## 2 Critical Biosynthetic Steps for Phytocannabinoid Formation
### The Formation of Olivetolic Acid
The later pathway enzymes (prenyltransferase and cyclases) were also promiscuous enough to adopt the different side-chains, since novel cyclized cannabinoids were formed by feeding different carboxylate acid starting molecules to yeast expressing these genes [37]. These results make it possible to further diversify cannabinoids by altering the side-chain. Unlike the membrane-bound prenyltransferase or the terminal cyclases requiring a eukaryotic protein folding system (discussed later in this article), expression of CsTKS and CsOAC is straight forward in both bacterial or eukaryotic systems. Currently, OA formation \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Expression Systems** & **Feedstock** & **Product** & **Titer** & **Sources** \\ \hline \multirow{8}{*}{_Saccharomyces cerevisiae_} & Olivetolic acid & CBGA & 96 mg/L & [34] \\  & Olivetolic acid & THCA & 84 mg/L & [34] \\  & Hexanoic acid & CBGA & 34 mg/L & [34] \\  & Hexanoic acid & THCA & 23 mg/L & [34] \\  & Olivetolic acid & CBGA & 216 mg/L & [35] \\  & Hexanoic acid & CBGA & 73 mg/L & [35] \\  & Olivetolic acid & CBDA & 234 mg/L & [36] \\  & Olivetolic acid & THCA & 320 mg/L & [36] \\  & de novo & 1-methyl-CBG & 66 mg/L & [30] \\  & de novo & Olivetol & 76 mg/L & [30] \\  & de novo & 1-methyl-bilvetol & 42 mg/L & [30] \\  & Hexanoic acid & CBDA & 4.2 mg/L & [37] \\  & Hexanoic acid & THCA & 8.0 mg/L & [37] \\  & Olivetolic acid & CBGA & 1.0 mg/L & [38] \\  & Olivetolic acid & CBGA & 1.0 mg/L & [38] \\ \hline \multirow{4}{*}{_Komagataella phiffi_} & CBGA & THCA & 32.6 mg/L & [39] \\  & CBGA & THCA & 0.36 g/L & [40] \\  & Olivetolic acid & THCA & 615 pmol/L & [41] \\  & CBGA & THCA & 3.05 g/L & [42] \\ \hline \multirow{4}{*}{_Escherichia coli_} & Olivetolic acid/GPP & CBGA & 1.2 mg/L & [43] \\  & de novo & Hexanoic acid & 140 mg/L & [44] \\  & de novo & Hexanoic acid & 190 mg/L & [45] \\  & de novo & CBGA & detectable & [46] \\  & Hexanoic acid & Olivetolic acid & 0.48 mg/L & [47] \\ \hline \multirow{4}{*}{_Nicotiana benthamiana_} & CBGA & CBDA & 34 ppm & [48] \\  & CBDA & CBDA-glucosidase & N/A & [48] \\  & CBDA & CBDA-glucose & N/A & [49] \\  & CBGA & THCA & N/A & [38] \\  & Hexanoic acid & Olivetolic acid & N/A & [38] \\  & Hexanoic acid & glucose & N/A & [38] \\  & CBGA & THCA & 82 ug/30 mL & [50] \\ \hline \multirow{4}{*}{_Kluyperomyces_} & \multirow{2}{*}{de novo} & CBDA, THCA & N/A & [51] \\  & & & & \\  & & & & \\ \hline \multirow{2}{*}{_Candida viswanathii_} & oleic acid & CBGA & 0.67 mg/L supernatant & [52] \\  & oleic acid & CBGA & 1.51 mg/L lysate & [52] \\  & oleic acid & Olivetolic acid & 13.1 mg/L & [52] \\ \hline \multirow{2}{*}{_Chlamydomonas_} & \multirow{2}{*}{de novo} & CBGA & detectable & [53] \\  & & & & \\ \hline \multirow{2}{*}{Cell free} & Olivetolic acid & CBGA & 744 mg/L & [54; 55] \\  & Olivetolic acid & CBGA & 1.25 g/L & [54; 56] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Summarized published work of heterologous systems for cannabinoid production. by CsTKS and CsOAC has not been actively engineered in yeast or _E. coli_, compared to the late prenylation and cyclization reactions and the early geranyl diphosphate (GPP) and hexanoyl CoA supply. However, a recent report indicated that OA strongly inhibited CsTKS activity at medium (0.25 mM) and high (5 mM) OA concentrations in their in vitro cannabinoid production system, suggesting protein engineering of CsTKS may be required to improve its catalysis [56]. When assayed in vitro, the recombinant CsTKS and CsOAC have always produced significant amounts of lactone by-products (TALs), which are not reported in _C. sativa_[22; 23; 24]. While the reason is unclear, it may be possible to engineer the recombinant proteins for improved product specificity.