title: Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System.
abstract: Clostridium saccharoperbutylacetonicumN1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized forClostridium beijerinckiiwas evaluated inC. saccharoperbutylacetonicumby targetingptaandbuk, two essential genes for acetate and butyrate production, respectively.ptaandbuksingle and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter PJ23119, we achieved a mutation rate of 75% forptadeletion without serial subculturing as suggested previously forC. beijerinckiiThus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing inC. saccharoperbutylacetonicumBatch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In thepta bukdouble deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations.IMPORTANCEAn efficient CRISPR-Cas9 genome engineering system was developed forC. saccharoperbutylacetonicumN1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in closely related microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the areas of metabolic engineering and synthetic biology. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production.
authors.affiliation: Department of Biosystems Engineering, Auburn University, Auburn, Alabama, USA.
authors.forename: Shaohua
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Biosystems Engineering, Auburn University, Auburn, Alabama, USA.
authors.forename: Sheng
authors.initials: S
authors.lastname: Dong
authors.affiliation: Department of Biosystems Engineering, Auburn University, Auburn, Alabama, USA.
authors.forename: Pixiang
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Biosystems Engineering, Auburn University, Auburn, Alabama, USA.|Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tao
authors.affiliation: Department of Biosystems Engineering, Auburn University, Auburn, Alabama, USA yiwang3@auburn.edu.|Center for Bioenergy and Bioproducts, Auburn University, Auburn, Alabama, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000440:Butanols / Q000378:metabolism; D002087:Butyrates / Q000378:metabolism; D064113:CRISPR-Cas Systems; D046970:Clostridium beijerinckii / Q000235:genetics* / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005285:Fermentation; D000072669:Gene Editing; D016680:Genome, Bacterial; D060847:Metabolic Engineering; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion
chemical_list: D001426:Bacterial Proteins; D000440:Butanols; D002087:Butyrates
keywords: CRISPR-Cas9; Clostridium saccharoperbutylacetonicum; buk; butanol; gRNA; genome engineering; pta