Design of a noncovalently linked bifunctional enzyme for whole-cell biotransformation

Abstract

Optical pure d-p-hydroxyphenylglycine (d-HPG) is a precursor for semi-synthetic antibiotics. It can be synthesized from d,l-hydroxyphenyl hydantoin (HPH) by a two-step reaction mediated by d-hydantoinase (HDT) and amidohydrolase (AHL). In this study, a bifunctional enzyme was originally created by in-frame fusion of AHL with HDT genes (AHL-HDT). However, the AHL-HDT fusion protein expressed in Escherichia coli was prone to aggregates, recognized as a frequently encountered problem for this conventional method. To address this issue, small interacting motifs, cohesin (Coh) and dockerin (Doc) domains of cellulosomes, were explored and illustrated to interact in vivo. Accordingly, Coh and Doc were fused with AHL and HDT, respectively. After co-expression in E. coli, Coh-tagged AHL and Doc-tagged HDT assembled into a soluble protein complex via the high-affinity interaction of Coh and Doc. Consequently, the protein assembly exhibited both AHL and HDT activities and a higher reaction rate than free counterparts. Whole cells expressing the protein assembly were more stable than ones with free proteins for d-HPG production, and they could be recycled six times with a conversion yield of d-HPG exceeding 90%.