D-Hydantoinase and D-carbamoylase genes from Agrobacterium radiobacter TH572 were cloned by polymerase chain reaction (PCR). The plasmid pUCCH3 with a polycistronic structure that is controlled by the native hydantoinase promoter was constructed to co-express the two genes and transformed into Escherichia coli strain JM105. To obtain the highest level of expression of the D-carbamoylase and avoid intermediate accumulation, the D-carbamoylase gene was cloned closer to the promoter and the RBS region in the upstream of it was optimized. This resulted in high active expression of soluble D-hydantoinase and D-carbamoylase that is obtained without any inducer. Thus, by the constitutive recombinant JM105/pUCCH3, D-p-hydroxyphenylglycine (D-HPG) was obtained directly with 95.2% production yield and 96.3% conversion yield. (c) 2008 Elsevier Inc. All rights reserved.