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To get a broader overview and validate that the planned modifications would not disrupt the basic functions of yeast a genome scale model of the envisioned system was constructed. The original model originates from Lu, H. et al.“A consensus S. cerevisiae metabolic model Yeast8 and its ecosystem for comprehensively probing cellular metabolism.” and was acquired through github. The model was run using Cobrapy 0.26.3 and Python 3.11.4 in a Jupyter notebook (2022.9.130).The model is based on the base Yeast8 model (8.7) forSaccharomyces CerevisiaeS288C and includes 4131 reactions and 2806 metabolites in the model but does not take enzyme constraints or protein 3D structure into account. Also worth pointing out here is that the modeled strain of yeast is not the same strain as the B12-dependent one that was originally planned to be used. In the end we stuck with Yeast8 since the models for S288C (including Yeast8) are more extensive so we decided to use this model over alternatives.
The model in question is a rather fleshed out model of the yeastSaccharomyces Cerevisiae and allowed us to mathematically model the pathway that our beta carotene was produced through before any lab work had taken place. This was done by manually adding the metabolites and reactions to the model and letting it find a new solution space with this new pathway present. The initial flux balance analysis yielded a flux of 0.082 (normalised to glucose uptake) through the biomass pseudo-reaction. A flux variability analysis then reported that at 90% efficiency for the pseudo-reaction the model could in theory produce a flux of up to 0.01 (normalised) of β-carotene. The simplified take on this is that, at least theoretically, it is possible for the yeast to have a yield of 1% β-carotene on glucose while also growing at 90% of its maximum growth rate, which was deemed sufficient for our use case.
While the model was not a major part of our project, it proved valuable as an early proof of concept.
All the files needed to run it are available on our GitLab at:https://gitlab.igem.org/2023/chalmers-gothenburg/-/tree/main/modeling