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Title: 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 » to manipulate and engineer R. opacus, making it a viable new chassis for bioproduction from renewable feedstocks.« less

Authors:
 [1];  [1];  [1]; ORCiD logo [1]
  1. 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) (SC-23)
OSTI Identifier:
1419636
Grant/Contract Number:
SC0012705
Resource Type:
Journal Article: 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. doi: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. doi:10.1021/acssynbio.7b00416.
@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}
}

Journal Article:
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This content will become publicly available on January 24, 2019
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