Study On D-p-hydroxyphenylglycine Production From L-p-hydroxyphenylglycine Using A Multi-enzyme Cascade

The non-proteinogenic amino acid D-p-hydroxyphenylglycine（D-HPG）is an important building block used in the pharmaceutical industry for the production of amoxicillin,penicillin,and cephalosporin.Several efficient synthesis methods have been developed,including chemical and enzymatic methods.Chemical methods typically synthesize racemate DL-HPG and then proceed to kinetic resolution to obtain D-HPG;the L-HPG obtained via resolution is discarded,wasting raw materials.However,the dual-enzyme（Hase and Case）cascade transformation in the enzymatic method has the problems of low substrate solubility and low yield.In this study,we constructed a three-enzyme cascade pathway using DL-HPG as substrate to produce D-HPG;then,the catalytic activity of PtDAPDH was further improved by protein engineering.Finally,under the optimum transformation conditions achieved the efficient synthesis of D-HPG.The major results are described as follows:（1）Design,construction and verification of a cascade pathway for production of D-HPG.First,we designed a three-enzyme cascade pathway using DL-HPG as substrate to produce D-HPG.Then,aromatic amino acid aminotransferase from Escherichia coli（Ec Aro AT）,diaminopimelate dehydrogenase from Prevorella timonensis（PtDAPDH）,and glucose dehydrogenase from Bacillus megaterium（Bm GDH）were selected to catalyzed the reaction as path enzymes.Subsequently,the production of the product was detected by in vitro catalytic reaction,and the feasibility of the pathway was verified by cationic mass spectrometry.Finally,PtDAPDH was identified as the rate-limiting enzyme of the cascade reaction by measuring the catalytic activities ratio of the three enzymes in vitro.（2）Protein crystallization and analysis of the structure and mechanism of PtDAPDH.First,the recombinant strain E.coli-p ET28a-PtDAPDH was expressed and purified,and the crystallization conditions were screened using a commercial crystal screening kit,we determined the structure of apo-PtDAPDH and the cofactor NADPH-PtDAPDH complex via X-ray diffraction with 2.47?（PDB:8HP0）and 3.07?resolution（PDB:8HP3）,respectively.Then,the secondary structure of the crystal was analyzed,and the mutation of key residues were verified according to the molecular docking model;finally,the reduction amination mechanism was deduced.（3）Rational design of PtDAPDH to improve catalytic ability.Based on the crystal structure and catalytic mechanism,a“binding pocket and conformation remodeling”strategy was developed for protein engineering.The best variant obtained following this approach,PtDAPDH^{M4（W121V/H227I/R181T/S70D/S160R）},the specific activity increased by25.6-fold and the k_cat/K_m values increased by 26.75-fold.Structure comparison and MD simulations showed that:（i）Enlarging substrate binding pocket shortened the hydride transfer distance between substrate and NADPH;MD simulations showed that mutant formed hydrogen bonds with the residues surrounding the active center were more extensive than wild type.（ii）The S72D and S160R mutation led to form new interaction and enhance the interdomain interaction network,enhance the conformational distribution toward closed state.（4）Cascade assembly of whole-cell to produce D-HPG.Replacing the PtDAPDH in the pathway with PtDAPDH^M4,transformation experiment showed that the rate-limiting step of reducing amination of HPGA by PtDAPDH was eliminated.After the conversion conditions were optimized,the reaction was amplified to 3 L fermenter and19.8 g·L^-1 D-HPG was produced from 40 g·L^-1 racemate DL-HPG with a total D-HPG titer of 39.8 g·L^-1,with 49.5%conversion and>99%ee within 10 h.