PCR isolation of catechol 2,3-dioxygenase gene fragments from environmental samples and their assembly into functional genes.

A method was developed to isolate central segments of catechol 2, 3-dioxygenase (C23O) genes from environmental samples and to insert these C23O gene segments into nahH (the structural gene for C23O encoded by catabolic plasmid NAH7) by replacing the corresponding nahH sequence with the isolated segments. To PCR-amplify the central C23O gene segments, a pair of degenerate primers was designed from amino acid sequences conserved among C23Os. Using these primers, central regions of the C23O genes were amplified from DNA isolated from a mixed culture of phenol-degrading or crude oil-degrading bacteria. Both the 5' and 3' regions of nahH were also PCR-amplified by using appropriate primers. These three PCR products, the 5'-nahH and 3'-nahH segments and the central C23O gene segments, were mixed and PCR-amplified again. Since the primers for the amplification of the central C23O gene segments were designed so that the 20 nucleotides at both ends of the segments are identical to the 3' end of the 5'-nahH segment and the 5' end of the 3'-nahH segment, respectively, the central C23O gene segments could anneal to both the 5'- and 3'-nahH segments. After the second PCR, hybrid C23O genes in the form of (5'-nahH segment-central C23O gene segment-3'-nahH segment) were amplified to full length. The resulting products were cloned into a vector and used to transform Escherichia coli. This method enabled divergent C23O sequences to be readily isolated, and more than 90% of the hybrid plasmids expressed C23O activity. Thus, the present method is useful to create, without isolating bacteria, a library of functional hybrid genes.