Abstract: Hydroxyproline is an industrially important compound with applications in the pharmaceutical, nutrition, and cosmetic industries. trans-4-Hydroxy-l-proline is recognized as the most abundant of the eight possible isomers (hydroxy group at C-3 or C-4, cis- or trans-configuration, and l- or d-form). However, little attention has been paid to the rare isomers, probably due to their limited availability. This mini-review provides an overview of recent advances in microbial and enzymatic processes to develop practical production strategies for various hydroxyprolines. Here, we introduce three screening strategies, namely, activity-, sequence-, and metabolite-based approaches, allowing identification of diverse proline-hydroxylating enzymes with different product specificities. All naturally occurring hydroxyproline isomers can be produced by using suitable hydroxylases in a highly regio- and stereo-selective manner. Furthermore, crystal structures of relevant hydroxylases provide much insight into their functional roles. Since hydroxylases acting on free l-proline belong to the 2-oxoglutarate-dependent dioxygenase superfamily, cellular metabolism of Escherichia coli coupled with a hydroxylase is a valuable source of 2-oxoglutarate, which is indispensable as a co-substrate in l-proline hydroxylation. Further, microbial hydroxyproline 2-epimerase may serve as a highly adaptable tool to convert l-hydroxyproline into d-hydroxyproline. Key points: • Proline hydroxylases serve as powerful tools for selectivel-proline hydroxylation. • Engineered Escherichia coli are a robust platform for hydroxyproline production. • Hydroxyproline epimerase convertsl-hydroxyproline intod-hydroxyproline.
ASJC Scopus subject areas