. dendrorhous. We use the bidirectional TDH3-TEF1 promoter and the terminators tCYC1 and tPGK.

Literature: Hery Rabeharindranto, Sara Castaño-Cerezo, Thomas Lautier, Luis F. Garcia-Alles, Christian Treitz, Andreas Tholey, Gilles Truan, 2019. Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae. Metab Eng Commun. 2019 Jun; 8: e00086.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1015
    Illegal PstI site found at 50
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 683
    Illegal NheI site found at 3586
    Illegal NheI site found at 4491
    Illegal NheI site found at 5834
    Illegal PstI site found at 50
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1641
    Illegal BglII site found at 3716
    Illegal BglII site found at 5725
    Illegal BglII site found at 5785
    Illegal BamHI site found at 3376
    Illegal XhoI site found at 705
    Illegal XhoI site found at 3415
    Illegal XhoI site found at 3547
    Illegal XhoI site found at 4366
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1015
    Illegal PstI site found at 50
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1015
    Illegal PstI site found at 50
    Illegal NgoMIV site found at 870
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None