The production of monoterpenes starts with the MVA pathway which is naturally found in Eukaryotes. Previous pathway engineering efforts have created a heterologous MVA pathway in E.coli which confers monoterpene production to this non-native producer. (Alonso-Gutiérrez et al., 2013) The MVA pathway starts with acetyl-CoA, a pivotal component in cell metabolism and a compound that should be readily available inside our cells. With various enzymes, limonene is first produced which is then oxidised by a cytochrome P450. From previous studies there are two plasmids that have been included into E.coli for PA production.
Figure 2 above shows a scheme of our solution. When E.coli undergoes the MVA pathway, toxic intermediates accumulate, such as IPP/DMAPP, GPP, and the products of the pathway, limonene and Perillyl alcohol. The accumulation of these toxic intermediates eventually leads to cell death (Helfrich et al., 2019), resulting in a low yield of Perillyl alcohol production. Our approach is to extend the lifespan of E.coli by introducing a protein envelope pathway, which allows E.coli to switch the pathway off when there are high levels of toxic intermediates.
By inducing our protein envelope pathway, it senses the membrane stress caused by the accumulation of toxic intermediates, releasing CPxR protein, which binds to the promoter region of the induced gene. This upregulates the release of TetR repressor protein, which binds to the pTet region of the 6410 plasmid promoter, repressing enzyme expression and shutting the pathway off. As a result, the cell defaults back to the Krebs cycle, reducing flux through the MVA pathway and preventing the production of toxic intermediates. This enables the cell to survive. On the other hand, the binding of the CPxR protein also upregulates the native E.coli efflux pump, which pumps out more toxic intermediates, keeping the toxicity low within the cell. This keeps the E.coli healthy and, in the long term, allows for the production of more Perillyl alcohol.
1. Alonso-Gutierrez J, Chan R, Batth TS, Adams PD, Keasling JD, Petzold CJ, Lee TS. Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metab Eng. 2013 Sep;19:33-41.
2. Figure 1 created in https://www.biorender.com/
3. Helfrich, E. J. N., Lin, G., Voigt, C. A., & Clardy, J. (2019). Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering. Beilstein Journal of Organic Chemistry, 15, 2889–2906.