# The Terpene Mini-Path, a New Promising Alternative for Ternpenoids Bio-Production
## 3 One Enzyme Access to DMAPP/IPP
### The TMP In Vitro--Purified Enzymes  
The first report dealing with the use of purified enzymes to access terpenids from C5 alcohols demonstrated that the combination of a phosphatase and an IPK could generate DMAPP starting from DMAOH [12]. As a proof of concept, the former was used as a prenyl donor to generate a cytotoxic prenylated diketopiperazine compound, tryprostatin B, using chemically synthesized brevianamide F (BF) and prenyl transferase FtmPT1 from _Aspergillus fumigatus_ as a catalyst (Scheme 5). A large optimization of the three enzymes cascade allowed to totally transform BF into tryprostatin B in 24 h on a 10 mM scale. The main influencing parameters were found to be the presence of an ATP recycling system, an increase in DMAOH concentration _versus_ BF concentration, a double ATP addition during the reaction, a decreased concentration in phosphatase, and a higher concentration in prenyl transferase FtmPT1 as compared to initial conditions. This optimization was made necessary by the use of a phosphatase for the first enzymatic step. Indeed, it was demonstrated that this phosphatase was able to quickly hydrolyze any of the phosphates or diphosphates added in the reaction medium or produced during the reaction (DMAP, DMAPP, PEP, AMP, ADP, ATP). As a general rule, influencing parameters acting either to slow down phosphate and diphosphate hydrolysis or to maintain a high level in phosphorylating agent (low phosphatase activity, ATP regeneration system, addition of ATP) or to promote the production and use of DMAPP (higher DMAOH concentration, higher preynl transferase activity) had to counteract phosphatase activity. While the proof of concept of the in vitro usefulness of the TMP was completed and a final concentration of tryprostatin B of ~3 g/L was obtained, the TMP using a phosphatase as the first enzyme did not appear to be the best option. It should be noted that the used enzymes were freshly purified and not frozen. Soon after [31], a paper described the use of the _Arabidopsis thaliana_ IPK in combination with a true kinase, the choline kinase from _S. cerevisiae_, as the first enzyme of the TMP, called here the IUP. The combination of these two kinases with IDI (isopentenyl diphosphate isomerase), FPPS from _E. coli_, and various terpene synthases (limonene synthase, amorphadiene synthase, and valencene synthase) allowed the in vitro production of the corresponding hydrocarbons (Scheme 6). Taxadiene synthesis was also attempted using the GGPPS (geranylgeranyl diphosphate synthase) from _Taxus canadensis_ and the taxadiene synthase from _Taxus brevifolia_. After the optimization of the various enzyme concentrations, as well as of ATP, DTT, and Mg\({}^{2+}\) concentrations and other parameters, such as using only IOH with IDI versus a mix of DMAOH and IOH without IDI, a final yield of 220 mg/L in taxadiene was achieved in 9 h with a 65% conversion of added IOH. In 2020, two papers appeared, both taking advantage of the use of another true kinase (the ThiM kinase from _E. coli_, see above) to phosphorylate DMAOH and IOH into DMAPP and IPP in conjunction with the _Methanocaldococcus jannaschii_ IPK [29; 30]. In the former case, the formed DMAPP and IPP were used as substrates of different linear preynl transferases (Scheme 6) to access (_E,E_)-FPP, (_Z,Z_)-FPP, GPP, and GGPP. Implementing a terpene synthase/cyclase further afforded, depending on the used enzyme, (_S_)-germacrene D, (-)-germacradiene-4-ol, amorpha-4,11-diene, and 7-epi-zingiberene (Scheme 7). The TMP has also been exploited to generate, from homologs of DMAOH and IOH, various linear diphosphates homologous to FPP. Some of them were then used as substrates for germacrene D and amorphadiene sesquiterpene synthases, providing the corresponding unnatural terpenoids [30]. The second article [29] deals with the generation of cannabinoids, implying many more enzymatic steps than previous reports (12 enzymes in total).