Biocatalytic production of D-p-hydroxyphenylglycine using cell surface display of D- hydantoinase and D-carbamoylase on Escherichia coli

D-p-hydroxyphenyl glycine (D-ρHPG) is an intermediate that is widely used in the production of semi-synthetic antibiotics. While it can be produced chemically, conventional chemical methods are associated with high production costs and environmental pollution, making them less desirable. As a result, biotechnological approaches have become increasingly attractive for D-ρHPG production. Among the various biocatalytic strategies, one enzymatic method that has gained considerable attention for the production of D-ρHPG is the two-step pathway in which D-hydantoinase (D-Hase) and D-carbamoylase (D-Case) sequentially convert DL-hydroxyphenylhydantoin (DL-HPH) into D-ρHPG, valued for its efficiency and selectivity. This pathway has been successfully implemented in microbial systems, particularly in Escherichia coli, for whole-cell bioconversion of racemic precursors into optically pure D-ρHPG. Whole-cell biocatalysis provides a convenient and cost-effective method for catalyst production. Nevertheless, the whole-cell approach has limitations such as restrained substrate solubility, transport obstacles, and intracellular degradation. To address these challenges, we developed a bacterial surface display system that express D-Hase and D-Case on the outer membrane of Escherichia coli using the ice nucleation protein (INP) as an anchoring motif. This design allows direct substrate access to the catalytic sites while minimizing intracellular transport and degradation issues. The successful surface localization of each enzyme was verified through SDS-PAGE and enzymatic activity assays following cell fractionation. The activities of surface-displayed enzymes were measured using colorimetric and HPLC techniques, yielding 0.72 and 1.13 mM/min, respectively. The engineered E. coli cells efficiently converted DL-HPH to D-ρHPG with a 90% yield. The bacterial surface display strategy presented in this study offers a novel, cost-effective approach for the production of D-ρHPG, with potential applications in the pharmaceutical and biotechnology industries.