초록
<P><B>Significance</B></P><P>We have demonstrated the design, prototyping, and implementation of a non-natural route to isoprenoid biosynthesis, termed the isoprenoid alcohol (IPA) pathway. Opposed to working within the confines of native metabolic pathways, our approach of utilizing all available biochemical reactions identified a synthetic route to isoprenoid compounds that is both different and provides a more energy-efficient pathway compared with native isoprenoid biosynthesis. By improving the overall pathway design, the IPA pathway has the potential to facilitate the synthesis of a diverse range of isoprenoid compounds at high product titers and yields.</P><P>The more than 50,000 isoprenoids found in nature are all derived from the 5-carbon diphosphates isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Natively, IPP and DMAPP are generated by the mevalonate (MVA) and 2-C-methyl-<SMALL>D</SMALL>-erythritol-4-phosphate (MEP) pathways, which have been engineered to produce compounds with numerous applications. However, as these pathways are inherently constrained by carbon, energy inefficiencies, and their roles in native metabolism, engineering for isoprenoid biosynthesis at high flux, titer, and yield remains a challenge. To overcome these limitations, here we develop an alternative synthetic pathway termed the isoprenoid alcohol (IPA) pathway that centers around the synthesis and subsequent phosphorylation of IPAs. We first established a lower IPA pathway for the conversion of IPAs to isoprenoid pyrophosphate intermediates that enabled the production of greater than 2 g/L geraniol from prenol as well as limonene, farnesol, diaponeurosporene, and lycopene. We then designed upper IPA pathways for the generation of (iso)prenol from central carbon metabolites with the development of a route to prenol enabling its synthesis at more than 2 g/L. Using prenol as the linking intermediate further facilitated an integrated IPA pathway that resulted in the production of nearly 0.6 g/L total monoterpenoids from glycerol as the sole carbon source. The IPA pathway provides an alternative route to isoprenoids that is more energy efficient than native pathways and can serve as a platform for targeting a repertoire of isoprenoid compounds with application as high-value pharmaceuticals, commodity chemicals, and fuels.</P>