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

DeLorenzo, Drew M.; Rottinghaus, Austin G.; Henson, William R.; Moon, Tae Seok

doi:10.1021/acssynbio.7b00416

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

Journal Article · Wed Jan 24 00:00:00 EST 2018 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.7b00416· OSTI ID:1419636

DeLorenzo, Drew M. ^[1]; Rottinghaus, Austin G. ^[1]; Henson, William R. ^[1];

^[1]

Washington Univ., St. Louis, MO (United States)

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.

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Research Organization:: Washington Univ., St. Louis, MO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0012705

OSTI ID:: 1419636

Journal Information:: ACS Synthetic Biology, Vol. 7, Issue 2; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 51 works

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