Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Rhodococcus opacus PD630 is a Gram-positive bacterium with high potential for lignocellulosic biomass conversion due to its growth rate, lipid production, and tolerance to toxic compounds.
• Underutilization of R. opacus is attributed to a lack of genetic tools and reliable engineering methods.

Purpose of the Study:

• To develop a comprehensive molecular toolbox for enhanced gene expression control and genome engineering in R. opacus.
• To establish R. opacus as a more accessible and versatile chassis for bioproduction.

Main Methods:

• Construction of a constitutive promoter library for tunable gene expression.
• Determination of plasmid copy numbers using quantitative PCR.
• Screening of antibiotic resistance markers and development of a curable plasmid backbone.
• Identification of neutral genomic integration sites.
• Implementation of CRISPR interference (CRISPRi) for gene repression.

Main Results:

• A promoter library with a 45-fold output range was created.
• Copy numbers for 13 plasmids were quantified.
• A new antibiotic resistance marker (HygR) and a curable plasmid (pB264) were developed.
• Three neutral integration sites were identified.
• The first system for targeted, tunable gene repression in Rhodococcus using CRISPRi was established.

Conclusions:

• The developed molecular toolbox significantly enhances the genetic manipulation capabilities of R. opacus.
• This advancement positions R. opacus as a more viable and engineerable platform for bioproduction from renewable resources.