Developing a Cell-free Protein Synthesis Platform for Anaerobic Gut Fungi

Advancements in cell-free protein synthesis (CFPS) have enabled the rapid prototyping of genetic circuits and biosynthetic pathways in E. coli, bypassing traditional time-intensive cloning workflows. This same technology stands to benefit non-model microbes that lack the genetic toolkits necessary for creating genetically modified mutants in implementing design-build-test-learn cycles. Our group is interested in the metabolic activity of anaerobic gut fungi (AGF) isolated from the guts of ruminant herbivores. AGF grow natively on lignin-rich substrates and could be used for microbial bioprocessing of lignocellulosic biomass into biofuels. To date, genetic engineering of AGF is hindered by a limited genetic toolkit, low efficacy for heterologous expression in E. coli, and lacks functional annotations for up to 75% of protein encoding genes. This project aims to develop an anaerobic cell-free system to analyze protein production and metabolic pathways in AGF, providing insight into the functionality of uncharacterized genes. Our work to date has focused on identifying and evaluating potential anaerobic fluorescent proteins (iRFP702 and mFAP2a) to serve as reporters of protein expression rate in anaerobic cell-free expression experiments. Initial screening conducted using E. coli shows that mFAP2a is fluorescent anaerobically in vivo with fluorescence 2.25-fold above background while fluorescence of iRFP702 is hindered by the cell-membrane permeability of its cofactor. Aerobic E. coli CFPS showed fluorescence from mFAP2a and iRFP702 to be 28.3-fold and 13.5-fold above background respectively. Work continues towards the optimization of anaerobic E. coli CFPS, to use this as a testbed to screen the activity of enzymes native to AGF. Preliminary data from sugar-reducing assays show enzymatic activity from select AGF carbohydrate-active enzymes (CAZymes) in aerobic E. coli CFPS. Future work will focus on optimization and refinement of techniques for the preparation of AGF’s native cellular lysate to see how enzymes expressed in AGF lysate compare to E. coli based CFPS. Ultimately, this system, when used in combination with UCSB’s incoming biofoundry, will allow for an automated workflow to more rapidly screen combinations of genetic parts. The development of a cell-free system for a non-model organism like AGF will expand our ability to study these strains while work continues for the development of genetic tools for strain development.