Abstract
BackgroundCorynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes.ResultsIn this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions.ConclusionsIn this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.
Highlights
Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities
Green fluorescent protein (GFP) was used as a model protein to examine the effect of different genedeletion strains produced by the CRISPR/Cas9 system on recombinant protein expression and the results showed varying GFP expression levels in different strains
Construction of the CRISPR/Cas9 system in two plasmids To establish a genome editing system in C. glutamicum based on CRISPR/Cas9, we designed and constructed a two-plasmid system that separated Cas9 and the synthetic guide RNA (sgRNA) into pFSC and pFST plasmid series, respectively (Fig. 1a and b). pFSC was constructed from pXMJ19 [36], and included the Cas9 gene, an SD sequence and the Ptac promoter
Summary
Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. Corynebacterium glutamicum, a gram-positive bacterium with a high G+C content, has been used for the industrial production of various amino acids for more than 50 years It has recently demonstrated strong potential for use as a protein expression system [1, 2] because of its excellent culture characteristics and because it is non-pathogenic, does not produce endotoxins, and is generally recognized as safe [3, 4]. A more efficient method for C. glutamicum genome editing is desirable
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
