Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production

Abstract

Isoprenoids are natural products that are all derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These precursors are synthesized either by the mevalonate (MVA) pathway or the 1-Deoxy-D-Xylulose 5-Phosphate (DXP) pathway. Metabolic engineering of microbes has enabled overproduction of various isoprenoid products from the DXP pathway including lycopene, artemisinic acid, taxadiene and levopimaradiene. To date, there is no method to accurately measure all the DXP metabolic intermediates simultaneously so as to enable the identification of potential flux limiting steps. In this study, a solid phase extraction coupled with ultra performance liquid chromatography mass spectrometry (SPE UPLC-MS) method was developed. This method was used to measure the DXP intermediates in genetically engineered E. coli. Unexpectedly, methylerythritol cyclodiphosphate (MEC) was found to efflux when certain enzymes of the pathway were over-expressed, demonstrating the existence of a novel competing pathway branch in the DXP metabolism. Guided by these findings, ispG was overexpressed and was found to effectively reduce the efflux of MEC inside the cells, resulting in a significant increase in downstream isoprenoid production. This study demonstrated the necessity to quantify metabolites enabling the identification of a hitherto unrecognized pathway and provided useful insights into rational design in metabolic engineering.

Engineering Escherichia coli coculture systems for the production of biochemical products

“…There are, however, situations where this approach may be limited, as in the cases where (i) a single host cell cannot provide an optimal environment for the functioning of all pathway enzymes, (ii) biosynthetic efficiency is reduced due to overwhelming metabolic stress from the overexpression of long and complex pathways (3,4), or (iii) pathway intermediates are secreted yielding undesired byproducts and reducing substrate utilization (5,6). The above limitations can potentially be overcome through the use of rationally designed microbial coculture systems.…”

Deoxyxylulose 5-Phosphate Synthase Controls Flux through the Methylerythritol 4-Phosphate Pathway in Arabidopsis

“…Our calculations of MEcDP partitioning between the plastid and nonplastid pools indicate that this diversion occurs in DXS-overexpressing plants, but not in wild-type plants under our growing conditions. DXS overexpression may inadvertently trigger a natural efflux process involved in stress signaling that occurs under certain environmental conditions (Xiao et al, 2012;Zhou et al, 2012).…”

“…MEcDP efflux was recently described for an Escherichia coli strain overexpressing DXS (Zhou et al, 2012), possibly as a response to oxidative stress. MEcDP diversion may also result from pathogen attack as part of a stress response mediated by salicylic acid (Gil et al, 2005).…”

Overexpression of key enzymes of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for improving squalene production in Escherichia coli

“…Previous report (Zhou et al, 2012) showed that overexpression of dxs, idi and ispDF could lead to over-production and accumulation of MECPP in cell to outflow into the broth that is toxic to MEP pathway, which further decreased the lycopene production. The same situation may also appear in the current study where the outflow of MECPP produced by overexpression of genes dxs, idi and ispDF reduced toxic effect to MEP pathway, thus increased transcriptional levels of dxs and idi genes in strain YSS8.…”

“…Overexpression of genes dxr (Lv et al, 2016), ispDF Yuan et al, 2006), ispG (Liu et al, 2014) and ispA (Han et al, 2016) were able to enhance terpenoids production. On the contrary, other studies have shown that overexpression of dxr coupled with dxs produced a similar isoprene level when compared with the dxs overproduction strain (Xue and Ahring, 2011), and overexpression of ispDF together with dxs and idi resulted in decrease in terpenoids production (Zhou et al, 2012). Meanwhile, genes ispH, ispE and ispA have not been overexpressed in combination with dxs and idi genes of MEP pathway in the production of terpenoids in E. coli.…”