Abstract
Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 ± 0.63 mM to 40.33 ± 1.03 mM, the highest amount achieved using biosynthesis without process modifications and product removal strategies. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules.
Original language | English (US) |
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Pages (from-to) | 89-95 |
Number of pages | 7 |
Journal | Metabolic Engineering |
Volume | 61 |
DOIs | |
State | Published - Sep 2020 |
Funding
We graciously thank the Department of Energy (BER grant: DE-SC0018249 ), the David and Lucile Packard Foundation ( 2011-37152 ), and the Camile Dreyfus Teacher-Scholar Program for support. B.J.R. is an NDSEG Fellow (Award ND-CEN-017-095 ). We also thank the Joint Genome Institute Community Science Program Project 503280. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
Keywords
- Biomanufacturing
- CFPS
- Cell-free
- In vitro
- Styrene
ASJC Scopus subject areas
- Biotechnology
- Bioengineering
- Applied Microbiology and Biotechnology