Difference between revisions of "Part:BBa K3570002"
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Biosynthesis of β-carotene in <i>X. dendrorhous</i> begins with the production of phytoene from GGPP by the domain B of bifunctional lycopene cyclase/phytoene synthase (CrtYB). Phytoene desaturase (CrtI) then catalyzes four successive desaturation reactions to form lycopene. In the end, the domain Y of CrtYB performs the cyclization on both sides of lycopene to produce β-carotene (fig.1). </p>
  
  

Revision as of 13:28, 22 October 2020


Provitamin A synthesis from GGPP in S. cerevisiae


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1297
    Illegal PstI site found at 2357
    Illegal PstI site found at 2627
    Illegal PstI site found at 3605
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2012
    Illegal NheI site found at 5006
    Illegal PstI site found at 2357
    Illegal PstI site found at 2627
    Illegal PstI site found at 3605
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 738
    Illegal BglII site found at 3364
    Illegal BglII site found at 5489
    Illegal BglII site found at 6585
    Illegal BamHI site found at 3157
    Illegal XhoI site found at 4943
    Illegal XhoI site found at 4984
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1297
    Illegal PstI site found at 2357
    Illegal PstI site found at 2627
    Illegal PstI site found at 3605
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1297
    Illegal PstI site found at 2357
    Illegal PstI site found at 2627
    Illegal PstI site found at 3605
    Illegal NgoMIV site found at 2018
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

This biobrick shall be used to boost the production of produce provitamin A (𝛽-carotene) in S. cerevisiae . 𝛽-carotene is one of the carotenoids produced in yeast. The metabolic pathway comprises multiple intermediate as well as side-products before reaching 𝛽-carotene (fig. 1). It starts with geranylgeranyl diphosphate (GGPP), which is a derivative from Mevalonate pathway. GGPP is importantly used in yeast since it is a precursor to carotenoids[1], tocopherols[2], and to geranylgeranylated proteins[3]. Therefore, for the best production yield of 𝛽-carotene production using this biobrick, it is best to use it in synergy with the "GGPP production enhancement in S. cerevisiae" biobrick (BBa_K3570000).

Fig. 1: From Rabeharindranto et al. 2019. Presumed biosynthetic pathway for the synthesis of β-carotene in X. dendrorhous (Verdoes et al., 1999). The bifunctional lycopene cyclase/phytoene synthase (CrtYB) enzyme is depicted in orange, the phytoene desaturase enzyme (CrtI) is depicted in red. Orange or red boxes represent the localization of the modifications introduced by the CrtYB or CrtI enzymes respectively.

Design

<p style="text-indent: 40px"> Biosynthesis of β-carotene in X. dendrorhous begins with the production of phytoene from GGPP by the domain B of bifunctional lycopene cyclase/phytoene synthase (CrtYB). Phytoene desaturase (CrtI) then catalyzes four successive desaturation reactions to form lycopene. In the end, the domain Y of CrtYB performs the cyclization on both sides of lycopene to produce β-carotene (fig.1).


Fig. 2: CrtYB(ek)I-pUC19. The integrative locus used in yeast is HO. The selective locus used is URA3. The genes coding for CrtYB and CrtI come from X. dendrorhous and is fused using the artificial peptide linker ek. We use the TDH1 promoter and the tCYC1 terminator.



Experiments

References

  • [1]- Rabeharindranto, H., Castaño-Cerezo, S., Lautier, T., Garcia-Alles, L. F., Treitz, C., Tholey, A., & Truan, G. (2019). Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae. Metabolic Engineering Communications, 8, e00086
  • [2]- DIPLOCK, A. T., GREEN, J., EDWIN, E. E., & BUNYAN, J. (1961). Tocopherol, Ubiquinones and Ubichromenols in Yeasts and Mushrooms. Nature, 189(4766), 749–750. https://doi.org/10.1038/189749a0
  • [3]- Ohya, Y., Qadota, H., Anraku, Y., Pringle, J. R., & Botstein, D. (1993). Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p. Molecular Biology of the Cell, 4(10), 1017–1025. https://doi.org/10.1091/mbc.4.10.1017