Part:BBa_K3570002

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[5]. 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

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.

Eukaryotic CrtY enzymes are predicted to be transmembrane proteins. On the contrary, CrtB and possibly CrtI are supposedly cytosolic[4]. Additionally, in some fungi, the phytoene CrtY and CrtB catalytic activities are fused within a multidomain protein named CrtYB in Xanthophyllomyces dendrorhous[5]. The complete biosynthetic pathway of X. dendrorhous, with a multidomain CrtYB, was efficiently expressed in S. cerevisiae[6]. Even though this heterologous system is the most productive to date, the accumulation of β-carotene precursors in the pathway, such as phytoene, was remarked. Nevertheless, the overexpression of CrtI to overcome the metabolic bottleneck turned out to be only partially successful[6].

Recently, the natural tridomain fusion (CrtIBY) from Schizotrium sp. was expressed in Yarrowia lipolytica[7] but unfortunately, the β-carotene production yields were considerably lower than those obtained with the yeast-expressed X. dendrorhous configuration [6], [8]. We then searched to design an enzyme(s), that would have at the same time high production yields of β-carotene precursors (as in X. dendrorhous) with the spatial proximity of both cytosolic enzymes (CrtB and CrtI).

A strategy for creating an enzyme that would have crtYB et crtI activities and every listed property above was adapted from Rabeharindranto, H et al., 2019. This way, a tridomain fusion protein CrtYBekI was expressed in S. cerevisiae, with ‘‘ek’’ being a peptidic linker. This spatial reorganization of the carotenoid enzymes should reduce the accumulation of intermediates and reorient the metabolic fluxes towards β-carotene production.

TDH1 promoter proved itself to be quite versatile under different carbon sources for yeast[9]. TDH1 promoter is a gene-specific promoter from the yeast TDH1 gene[10]. The sequence was extracted from TDH1 gene in SGD[10]. CYC1 terminator was chosen because of its large usage in yeast biotechnological manipulations[11]. The sequence was identified from the personal communication with Dr. Anthony Henras.

HO upstream and downstream homology arms (BBa_K3570009 and BBa_K3570010) are used to target a functional yeast integration locus. This will result in homologous recombination within the Diacylglycerol pyrophosphate phosphatase 1 (DPP1) gene and thus integration into the S. cerevisiae's genome[12]. The sequence was identified from personal communication with Dr. Gilles Truan.

URA3 selection marker (BBa_K3570013) is a gene commonly used as a selection marker for yeast. Only the cells that have integrated the biobrick (and the URA3 gene in it) would be able to grow without histidine addition in the medium. The sequence was taken from FL38 plasmid [13].

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
• [4]- Schaub, P., Yu, Q., Gemmecker, S., Poussin-Courmontagne, P., Mailliot, J., McEwen, A.G., et al., 2012. On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/ isomerase. PLoS One 7, e39550.
• [5]-Verdoes, J.C., Krubasik, P., Sandmann, G., Van Ooyen, A.J.J., 1999. Isolation and functional characterization of a novel type of carotenoid biosynthetic gene from Xanthophyllomyces dendrorhous. Mol. Gen. Genet. MGG 262, 453–461.
• [6]-Verwaal, R., Wang, J., Meijnen, J.-P., Visser, H., Sandmann, G., Berg, J.A. van den, et al., 2007. High-level production of beta-carotene in saccharomyces cerevisiae by successive transformation with carotenogenic genes from xanthophyllomyces dendrorhous. Appl. Environ. Microbiol. 73, 4342–4350.
• [7]-Gao, S., Tong, Y., Zhu, L., Ge, M., Jiang, Y., Chen, D., et al., 2017. Production of βcarotene by expressing a heterologous multifunctional carotene synthase in Yarrowia lipolytica. Biotechnol. Lett. 39, 921–927
• [8]-Xie, W., Ye, L., Lv, X., Xu, H., Yu, H., 2015. Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae. Metab. Eng. 28, 8–18.
• [9]- Monfort, A., Finger, S., Sanz, P., & Prieto, J. A. (1999). Evaluation of different promoters for the efficient production of heterologous proteins in baker's yeast. Biotechnology Letters, 21(3), 225–229. https://doi.org/10.1023/a:1005467912623
• [10]- SGD:S000003588
• [11]- Curran, K. A., Karim, A. S., Gupta, A., & Alper, H. S. (2013). Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metabolic Engineering, 19, 88–97. https://doi.org/10.1016/j.ymben.2013.07.001
• [12]- S. cerevisiae genome, chromosome IV, HO gene. GenBank: CP046084.1
• [13]- FL38 plasmid