Partnership
GastonDay-Shangde iGEM
Overview
For our partnership, GastonDay-Shangde iGEM provided us with information about their genetic circuit, an E. coli construct designed to convert L-phenylalanine to cinnamaldehyde, which we modeled for them using a genome-scale metabolic model (GEM) and, in the process, explored ways of incorporating synthetic circuits into our software.
Detailed Information
Gaston Day School is a high school located in North Carolina and Shangde Experimental School is a boarding school located in Shanghai, China. GastonDay-Shangde iGEM’s 2022 project involves engineering E. coli to more readily produce cinnamaldehyde from L-phenylalanine, as this can be used in therapeutics to counter the effects of attenuated nonketotic hyperglycinemia, a metabolic disorder caused by a deficiency in the glycine cleavage system (Kure et al., 1997; Tada et al., 1969). They are increasing production by promoting heightened transcription of the three enzymes that control this reaction: phenylalanine-ammonia lyase, 4-coumarate:CoA ligase, and cinnamoyl-CoA reductase (Bang et al., 2016). In our first meeting, they communicated that they worried that increasing production of these enzymes was taking metabolites away from necessary processes in the cell, and thus was resulting in their cells dying.
We contributed to their project by incorporating their genetic circuit into a genome-scale metabolic model through the use of a COBRA toolkit. Specifically, we modeled their system within COBRA model iJO1366 of the BIGG Database (King et al., 2016), which is a genome-scale metabolic model for E. coli strain K-12, substrain MG1655 (King et al., 2016). Since GEMs take into account the inputs and outputs of thousands of reactions occurring within a cell at once, this allowed us to model the metabolic burden of their circuit. In order to achieve an accurate model, we met with them three times over zoom and communicated back and forth multiple times over email, resulting in a season-long partnership.
For this partnership, we modeled their genetic system by incorporating these three reactions into an existing genome-scale metabolic model (Bang et al., 2016):
- Conversion of L-phenylalanine to cinnamic acid
- Reactant: L-phenylalanine
- Enzyme: PAL (phenylalanine-ammonia lyase)
- Products: Cinnamic acid, NH3
- Conversion of cinnamic acid to cinnamoyl-CoA
- Reactants: Cinnamic acid, ATP, CoA-SH
- Enzyme: 4CL (4-coumarate:CoA ligase)
- Products: Cinnamoyl-CoA, ADP, inorganic phosphate, H2O
- Conversion of cinnamoyl-CoA to cinnamaldehyde
- Reactants: Cinnamoyl-CoA, NADPH
- Enzyme: CCR (cinnamoyl-CoA reductase)
- Products: Cinnamaldehyde, NADP+, CoA-SH
Image adapted from Figure 1 of Bang et al., 2016.
In addition to these reactions, we also added four new metabolites to model iJO1366: ammonia, cinnamic acid, cinnamoyl-CoA, and cinnamaldehyde.
Upon finishing the code, we provided their team with the model by sharing the link to a Google Collaboratory file containing the iJO1366 model with the new reactions and metabolites we incorporated. After providing them with the code for this model, a member of our modeling team met with members of Gaston Day School iGEM over Zoom to provide a general overview of the code, answer questions regarding the model, and recommend Documentation for COBRApy (Documentation for COBRApy — Cobra 0.25.0 Documentation, n.d.) as a resource for learning more about building genome-scale metabolic models. This partnership provided our team with an opportunity to explore ways of incorporating genetic circuits directly into genome-scale metabolic models, which was an important starting point in accounting for the effect of genetic circuits on the metabolism of a chassis, a key future direction for our project.
Image of Google Collaboratory code incorporated into a genome-scale metabolic model to represent some of the reactions needed to convert L-phenylalanine to cinnamaldehyde.
To view our full code for the Gaston Day School Collab, please visit the Gitlab here.