Simultaneously improving the activity and thermostability of a new proline 4-hydroxylase by loop grafting and site-directed mutagenesis
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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.
Keywordstrans-Proline 4-hydroxylase Loop grafting trans-4-Hydroxy-L-proline Fed-batch fermentation Site-directed mutagenesis
We thank Xingchu Wang (Tianjin Institute of Industrial Biotechnology) for helpful discussions. We thank Dr. Timothy C. Cairns and Taiwo Dele-Osibanjo for critical reading and editing of the manuscript.
We are grateful for the financial support from Tianjin Natural Science Foundation (18JCQNJC10300), National Natural Science Foundation of China (No. 21606251), the Key Research Program of the Chinese Academy of Sciences (No. KFZD-SW-212), Youth Innovation Promotion Association of CAS (2015137), and Science and Technology Project of Tianjin (Nos. 15PTCYSY00020 and 14ZCZDSY00058).
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors. All authors confirm that ethical principles have been followed in the research as well as in manuscript preparation, and approved this submission.
Conflict of interest
The authors declare that they have no conflict of interest.
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