Utilizing a Cell-free Protein Synthesis Platform for the Biosynthesis of a Natural Product, Caffeine

Natural products are a valuable source of pharmaceuticals, providing a majority of the small molecule drugs in use today. However, their production through organic synthesis or in heterologous hosts can be difficult and time-consuming. Therefore, to allow for easier screening and production of natural products, we demonstrated the use of a cell-free protein synthesis (CFPS) system to partially assemble natural products in vitro using SAM-dependent methyltransferase enzyme reactions. The tea caffeine synthase TCS1 was utilized to synthesize caffeine within a CFPS system. Cell-free systems also provide the benefit of allowing the use of substrates that would normally be toxic in a cellular environment to synthesize novel products. However, TCS1 is unable to utilize a compound like AdoEt as a cofactor to create ethylated caffeine analogs. The automation and reduced metabolic engineering requirements of CFPS systems, in combination with other synthesis methods, may enable the more efficient generation of new compounds.