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Amino acid production today is by far mostly done for feeding livestock for animal protein production or as a direct food additive. Nowadays, amino acid production relies mostly on fermentation of sugars, and the amino acid market for 2022 is estimated to reach ten million tons with over $13 billion USD market value. However, to make amino acid production more sustainable and ecologically viable, alternative resources and waste streams have to be considered. To contribute to a sustainable future vision, here we show a synthetic methanol alanine pathway (MAP) as a cell-free enzymatic cascade. The pathway consists of nine enzymes with an intrinsic cofactor recycling system and produces L-alanine from methanol with a maximum of 90% theoretical yield. Due to the increasing possibilities of sustainably producing methanol from CO2, this study paves the way to C1 building-block-based amino acid synthesis at reduced environmental burden.