Enhancing control of cell-free metabolism through pH modulation
Engineering metabolism for the synthesis of bio-based products in non-model organisms can be challenging. One specific challenge is that biosynthetic pathways are often built from enzyme candidates sourced from diverse organisms, which can prove difficult to implement in recombinant hosts due to differences in their cellular environments (e.g. pH, cofactor balance). To address this problem, in this work we report a cell-free synthetic biology approach for understanding metabolism in a range of environmental conditions, specifically under varied pH. The key idea is to control the pH of Escherichia coli-based cell-free systems for assessing pathway performance using enzymes sourced from organisms other than E. coli. As a model, we apply this approach to study the impact of pH on the n-butanol biosynthesis pathway derived from clostridia in E. coli lysates. Specifically, we exploit the open, cell-free reaction environment to explore pH outside the habitable range of E. coli, revealing insights into how chemical context impacts the interaction between native metabolism and heterologous enzymes. We find that the pH optimum for butanol production from acetyl-CoA is substantially lower than the optimal pH of glycolysis in E. coli-based crude lysates. In addition, pH is an essential factor to consider when activating metabolic pathways in the cell-free environment due to its effect on reaction yield or enzyme activity, the latter of which is demonstrated in this work for alcohol dehydrogenases from a range of extremophiles. Ultimately, altering metabolism through pH control will allow cell-free systems to be used in studying the metabolic state of organisms and identify suitable enzymes for pathway engineering.
- Research Organization:
- Northwestern Univ., Evanston, IL (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division; National Science Foundation (NSF); National Defense Science and Engineering
- Grant/Contract Number:
- SC0018249; DGE-1324585-008; ND-CEN-017-095
- OSTI ID:
- 2298972
- Alternate ID(s):
- OSTI ID: 1659131
- Journal Information:
- Synthetic Biology, Vol. 5, Issue 1; ISSN 2397-7000
- Publisher:
- Oxford University PressCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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