Efficient production of trans-3-hydroxyproline by a bacterial trans-3-proline hydroxylase and characterization of enzymatic properties

• trans -3-Hyp was firstly bioproduced from glucose with high titer and purity. • UbP3H showed high activity and selectivity for producing trans -3-Hyp. • UbP3H-Da-E112 P mutant dramatically inverted enzyme regioselectivity. • Structure basis for proline hydroxylase' regioselectivity was identified. Hydroxyprolines (Hyp) are valuable building blocks for important pharmaceuticals like carbapenem antibiotics. To date, one of four possible stable L-Hyp isomers, trans -3-Hyp, has not been specifically produced by microbial hydroxylation of L-proline, which is due to the absence of regio- and stereo-specific trans -proline 3-hydroxylases ( trans -P3Hs). Here, we report the efficient bioproduction of trans -3-Hyp (˜30 g/L, 0.74 g/L/h and 0.104 g/g glucose) at an 82% (w/w) proportion by fed-batch fermentation from glucose using an engineered E. coli strain. This isolate expressed a bacterial trans -P3H from an uncultured bacterium esnapd13 (UbP3H). UbP3H exhibited a 1.6-fold and 2.6-fold increase in activity (566.2 vs. 362 U/mg) and proportion of trans -3-Hyp (85% vs. 33%, w/w) when compared with reported fungal L-proline hydroxylase (PH) from Aspergillus pachycristatus (HtyE), respectively. The trans -3-Hyp proportions for UbP3H at different temperatures (25, 37 and 45 °C) and pH buffers (6.5 and 8.0) remained largely unchanged. Additionally, the substrate specificity of UbP3H was different when compared to HtyE. Next, we elucidated the structural basis behind the high trans -3-selectivity of UbP3H towards L-proline by analyzing UbP3H and its three mutants (UbP3H-Da, E112 P and UbP3H-Da-E112 P). The results show that the engineering of loops near an active site seems to be an effective method to alter the regioselectivity of enzymes, specifically in the Fe(II)/α-ketoglutarate-dependent dioxygenase family.