反式-脯氨酸4-羟化酶(反式-P4Hs)将游离的L-脯氨酸羟基化为反式-4-羟基-L-脯氨酸(反式-4-Hyp)是重要药物如碳青霉烯类抗生素的有价值的手性合成子。然而,只有很少的微生物反式-P4Hs被鉴定出来,和反式-4-Hyp发酵使用表达反式-P4Hs的工程大肠杆菌菌株通常在低于37°C的温度下进行,这可能是由于稳定性差和低活性。在本研究中,报道了一种来自未培养细菌esnapd13(UbP4H)的新反式P4H,具有在37°C下发酵生产反式4-Hyp的潜力。为了增强UbP4H的活性和热稳定性,将其推定的“lid”环与定点诱变相结合进行替换。因此,通过用四个其他已知的反式-P4Hs的相应序列取代UbP4H(A162-K178)的环来设计四环杂交体,分别。其中,与野生型相比,UbP4H-Da表现出加倍的活性(81.6±1.9vs.40.4±4.6U/mg),但热稳定性降低(t1/2,11vs.47分钟)。同时,通过序列比对和折叠自由能计算设计10个单一变体。三个最佳点替换分别与UbP4H-Da组合,产生UbP4H-Da-R90G,UbP4H-Da-E112P,和UbP4H-Da-A260P。UbP4H-Da-E112P表现出1.8倍的活性(85.2±0.6vs.46.6±4.0U/mg),t1/2增加7.6倍(359vs.47分钟),Tm上升3°C(46vs.43°C)当与UbP4H相比时。评估了使用表达UbP4H或其变体的反式4-Hyp生产底盘细胞在37°C下的反式4-Hyp的补料分批发酵,对于UbP4H-Da-E112P,反式-4-Hyp滴度增加了3.3倍(12.9±0.1vs.UbP4H为3.9±0.0g/L)。这些结果证明了UbP4H-Da-E112P在反式-4-Hyp的工业生产中的潜在应用。
trans-Proline 4-hydroxylases (trans-P4Hs) hydroxylate free L-proline to trans-4-hydroxy-L-proline (trans-4-Hyp) is a valuable chiral synthon for important pharmaceuticals such as carbapenem antibiotics. However, merely few microbial trans-P4Hs have been identified, and trans-4-Hyp fermentations using engineered Escherichia coli strains expressing trans-P4Hs are usually performed at temperatures below 37 °C, which is likely due to poor stability and low activities. In the present study, a new trans-P4H from uncultured bacterium esnapd13 (UbP4H) with potential in the fermentative production of trans-4-Hyp at 37 °C was reported. In order to enhance the activity and thermostability of UbP4H, the replacement of its putative \"lid\" loop in combination with site-directed mutagenesis was performed. Consequently, four loop hybrids were designed by substituting a loop of UbP4H (A162-K178) with the corresponding sequences of four other known trans-P4Hs, respectively. Among them, UbP4H-Da exhibited a doubled activity when compared to the wild type (81.6 ± 1.9 vs. 40.4 ± 4.6 U/mg) but with reduced thermostability (t1/2, 11 vs. 47 min). Meanwhile, 10 single variants were designed through sequence alignments and folding free energy calculations. Three best point substitutions were respectively combined with UbP4H-Da, resulting in UbP4H-Da-R90G, UbP4H-Da-E112P, and UbP4H-Da-A260P. UbP4H-Da-E112P exhibited a 1.8-fold higher activity (85.2 ± 0.6 vs. 46.6 ± 4.0 U/mg), a 7.6-fold increase in t1/2 (359 vs. 47 min), and a 3 °C rise in Tm (46 vs. 43 °C) when compared to UbP4H. The fed-batch fermentations of trans-4-Hyp at 37 °C using trans-4-Hyp producing chassis cells expressing UbP4H or its variants were evaluated, and a 3.3-fold increase in trans-4-Hyp titer was obtained for UbP4H-Da-E112P (12.9 ± 0.1 vs. 3.9 ± 0.0 g/L for UbP4H). These results demonstrate the potential application of UbP4H-Da-E112P in the industrial production of trans-4-Hyp.