Team:KEYSTONE/Improvement
GreatBay_China 2018 has constructed a pMVA-GPPS-GES which was used to successfully synthesize geraniol, which is a sweet, citrus, and floral tasting monoterpenoid and an alcohol that is the primary component of rose oil [1]. Simple sugars are made into Acetyl-CoA through glycolysis, and it is the first part of the MVA pathway. Through varies reactions, geranyl pyrophosphate (GPP), which can be used to produce most monoterpenes, sesquiterpenes, and diterpenes [2] is synthesize by the GPPS (geranyl pyrophosphate synthase) gene from dimethylallyl pyrophosphate, and the GES (geraniol synthase gene) was able to synthesize geraniol from geranyl pyrophosphate. GreatBay_China was successful in geraniol product with the addition of other plasmids. See Part:BBa_K2753015.
Figure 1 A combined plasmid with GPPS&GES operon and MVA pathway designed by GreatBay_China (2018) [3]
We applied the pathway to a new fragrance synthase. We aimed to produce linalool, a monoterpene that is commonly used as a scent and flavoring agent. The linalool synthase used in our experiments is the linalool synthase from Streptromyces clavuligerus that is called bLIS. It is a very effective linalool synthase. The synthesis of linalool can also be increased by the optimization of the fusion tags and the ribosome binding sites of the protein [2]. We applied the Gibson Assembly method to substitute the GES gene with bLIS ( (3R)-linalool synthase) from Streptomyces clavuligerus [4] and was able to synthesize linalool from geranyl pyrophosphate. See Part:BBa_K3478889
Figure 2 Two plasmids are used for linalool production. (a) p15A-MVA allows the production of IPP and DMAPP, which are the substrates for GPP, from Acetyl-CoA
(b) pR6K-ptac-GPPS-bLIS then produces GPP synthase and linalool synthase which synthesizes GPP and, subsequently, linalool.
Form our experimental results, the characterization of this new part is successful. A significant linalool fragrance is produced from the E.coli which we’ve cotransformed the two plasmids into. We were able to distinguish that we did produce linalool instead of geraniol since based on our observation, the aroma of geraniol contains sweetness, whereas the aroma of standard linalool sample has a slight peppery smell, which was identifiable in our samples. The original smell of E.coli has been overcome by the fragrance of linalool and is almost neglectable.
See https://2020.igem.org/Team:KEYSTONE/Engineering for more information
References
[1] PubChem. (n.d.) Geraniol. PubChem. https://pubchem.ncbi.nlm.nih.gov/compound/geraniol.
[2] Alonso-Gutierrez, J., Chan, R., Batth, T. S. et al (2013). Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metabolic Engineering, volume 19, pp.33-41. https://doi.org/10.1016/j.ymben.2013.05.004.
[3] GreatBay_China. (2018). CATNIP-Result. https://2018.igem.org/Team:GreatBay_China/Demonstrate.
[4] Xun, W., Jing, W., Jiaming, C. et al (2019). Efficient biosynthesis of R-(-)-linalool through adjusting expression strategy and increasing GPP supply in Escherichia coli. https://doi.org/10.21203/rs.2.18761/v1.
