Direct C-H hydroxylation catalyzed by non-heme iron-dependent oxygenases is a rapid and efficient way to access various complex hydroxylated amino acids and pharmaceutical intermediates , but until recently enzymes generating trans-3-hydroxyproline were not known in nature, and those that have been discovered in recent years do not possess the selectivities or performance required for large-scale synthesis . Faced with this challenge, we explored the functions of uncharacterized fungal hydroxylases and identified an enzyme that slightly favors production of the desired hydroxyproline, generating a 1.2:1 ratio of the trans-3 to trans-4 isomers. This non-heme, a-ketoglutarate-dependent enzyme, which we termed ANOh, was then engineered to achieve significantly higher levels of stability and activity, while improving the selectivity to >20:1 trans-3:trans-4 . Using the resulting engineered ANOh enzyme CDX-090, a process was developed that produces trans-3hydroxyproline under scalable conditions at high substrate concentrations (>30 g/L-proline 3).