Improving D-carbamoylase thermostability through salt bridge engineering for efficient D-p-hydroxyphenylglycine production

Abstract

D-p-hydroxyphenylglycine (D-HPG) is an important intermediate in the pharmaceutical industry, and it is commonly synthesized by cascading D-hydantoinase (DHase) and D-carbamoylase (DCase). In this study, the stability of DCase was identified as the main problem that limits its application. Therefore, the complexed structure of AkDCase (DCase from the Agrobacterium sp. strain KNK712) with the substrate N-carbamoyl-D-p-hydroxyphenylglycine (CpHPG) (with 2.52 Å resolution) and catalytic mechanism were resolved. Based on the catalytic mechanism and electrostatic stabilization, salt bridge engineering was adopted to improve AkDCase thermostability. The best variant, AkDCase^D30A, increased the T_m by 2.91 °C and half-life (t_1/2) at 40 and 60 °C by 18.43 h and 23.21 min, respectively. After AkDCase^D30A was assembled with GsDHase (DHase from Geobacillus stearothermophilus SD-1) in a single Escherichia coli cell, the recombinant strain could produce 29.53 g/L D-HPG within 12 h, with a 97% conversion and a 2.46 g/(L·h) space–time yield (STY). The titer of D-HPG increased by 40.55% compared to the E. coli cell harboring pETduet-1-AkDCase- GsDHase. The recombinant strain could be used for two cycles. Our research provides a basis for the industrial production of D-HPG.

Keywords

D-p-hydroxyphenylglycine / D-carbamoylase / Thermostability / Salt bridge engineering

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Funding

National Key R & D Program of China(2021YFC2100100); Innovative Research Group Project of the National Natural Science Foundation of China(22178146); Ten Thousand Talent Plans for Young Top-notch Talents of Yunnan Province