Tags

Type your tag names separated by a space and hit enter

A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH.
Metab Eng 2019; 57:13-22ME

Abstract

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.

Authors+Show Affiliations

Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China; Key Laboratory of Industrial Biocatalysis, Tsinghua University, Ministry of Education, Beijing, 100084, China.Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China; Key Laboratory of Industrial Biocatalysis, Tsinghua University, Ministry of Education, Beijing, 100084, China.Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China; Key Laboratory of Industrial Biocatalysis, Tsinghua University, Ministry of Education, Beijing, 100084, China.Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China; Key Laboratory of Industrial Biocatalysis, Tsinghua University, Ministry of Education, Beijing, 100084, China; Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China. Electronic address: yuhm@mail.tsinghua.edu.cn.Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China; Key Laboratory of Industrial Biocatalysis, Tsinghua University, Ministry of Education, Beijing, 100084, China.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31610242

Citation

Liang, Youxiang, et al. "A CRISPR/Cas9-based Genome Editing System for Rhodococcus Ruber TH." Metabolic Engineering, vol. 57, 2019, pp. 13-22.
Liang Y, Jiao S, Wang M, et al. A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH. Metab Eng. 2019;57:13-22.
Liang, Y., Jiao, S., Wang, M., Yu, H., & Shen, Z. (2019). A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH. Metabolic Engineering, 57, pp. 13-22. doi:10.1016/j.ymben.2019.10.003.
Liang Y, et al. A CRISPR/Cas9-based Genome Editing System for Rhodococcus Ruber TH. Metab Eng. 2019 Oct 11;57:13-22. PubMed PMID: 31610242.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH. AU - Liang,Youxiang, AU - Jiao,Song, AU - Wang,Miaomiao, AU - Yu,Huimin, AU - Shen,Zhongyao, Y1 - 2019/10/11/ PY - 2019/06/27/received PY - 2019/10/10/revised PY - 2019/10/10/accepted PY - 2019/10/15/pubmed PY - 2019/10/15/medline PY - 2019/10/15/entrez KW - Acrylamide bio-production KW - CRISPR/Cas9 KW - Genome editing KW - Recombinase KW - Restriction-modification system KW - Rhodococcus SP - 13 EP - 22 JF - Metabolic engineering JO - Metab. Eng. VL - 57 N2 - 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. SN - 1096-7184 UR - https://www.unboundmedicine.com/medline/citation/31610242/A_CRISPR/Cas9-based_genome_editing_system_for_Rhodococcus_ruber_TH L2 - https://linkinghub.elsevier.com/retrieve/pii/S1096-7176(19)30266-6 DB - PRIME DP - Unbound Medicine ER -