Host-vector system for phenol-degrading Rhodococcus erythropolis based on Corynebacterium plasmids.

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Rhodococcus erythropolis CCM2595 is known for its phenol degradation capabilities.
• Genetic manipulation is crucial for improving microbial strains for industrial applications.
• Existing genetic tools for R. erythropolis were limited, hindering strain development.

Purpose of the Study:

• To develop genetic manipulation techniques for Rhodococcus erythropolis CCM2595.
• To construct and characterize novel cloning vectors for R. erythropolis.
• To enable efficient gene cloning and expression in R. erythropolis for strain improvement.

Main Methods:

• Optimization of electrotransformation protocols for R. erythropolis.
• Construction of Escherichia coli-Rhodococcus erythropolis shuttle vectors using replicons from Corynebacterium glutamicum.
• Characterization of the shuttle vector pSRK21 for cloning sites and stability.
• Cloning and expression of the green fluorescent protein (gfpuv) gene.

Main Results:

• An efficient electrotransformation procedure yielding up to 7x10(4) transformants/microg DNA was established.
• Novel shuttle vectors were constructed, including the small, versatile pSRK21 (5.8 kb).
• The pSRK21 vector demonstrated high segregational stability in R. erythropolis and facilitated efficient gfpuv gene expression.

Conclusions:

• The developed genetic tools and shuttle vector pSRK21 significantly advance the genetic manipulation of Rhodococcus erythropolis.
• These advancements facilitate strain improvement for enhanced phenol degradation and other biotechnological applications.
• Efficient cloning and expression capabilities open new avenues for metabolic engineering of R. erythropolis.