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.