Improving D-carbamoylase thermostability through salt bridge engineering for efficient D-p-hydroxyphenylglycine production
Landong Zhang, Changzheng Gao, Wei Song, Wanqing Wei, Cong Gao, Xiulai Chen, Jia Liu, Liming Liu, Jing Wu
Systems Microbiology and Biomanufacturing ›› 2023, Vol. 4 ›› Issue (1) : 250-262.
Improving D-carbamoylase thermostability through salt bridge engineering for efficient D-p-hydroxyphenylglycine production
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, AkDCaseD30A, increased the Tm by 2.91 °C and half-life (t1/2) at 40 and 60 °C by 18.43 h and 23.21 min, respectively. After AkDCaseD30A 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.
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