Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

2024 Abstracts

Using Cell-Free Systems to Accelerate Biosystems Design for Carbon-Negative Manufacturing


Yun-nam Choi1*, Kyle Zolkin1*, Grant Landwehr1,2, Ashty S. Karim2, Michael C. Jewett1,2


1Stanford University; 2Northwestern University


The accelerating climate crisis combined with rapid population growth poses some of the most urgent challenges to humankind, all linked to the unabated release and accumulation of carbon dioxide (CO2) across the biosphere. By harnessing the capacity to partner with biology, the abundance of available CO2 can be leveraged to transform the way the world produces and uses carbon. Yet, designing, building, and optimizing non-model CO2-fixing biosystems to achieve a broader range and more complex biofuels, bioproducts, and biomaterials remains a formidable challenge.

To address this challenge, the research team is developing a cell-free protein synthesis approach for high-throughput engineering of natural and novel enzymes for CO2 assimilation and biosynthetic product pathways. In one example, the team uses cell-free systems to study natural enzymes like ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which is the key enzyme of the Calvin cycle. Various types of RuBisCO were successfully expressed, including form I and form II, via cell-free protein synthesis, and their activity was confirmed by NADH-linked assay and liquid chromatography-mass spectroscopy.

In another example, hydroxyacyl-CoA lyases (HACLs) are being engineered. HACLs have become increasingly relevant due to their ability to form carbon-carbon bonds between formyl-CoA (C1 donor) and a larger carbonyl-containing molecule (C1 acceptor). The research team has expressed, purified, and characterized over 60 homologs selected by sequence similarity, uncovering the high promiscuity of these enzymes.

Collectively, these efforts demonstrate how cell-free systems can be used as a screening tool to explore the wide scope of natural enzyme diversity. The newly characterized enzymes can contribute to the engineering of C1 assimilation routes and expand branches of synthetic metabolism to enable a diverse set of enzymatic reactions for sustainable bioproduction.