Abstract
Trans-4-hydroxy-l-proline is produced by trans-proline-4-hydroxylase with l-proline through glucose fermentation. Here, we designed a thorough "from A to Z" strategy to significantly improve trans-4-hydroxy-l-proline production. Through rare codon selected evolution, Escherichia coli M1 produced 18.2 g L(-1) l-proline. Metabolically engineered M6 with the deletion of putA, proP, putP, and aceA, and proB mutation focused carbon flux to l-proline and released its feedback inhibition. It produced 15.7 g L(-1) trans-4-hydroxy-l-proline with 10 g L(-1) l-proline retained. Furthermore, a tunable circuit based on quorum sensing attenuated l-proline hydroxylation flux, resulting in 43.2 g L(-1) trans-4-hydroxy-l-proline with 4.3 g L(-1) l-proline retained. Finally, rationally designed l-proline hydroxylase gave 54.8 g L(-1) trans-4-hydroxy-l-proline in 60 hours almost without l-proline remaining-the highest production to date. The de novo engineering carbon flux through rare codon selected evolution, dynamic precursor modulation, and metabolic engineering provides a good technological platform for efficient hydroxyl amino acid synthesis.