# The Terpene Mini-Path, a New Promising Alternative for Ternpenoids Bio-Production
## 3 One Enzyme Access to DMAPP/IPP
### One Enzyme Double Phosphorylation of DMAOH and IOH  
The one enzyme double phosphorylation of DMAOH and IOH is obviously the shortest route to access the universal precursors DMAPP and IPP. Two papers have been devoted to such a restricted cascade, both taking advantage of the ability of IPKs to catalyze this double phosphorylation. In one case, the report is based on protein engineering of IPKs by analysis of amino acid coevolution [14]; the other is focused on screening of IPK biodiversity [15]. [PERSON] et al. were the first to use the ability of IPKs to catalyze, in addition to their normal IP kinase activity leading to IPP, another kinase activity on DMAOH, potentially leading to the construction of a one enzyme two step access to DMAPP [14]. It should be noted that the DMAOH kinase catalytic efficiency of _Thermoplasma acidophilum_ IPK (IPK\({}_{In}\)) was three orders of magnitude lower than the normal IP kinase activity [14; 23]. In order to improve the DMAOH kinase activity, the coevolution of the IPK protein sequences was analyzed and potential amino acid positions were detected that can, if mutated, increase the catalytic activity of IPK\({}_{In}\) towards DMAOH. Single, double, or triple mutants were generated, the best ones showing up to an eight-fold increase in catalytic activity. This protein engineering approach was used to produce in vivo (_E. coli_) \(\beta\)-carotene. A maximum of 3.78 mg of \(\beta\)-carotene per gram of dry cell weight was obtained in the best case, involving a triple mutant of IPK\({}_{In}\). As part of a larger project, various IPKs extracted from biodiversity were tested as potential catalysts to be produced in _E. coli_ in a soluble active form, usable at pH 7, 37 \({}^{\circ}\)C, and with ATP as a phosphorylating agent. From the 93 over-expressed proteins, 66 were correctly produced after IPTG addition, but only 24 were soluble. Five IPKs exhibiting high isopentenyl phosphate kinase activity with low sequence similarity to IPK\({}_{In}\) were further chosen and their genes combined individually with the genes encoding the enzymes involved in the generation of lycopene (crtE, crtB, and crtl). When adding DMAOH and IOH to the culture medium, a maximum 11-fold increase in carotenoid content was obtained with the IPK of _Methanococcus marip  
agent [27]. Of particular relevance was the fact that such an enzyme was found to be able to catalyze the formation of isopentanyl phosphate from isopentanol, the hydrogenated equivalent of DMAOH and IOH [27]. Obviously, the use of a phosphatase poses the problem of the unwanted reverse hydrolysis of the newly formed monophosphate. However, if the monophosphate is immediately transformed into the corresponding diphosphate, double phosphorylation can be secured, especially if the enzymatic cascade can be pushed forward by coupling with a prenyl transferase consuming the formed diphosphates. In another pioneering work [10], _Saccharomyces cerevisiae_ choline kinase was found, when screening potentially useful kinases for DMAOH/IOH phosphorylation, to be the one with the most interesting kinetic parameters. It was subsequently used further to develop the so-called isopentenol utilization pathway (IUP). Very recently, a kinase described as acting on 4-methyl-5-hydroxyethylthiazol (ThiM kinase) was proved to be promiscuous enough to catalyze the phosphorylation of DMAOH into DMAP [28]. The latter is the prenyl donor in the generation of prenylated FMN, a recently discovered cofactor used by the UbiD family of reversible decarboxyllases [28]. In _E. coli_, DMAP can be generated either by Nudx phosphatase from DMAPP or by ThiM kinase from DMAOH. The presence of both ThiM and IP kinases in _E. coli_ could, by analogy, explain the reported ability of some microorganisms to use added DMAOH and IOH as substrates for the generation of DMAPP/IPP, as shown above.