期刊
ACS SYNTHETIC BIOLOGY
卷 7, 期 2, 页码 727-738出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.7b00416
关键词
Rhodococcus opacus; promoter library; plasmid copy number; CRISPR interference; genome modification; neutral integration site
资金
- U.S. Department of Energy [DE-SC0012705, DE-SC0018324]
- NSF Graduate Research Fellowship [DGS-1143954]
- U.S. Department of Energy (DOE) [DE-SC0012705, DE-SC0018324] Funding Source: U.S. Department of Energy (DOE)
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 because of 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 similar to 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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