## 4 Precursor Hexanoyl CoA Production  
The origin of hexanoyl CoA in _C. sativa_ has not been clearly determined. It is possible that hexanoate is produced via an early termination of chain-elongation during long-chain fatty acid biosynthesis. However, transcriptomic data supports the biogenesis of hexanoate through the fatty acid degradation pathway, in which a long-chain fatty acid is desaturated by a desaturase, oxidized by a lipoxygenase, and finally cleaved by a hydroperoxide lyase to give the free hexanoate (Figure 2). Members of these three enzymes were found to be highly represented in trichome transcriptomes [8; 94]. Hexanoate subsequently forms the CoA thioester by one or more CoA ligases termed acyl-activating enzyme (AAE) or hexanoyl CoA synthase (HCS) found in _C. sativa_, such as CsAAE1-3 (CsHCS1-3) (Figure 2) [94; 95]. CsAAE1 is likely responsible for hexanoic CoA biosynthesis in planta owing to its high transcripts and preference for medium-chain fatty acids [94]. While CsAAE1 and CsAAE2 appear to reside in the cytoplasm, CsAAE3 contains a peroxisome targeting sequence, which may suggest other in planta function [94; 95].  
Engineering the production of medium-chain fatty acids and alcohols in microorganisms predated the engineering of cannabinoid production, due to their implication as biofuels to replace fossil fuels. For example, the bacteria _Clostridium acetobutylcium_ naturally produce 1-butanol (C4). The fermentative production of butanol is well understood in these bacteria and has been engineered either in _Clostridium_ or _E. coli_ using their enzymes, reaching titers of 10-30 g/L [96; 97]. The pathway proceeds via acetyl CoA acetylation, ketone reduction, dehydration, and reduction reactions to form butyryl CoA, followed by thioesterification and carboxylate reduction to butanol [97]. Inclusion of a _Ralstonia eutropha_\(\beta\)-ketothiolase (RsBkkB) allowed further extension of the C4 CoA ester to afford hexanoyl CoA (C6) from the same set of enzymes (Figure 2) [98]. Engineered _E. coli_ afforded \(\sim\)400 mg/L hexanol [99], while engineered yeast _Kluyveromyces marxianus_ in a similar fashion was able to produce 140 mg/L hexanoic acid [44]. The same pathway was also used in a patent for cannabinoid production [51].  
Compared to engineering GPP supply, engineering de novo hexanoyl CoA supply has not been actively explored for cannabinoid production in microorganisms. Several reasons may have contributed to the engineering decisions. Firstly, the production of hexanoyl CoA and IPP may compete for the same acetyl CoA pool if the MVA pathway is used. Secondly, exogenous hexanoate supply are sufficient for hexanoyl CoA biosynthesis with CsAAE overexpression seen in several aforementioned reports. Considering the lower price of hexanoate compared to IPP, it is more economically viable to use hexanoate as feedstock. Thirdly, the cannabinoid pathway enzymes (AAE, TKS, OAC, preryltransferase and cyclases) are promiscuous enough to allow various short to medium aliphatic acids as starter molecules, rendering a repertoire of diverse cannabinoid analogs to be synthesized. Therefore, aliphatic acids feeding may be a wise choice for producing specialty cannabinoids.