Efficient production of trans-4-hydroxy-l-proline from glucose using a new trans-proline 4-hydroxylase in Escherichia coli

Abstract

trans-4-Hydroxy-l-proline (trans-4Hyp) is widely used as a valuable building block for the organic synthesis of many pharmaceuticals such as carbapenem antibiotics. The major limitation for industrial bioproduction of trans-4Hyp is the low titer and productivity by using the existing trans-proline 4-hydroxylases (trans-P4Hs). Herein, three new trans-P4Hs from Alteromonas mediterranea (AlP4H), Micromonospora sp. CNB394 (MiP4H) and Sorangium cellulosum (ScP4H) were discovered through genome mining and enzymatic determination. These trans-P4Hs were introduced into an l-proline-producing chassis cell, and the recombinant strain overexpressing AlP4H produced the highest concentration of trans-4Hyp (3.57 g/L) from glucose in a shake flask. In a fed-batch fermentation with a 5 L bioreactor, the best strain SEcH (pTc-B74A-alp4h) accumulated 45.83 g/L of trans-4Hyp within 36 h, with the highest productivity (1.27 g/L/h) in trans-4Hyp fermentation from glucose, to the best of our knowledge. This study provides a promising hydroxylase candidate for efficient industrial production of trans-4Hyp.

Keywords: Escherichia coli; Fed-batch fermentation; Genome mining; trans-4-Hydroxy-l-proline; trans-Proline 4-hydroxylase.