Developing an Anaerobic Cell-Free Protein Expression Platform for Discovery of Novel Enzyme Function

From the human gut to the soils that support plant growth, many of the microbes that support life thrive in anaerobic environments. These microbes often thrive in these environments by producing proteins that facilitate unique chemical transformations; notable examples include lignocellulosic biomass degradation and nitrogen fixation. To date, however, our ability to harness and engineer these functionalities has been stymied by challenges associated with mapping gene to function relationships in anaerobic organisms. These include a lack of genetic tools for non-model, anaerobic organisms and difficulties with heterologous expression systems (e.g., limited access to relevant post-translational modifications or maintenance of anaerobic conditions for oxygen-sensitive proteins). To address these challenges, we propose the development of anaerobic cell-free protein synthesis (CFPS) systems derived from these anaerobic organisms.

In this talk, I will describe I work towards this goal. First, we have identified fluorescent proteins compatible with anaerobic conditions—iRFP702 and mFAP2a—that can serve as reporters of protein expression in anaerobic cell-free expression experiments. We find that both reporters function aerobically and anaerobically in CFPS, producing good signal above background, in contrast to an sfGFP control, which only produces signal in the presence of oxygen. To enable future high-throughput screening of genes of interest in the future, we have also investigated miniaturization of CFPS reactions using acoustic liquid handling. Experiments with our two fluorescent reporters—iRFP702 and mFAP2a—as well as the sfGFP reporter commonly used for optimizing CFPS indicate that total reaction volume can be scaled down to 0.5 µL while still maintaining adequate signal-to-noise for all three reporters. This work established an accurate and reproducible liquid dispensing method for future exploratory experiments and will allow for fully automated workflows. Ultimately, we envision coupling this workflow with other automated processes in the NSF Biofoundry for Extreme and Exceptional Fungi, Archaea and Bacteria (ExFAB) housed at UC Santa Barbara to efficiently optimize CFPS in lysates derived from non-model organisms and, eventually, screen genes of unknown function. These developments will expand our ability to study these strains while work continues for the development of genetic tools for strain development.