Construction of a 3NBA-degrading recombinant Cupriavidus strain.

In Comamonas sp. JS46, the oxygenase component (MnbA) and the oxidoreductase component (MnbB) were identified as the enzymes responsible for ring dioxygenation of 3NBA (10). A 2.8-kb DNA fragment that carried the mnbB, mnbR (a regulator), and mnbA genes was PCR amplified from the genomic DNA of strain JS46 by using primers mnbF and mnbR (Table 2). The amplified DNA fragment was cloned into the broad-host-range shuttle vector pBBR1MCS2_START (24), followed by electroporation into wild-type Cupriavidus sp. ST-14 cells. The transformants were selected on LB-kanamycin agar plates. Expression of 3NBA ring-hydroxylating dioxygenase was confirmed based on the growth of the selected clones (recombinant strain ST-14::3NBA) on 3NBA (0.5 g/liter) as the sole carbon source in MSM supplemented with kanamycin. Resting-cell incubations for the transformation of 3NBA and preparation of cell extracts to analyze in vitro enzyme activity using ST-14::3NBA cells that were grown on 3NBA were conducted as described above for the wild-type strain.