Reverse engineering of fatty acid-tolerant Escherichia coli identifies design strategies for robust microbial cell factories
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...
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| Published in: | Metabolic engineering Vol. 61; pp. 120 - 130 |
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| Main Authors: | , , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Belgium
Elsevier Inc
01-09-2020
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| Subjects: | |
| Online Access: | Get full text |
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| Summary: | 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.
•RpoC H419P mutation increases fatty acid tolerance and production, lipid length.•BasS-BasR mutation improves tolerance of fatty acids at higher concentrations.•WaaG impacts EPS sugar abundance, membrane permeability and membrane rigidity.•Reverse engineering identifies timing, contribution of mutations to evolved phenotype. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Yingxi Chen: conceptualization, methodology, validation, formal analysis, investigation, resources, writing – original draft, review & editing; Erin E. Boggess: conceptualization, methodology, software, formal analysis, resources, data curation, writing – original draft, review & editing; Efrain Rodriguez Ocasio: conceptualization, methodology, formal analysis, investigation; Aric Warner: methodology, formal analysis, investigation, resources; Lucas Kerns: investigation, resources; Victoria Drapal: methodology, investigation, resources; Chloe Gossling: investigation, resources; Wilma Ross: conceptualization, methodology, software, validation, formal analysis, investigation, resources, writing – original draft, review & editing; Richard L. Gourse: conceptualization, methodology, writing – original draft, review & editing; Zengyi Shao: conceptualization, methodology, writing – original draft; Julie Dickerson: conceptualization, methodology, writing – original draft; Thomas J. Mansell: conceptualization, methodology, writing – original draft, review & editing; Laura R. Jarboe: conceptualization, methodology, writing – original draft, review & editing |
| ISSN: | 1096-7176 1096-7184 |
| DOI: | 10.1016/j.ymben.2020.05.001 |