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CrtYB(ek)I - Tridomain fusion protein for β-carotene production in yeast

Usage

Nowadays, the supplement of food in vitamins is done routinely. New biotechnological processes are developed in order to allow cheaper, faster, and more accessible production of vitamins. Our goal was to enrich Saccharomyces Cerevisiae in β-carotene.

Although the part of the biosynthetic pathway from geranylgeranyl diphosphate (GGPP) to β-carotene is made up of only three enzymes, multiple enzymatic reactions are possible inside the pathway between the precursors. This phenomenon leads to multiple side-products and precursors on the way to β-carotene. (add a scheme) Fusing enzymes of this pathway and thus colocating the metabolic reactions, would allow a higher yield of β-carotene production. See “Biology” part to get more details.

Biology

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. 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.

Eukaryotic CrtY enzymes are predicted to be transmembrane proteins. On the contrary, CrtB and possibly CrtI are supposedly cytosolic (Schaub et al., 2012). Additionally, in some fungi, the phytoene CrtY and CrtB catalytic activities are fused within a multidomain protein named CrtYB in Xanthophyllomyces dendrorhous (Verdoes et al., 1999). The complete biosynthetic pathway of X. dendrorhous, with a multidomain CrtYB, was efficiently expressed in S. cerevisiae (Verwaal et al., 2007). 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 (Verwaal et al., 2007).

Recently, the natural tridomain fusion (CrtIBY) from Schizotrium sp. was expressed in Yarrowia lipolytica (Gao et al., 2017) but unfortunately, the β-carotene production yields were considerably lower than those obtained with the yeast-expressed X. dendrorhous configuration (Verwaal et al., 2007; Xie et al., 2015). We then searched to design an enzyme(s), that would have at the 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 taken 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 toward β-carotene production.

References

• 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
• 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.
• 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.
• 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.
• 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
• 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.

Characterisation