Project Abstract – Invizyne Technologies, Inc Regio- and stereo-specific methylation of natural products and synthetic pharmaceuticals can dramatically improve their physiochemical, biological, and pharmacological properties. Biocatalytic methylations are currently limited by the availability of efficient, scalable regeneration systems for the cofactor S-adenosyl methionine (SAM). We propose a universal in vitro platform for the regeneration of SAM using only methionine and ribose as inexpensive feedstocks. Efficient, low-cost SAM regeneration bridges the gap between small scale high- throughput screening of methyltransferases for novel methylated products and industrial production of lead compounds, which is hampered by the availability of large amounts of SAM. Invizyne has developed a bench-scale, enzyme-based, proof-of-concept SAM regeneration system called SimpleSAM that is able to efficiently convert luteolin to chrysoreriol. In Phase I of this project we will i) optimize this enzyme cascade for longevity, stability, SAM turnover numbers and product yield, ii) expand the system to include C-, N-, and O-methylated targets of interest and iii) replace the currently inefficient ATP regeneration system with a new cell-free cascade to regenerate ATP from low-cost feedstock. The resulting GlyCoSAM platform will expand the current toolkit for sustainable cell-free biomanufacturing and enable the scalable production of methylated natural products, nutraceuticals, and synthetic pharmaceuticals.