Difference between revisions of "Part:BBa K3570001"

Revision as of 14:16, 23 October 2020

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.

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

Sequence and Features