2019 AIChE Annual Meeting
(175c) Development of a Cripsr/Cas9-Based Recombineering System for the Genome Editing of Rhodococcus
Authors
Liang, Y. - Presenter, Tsinghua University
Yu, H., Department of Chemical Engineering, Tsinghua University
Jiao, S., Department of Chemical Engineering, Tsinghua University
Shen, Z., Tsinghua University
Rhodococcus are organic-solvent tolerant strains with adaption abilities and diverse metabolic activities, which have been widely utilized in bioconversion, biosynthesis and bioremediation. In particular, the bio-production of acrylamide using Rhodococcus cells harboring nitrile hydratase has been one of the most successful cases of industrial biotechnology. However, the genome editing of Rhodococcus mainly relied on traditional homologous recombination, and was limited by the low transformation efficiency and recombination efficiency. In this study, a CRISPR/Cas9-mediated genome editing tool was established for R. ruber for the first time. With the bypass of restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, which facilitated to obtain colonies from the cleavage by Cas9. However, the editing efficiency was only 1/96 due to the insufficient recombination efficiency of R. ruber. Thus a pair of bacteriophage recombinases Che9c60 and Che9c61 were introduced and characterized in R. ruber. The CRISPR/Cas9-based recombineering system improved the editing efficiency up to 12/16, and achieved gene deletion, insertion and mutation efficiently. Moreover, an iterative genome editing procedure was performed to engineer R. ruber for the bio-production of acrylamide. One nitrilase gene and two aliphatic amidase genes were deleted to reduce the formation of byproduct acrylic acid by 60%, and the native nitrile hydratase gene was replaced by a mutant one for 2-fold enhanced enzyme stability. The engineered R. ruber cells showed great potential in the green production of acrylamide with reduced energy and substrate consumption.
Acknowledgements: This work was supported by the National Natural Science Foundationof China (No. 21776157; No. 21476126).