## 3 Precursor Geranyl Pyrophosph Production
ERG20 catalyzes consecutive additions of IPP onto DMAPP to form GPP then FPP, while the GPP intermediate remains enzyme bound. The effect of GPPS overexpression in yeast is hindered by ERG20 that effectively converts GPP further to FPP [90]. The double mutant _erg20_ F96W_N127W and single mutant K197E have been developed to augment ERG20's GPPS activity while reducing its FPPS activity, which led to several magnitudes of monoterpene titer improvement to mg per litre levels [91; 90]. This is pertinent to GPP supply in yeast cannabinoid production, since it was reported that overexpressing mutant _erg20_ outperformed overexpressing plant GPPS [35]. In the several related reports for cannabinoid production in yeast [35; 36; 37], the GPP production has been heavily engineered with one of the variations shown in Figure 2. In general, all MVA pathway genes were overexpressed with strong inducible promoters, and several could be replaced by enzymes from other organisms for higher activity or less inhibition in yeast. With CsPT4 and enhanced expression of terminal cyclases, these yeast strains were shown produce 200-300 mg/L CBGA, THCA or CBDA in shake flasks after 3-5 days when fed with exogenous OA (Table 1). Roughly 70-90 mg/L C10 precursor was produced based on stoichiometry. While it is difficult to unambiguously identify the rate limiting step based on the available reports, it is likely that GPP supply could still be a limiting factor in these strains considering the reported monoterpene titers in literature are in similar range [84]. An alternative solution has been devised to circumvent ERG20's intrinsic GPP isopentanyltransferase activity, by introducing a neryl diphosphate synthase (NPPS) [88]. NPP is the _cis_-isomer of GPP and no longer a substrate for ERG20. Protein engineering on a terpene synthase permitted its use of NPP as substrate [88], which provides an interesting route for monoterenoid production in yeast. CsPT1 does not accept NPP substrate [60], whereas the NPP preference of CsPT4 or StNphB has not been reported. The cyclases, however, are able to produce respective final cannabinoids from nerylated OA: CBNRA. As such, it may be possible to produce cannabinoids via NPP, if the prenyltransferases are found or engineered to use NPP or alternative prenyltransferases could be sourced. Another alternative route for IPP or GPP supply is to provide exogenous isoprenol and preenol then convert them to IPP and DMAPP by overexpression of several kinases [92]. While it circumvents the endogenous regulation and perhaps limitation of isoprenoid precursor supply, its application in microbial cannabinoid production may be limited by other factors including GPP production, downstream enzyme activities, and feedstock cost. There are few publicly available reports on engineering _E. coli_ for cannabinoid production, although de novo synthesis of CBG or CBGA is possible since all enzymes to this point are either native to _E. coli_ or can be effective expressed. For example, the monoterpene limonene could be produced in _E. coli_ at 400 mg/L in shake-flasks enabled by the overexpression of MVA pathway and truncated plant GPPS [93]. A patent application describes overexpression of several MEP pathway genes including a bifunctional IspDF gene in _E. coli_ towards IPP production, however the production of cannabinoids was not reported despite demonstrating functional expression of CsPT1 [43]. In another patent, detectable CBGA was claimed de novo in _E. coli_ with cannabis enzymes and CsPT1 [46]. There are a number of companies claiming technology for cannabinoid production in _E. coli_, which may suggest adoption of the described approaches or inventions that are not made public.