4.2. The TMP In Vitro—Purified Enzymes
The first report dealing with the use of purified enzymes to access terpenoids from C5 alcohols demonstrated that the combination of a phosphatase and an IPK could generate DMAPP starting from DMAOH 
. 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
First in vitro use of the TMP to access tryprostatin B in a three enzymes-one pot cascade 
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 prenyl 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 
, 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
In vitro access to various terpenes thanks to the use of the TMP constitute of the choline kinase from S. cerevisiae
and the Arabidopsis thaliana
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 Mg2+ 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
. In the former case, the formed DMAPP and IPP were used as substrates of different linear prenyl transferases (Scheme 6
) to access (E
)-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 
Scheme 7. In vitro access to various sesquiterpenes thanks to the use of the TMP constituted of the ThiM kinase from E. coli and the Methanocaldococcus jannaschii IPK.
The second article 
deals with the generation of cannabinoids, implying many more enzymatic steps than previous reports (12 enzymes in total). This demonstrated that the in vitro enzymatic production of natural products of mixed biosynthetic origin (polyketide/terpene in that case) could be envisioned seriously thanks to the reduced number of enzymes of the TMP. As far as the terpene part is concerned, GPP was produced with a four enzymes cascade starting from IOH using ThiMEc
, and FPPSGs
S82F. The latter, a point mutant of FPPS from Geobacillus stearothermophilus
, no more catalyzed the formation of FPP but rather of GPP. This so-called ISO module was combined with an ATP regeneration module employing acetyl-phosphate as a sacrificial phosphate donor. The aromatic polyketide module affords either the olivetolic or divarinic acids depending on the length of the hydrocarbon side chain and, when combined with GPP through the cannabinoid module, they afford cannabigerolic acid at 480 mg/L or cannabigerovarinic acid at 580 mg/L titers on a 1 mL scale in 10 h (Scheme 8
In vitro access to cannabinoids thanks to the TMP involving the ThiM kinase from E. coli
and the Methanocaldococcus jannaschii
IPK, as well as an aromatic polyketide module and a cannabinoid module. The ATP regeneration module, as well as ATP consumption for the various modules, except for kinases, are omitted (adapted from 
These four examples prove that the TMP offers, compared to the MVA and MEP pathways, the possibility to easily synthesize terpenes and terpenoids in vitro using purified enzymes. Although the enzyme purification is a time-consuming and costly process, the drastic reduction in the number of enzymes needed to access DMAPP and IPP thanks to the TMP (two instead of eighteen) can be exploited to access various terpenoids and could be of interest in the discovery of new terpene synthases as well as in the determination of their substrate promiscuity.