Abstract: In this study, we obtained a novel L-proline-4-hydroxylase with high activity from Micromonospora sp. CNB394. Then we successfully constructed a trans-4-hydroxy-L-proline-producing strain named HYP15 by enhancing the L-proline synthesis pathway and introducing the high-activity L-proline-4-hydroxylase into Escherichia coli using clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9)-mediated gene editing. The strain could efficiently produce trans-4-hydroxy-L-proline utilizing glucose as the carbon source. HYP15 produced 13.6 g/L trans-4-hydroxy-L-proline after 30 h of fermentation in shake flasks and produced 48.6 g/L trans-4-hydroxy-L-proline with conversion ratio of sugar to acid of 21.6% and average productivity of 1.22 g/(L·h) after 40 h of fed-batch fermentation in a 5-L bioreactor and, therefore, could be promising for industrial application.

Key words: L-proline-4-hydroxylase, trans-4-hydroxy-L-proline, Escherichia coli, CRISPR-Cas9, fermentation