## 3 Discussion
It was also reported that the gene following the T2A sequence would be affected on the expression level [46] and, thus, would yield fewer _Cs_OAC transcripts, but the effect of this phenomenon would be validated if the C10-C12 polyketide product was available as a standard for HPLC detection, which is not the case. When compared to previous studies, the production of OA in _Yarrowia lyolytica_ slightly changed when _Cs_TKS and _Cs_OAC were expressed under different promoters, compared to when the two genes were fused [42]; in _P. tricornutum_, the _P_tOA1 showed a higher quantity of OA (Figure A3) compared to there being none in _P_tOA3 when the two genes were fused by a glycine-serine linker. This is not applicable in _P_tOA2 since the Western blot did not show cleaved proteins, which could indicate that the cleaved proteins were below detection levels. To our knowledge, this is the first time that a brown microalgae host has demonstrated the capacity to accumulate OA and use it for the synthesis of CB-like metabolites. The heterologous production of OA was observed in the different transconjugants of _P. tricornutum_, transformed with episomal constructions and harboring various combinations of _C. sativa TKS_ and _OAC_ genes (Figure 2; Appendix A, Figure A1 and Table A2). The cassette designs in this study were fairly simple and direct, with only two main cannabis genes being introduced. When compared to the literature, the first approaches regarding yeast achieved the production of only 0.48 mg/L of OA by expressing _Cs_TKS and _Cs_OAC, and by feeding the culture with sodium hexanoate [9]. Here, we obtained higher yields (0.56-2.6 mg/kg) compared to yeast with and without hexanoate supplementation [9]. In _Y. lipolytica_, it was bioengineered with _Pseudomonas_ sp. _LvaE_, encoding a short-chain acyl-CoA synthetase, acetyl-CoA carboxylase, pyruvate dehydrogenase bypass, NADPH-generating malic enzyme, as well as the activation of the peroxisomal \(\beta\)-oxidation pathway and the ATP export pathway to redirect the carbon flux toward OA synthesis [47]; the yield was also less than 0.5 mg/L until batch culture optimization. Further optimization of the _P. tricornutum_ transconjugant strains by boosting the pool of precursors could increase the production of OA. In _E. coli_, besides introducing _Cs_TKS and _Cs_OAC, metabolic engineering consisted of the overexpression of the acetyl-CoA carboxylase subunits A, B, C, and D from the same host to ensure the high production of malonyl-CoA, and a reversal of beta-oxidation that allowed the accumulation of hexanoyl-CoA [33]. This led to a higher quantity of OA (46.3 mg/L) after the optimization of culture conditions and induction parameters [33]. In _P. tricornutum_, hexanoyl-CoA and malonyl-CoA occur naturally, due to the long-chain acyl-CoA synthase (_Pt_ACS3 and _Pt_ACS4) for hexanoyl-CoA and to the acetyl-CoA ATP-dependent carboxylase (_Pt_ACCase) for malonyl-CoA [43]. _P. tricornutum_ was also shown to accumulate intracellular amountsof GPP, along with neryl diphosphate (NPP) [36; 48]; both precursors are donors of the prenyl moiety of several cannabinoids, such as CBGA and cannabinoid acid (CBNA) in cannabis [46]. However, overexpression of these upstream genes could eliminate the need for supplementation while maintaining the necessary pools of precursors for more stable production of cannabinoids, which is similar to the process used with yeast [31; 34]. Along with precursor bioavailability, endogenous enzymes are required to enable the conversion of OA into CB or CB-like compounds, as observed in this study, either by direct production (Figure A4) or by supplementation assays (Table 1). Bioinformatic analyses led to the identification of two putative enzymes (Appendix B, Figure A6) that are encoded by Phatr3_J37858 and Phatr3_J2738.t1 and are capable of prenyltransferase activity, thereby allowing the production of CBGA. Both enzymes are UbiA prenyltransferase family members that could be used originally by the diatom to produce terpen or flavonoids [49; 50].