The Non-Mevalonate Pathway Requires a Delicate Balance of Intermediates to Maximize Terpene Production

Author:

Raghavan Indu,Wang Zhen Q.

Abstract

AbstractTerpenes are valuable industrial chemicals whose demands are increasingly being met by bioengineering microbes such asE. coli. Although the bioengineering efforts commonly involve installing the mevalonate (MVA) pathway inE. colifor terpene production, the less studied methylerythritol phosphate (MEP) pathway is a more attractive target due to its higher energy efficiency and theoretical yield, despite its tight regulation. In this study, we integrated an additional copy of the entire MEP pathway into theE. coligenome for stable, marker-free terpene production. The genomically integrated strain produced more monoterpene geraniol than a plasmid-based system. The pathway genes’ transcription was modulated using different promoters to produce geraniol as the reporter of the pathway flux. Pathway genes, includingdxs, idi, andispDF, expressed from a medium-strength promoter, led to the highest geraniol production. Quantifying the MEP pathway intermediates revealed that the highest geraniol producers had high levels of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), but moderate levels of the pathway intermediates upstream of these two building blocks. A principal component analysis demonstrated that 1-deoxy-D-xylulose 5-phosphate (DXP), the product of the first enzyme of the pathway, was critical for determining the geraniol titer, whereas MEP, the product of DXP reductoisomerase (Dxr or IspC), was the least essential. This work shows that an intricate balance of the MEP pathway intermediates determines the terpene yield in engineeredE. coli. The genetically stable and intermediate-balanced strains created in this study will serve as a chassis for producing various terpenes.Key PointsGenome-integrated MEP pathway afforded higher strain stabilityGenome-integrated MEP pathway produced more terpene than the plasmid-based systemHigh monoterpene production requires a fine balance of MEP pathway intermediates

Publisher

Cold Spring Harbor Laboratory

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