All naturally produced terpenes are derived from two universal C5 diphosphate precursors, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Various prenyl transferases use DMAPP to prenylate aromatic compounds, while others, in combination with IPP, lead to the enzymatic formation of geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and geranylfarnesyl diphosphate, the direct precursors of monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), and sesterterpenes (C25), respectively. FPP and GGPP are also the basis for the biosynthesis of triterpenes (steroids) and tetraterpenes (carotenoids), respectively. Nature has developed two biosynthetic pathways to produce DMAPP and IPP, the mevalonate (MEV) pathway and the methylerythritol phosphate (MEP) pathway. Both use compounds derived from glucose through glycolysis, and 18 enzymes are involved to generate both DMAPP and IPP. Here, we sought to simplify biochemical access to these two universal diphosphates using the two commercially and industrially available C5-OHs, dimethylallyl alcohol and isopentenol (IOH), as starting substrates, as well as two enzymes, selected from a diverse choice, able to carry out the double phosphorylation of these two C5-OHs at room temperature using ATP as a phosphate donor. The first phosphorylation is performed by a promiscuous acid phosphatase (AP), used in the reverse reaction mode, whereas the second is performed by the recently described isopentenyl phosphate kinase (IPK). We show the interest of this artificial biosynthetic terpene mini-path (TMP) by testing it in a three-enzyme cascade, leading to the formation of the cytotoxic prenylated diketopiperazine tryprostatin B (TB) from chemically synthesized brevianamide F (BF), using FtmPT1 prenyltransferase as a biocatalyst, in addition to the two previously mentioned kinases. We first performed the proof of concept of this simplified pathway in vivo (Escherichia coli), using already described enzymes, that is, an AP from Salmonella enterica and an IPK from Thermoplasma acidophilum. The complete conversion of BF (3.3 mM, 1 g/L) to TB was obtained after optimization of culture conditions and process parameters. Following this first success, we performed a screen in search of highly active phosphatases and IPKs to develop the TMP in vitro. A highly active AP from Xanthomonas translucens and an IPK from Methanococcus vannielii were selected from these screens, allowing the in vitro development of the TMP. Under optimized conditions, the three-enzyme cascade led to the total transformation of BF (10 mM, 3.3 g/L) to TB in less than 24 h, establishing the in vitro utility as well as the in vivo utility of the TMP. The implementation of this biosynthetic TMP offers thus the possibility to access virtually any terpene structure using two easily commercially and industrially available compounds in bulk, either in vivo or in vitro, and is thus a viable alternative to the natural MEV and MEP pathways for bioaccess to terpenes.
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