Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630
Abstract
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 »
- Authors:
-
- Washington Univ., St. Louis, MO (United States)
- Publication Date:
- Research Org.:
- Washington Univ., St. Louis, MO (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1419636
- Grant/Contract Number:
- SC0012705
- Resource Type:
- Accepted Manuscript
- Journal Name:
- ACS Synthetic Biology
- Additional Journal Information:
- Journal Volume: 7; Journal Issue: 2; Journal ID: ISSN 2161-5063
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 09 BIOMASS FUELS; Rhodococcus opacus; promoter library; plasmid copy number; CRISPR interference; genome modification; neutral integration site
Citation Formats
DeLorenzo, Drew M., Rottinghaus, Austin G., Henson, William R., and Moon, Tae Seok. Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630. United States: N. p., 2018.
Web. doi:10.1021/acssynbio.7b00416.
DeLorenzo, Drew M., Rottinghaus, Austin G., Henson, William R., & Moon, Tae Seok. Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630. United States. https://doi.org/10.1021/acssynbio.7b00416
DeLorenzo, Drew M., Rottinghaus, Austin G., Henson, William R., and Moon, Tae Seok. Wed .
"Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630". United States. https://doi.org/10.1021/acssynbio.7b00416. https://www.osti.gov/servlets/purl/1419636.
@article{osti_1419636,
title = {Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630},
author = {DeLorenzo, Drew M. and Rottinghaus, Austin G. and Henson, William R. and Moon, Tae Seok},
abstractNote = {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 ability to manipulate and engineer R. opacus, making it a viable new chassis for bioproduction from renewable feedstocks.},
doi = {10.1021/acssynbio.7b00416},
journal = {ACS Synthetic Biology},
number = 2,
volume = 7,
place = {United States},
year = {Wed Jan 24 00:00:00 EST 2018},
month = {Wed Jan 24 00:00:00 EST 2018}
}
Web of Science
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