## 2 Results  
### Heterologous Protein Expression and Localization in the Diatom P. tricornutum  
#### 2.1.1 Transformation Validation and Transconjugant Characterization  
To inform and design the metabolic engineering strategy, we first performed computational analyses to assess the availability of the lipid-derived precursors required to produce OA and cannabinoids in _P. tricornutum_ with in silico tools and showed that upstream pathways existed in the host organism. According to the Biocyc database [40], hexanoyl-CoA can be formed in _P. tricornutum_ in the peroxisome via the fatty acid \(\beta\)-oxidation pathway, mainly by long-chain acyl-CoA synthase (_Pt_ACS3 and _Pt_ACS4), while malonyl-CoA is formed via the activity of an ACYL-CoA carboxylase (ACCase). Moreover, the geranyl pyrophosphate (GPP) pool is present at detectable levels in _P. tricornutum_[36].  
To produce OA in _P. tricornutum_, three different expression cassettes were designed using p_Pt_GE30 as a backbone plasmid DNA (Figure 2A), namely, _Pt_OA1, _Pt_OA2, and _Pt_OA3, with various combinations of _C. sativa_ genes encoding for _Cs_TKS and _Cs_OAC (Figure 2B). Each expression cassette was codon-optimized for _P. tricornutum_ and assembled (Appendix A, Figure A1, Tables A1-A3) according to the HIVE lab codon usage table (CUT) [41].  
The transconjugant strains _Pt_OA1, _Pt_OA2, and _Pt_OA3 did not present significant differences in growth and shape compared to _P. tricornutum_ when transformed with the p_Pt_GE30 empty vector or wild-type strains (Figure 3A-C). In addition, morphological properties such as morphotype, size, granulosity, and chlorophyll levels did not significantly differ between the studied strains, as evaluated by microscopy (Figure 3A) and flow cytometry (Figure 4).  
Figure 3: Cell shapes and growth curves of the transformed clones in comparison with wild-type _P. tricornutum_. (**A**) Optical microscope images of wild-type (WT) _P. tricornutum_ and the transformed cells _Pt_OA1, _Pt_OA2, and _Pt_OA3. (**B**,_C_) _P. tricornutum_ WT, transformed with pPtGE30, _Pt_OA1, _Pt_OA2, and _Pt_OA3 cell culture growth curves, followed by absorbance at 680 nm (**B**) or via cell count (**C**) during the 15 days of the cycle. The plotted values represent the means of three replicates and error bars represent the standard deviation.  
Figure 2: Design of the expression vectors _Pt_OA1, _Pt_OA2, and _Pt_OA3. (**A**) Schematic representation of the p_P_FOE30 expression vector. (**B**) Description of the recombinant cassettes used to express _C. sativa_ polyketide synthase (_Cs_TKS) and olivetolic acid cyclase (_Cs_OAC) under _P. tricornutum_’s constitutive promoter 40 SRPS8, the fucoxanthin-chlorophyll binding protein A (FcpA) terminator, with or without tags (His = histidine, eYFP = enhanced yellow fluorescent protein, Seq = sequence, T2A = the _Thosea astigna_ virus 2A cleavable sequence), and reporter genes of different sizes (base pair = bp).  
Figure 4: Flow cytometry observation for cell size and chlorophyll auto-fluorescence among _P. tricornutum_ transconjugants and wild-type cultures. (**A**) Scatter plots for each strain and (**B**) a graphical  
representation of chlorophyll and size percentages among gated cells of wild-type _P. tricornutum_ (WT), and _P. tricornutum_, harboring the empty vector p_PI_GE30, _PTOA_1, _PTOA_2, and _PI_OA3. (**C**) Histogram representing the mean chlorophyll fluorescence intensity, mean granulosity, and mean size scatter.