Cell-free protein synthesis for biocatalysis.

Cell-free protein synthesis (CFPS) serves as an innovation booster in the field of biocatalysis. By integrating CFPS into the design and development of biocatalysts, the discovery, synthesis, and screening of previously untapped enzymes and their engineered variants can be improved. The high-throughput capability of CFPS accelerates the identification of optimal synthesis conditions, including expression hosts, chaperone sets, temperature, and codon optimization. Moreover, the availability of various CFPS systems facilitates the incorporation of non-canonical amino acids and enables native post-translational modifications. Using CFPS in combination with enzymatic activity assays also helps to determine the best conditions for biocatalytic reactions, such as temperature, pH, substrate, and choice of cofactor. The compatibility of CFPS with robotic and microfluidic systems, along with artificial intelligence, further enhances its high-throughput capabilities. However, challenges remain regarding scalability, the low concentration of the target protein, and the applicability of a generalized CFPS system for the synthesis of any protein. While these challenges impede the incorporation of CFPS in industrial scale biocatalytic processes, its applicability as screening tool is validated to improve biocatalytic reactions. This knowledge can then be transferred to in vivo synthesis systems to improve the overall production outcomes.