## 3 Discussion  
The bioengineering of _P. tricornutum_ with cannabis genes represents a promising approach to addressing the challenges associated with traditional cannabinoid production. By harnessing the unique capabilities of marine diatoms, this study paves the way for sustainable and controlled cannabinoid biosynthesis. Furthermore, the successful expression of cannabis genes in a non-native host highlights the versatility of synthetic biology in the context of metabolic engineering. This work represents the first report of cannabinoid engineering in brown microalgae and provides a proof-of-concept evaluation of _P. tricornutum_ for the heterologous production of OA and cannabinoids. We provide evidence that the natural metabolism of the marine diatom _P. tricornutum_ is well-suited for use in metabolic engineering to produce the cannabinoid precursor, OA, and CB-like metabolites. The episomal vectors, _Pt_OA1 and _PI_OA2, contained both _CsTKS_ and _CsOAC_ genes, separated by a self-cleaving peptide sequence and under the control of the same promoter, to ensure similar levels of expression of both genes and, subsequently, comparable levels of accumulated enzymes for performing the two-step reactions. The transconjugants _Pt_OA1 and _Pt_OA2 only differed in terms of the addition of a 6 xHis tag on _Cs_TKS for easier detection of the protein. In _Pt_OA3, both genes were linked together with a linker and tagged with YFP for visualization and localization studies. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multicolumn{3}{c}{**Supplemented mM**} & \multicolumn{3}{c}{**Detected**} \\
**OA** & **GPP** & **CBGA** & **HPLC** & **MS (_m/z_)** & **Confirmation** & **HPLC** & **MS (_m/z_)** & **Confirmation** \\ \hline
0.45 & 1 & 0 & CBGA & yes & CBDA, CBNA & No \\
0 & 0 & 0.27 & CBDA & yes & THCA, CBNA & No \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Results of a supplementation assay of _P. tricornutum_ with olivetolic acid (OA), geranyl diphosphate (GPP), and cannabinoid acid (CBGA). The results show the mean detection levels that were found in three different strains of _P. tricornutum_ transconjugants harboring p_Pt_GE30. Although a previous study demonstrated the occurrence of episomal rearrangements [42], in this study, the _P. tricornutum_ transconjugant strains obtained for each episomal vector did not show significant DNA sequence rearrangement after 1 year (Appendix A, Figure A1 and Table A4). This suggests that episomal rearrangements could be sequence- or DNA-motif-specific. Also, the _P_IOA1-3 transconjugant strains grew similarly to the WT and EV strains (Figures 3 and 4). This suggests that the presence and expression of the transgenes and the produced metabolites did not significantly affect the growth and division of _P. tricornutum_. Genome analysis of green algae, diatoms, and higher plants revealed similarities in lipid metabolism [43; 44]. Therefore, the fact that there is little to no effect of CsTKS and CsOAC expression on _P. tricornutum_ growth and fitness could be a result of the nature of the pathway, as an extension of fatty acid metabolism that is fairly close to the diatom chassis, and a preference for energy conversion into lipids. Heterologous protein detection was not consistent among the different transconjugant strains (Figure 5 and Appendix A, Figure A2). For example, the four analyzed transconjugant strains of _P_tOA1 show a similar pattern of accumulation, whereas, in the _P_tOA2 strains, the T2A sequence did not appear to be cleaved. This could be explained either by the possible presence of negatively acting _cis_ elements in the sequence of _P_tOA2 transgenes [43] or by the nature of the amino acid environment that surrounds the T2A sequence cleavage site [45].