## 3 Discussion
The in silico identification of THCAS and CBDAS-like endogenous candidate genes is more intricate, given the specificity of these enzymes. Our approach can also be used to further characterize endogenous diatom enzymes with multiple functions by testing the reactivity of diatoms to drug precursors in higher-level plants. The episomal sequences were well-conserved in the timeframe of this study in all three constructions of interest (in DNA and proteins). However, after such a timeframe, the production of OA was not detectable, perhaps due to silencing via an epigenetic mechanism [42], mRNA or protein stability [37], and, less probably, episomal rearrangements, as seen in other cases [42]. It would be useful to characterize a larger number of transformed _P. tricornutum_ using a method similar to the process used with yeast [31; 34], or to try randomly integrated chromosomal expression (RICE). Interestingly, a loss of production was observed in parallel with a change in cell morphotype from a triradiate to a fusiform population, which could also be associated with changes in the intracellular levels of certain precursors related to general metabolism [51]. A loss of heterologous production of specialized metabolites was previously observed for _E. coli_, where, after the 31 st generation, lycopene production decreased from 3.3% of its initial yield [52] due to segregational plasmid instability. Similarly, the use of cyanobacteria as a heterologous production platform faces many challenges, such as a loss of productivity due to the burden or loss of the metabolic pathway and genetic instability [52; 53]. In yeast, low expression-cassette instability in large fermentation batches led to a decrease in recombinant protein production [54]. Taken together, these studies show that a loss of production seems to occur elsewhere in other systems, as a result of different underlying problems. In addition, these further demonstrate that like other systems, diatoms need further optimization of the metabolic pathway-encoding constructs discussed below to ensure a suitable platform for the heterologous production of specialized metabolites. Recently, cloning the _Metarhizium anisopliae ARSEF23_ highly reducing PKS enzyme (_MaOvaA_), in addition to a non-reducing PKS (_MaOvaB_) and a domain PKS (_MaOvaC_) consisting of an acyl carrier protein and a thioesterase, in the fungus _Aspergillus nidulans_ yielded high titers of OA (80 mg/L) [55]. Thus, it would be interesting to compare the expression and behavior of cannabis enzymes regarding fungal enzymes such as highly reducing and non-reducing PKS, along with thioesterase and acyl carrier enzymes [50]. Another approach to increase OA titers could be the construction of alternative pathways using enzymes found in other OA-producing plants than cannabis, in order to achieve OA and other CB precursors while using fungal enzymes or mutation-enhanced enzymes. It is possible, from an evolutionary point of view, that other sources of transgenes are more suitable for heterologous expression in diatoms. Moreover, in this study, low OA yield could be explained by the detection of cannabinoid-id-like compounds in higher titers (Figure A4) similar to those observed in other studies, where flavonoid precursors were consumed to form flavonoids in bioengineered yeast [56]. Further steps of compartmentalization of the products would be an interesting strategy, given the possible cytotoxicity of the cannabinoids in many systems [12; 57]. The application of _P. tricornutum_ as a production platform for OA raises intriguing questions about its metabolic compatibility with the broader CB biosynthesis pathway. Further research is warranted to understand how the engineered diatom handles the downstream conversion of OA into various cannabinoids. Additionally, optimizing the growth conditions, such as light intensity, nutrient availability, and cultivation scale, will be crucial for maximizing production yields [51; 58; 59]. The successful synthesis of olivetolic acid in _P.