Campus Units
Chemical and Biological Engineering, Electrical and Computer Engineering, Bioinformatics and Computational Biology, Microbiology, Ames Laboratory, NSF Engineering Research Center for Biorenewable Chemicals
Document Type
Article
Research Focus Area
Renewable Energy
Publication Version
Accepted Manuscript
Publication Date
5-28-2020
Journal or Book Title
Metabolic Engineering
DOI
10.1016/j.ymben.2020.05.001
Abstract
Adaptive laboratory evolution is often used to improve the performance of microbial cell factories. Reverse engineering of evolved strains enables learning and subsequent incorporation of novel design strategies via the design-build-test-learn cycle. Here, we reverse engineer a strain of Escherichia coli previously evolved for increased tolerance of octanoic acid (C8), an attractive biorenewable chemical, resulting in increased C8 production, increased butanol tolerance, and altered membrane properties. Here, evolution was determined to have occurred first through the restoration of WaaG activity, involved in the production of lipopolysaccharides, then an amino acid change in RpoC, a subunit of RNA polymerase, and finally mutation of the BasS-BasR two component system. All three mutations were required in order to reproduce the increased growth rate in the presence of 20 mM C8 and increased cell surface hydrophobicity; the WaaG and RpoC mutations both contributed to increased C8 titers, with the RpoC mutation appearing to be the major driver of this effect. Each of these mutations contributed to changes in the cell membrane. Increased membrane integrity and rigidity and decreased abundance of extracellular polymeric substances can be attributed to the restoration of WaaG. The increase in average lipid tail length can be attributed to the RpoCH419P mutation, which also confers tolerance to other industrially-relevant inhibitors, such as furfural, vanillin and n-butanol. The RpoCH419P mutation may impact binding or function of the stringent response alarmone ppGpp to RpoC site 1. Each of these mutations provides novel strategies for engineering microbial robustness, particularly at the level of the microbial cell membrane.
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Copyright Owner
Published by Elsevier Inc. on behalf of International Metabolic Engineering Society
Copyright Date
2020
Language
en
File Format
application/pdf
Recommended Citation
Chen, Yingxi; Boggess, Erin E.; Rodriguez Ocasio, Efrain; Warner, Aric; Kerns, Lucas; Drapal, Victoria; Gossling, Chloe; Ross, Wilma; Gourse, Richard L.; Shao, Zengyi; Dickerson, Julie; Mansell, Thomas J.; and Jarboe, Laura R., "Reverse engineering of fatty acid-tolerant Escherichia coli identifies design strategies for robust microbial cell factories" (2020). Chemical and Biological Engineering Publications. 432.
https://lib.dr.iastate.edu/cbe_pubs/432
Included in
Electrical and Computer Engineering Commons, Membrane Science Commons, Microbial Physiology Commons
Comments
This is a manuscript of an article published as Chen, Yingxi, Erin E. Boggess, Efrain Rodriguez Ocasio, Aric Warner, Lucas Kerns, Victoria Drapal, Chloe Gossling, Wilma Ross, Richard L. Gourse, Zengyi Shao, Julie Dickerson, Thomas J. Mansell, and Laura R. Jarboe. "Reverse engineering of fatty acid-tolerant Escherichia coli identifies design strategies for robust microbial cell factories." Metabolic Engineering (2020). DOI: 10.1016/j.ymben.2020.05.001. Posted with permission.