Composite

Part:BBa_K3570002

Designed by: Anton Mykhailiuk   Group: iGEM20_Toulouse_INSA-UPS   (2020-10-15)
Revision as of 16:11, 16 October 2020 by Antonmykhailiuk (Talk | contribs)


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 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 "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

According to Rabeharindranto et al. 2019, the enhancement of the mevalonate pathway can be achieved by overexpressing the HMG1 and CrtE genes. The construction as it is presented here differs from the publication in the choice of the promoter. We thus created the plasmids containing a truncated version of the HMG1 (tHMG1) gene from S. Cerevisiae and the CrtE gene from X. Dendrorhous as on figure 2.

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.

The HMG1 (3-hydroxy-3-methylglutaryl coenzyme A) enzyme is considered as a rate-limiting step in the mevalonate pathway. To counteract this, authors [2] amplified it's catalytic domain and named it tHMG1. The overexpression of tHMG1 and CrtE (GGPP synthase) in S. Cerevisiae led to a significant improvement of carotenoid production because the direct precursor GGPP was increased[3].

The choice of a couple of promoters was essential for the optimal functioning of our construct since tHMG1 and CrtE needed to be expressed at a constant level under different conditions (such as carbon source, for example). TDH3 and TEF1 promoters proved themselves to have a non-significant difference in the expression level of the downstream gene, and to be quite versatile under different carbon sources for yeast[4]. TDH3 promoter is a gene-specific promoter from the yeast TDH3 gene[5], in parallel, TEF1 promoter is a gene-specific promoter from the yeast TEF1 gene[6]. The bidirectional TDH3-TEF1 promoter was designed for this construction. The sequence was identified from personal communication with Dr. Gilles Truan.

CYC1 and PGK1 terminators are chosen because of their large usage in yeast biotechnological manipulations[7] and from the personal communication with Dr. Anthony Henras.

DPP1 upstream and downstream homology arms (BBa_K3570006 and BBa_K3570007 are used target a functional yeast integration locus. This will result in homologous recombination within the Diacylglycerol pyrophosphate phosphatase 1 (DPP1) gene and thus integration in into the S. cerevisiae's genome[8]. The sequence was identified from personal communication with Dr. Gilles Truan.

Finally, HIS3 selection marker (BBa_K3570008) is a gene that is commonly used as a selection marker for yeast. Only the cells that have integrated the biobrick (and HIS3 gene in it) would be able to grow without histidine addition in the medium.

Experiments

Refernces

  • [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


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