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

• Published in: Metabolic engineering 2020-01, Vol.57, p.13-22
• Main Authors: Liang, Youxiang, Jiao, Song, Wang, Miaomiao, Yu, Huimin, Shen, Zhongyao
• Format: Article
• Language: English
• Subjects: Acrylamide - metabolism; Acrylamide bio-production; Biocatalysis; CRISPR-Cas Systems; CRISPR/Cas9; Gene Editing; Genome editing; Metabolic Engineering; Recombinase; Restriction-modification system; Rhodococcus; Rhodococcus - genetics; Rhodococcus - metabolism
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2019.10.003

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. •Bypassing RM system of Rhodococcus enhanced the transformation efficiency by 89-fold.•Introducing bacteriophage recombinases enabled dsDNA recombineering of Rhodococcus.•A CRISPR/Cas9 method was developed for Rhodococcus genome editing for the first time.•The engineered Rhodococcus could achieve 50% acrylamide production for 4 batches.