Reverse engineering of fatty acid-tolerant Escherichia coli identifies design strategies for robust microbial cell factories |
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| Institution: | 1. School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China;2. Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China;1. CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China;2. University of Chinese Academy of Sciences, Beijing, China;3. State Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China;1. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, 117599, Singapore;2. Synthetic Biology Research Consortium, National University of Singapore, 28 Medical Drive, 117456, Singapore;3. School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, 637459, Singapore;4. Singapore Institute of Technology, 10 Dover Drive, 138683, Singapore;1. Department of Biosystems Engineering, Auburn University, Auburn, AL 36849, USA;2. School of Chemistry, National University (UNA), Heredia, Costa Rica;3. Center for Bioenergy and Bioproducts, Auburn University, Auburn, AL 36849, USA;4. Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), Av. IPN 195, La Paz, B.C.S. 23096, Mexico;5. The Bashan Institute of Science, 1730 Post Oak Court, Auburn, AL 36830, USA;6. Department of Entomology and Plant Pathology, Auburn University, 301 Funchess Hall, Auburn, AL 36849, USA;7. College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China |
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| 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. |
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| Keywords: | Adaptive evolution Reverse Engineering Cell Membrane Stringent response Octanoic acid Butanol |
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