Development of a homologous expression system for rubber oxygenase RoxA from Xanthomonas sp

Area of Science:

• Biochemistry and Microbiology
• Enzymology
• Biotechnology

Background:

• Natural rubber degradation is catalyzed by rubber oxygenase A (RoxA), a novel dihaem dioxygenase from Xanthomonas sp.
• The exact mechanism of rubber backbone cleavage by RoxA remains unelucidated.
• Previous attempts to express functional RoxA in heterologous hosts like E. coli and P. putida were unsuccessful.

Purpose of the Study:

• To establish a system for expressing functional recombinant RoxA in its homologous host, Xanthomonas sp.
• To facilitate the investigation of RoxA's catalytic mechanism through mutagenesis studies.
• To enable efficient production and purification of active RoxA for biochemical analysis.

Main Methods:

• Development of a transformation system (electroporation) and optimization of a conjugation system for Xanthomonas sp.
• Inactivation of the chromosomal roxA gene via insertional mutagenesis to confirm its role in rubber utilization.
• Cloning of an intact roxA gene into a broad host range vector under a rhamnose-inducible promoter for high-level expression in Xanthomonas sp.

Main Results:

• Successful establishment of genetic manipulation tools for Xanthomonas sp.
• Demonstrated that roxA gene inactivation renders Xanthomonas sp. unable to utilize rubber.
• Achieved high expression of functional RoxA in Xanthomonas sp. using a rhamnose-inducible system, simplifying purification and yielding approximately 6 mg/L.

Conclusions:

• The developed methods provide a robust platform for molecular biological studies in Xanthomonas sp.
• Recombinantly produced RoxA exhibits high activity and comparable spectral properties to the wild-type enzyme.
• This work lays the foundation for detailed biochemical investigations into the biodegradation of natural rubber by RoxA.