Cloning and expression of the benzoate dioxygenase genes from Rhodococcus sp. strain 19070

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Gram-positive bacteria like Rhodococcus sp. strain 19070 possess diverse metabolic capabilities.
• Benzoate dioxygenases are key enzymes in the degradation of aromatic compounds.
• Understanding these enzymes aids in bioremediation and metabolic engineering.

Purpose of the Study:

• To characterize the bopXYZ genes from Rhodococcus sp. strain 19070.
• To investigate the substrate specificity and activity of the encoded benzoate dioxygenase.
• To compare the bopXYZ genes with known dioxygenase systems.

Main Methods:

• Gene expression in Escherichia coli.
• Enzyme activity assays (dioxygenase, reductase).
• Sequence homology analysis.
• Pulsed-field gel electrophoresis.

Main Results:

• Expression of bopXYZ in E. coli enabled conversion of benzoate and anthranilate to cis-diol and catechol, respectively.
• m-toluate was transformed to an unidentified product; 2-chlorobenzoate was a poor substrate.
• The BopXYZ dioxygenase showed homology to BenABC and XylXYZ systems from gram-negative bacteria.
• Strain 19070 possesses both meta- and ortho-cleavage pathways.
• The reductase component, BopZ, exhibited unique C-terminal sequence characteristics.

Conclusions:

• The bopXYZ genes encode a broad-substrate benzoate dioxygenase with potential applications.
• The enzyme's activity and homology suggest a role in aromatic compound degradation.
• The unique features of BopZ warrant further investigation into its catalytic mechanism.