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

As an extension of our goal to characterize our fungal toolkit with fluorescent proteins, we decided to show that a series of genes in a metabolic pathway could also be modified and engineered into fungi. For this, 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 Edinburgh team in 2007, 2008, and 2011, and we modified their biobricks for our purposes.
We used the section of the carotenoid pathway composed of 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 naturally produce FPP, including our chassis. 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].

Adapted from KEGG reference pathways

1. Fungal Expression with T7 promoters

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. Each gene would be transcribed individually by T7, then translated by the fungus.

2. Bacterial Expression with T7 promoters

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. However, we needed at least three different fungal promoters because E. coli considers large repeated sequences, such as eukaryotic promoters, to be duplications, and will get delete them. So, different fungal promoters must be used with our carotenoid genes. This also allows us to easily swap out promoters to study the carotenoid pathway and combinations of promoters in further detail.