## 2 Results
### Temporal Metabolite Production  
#### 2.2.1 Metabolite Detection and Identification  
Selected positive transconjugant strains, displaying protein accumulation via Western blot analysis, were cultivated, extracted, and used for targeted metabolite analysis. OA was detected at 2.6 mg/kg in _Pt_OA1 and 0.54 mg/kg in _Pt_OA2, using HPLC-UV, whereas no peaks at the OA retention time were detected in the _Pt_OA3 extracts (Figure 6 and Appendix A, Figure A3).  
Interestingly, additional peaks with similar retention times to the cannabinoid standards of CBN, CBD, and THC, and their corresponding acid forms were detected in the extracts of OA-producing transconjugants (_Pt_OA1C1 and _Pt_OA2C2) (Appendix A, Figure A4). Liquid chromatography-mass spectrometry (LC-MS) analyses using electrospray ionization in the positive mode (ESI+) were performed to validate if the mass-to-charge ratio (\(m/z\)) of these peaks corresponded to protonated CBs (M + H)\({}^{+}\). The compounds were first scanned for specific masses corresponding to the metabolites of interest, then, in the case of a positive scan, another LC-MS analysis was performed to make sure that the fragments corresponded to the target compound. Unexpectedly, the daughter \(m/z\) obtained after employing collision-induced energy (CID) at 40 V of the selected RT and \(m/z\) corresponded to the cannabinoid standards (Appendix A, Figure A4). This suggests that _P. tricornutum_ may contain endogenous enzymes that are capable of converting OA into CB-like metabo  
Figure 6: Olivetolic acid production, obtained via HPLC-UV analysis at 280 nm. (**A**) Curves represent the peak at the OA retention time of chromatograms, obtained following the injection of 10 \(\upmu\)L of each extracted sample. At the bottom, a concentration of 10 \(\upmu\)g/mL of commercial standards (STD) is shown: metabolite extraction from the _P. tricornutum_ wild type (WT); _P. tricornutum_, transformed with an empty vector (EV = p_Pt_GE30), _Pt_OA1, _Pt_OA2, and _Pt_OA3. The peaks represent olivetolic acid. (**B**) Schematic representation of the bioengineered _P. tricornutum_ for the production of cannabinoid precursors.  
lites such as CBGA, CBDA, and THCA. Such enzymes could be mimicking the activity of cannabis APT (using the endogenous pool of GPP) and CBDAS/THCAS (Figure 1). Candidate enzymes from the diatom database were searched and low-similarity sequences for APT and THCAS were identified (Appendix B, Figure A6). However, further characterization is needed and might help find closer candidates among microalgae. Alternatively, candidates from a completely different type of enzyme could be involved.  
To test for the presence of such endogenous enzymes, we performed a supplementation assay with _P. tricornutum_ transconjugants harboring the p_Pt_GE30, which were devoid of cannabis genes (Table 1). We first added OA and GPP for 16 h and could subsequently detect small amounts (circa 3-5 mg/kg) of CBGA and other CB-like metabolites using HPLC analysis. This suggests the presence of an APT-like enzyme in _P. tricornutum_ (Table 1). Next, we supplemented the sample with CBGA and detected metabolites that behaved in a similar way to CB on an HPLC chromatogram, but we were unable to confirm their identity (not all were appropriate \(m/z\) fragments) via LC-MS.