Research ArticleMICROBIOLOGY

Significantly enhancing production of trans-4-hydroxy-l-proline by integrated system engineering in Escherichia coli

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Science Advances  22 May 2020:
Vol. 6, no. 21, eaba2383
DOI: 10.1126/sciadv.aba2383


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.

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