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

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Initial research was based on finding the simplest aromatic compound to biosynthesize. Identifying compounds began with a search for a mint-scent. However, the pathway to produce the desired spearmint scent (-)-Carvone required two additional steps beyond the single step used to produce (-)-Limonene. Given that the precursors for (-)-Limonene found naturally in <i>G. lucidum</i> are only a small number of enzymatic reactions away from becoming limonene, we opted to work with the shorter pathway [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.  
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Initial research was based on finding the simplest aromatic compound to biosynthesize. Identifying compounds began with a search for a mint-scent. However, the pathway to produce the desired spearmint scent (-)-Carvone required two additional steps beyond the single step used to produce (-)-Limonene. Given that the precursors for   (-)-Limonene found naturally in <i>G. lucidum</i> are only a small number of enzymatic reactions away from becoming limonene, we opted to work with the shorter pathway [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.  
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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.   
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.   

Revision as of 04:21, 28 September 2013

Cornell University Genetically Engineered Machines

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 of the 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 byproduct. 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/degrading polystyrene.

Initial research was based on finding the simplest aromatic compound to biosynthesize. Identifying compounds began with a search for a mint-scent. However, the pathway to produce the desired spearmint scent (-)-Carvone required two additional steps beyond the single step used to produce (-)-Limonene. Given that the precursors for (-)-Limonene found naturally in G. lucidum are only a small number of enzymatic reactions away from becoming limonene, we opted to work with the shorter pathway [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.

References

1. Carter, Ora A., Reuben J. Peters, and Rodney Croteau. "Monoterpene Biosynthesis Pathway Construction In Escherichia Coli." Phytochemistry 64.2 (2003): 425-433. Print.

2. Technical University Munich's Genetically Engineered Machines Team. (2012). https://2012.igem.org/Team:TU_Munich/Project/Limonene

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