Developing in vitro tools for engineering allosteric transcription factors, enzymes and metabolic systems

Anthropogenic climate change, caused by the emission of more greenhouse gases such as carbon dioxide than the global carbon cycle can fix, is drastically affecting our planet and life on it. While humanity can decarbonize its energy needs, we will continue to rely on carbon-based molecules for food, medicine, and materials of all kinds. 34% of anthropogenic greenhouse gas emissions come from industrial processes such as the production of chemicals, plastics and fertilizers, and are primarily caused by the need for high heat and pressure in industrial processes. Biocatalysis allows the production of chemicals at ambient pressure and lower temperatures, as well as the use of substrates derived from waste streams, biomass or atmospheric carbon dioxide. In recent years, many methods have been developed to tailor enzymes to the needs of industrial biocatalysis, but low throughput in testing enzyme variants limits progress. The aim of this work was to develop tools to increase the throughput of enzyme engineering and the testing of metabolic systems such as enzyme cascades. Allosteric transcription factor (aTF)-based biosensors allow the rapid and label-free detection of metabolites using common laboratory equipment, making them attractive for highthroughput enzyme assays. aTFs transduce the formation of the aTF ligand by the enzyme variant tested (the input) into the generation of a reporter signal such as fluorescence or DNA replication (the output). To demonstrate the rapid and inexpensive testing of a complex enzyme cascade and its individual enzymes, we developed an in vitro transcription (IVT)-based biosensor to read out the product formation of the CETCH cycle, a complex synthetic CO2 fixation cycle that produces glycolate from CO2. Notably, several cofactors of the CETCH cycle inhibited IVT, but we still achieved high correlations of r = 0.94-0.98 between IVT output and LC-MS quantification. However, suitable aTFs that recognize metabolites of interest in the required concentration range are rare, limiting the widespread application of biosensing. To address this deficiency, I have developed a new concept for rapidly engineering aTFs for new substrate specificities and other optimized properties such as sensitivity. The concept is based on one major principle. Despite the many advantages of living cells,2 their active metabolism, metabolome and membrane pose challenges to the engineering of aTFs, such as product degradation, interference with the native metabolome leading to low sensitivity and crosstalk with structurally similar compounds, and exclusion of membrane-impermeable ligands. To overcome these limitations, the PURE system, an in vitro transcription-translation system reconstituted from purified components, allows the testing of protein variants without competing metabolism, metabolome and membrane, allowing the use of metabolites that cannot be tested in living cells. Recently, a powerful in vitro compartmentalization method for engineering transcription-translation-related enzymes, called compartmentalized partnered replication (CPR), has been demonstrated. In CPR, the activity of an aTF is coupled to the production of a thermostable DNA polymerase in Escherichia coli, the cells are encapsulated in water-in-oil emulsions, and functional aTF genes are replicated by emulsion PCR. The better the encoded aTF, the more DNA polymerase is produced and the more the aTF gene is enriched in the gene population. However, the previously published CPR approach is not compatible with the PURE system. We have laid the foundation for CPR in the PURE system by establishing the genetically encoded control of DNA replication in the PURE system, called transcription-translation coupled DNA replication (TTcDR). We present data on several TTcDR systems, general design rules for genetic circuits in the PURE system, and improved DNA polymerase mutants that yield a genetic circuit based on the model aTF TetR with >1000-fold DNA replication under non-repressing conditions, ~150-fold repression by TetR, and ~4-fold derepression by the ligand anhydrotetracycline. We will use the genetic circuit-controlled TTcDR system to establish selection for the evolution of allosteric transcription factors in the first place, and enzymes and metabolic systems in the future.