Natural products are a rich source of pharmaceuticals, constituting the majority of small molecule drugs in use today. However, their production through organic synthesis or heterologous hosts can be difficult and time-consuming. To enable easier screening and production of natural products, a cell-free protein synthesis (CFPS) system was utilized to partially assemble natural products in vitro using SAM-dependent methyltransferase enzyme reactions. Caffeine synthesis by the tea caffeine synthase TCS1 was demonstrated within the CFPS system, and production of caffeine and intermediate products such as theobromine was confirmed via LC-MS. One major benefit of cell-free systems is the ability to use toxic substrates that would normally be difficult to use in a cellular environment to synthesize novel products, however we found that TCS1 and its homologs were unable to use cofactors such as SAE, the ethyl analogue of SAM.

Therefore, protein engineering is essential to expand the repertoire of SAM-dependent methyltransferases for synthesizing new products, as some enzymes are unable to use SAM analogues. A structure-guided approach was employed to modify the tyrosine O-methyltransferase MfnG to use SAE in addition to SAM while also reducing its preference for SAM. A series of MfnG mutants were generated, and one mutant exhibited significantly higher capacity to utilize SAE for tyrosine O-ethylation. Further investigation of this mutant was carried out through predicted structural analysis and molecular dynamics simulations. This work demonstrates the potential of cell-free systems for natural product synthesis and the power of protein engineering to expand the capabilities of SAM-dependent methyltransferases for novel product synthesis.