Next, we will apply the now proven plasmid-retaining capacity of the Hok-Sok system to a true synthetic biology experiment. As the summer concluded, 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 (Figure 1). 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.  

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Our aim is to introduce a system capable of producing lutein from carotenoid precursors into a bacterial system. As these pathways are native to plants and nonexistent in bacteria, the main challenge is obtaining every enzyme necessary to allow the pathway to occur. Strains of lycopene (a carotenoid precursor) producing bacteria already exist, and we expect to begin the synthesis from this point. Another main challenge is the regulation of genes required to proceed from lycopene to lutein. Lycopene is the precursor for a multitude of carotenoids, all of which are produced in plants due to necessity. To produce lutein, the lycopene must first undergo a reaction with the specific enzymes in order, ε-cyclase then β-cyclase. Having both enzymes in the system, though, allows reactions between β-cyclase and lycopene, which yields a product unable to be converted into lutein. Through regulatory measures such as altering gene expression levels, we plan to optimize the efficiency of the lutein synthesis pathway. We also aim to create a mathematical modeling system capable of correlating the expression levels of proteins to relevant efficiencies in similar synthesis pathways.

<p>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. As shown above, lutein requires α-carotene as precursor in its biosynthesis. In most photosynthetic organisms, independent activity of LYCB and LYCE produces stoichiometric levels of α- carotene, and β-carotene, a useful molecule in its own right, but a contaminating side-product in lutein synthesis.

 

First, we will transform 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. In most photosynthetic organisms, independent activity of LYCB and LYCE produces stoichiometric levels of α- carotene, and 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 then tailor associated promoters as well as induction conditions in order to maximize the α-carotene/β-carotene ratio.

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 each enzyme in their pathway, as well as candidate promoters in the iGEM registry that may approximate this level of expression.