Some projects we worked on not directly related to our main ones
Though significantly delayed by the acquisition of relevant sequences, we laid the groundwork to engineer a biosynthetic pathway to optimize lutein production in E. coli. Lutein, one of many xanthophyll pigments produced by photosynthetic organisms, has been shown to delay the onset and severity of Age-related Macular Degeneration (AMD) in a dose-dependent manner (Liu, 2014). Over 8% of all adults between the ages of 45-85 exhibit impaired vision due to AMD, and the number of people with AMD symptoms is projected to reach 288 million by 2040 (Wong, 2014). Currently, lutein is extracted in small quantities from marigold flowers and microalga that contain an abundance of related molecules. Current efforts to synthesize lutein via organic chemistry typically involve the generation of toxic byproducts. We intend to engineer a synthetic biological system capable of producing lutein with increased resource- and time-efficiency as to be amenable to straightforward extraction techniques.
To this end, we will transform pre-existing lycopenic E. coli with a novel plasmid engineered for the preferential production of lutein relative to other products in the carotenoid pathway. Lycopene, a precursor in lutein biosynthesis, is produced in E. coli capable of rerouting farnesyl pyrophosphate into carotenoid biosynthesis. The genes for lycopene producing enzymes have previously been introduced by both outside researchers (Kim 2009) as well as previous iGEM teams (Cambridge 2009). Through a small number of inducible enzymatic reactions encoded on a second plasmid, lycopene can then converted to lutein. This requires cyclization by a β-cyclase (LYCB) and an ε- cyclase (LYCE) to form α-carotene, followed by a hydroxylation event on each ring by a β-hydroxylase and an ε- hydroxylase.
Lutein requires α-carotene as a precursor in its biosynthesis. In most photosynthetic organisms, independent activity of LYCB and LYCE produces stoichiometric levels of α- carotene as well as β-carotene (a useful molecule in its own right, but a contaminating side-product in lutein synthesis). To increase the ratio of α-carotene to β-carotene, we plan on producing the two cyclases separately in one E. coli, with the LYCE gene under a stronger promoter than the LYCB gene. This attempts to force carotenoid synthesis through α-carotene via increased specific activity of the ε-cyclase. We will tailor associated promoters as well as induction conditions in order to maximize the α-carotene/β-carotene ratio.
Thus far, we have cloned and submitted the ε-cyclase and -hydroxylase to the iGEM registry (see parts page), to complement pre-existing β-cyclase and hydroxylase biobricks. We have also developed a basic version of a mathematical modeling system capable of predicting output of the enzymes contributing to α-carotene production. After tuning its predictive ability to the experimentally determined output of the biosynthetic pathway, we intend to create an easy-to-use interface so that other iGEMers may input basic kinetic data and production goals and receive information on appropriate expression levels for the enzymes in their pathway of interest, as well as candidate promoters in the iGEM registry that may approximate this level of expression.
1. Blatt, A., Bauch, M.E., Porschke, Y., and Lohr, M. (2015) A lycopene β-cyclase/lycopene ε-cyclase/light-harvesting complex-fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α-carotene and β-carotene at different ratios. Plant Journal, doi: 10.1111/tpj.12826
2. Kim, S., Kim, J., Ryu, J., Jung, J., Kim, J. (2009) High-level production of lycopene in metabolically engineered E. coli. Process Biochemistry, 44 (8), 899-905
3. Liu, R., Wang, T., Zhang, B., Qin, L., Wu, C., Li, Q., and Ma, L. (2014) Lutein and zeaxanthin supplementation and association with visual function in age-related macular degeneration. Invest. Opthalmol. Vis. Sci (1):252-8. doi: 10.1167/iovs.14-15553
4. Wong, W., Su, X., Li, X., Cheung, C., Klein, R., Cheng, C., and Wong, T. (2014) Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and metaanalysis. Lancet Glob. Health (2):e106-16. doi: 10.1016/S2214-109X(13)70145-1
One of our primary goals at the start of the summer was to train new members in basic molecular biology techniques and familiarize them with the general process of biobrick construction and amendment. We looked for a small project that might be a fruitful endeavor for our budding scientists and that would improve a part in the registry. We settled on adding a promoter to the miraculin coding region.
Miraculin is an approximately 100kDa glycoprotein found naturally in Synsepalum dulcificum (the miracle fruit) that binds sweet receptors, making acidic foods be perceived as sweet. To elicit and verify protein production, we added a pBAD promoter to the miraculin coding region, induced expression with 0.1% arabinose for 18 hours, and verified protein expression using SDS-PAGE.
Matsuyama T, Satoh M, Nakata R, Aoyama T, Inoue H. Functional expression of miraculin, a taste-modifying protein in Escherichia coli. J Biochem. 2009;145(4):445-50.