Cell-Free Technology Accelerates Industrial Biotechnology

New in vitro platform prototypes and rapidly optimizes synthetic enzymes for cellular design.

Image courtesy of Ashty Karim
The cell-free prototyping framework can down-select enzymatic pathway candidates to accelerate synthetic biological design in cells.

The Science

Industrial biotechnology aims to use microbes, such as bacteria, as miniature factories. These factories would convert molecules, the smallest packages of chemicals, into desirable products. They rely on enzymes, which are proteins that accelerate specific chemical reactions. However, designing, building, and optimizing sets of enzymes to carry out biosynthetic pathways in cells is complex and slow. Scientists have now developed a cell-free framework to rapidly select from hundreds of enzyme pathways. The framework eliminates the need to use intact cells to facilitate the design of microbial factories.

The Impact

Speeding up biotechnology research and development could have important benefits for many industries. Biotech could lead to advances in areas ranging from clean energy to consumer products. This new cell-free approach has already accelerated the design of enzyme pathways to produce industrial chemicals in the microbe Clostridium autoethanogenum. This microbe is hard to manipulate but great at consuming waste carbon in the environment. This framework can be easily adopted and will decrease the number of microbes that need to be engineered and tested. It also reduces the time required to achieve specific objectives. 


The cell-free framework, termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (iPROBE), enabled the construction of a Clostridium strain that produces 14.63 ± 0.48 g/L of 3-hydroxybutyrate (approximately 20 times more than the previous highest report). In addition, using data-driven design, the researchers tested 205 butanol-producing enzymatic pathway combinations in vitro and increased cellular production by testing selected designs in Clostridium. They also identified a new route to synthesize 1,3-butanediol and the first biological production of the (S)-isomer of this molecule. The iPROBE cell-free framework shows a strong correlation with cellular performance.


Scientists are curating a growing suite of enzyme sets for the production of 2,3-butanediol, butanol, 3-hydroxybutyrate, styrene, the monoterpenes limonene, bisabolene, and pinene, and more. With the establishment of new cell-free systems built from other microbes such as Clostridium autoethanogenum, this approach offers a powerful tool to accelerate biological design.


Michael C. Jewett
Northwestern University


Michael Köpke
LanzaTech Inc.


This work is supported by the Department of Energy Office of Science, Office of Biological and Environmental Research, by the David and Lucile Packard Foundation, and by the Camille Dreyfus Teacher–Scholar Program. The scientists also thank the following investors in LanzaTech’s technology: BASF, CICC Growth Capital Fund I, CITIC Capital, Indian Oil Company, K1W1, Khosla Ventures, the Malaysian Life Sciences, Capital Fund, L. P., Mitsui, the New Zealand Superannuation Fund, Petronas Technology Ventures, Primetals, Qiming Venture Partners, Softbank China, and Suncor.


A. Karim, et al., “In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.” Nature Chemical Biology 16, 912–919 (2020). [DOI: 10.1038/s41589-020-0559-0]

A. Krüger, et al., “Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways.” Metabolic Engineering, in press (2020). [DOI:  10.1038/s41589-020-0559-0]

Q. Dudley, et al., “Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis.” Metabolic Engineering 61, 251-260 (2020). [DOI:  10.1016/j.ymben.2020.05.006]

W. Grubbe, B. Rasor, et al., “Cell-free styrene biosynthesis at high titers.” Metabolic Engineering 61, 89-95 (2020). [DOI:  10.1016/j.ymben.2020.05.009]

Related Links

Accelerating Biological Systems Design for Sustainable Biomanufacturing, Northwestern McCormick School of Engineering News

The perfect collaboration to accelerate biological design, Nature Research Bioengineering Community

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