## 4 Discussion  
### GEM Reconstruction, Functional Annotation and Secondary Metabolism of C. sativa  
The whole-genome assembly of _C. sativa_ (CBDRx:18:580) obtained by [PERSON] et al. [44] serves as the main input for the GEM reconstruction. CBDRx:18:580 was obtained from the leaf of a female plant grown indoors at 20-25 \({}^{\circ}\)C [44]. The plant belongs to chemotype III, which is associated with a high content of cannabinoid [26]. While this plant chemotype is widely recognized for its applications in the textile and paper industry, there exist other significant avenues that present potential opportunities to diversify and enhance its value chain [6]. Some of these potential uses are in the food industry (thanks to its nutraceutical value), medicine (thanks to the unique properties of cannabinoid), and cosmetics (thanks to the possible effects of cannabinoids in synergy with terpenes) [17]. As for the reconstruction, analysis of the corresponding stoichiometric matrix enables the identification of topological features of the network. The sparsity pattern is illustrated in Figure 2. The stoichiometric matrix consists of 1314 rows (metabolites) and 2101 columns (reactions). Out of a total of 2760714 entries, 8361 (0.302%) are non-zero (nz). Generally, fewer than 1% of the elements in a genome-scale stoichiometric matrix are non-zero. This value is particularly useful for comparing models based on the number of metabolites involved in each reaction. The double upper diagonal appearance observed in the stoichiometric matrix is primarily a result of the ordering of reactions, rather than an intrinsic feature [62; 65]. GEM reconstruction of _C. sativa_ incorporates various compartments, including the cytosol, stroma, Golgi, vacuole, cell wall, peroxisome, mitochondria, nucleus, and endoplasmic reticulum (Supplementary Figure S1). Notably, around 50% of the model reactions specifically pertain to compartments that play crucial roles in primary metabolism, such as the cytosol, mitochondria, and plastids. Numerous studies have demonstrated the relationship between primary and secondary metabolism in plants and how despite the great variety of secondary metabolites, only some basic pathways of primary metabolism function as their precursors [66]. Glycolysis is the precursor of fatty acid biosynthesis, the mevalonate pathway, and the DXP-MEP pathways, which give rise to a variety of important terpenes and phenolic compounds such as cannabinoids, flavonoids, and fatty acids. On the other hand, the Krebs cycle is a primary precursor in the biosynthesis of glutamate and aspartate, while the shikimate pathway is a precursor in the biosynthesis of phenylalanine, tyrosine, and tryptophan. This part of the relationship between primary and secondary metabolism is native to N-containing compounds. Thus, it is possible to affirm that glycolysis, Krebs cycle and shikimate pathways are the most important precursors in the reconstruction of the secondary metabolism of _C. sativa_ when researching its potential as a cosmeceutical, cosmetic, or additive in the food industry (thanks to its properties derived from terpenes), therapeutic and nutraceutical (thanks to its properties derived from phenolic compounds such as flavonoids, cannabinoids, alkaloids and N-containing compounds), potential phytonutrient (thanks to its properties derived from fatty acids), and many other applications previously mentioned. A significant number of transport reactions were evidenced in the reconstruction, corresponding to the high flux of metabolites passing from one compartment to another. These reactions are linked to alkaloids, furanocumarins, terpenes, and carotenoids formed in the chloroplast; similarly, sesquiterpenes, sterols, and hydroxylation steps together with fatty acid synthesis take place in a constant exchange between the cytosol and the endoplasmic reticulum.