## 5 Choice of Chassis and Further Technical Development  
Various organisms have been attempted for the heterologous production of cannabinoids or precursors, including bacteria _E. coli_[43; 45; 46; 47; 65; 66] and _Zymomonas mobilis_[100], cyanobacteria _Synechocystis_ spp. [101] and _Synechococcus elongatus_[53], fungal species _S. cerevisiae_[33; 34; 35; 36; 37; 40; 41; 51], _Kluyveromyces marxianus_[51], _Komagataella phaffii_ (formerly _Pichia pastoris_) [40; 41; 42; 72] and _Candida viswanathii_[52], and plant species _Chlamydomonas reinhardtii_ (green algae) [53] and _Nicotiana benthamiana_ (tobacco) [38; 48; 49; 74] (Table 1). For metabolic engineering, _E. coli_, _S. cerevisiae_, and tobacco are among the most studied species. There are abundant molecular and genetic tools for their manipulation and decades of knowledge on their uses in biotechnology. In these three species, _E. coli_ is disadvantaged because the terminal cannabinoid cyclases could not be successfully expressed due to the requirement of eukaryotic post-translational modification system for proper protein folding. However, the prenyl and aliphatic precursors could be produced at gram per liter levels in _E. coli_, and CBGA production was possible with CsPT1 and StNphB. The future of using _E. coli_ to produce cannabinoids relies on whether medical or nutraceutical uses of the non-cyclized cannabinoids will be developed, or if novel _E. coli_ compatible enzymes that are able to cyclize cannabinoids are discovered. Alternatively, _E. coli_ may be co-cultured with other organisms such as yeast to carry out the complete biosynthesis. The modular co-culturing strategy may overcome the limitations of the respective organisms, which has been demonstrated for the biosynthesis of several natural products [102]. However, significant engineering is needed for shuttling the intermediates between organisms and studying the co-culturing conditions to achieve high titers for industry use. The yeast _S. cerevisiae_ is well positioned for cannabinoid production. Extensive engineering of _S. cerevisiae_ has allowed for 200-300 mg/L cannabinoid production from galactose and OA feeding, and 30-80 mg/L cannabinoid production if OA is replaced with hexanoic acid (Table 1). The lowered production suggest improvement in intracellular hexanoic acid availability as well as the improvement of CsTKS/OAS activity. The production of olivetol and methyl olivetol by a type I polyketide synthase provided an interesting route for cannabinoid biosynthesis and will certainly be viable to produce non-cyclized cannabinoids. However, the wild-type terminal cyclases do not accept CBG as a substrate, therefore this route is limited by the availability of a novel or engineered cyclase for converting the decarboxylated precursors. In addition, it appeared that cannabinoids were secreted to the growth media by the engineered yeast, since the analyses in the above reports were performed using whole culture extraction without mechanical cell lysis, however this requires further confirmation. Plants are able to photosynthesize and therefor do not require an exogenous carbohydrate feedstock. They contain the necessary cellular machinery for expressing plant proteins that may need membranous systems or post-translational modifications that may not be provided by _E. coli_ or yeast. Plants also contain plastids and vacuoles that may be explored for specialized metabolite production and accumulation. On the other hand,plant metabolism is more complex, which may require more engineering efforts for metabolite stability and storage to avoid toxicity and to improve yield. When CsPKS, CsOAC, and CsAAE1 were transiently expressed in tobacco, feeding hexanoic acid led to the formation of OA-4-\(O\)-glucoside as a major product. Unfortunately, further inclusion of CsPT4 and THCAS did not lead to detectable amounts of CBGA or THCA, despite all enzymes proving functional in tobacco [38].