(676e) Optimization of the Escherichia coli Methyl Erythritol Phosphate Pathway

Isoprenoid pathways present attractive solutions for the production of biofuels, biomaterials, and biopharmaceuticals. In the current study, we report the production of isopentenol in Escherichia coli via the non-mevalonate pathway by the stable expression of the pyrophosphohydrolase enzyme NudB fused with N-terminal Flag tag. The expression of deoxy-xylulose-phosphate (DXP) synthase gene dxsthat condenses pyruvate and glyceraldehyde 3-phosphate into the MEP pathway was limited by the formation of insoluble inclusion bodies and cell lysis. Upon optimization of DXS expression and solubility, DXP concentrations and culture physiology were significantly enhanced thereby improving the isopentenol titers in the culture.

The carbon-efficient non-mevalonate pathway is limited by the flux imbalance of its intermediate metabolites due to the technical issues related to the post-translational modification of its enzymes with iron-sulfur clusters in E. coli. Due to this, the intermediate metabolite methyl erythritol cyclopyrophosphate (MECpp) of the MEP pathway which is utilised by the enzyme IspG, an Fe-S protein; was found to be effluxed into the culture supernatant, thereby resulting in significant carbon loss. Since anaerobic conditions have been observed to be better redox mediators for higher IspG activity, when isopentenol-producing E. coli strains were cultivated under anaerobic conditions, the specific productivity of isopentenol improved to 6 mg/L/ODunitcells as compared to 4 mg/L/ODunitcells under aerobic conditions. Supplementation of Fe-S cluster assembly proteins by the expression of the ISC operon further improved the isopentenol production levels. Through these multi-step optimizations, 64 mg/L isopentenol was obtained from 20 g/L glucose in E. colicultures.

The balance of the MEP-intermediate fluxes via the active expression of Fe-S cluster proteins IspG and IspH would be critical for further optimization of isopentenol production. To achieve this, we have further explored the expression of apoproteins IspG and IspH in Escherichia coli and their maturation into their holo-forms. The Fe-S cluster assembly proteins were supplemented by the co-expression of the ISC (Iron Sulfur Cluster) operon with the target apoproteins. We further investigated the expression of four proteins CyaY, ErpA, RIC and NfuA for their possible role as iron-donors or Fe-S cluster assembly/repair. It was interesting to observe that ErpA expression enhanced the labile iron and labile sulfide content in purified IspG as well as IspH. When the effect of these helper proteins were further studied under anaerobic culture conditions, the RIC protein which has been reported to be involved in repair of iron centres significantly improved the labile iron content in IspG and IspH. The increased iron and sulfide content in the purified target proteins was also accompanied by higher intensity bands on Western blots from NATIVE protein gels of the crude cell extract, clearly indicating higher level of expression of soluble IspG and IspH.

These studies also serve as model proteins to explore biogenesis of Fe-S cluster proteins that are involved in a variety of biological roles including electron transfer, catalysis, sensor and regulatory mechanisms, protein stabilizations etc. Further studies are being conducted to quantify the active Fe-S cluster in the target holo-proteins and investigate their effect in the in-vivo cultures and balance the fluxes of the MEP pathway.