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

d-Limonene is a chemical compound derived from the rind of citrus fruit.Motivation to incorporate d-limonene into the fungal strain was to improve the marketability of the styrofoam product. The current design alternative biodegradable styrofoam incorporates fungal mycelium along with agricultural byproducts that have a less than appealing odor. Incorporating the limonene synthase into the strain of interest allows the strain to express limonene, releasing the desired citrusy odor to mask the smell of the agricultural by-product.. In addition, besides the already well-known anticancerous, anti-inflammatory and bacteriocidal properties of d-limonene, further research has revealed that d-limonene substance is highly effective in recycling/degrade polystyrene.

Initial research was based on finding the simplest aromatic compound. Identifying compounds began involved searching for a mint-scent. However, the pathway to produce the produce the desired spearmint scent (-)-Carvone was 3 steps and far too complex to obtain. Luckily, we identified that (-)-Limonene was the first step we opted for a single-step protein pathway instead. [1] Working to identify the correct protein, we looked on the Bio-brick registry, we saw that the the limonene synthase was used by the Munich iGEM team in 2012. [2] Taking the construct from the kit-plate we ligated the limonene synthase into the backbone. Once that was successful, we transformed into a backbone upstream of a T7 promoter.

We believe that this system will be more than effective in masking the agricultural byproduct used in the biodegradable styrofoam. The fungal strains we will be transforming the limonene constructs all have the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that are needed to produce the Geranyl Diphosphate used by the limonene synthase to create d-limonene.