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 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 ∼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.

A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630

“…Third, 13 C analysis of the adaptively-evolved strains shows that their genetic differences do not lead to different flux networks. Recently, reliable genetic parts and engineering tools have been developed for R. opacus, allowing for gene expression control and genome modification in this strain (Delorenzo et al, 2017;Delorenzo et al, 2018). Future work will investigate how model-guided perturbations of its metabolism (e.g., knockouts and overexpression), as opposed to adaptive evolution, affects R. opacus's flux network.…”

Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630

“…R . opacus has been previously engineered to facilitate lignocellulose conversion 3 , 4 , 7 , 8 and a substantial genetic toolbox has recently been developed 9 , 10 . However, a deep understanding of this organism’s metabolism and any heterologous pathways being expressed is required to maximize its potential.…”

Impact of CRISPR interference on strain development in biotechnology

“…1). CRISPRi has been developed in a diverse group of bacteria, including E. coli [5] , Corynebacterium glutamicum [7] , Lactococcus lactis [18] , Rhodococcus opacus [19] , Burkholderia [20] , Clostridia [21–25] , Bacillus subtilis [26–29] , Bacillus methanolicus [30] , Streptomyces [31,32], and Mycobacteria [33–36], and applied for gene repression to interrogate their physiology or to identify gene targets for biotech applications (see Sections 3 and 4).…”