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.
Highlights
Isoprenoids are a large family of compounds comprising numerous products used as fragrances, insecticides, nutraceuticals and pharmaceuticals [1,2]
Microbial metabolic engineering has been intensively explored in the past decade for isoprenoid production and gram per liter production levels has been achieved for certain isoprenoid precursors [3]
To address the unmet need of a method for the simultaneous quantification of D-Xylulose 5Phosphate (DXP) metabolites, we have developed an integrated preanalytical solid phase extraction (SPE) procedure with the use of ultra performance liquid chromatography mass spectrometry (UPLC-MS)
Summary
Isoprenoids are a large family of compounds (more than 55,000) comprising numerous products used as fragrances, insecticides, nutraceuticals and pharmaceuticals [1,2]. Of these molecules has been limited by scarce plant resources from which they were originally extracted. Despite the structural diversity of isoprenoids, they are all derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Both metabolites are synthesized either by the mevalonate (MVA) pathway or the 1-Deoxy-D-Xylulose 5Phosphate (DXP) pathway (Figure 1) [4]. To better understand the mechanisms underlying the inverse correlation of lycopene production with overexpression of the DXP genes, a direct measurement of the metabolites is desirable
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