Abstract
The reg-psald gene is located immediately upstream of the psald gene encoding phenylserine aldolase in Pseudomonas putida 24-1. The amino acid sequence of Reg-psald showed a high homology with that of AraC family proteins. The Pfam database indicated that Reg-psald contained an AraC-like ligand binding domain in the N-terminal region and two bacterial regulatory helix-turn-helix domains in the C-terminal region. A DNA fragment containing reg-psald and psald genes was ligated into the EcoRI-HindIII site of pUC18. A crude extract of Escherichia coli JM109 cells harboring the resultant plasmid cultured in LB medium containing DL-threo-β-phenylserine showed phenylserine aldorase activity. Instead of the psald gene, the alanine dehydrogenase gene from Bacillus sp. DSM730 was ligated downstream of the reg-psald gene. Alanine dehydrogenase activity was detected in a crude extract of E. coli JM109 cells harboring the resultant plasmid, pUpstrPAla by cultivation in DL-threo-β-phenylserine-containing LB medium. These results suggest that the reg-psald gene DL-threo-β-phenylserine dependency regulates the expression of the downstream psald gene. Therefore, we considered that the reg-psald gene was useful for construction of a gene expression system using DL-threo-β-phenylserine as an inducer.
