A CRISPR/Cas9-based single-stranded DNA recombineering system for genome editing of Rhodococcus opacus PD630.

Genome engineering of Rhodococcus opacus PD630, an important microorganism used for the bioconversion of lignin, is currently dependent on inefficient homologous recombination. Although a CRISPR interference procedure for gene repression has previously been developed for R. opacus PD630, a CRISPR/Cas9 system for gene knockout has yet to be reported for the strain. In this study, we found that the cytotoxicity of Cas9 and the deficiency in pathways for repairing DNA double-strand breaks (DSBs) were the major causes of the failure of conventional CRISPR/Cas9 technologies in R. opacus, even when augmented with the recombinases Che9c60 and Che9c61. We successfully developed an efficient single-stranded DNA (ssDNA) recombineering system coupled with CRISPR/Cas9 counter-selection, which facilitated rapid and scarless editing of the R. opacus genome. A two-plasmid system, comprising Cas9 driven by a weak Rhodococcus promoter Pniami, designed to prevent cytotoxicity, and a single-guide RNA (sgRNA) under the control of a strong constitutive promoter, was proven to be appropriate with respect to cleavage function. A novel recombinase, RrRecT derived from a Rhodococcus ruber prophage, was identified for the first time, which facilitated recombination of short ssDNA donors (40-80 nt) targeted to the lagging strand and enabled us to obtain a recombination efficiency up to 103-fold higher than that of endogenous pathways. Finally, by incorporating RrRecT and Cas9 into a single plasmid and then co-transforming cells with sgRNA plasmids and short ssDNA donors, we efficiently achieved gene disruption and base mutation in R. opacus, with editing efficiencies ranging from 22 % to 100 %. Simultaneous disruption of double genes was also confirmed, although at a lower efficiency. This effective genome editing tool will accelerate the engineering of R. opacus metabolism.

Genetic toolkits for engineering Rhodococcus species with versatile applications.

Rhodococcus spp. are a group of non-model gram-positive bacteria with diverse catabolic activities and strong adaptive capabilities, which enable their wide application in whole-cell biocatalysis, environmental bioremediation, and lignocellulosic biomass conversion. Compared with model microorganisms, the engineering of Rhodococcus is challenging because of the lack of universal molecular tools, high genome GC content (61% ~ 71%), and low transformation and recombination efficiencies. Nevertheless, because of the high interest in Rhodococcus species for bioproduction, various genetic elements and engineering tools have been recently developed for Rhodococcus spp., including R. opacus, R. jostii, R. ruber, and R. erythropolis, leading to the expansion of the genetic toolkits for Rhodococcus engineering. In this article, we provide a comprehensive review of the important developed genetic elements for Rhodococcus, including shuttle vectors, promoters, antibiotic markers, ribosome binding sites, and reporter genes. In addition, we also summarize gene transfer techniques and strategies to improve transformation efficiency, as well as random and precise genome editing tools available for Rhodococcus, including transposition, homologous recombination, recombineering, and CRISPR/Cas9. We conclude by discussing future trends in Rhodococcus engineering. We expect that more synthetic and systems biology tools (such as multiplex genome editing, dynamic regulation, and genome-scale metabolic models) will be adapted and optimized for Rhodococcus.

Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber.

For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones RrGroEL and RrGroES from Rhodococcus ruber, a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, E. coli and R. ruber. In the E. coli host, fusion expression of nitrilase with either RrGroES or RrGroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32-47% reduction). The co-expression strategy was applied in R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of RrGroES or RrGroEL with a plasmid, the engineered strains R. ruber TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroES) and TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the RrGroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-Pami-RrNit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.

A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH.

Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive abilities and diverse metabolic activities, and are therefore widely utilized in bioconversion, biosynthesis and bioremediation. However, due to the high GC-content of the genome (~70%), together with low transformation and recombination efficiency, the efficient genome editing of Rhodococcus remains challenging. In this study, we report for the first time the successful establishment of a CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, making it feasible to obtain enough colonies for screening of mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61, the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated triple-plasmid recombineering system was developed with high efficiency of gene deletion, insertion and mutation. Finally, this new genome editing method was successfully applied to engineer R. ruber for the bio-production of acrylamide. By deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained with reduced byproduct formation and enhanced catalytic stability. Compared with the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells could be recycled from 1 batch to 4 batches.