# The Terpene Mini-Path, a New Promising Alternative for Ternpenoids Bio-Production
## 5 Discussion and Perspectives  
This review summarized the recent development of the artificial TMP to (bio)synthesize terpenids starting from DMAOH and IOH. Numerous advantages are associated with such a reduced pathway. The first and most obvious one is the drastic reduction in the number of enzymes needed to access the two universal precursors, DMAPP and IPP, from 18 for the MVA and MEP pathways to only two (or three if IDI is considered) in the TMP. The limited number of enzymes to deal with offers the opportunity to construct more easily in vitro enzymatic cascades to access terpenoids. The successful productions of a prenylated diketopiperazine, tryprostatin B at a titer of 3 g/L, of a precursor of taxol, taxol, taxolate at 220 mg/L, and of cannabigerolic acid at 480 mg/L or cannabigerovarinic acid at 580 mg/L are great achievements in the field. Another important point to be raised is the fact that the TMP only needs ATP as a cofactor and no reducing cofactors to the contrary of the MVA and MEP pathways, hence simplifying the buildup of the cascade. Furthermore, DMAOH and IOH are cheap industrial compounds, available in bulk and partially soluble in water, all important things from both an environmental and an industrial point of view. Another interesting point with the use of the TMP is its ability to easily access, for the first time, non-natural terpenoids by using analogs of DMAOH or IOH or of both [30]. Indeed, the two enzymes of the TMP used by [PERSON] et al. have been shown to be promiscuous enough to generate modified DMAPPs and IPPs, which can subsequently be incorporated into different terpenoids [30]. All these points are also beneficial when considering the in vivo use of the TMP. Indeed, only two genes are needed to construct the TMP, their introduction being easily completed in the chassis of choice through plasmid transformation. The only need for the TMP in vivo is the supply of ATP, something provided by the microorganism used as a chassis. As DMAOH and IOH are small, neutral, and rather hydrophobic chemicals, they easily pass the biological membranes and, thus, can be internalized without the need for a transporter. Probably, the main in vivo advantage of the TMP is its total decoupling (except for ATP production) from the central metabolism, with no regulation being associated with the use of the two enzymes. The TMP can thus be optimized on its own to adjust the flow of DMAPP/IPP on demand. Another important point is that the DMAOH/IOH ratio to be added to the culture medium can be adjusted at will using different ratios suitable for the production of monoterpenes (theoretical ratio 1/1), sesquiterpenenes (theoretical ratio 1/2), diterpenes (theoretical ratio 1/3), triterpenes (theoretical ratio 1/5), and tetraterpenes (theoretical ratio 1/7). Another option in this context is the use of an IDI-like enzyme to interconvert DMAPP to IPP and vice versa. The addition of DMAOH/IOH can also be done at will in the case of the TMP, particularly after induction of the latter, which is not possible with the MVA and MEP pathways where the source of carbon and energy (glucose, glycerois also the source of biosynthetic carbon. Some of these advantages have been exploited to in vivo access lycopene (190 mg/L), tryprostatin B (860 mg/L), limonene (500 mg/L), and geranoids (2 g/L) using _E. coli_ as a chassis. Higher yields were seen when a yeast chassis was used to produce terpenoids, but, in all the reported cases, both the TMP and the MVA pathways were used together to produce different terpenoids, such as lycopene (_Yarrowia lipolytica_, 4.2 g/L, 11 days), geranylgeraniol (_Saccharomyces cerevisiae_, 5.07 g/L, 7 days), and \(\alpha\)-farnesene (_Pichia pastoris_, 2.56 g/L, 3 days). Besides all the advantages described above, some disadvantages can be drawn when it comes to the use of the TMP either in vitro or in vivo. The first point is that DMAOH and IOH are, for now, chemically synthesized starting from isobutene and formaldehyde through the Prins reaction.