# The Terpene Mini-Path, a New Promising Alternative for Ternpenoids Bio-Production
## 3 One Enzyme Access to DMAPP/IPP
### The TMP In Vivo  
#### 4.3.1 Escherichia coli  
The very first report on a two-step access to DMAPP/IPP (IUP) was mainly dedicated to the in vivo synthesis of lycopene and taxadiene (valencene, limonene, miltiradiene, and amorphadiene synthesis were also attempted) in _E. coli_ by the use of a choline kinase from _Saccharomyces cerevisiae_ and the _Arabidopsis thaliana_ IPK as the two kinases of the artificial pathway [10]. The two corresponding genes were incorporated in an operon in combination with an _idi_ gene to balance the DMAPP/IPP ratio. Two plasmids were thus generated, one with a constitutive promoter, the second with an inducible promoter. When combined with a plasmid bearing a lycopene operon, the former outperformed, the best conditions being the use of 25 mM IOH. A further optimization of the plasmid copy number, as well as the promoter strength of the lycopene operon, led to lycopene content of 8000 ppm in a short time (12 h) following induction (Scheme 9A). When taxadiene production was attempted, a 15 mg/L concentration was obtained after 48 h of culture (Scheme 9A). A very interesting result has come from this pioneer work. By analyzing the intracellular concentration of various phosphates and diphosphates (IP, IPP, GPP, FPP, GGPP), it was demonstrated that the flux to IPP using the so-called IUP pathway is larger than that of the native MEP pathway. This also led to the intracellular accumulation of IPP and GGPP, the flux of the IUP being larger than the ones of the downstream lycopene and taxadiene operons. Soon after this first report, two other papers [11; 12] described the access to DMAPP /IPP from DMAOH/IOH using a phosphatase as the first enzyme of the artificial path in combination with an IPK. In one case [11], the genes coding the PhoN phosphatase from _Sligella flexneri_ and the _Theroplasma acidophulum_ IPK were cloned into a pET plasmid (forming the so-called alcohol-dependent heriterpene (ADHP) pathway). This plasmid was then combined in _E. coli_ with a plasmid bearing the genes encoding a lycopene biosynthetic path as well as the _idi_ gene allowing, at the end of the fermentation, a titer in lycopene of 150 mg/L. Optimization of the concentration of added alcohols (DMAOH and IOH, 5 mM each) further increased the lycopene titer to 190 mg/L. The same genetic construction but replacing the lycopene operon by the gene of a tryptophan phenyl transferase allowed an approximately 20 mg/L production of dimethylallyltyrtyptophan (Scheme 9B). In the other case [12], the V78L mutant of the _Salmonella enterica_ phosphatase and the IPK from _Theroplasma acidophilum_ were used in order to transform the chemically synthesized cyclo-dipeptide breevianamide F into the prenylated and cytotoxic derivative tryprostatin B. Based on added breevianamide F and after optimization, a 63% isolated yield in tryprostatin B was obtained after 24 h, at a titer of 860 mg/L (Scheme 9C). The same year, another report employing the _E. coli_ ThiM kinase in combination with _Methanothermobacter thermautotrophicus_ IPK, forming the so-called isoprenoid alcohol (IPA) pathway, described the production of various mono-, sesqui-, tri-, and tetraterpenoids [13]. When a truncated version of the geranyl diphosphate synthase from _Abies grandis_ was combined with either a geraniol synthase from _Ocinum basilicum_ or a limonene synthase from _Mentha spicata_, a mixture of monoterpene alcohols (geraniol, nerol, and citronellol)  
Figure 9: Initial demonstration of the in vivo access to terpenoids (_E. coli_) thanks to the so-called isopentenol utilization pathway (**A**), the alcohol-dependent hamiterpene pathway (**B**), the terpene mini-path (**C**), or the isoprenoid alcohol pathway (**D**) using a two enzymes cascade and starting from DMAOH/IOH to access DMAPP/IPP. was obtained at a titer of 2 g/L, as well as limonene at a titer of 0.5 g/L, respectively (Scheme 9D). By using dedicated enzymes, farnesol, diaponeurosporene (a C30 carotenoid), and lycopene were also obtained at titers of 80, 15, and 8 mg/L, respectively.