CONCLUSION

In this study, we successfully constructed geranate-producing E. coli strains via IUP. We applied a two-stage fermentation protocol to increase the product titer. We found a competing pathway for geranate production, and optimized the expression level of the oxidoreductases to reduce by-product formation. The best strains produced 764 mg/L of geranate from 2 g/L isopentenols within 24 h, among the highest geranate titers reported in the literature. To our knowledge, this is the first study demonstrating the engineering of E. coli as microbial cell factory for de novo production of geranate using simple and cheap isopentenol substrates. The optimization approaches followed in this study may be applied to similarly reduce by-product formation in the production of other value-added isoprenoid compounds. We suggest that follow up studies improve the substrate specificity of CdGeDH and CdGaDH via protein engineering, and thus provide a basis host cell for further increasing the geranate titer through genetic and process engineering. A potential challenge might be the toxicity of geranate to E. coliat higher concentrations. This issue could be addressed by using a more robust host (e.g., Pseudomonas species), evolving the engineeredE. coli in the presence of geranate, and/or sequestering geranate on charged resin or in a solvent.

ACKNOWLEDGMENTS

This work was financially supported by National Research Foundation Singapore through a Singapore-MIT Alliance for Research and Technology project (funding identifier: R-279-000-587-592) and an Intra-CREATE project (funding identifier: A-0001440-00-00).

AUTHOR CONTRIBUTIONS

Qiuchi Pan and Kang Zhou conceived and designed the research. Xiaoqiang Ma contributed key materials and analysis tools. Qiuchi Pan, Xiaoqiang Ma, Yurou Liu, Hong Liang performed the analysis. Qiuchi Pan wrote the original draft of the manuscript. Qiuchi Pan, Xiaoqiang Ma, Kang Zhou and Gregory Stephanopoulos edited the manuscript.

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.

DATA AVAILABILITY STATEMENT

All of the data used to support the claims of this study have been presented in the form of figures and/or tables, which are available in this manuscript.