Difference between revisions of "Part:BBa K3570001"

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Sweet rose taste induction system in S. cerevisiae

Introduction

This biobrick is purposed to make S. cerevisiae to have a sweet rose taste. This is obtained by producing a brazzein, a sweet taste protein. The rose flavor is obtained by expressing the geraniol synthase (GES), which produces geraniol, a rose odorant monoterpenoid. This biobrick can be directly integrated into the yeast genome thanks to YPRcdelta15 homology arms. The experimentator will be able to counter-select the cells that have integrated the construction since they are ment to acquire a prototroph character for leucine.

Design

Fig. 1: Brazzein-CrGES-pUC19. The integrative locus used is yprcΔ15. The selective locus used is LEU2. The gene coding for geraniol synthase (CrGES) comes from C. roseus. The gene coding for brazzein comes from an artificial sequence (LG263246.1). CYC1 and PGK terminators are used. The bidirectional promoter Gal 1/10 is used.

Geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol), an acyclic monoterpene alcohol, is widely used in the flavor and fragrance industries[1]. It is produced by GES, which is a monoterpene synthase, that catalyzes a reaction from geranyl pyrophosphate (GPP) to geraniol. Most of the GES come from Plantae and contain N-terminal transit peptide that targets the translation product towards the plastids[2]. This transit peptide would be catalytically removed when the translated preprotein is imported to the plastids to promote the formation of mature enzyme[3]. The main issue is that S. cerevisiae does not have a mechanism that would allow to get rid of the plastidial transit peptide which might result in decreased enzymatic activity. To overcome this bottleneck, it has been shown that if the experimenter truncates the GES at its N-terminus, the enzymatic activity would be restored or even augmented[4]. The sequence of GES for this part comes from Catharanthus roseus[5], hence the name CrGES. The sequence has been truncated to 43 amino acids[6] in order to overcome the cited issue above, as well as codons were adapted for S. cerevisiae.

Brazzein is a sweet taste protein that is initially found in West African fruit of the climbing plant Oubli (Pentadiplandra brazzeana Baillon). Brazzein can be used to give the yeast a sweat taste since it is 500 to 2000 times sweeter than sucrose, compared to 10% sucrose and 2% sucrose solution respectively[7]. The brazzein sequence have been taken from Genbank [8] then codon-optimized and 3 times Flag sequence was added to C-terminal in translational fusion.

While designing a construction that includes the expression of two coding sequences, the choice of promoters is essential especially because both proteins come from heterologous systems. The choice of GAL1/10 bidirectional promoter bases on multiple properties of GAL1 and GAL10 promoters. Firstly, the activities of the GAL1 and GAL10 are highly similar to one another. The sequence of these two promoters is also well conserved among Saccharomyces clade, which can come in handy for future utilisations of this part[9].

Experiments

Team iGEM Toulouse 2020 did not have sufficient time to complete the cloning and hence, to test this part functionality.

References

• [1]- Chen, W., & Viljoen, A. M. (2010). Geraniol — A review of a commercially important fragrance material. South African Journal of Botany, 76(4), 643–651. https://doi.org/10.1016/j.sajb.2010.05.008
• [2]- Turner, G., Gershenzon, J., Nielson, E. E., Froehlich, J. E., & Croteau, R. (1999). Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells. Plant Physiology, 120(3), 879–886. https://doi.org/10.1104/pp.120.3.879
• [3]- Bohlmann, J., Meyer-Gauen, G., & Croteau, R. (1998). Plant terpenoid synthases: Molecular biology and phylogenetic analysis. Proceedings of the National Academy of Sciences, 95(8), 4126–4133. https://doi.org/10.1073/pnas.95.8.4126
• [4]- Zhao, J., Bao, X., Li, C., Shen, Y., & Hou, J. (2016). Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 100(10), 4561–4571. https://doi.org/10.1007/s00253-016-7375-1
• [5]- JN882024
• [6]- Jiang, G.-Z., Yao, M.-D., Wang, Y., Zhou, L., Song, T.-Q., Liu, H., Xiao, W.-H., & Yuan, Y.-J. (2017). Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae. Metabolic Engineering, 41, 57–66. https://doi.org/10.1016/j.ymben.2017.03.005
• [7]- Birch, Gordon Gerard (2000). Ingredients Handbook - Sweeteners (Ingredients Handbook Series). Leatherhead Food Research Association
• [8]- LG263246.1
• [9]- Elison, G. L., Xue, Y., Song, R., & Acar, M. (2018). Insights into Bidirectional Gene Expression Control Using the Canonical GAL1/GAL10 Promoter. Cell Reports, 25(3), 737-748.e4. https://doi.org/10.1016/j.celrep.2018.09.050

Sequence and Features