Team:Cornell/project/wetlab/fungal toolkit/carotenoids

As a proof of concept to show that a metabolic pathway could be modified and engineered into fungi, we decided to use the carotenoid pathway. Carotenoids are organic pigments that are found in a wide variety of organisms used for functions such as photosynthesis and protection from light. The relevant carotenoid genes have been previously biobricked by the Cambridge team in 2009, and we modified their biobricks for our purposes.

We used crtE, crtB, crtI, and crtY. The starting compound in this carotenoid pathway is farnesyl-pyrophosphate (FPP). FPP is a compound critical to the production terpenes and sterols, and so many organisms will naturally produce FPP. crtE is responsible for converting FPP into geranylgeranyl-pyrophosphate (GGPP). Then crtB converts GGPP into phytoene. Phyotene is then converted into lycopene, which is the first compound in this pathway to provide a color, by crtI. From lycopene, the red pigment can then become β-carotene and produce an orange color with the use of crtY.

1. T7 Fungal Expression

For the first carotenoid pathway assembly, our goal was to test the efficacy of the T7 promoter and polymerase system within fungi. Each gene was individually appended with a T7 promoter for viral polymerase transcription.

2. T7 Bacterial Expression

For the second assembly, our goal was to test the efficacy of the T7 system in bacteria. Each individual carotenoid gene was appended with both T7 and rbs, for effective viral transcription and bacterial translation.

3. Fungal Expression with Native Promoters

For the third assembly, our goal was to test and characterize native fungal promoters with our crt genes.