Part:BBa_K3570001

Sweet rose taste induction system in S. cerevisiae

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4894
  Illegal XbaI site found at 3746
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4894
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4894
  Illegal BamHI site found at 3752
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4894
  Illegal XbaI site found at 3746
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4894
  Illegal XbaI site found at 3746
  Illegal AgeI site found at 1551
  Illegal AgeI site found at 4507
  
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

This biobrick purpose is to give S. cerevisiae a sweet rose taste. This is performed by producing 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 sequences. The experimentator will be able to counter-select the cells that have integrated the construction since they are meant 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 was chosen.

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, a monoterpene synthase which catalyzes the reaction from geranyl pyrophosphate (GPP) to geraniol. Most of the GES come from plants 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 the 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 a 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 of 43 amino acids[6] in order to overcome the cited issue above, and codons were adapted for S. cerevisiae.

Brazzein is a sweet taste protein that was 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[7]. The brazzein sequence have been taken from Genbank[8] then codon-optimized and 3 Flag sequences were added to C-terminal in translational fusion.

The GAL1/10 bidirectional promoter was chosen based on its multiple properties (similar activities of GAL1 and GAL10 promoters, induction by the many version of the GAL4 transcriptional factor[9]. The sequence was identified from Dr. Gilles Truan and taken from here [11]. CYC1 and PGK1 terminators were chosen because of their large usage in yeast biotechnological manipulations[10]. The sequence was identified from the personal communication with Dr. Anthony Henras.

YPRCdelta15 upstream and downstream homology sequences(BBa_K3570014 and BBa_K3570015) are used to target a functional yeast integration locus. This will result in homologous recombination within a region of type 1 long terminal repeat (Ty1 LTR) in chromosome XVI of the S. cerevisiae's genome[12]. The sequence was identified from personal communication with Dr. Gilles Truan.

LEU2 selection marker is a gene commonly used as a selection marker for yeast. Only the cells that have integrated the biobrick (and the LEU2 gene in it) would be able to grow without leucine addition in the medium. The sequence was taken from FL36 plasmid [13].

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
• [10]- 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
• [11]- MRI-34 plasmid
• [12]- Bai Flagfeldt, D., Siewers, V., Huang, L., & Nielsen, J. (2009). Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae. Yeast, 26(10), 545–551. https://doi.org/10.1002/yea.1705
• [13]- FL36 plasmid