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

Rhodococcus opacus PD630 is a non-model, gram-positive bacterium that possesses desirable traits for lignocellulosic biomass conversion. In particular, it has a relatively rapid growth rate, exhibits genetic tractability, produces high quantities of lipids, and can tolerate and consume toxic, lignin-derived aromatic compounds. Despite these unique, industrially relevant characteristics, R. opacus has been underutilized due to a lack of reliable genetic parts and engineering tools. In this work, we developed a molecular toolbox for reliable gene expression control and genome modification in R. opacus. To facilitate predictable gene expression, a constitutive promoter library spanning ~45-fold in output was constructed. To improve the characterization of available plasmids, the copy numbers of four heterologous and nine endogenous plasmids were determined using quantitative PCR. The molecular toolbox was further expanded by screening a previously unreported antibiotic resistance marker (HygR) and constructing a curable plasmid backbone for temporary gene expression (pB264). Furthermore, a system for genome modification was devised, and three neutral integration sites were identified using a novel combination of transcriptomic data, genomic architecture, and growth rate analysis. Finally, the first reported system for targeted, tunable gene repression in Rhodococcus was developed by utilizing CRISPR interference (CRISPRi). Overall, this work greatly expands the abilitymore » to manipulate and engineer R. opacus, making it a viable new chassis for bioproduction from renewable feedstocks.« less
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  1. Washington Univ., St. Louis, MO (United States)
Publication Date:
Grant/Contract Number:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Name: ACS Synthetic Biology; Journal ID: ISSN 2161-5063
American Chemical Society (ACS)
Research Org:
Washington Univ., St. Louis, MO (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
09 BIOMASS FUELS; Rhodococcus opacus; promoter library; plasmid copy number; CRISPR interference; genome modification; neutral integration site
OSTI Identifier: