Enhanced production of d-pantothenic acid in Corynebacterium glutamicum using an efficient CRISPR–Cpf1 genome editing method

Abstract

Background Corynebacterium glutamicum has industrial track records for producing a variety of valuable products such as amino acids. Although CRISPR-based genome editing technologies have undergone immense developments in recent years, the suicide-plasmid-based approaches are still predominant for C. glutamicum genome manipulation. It is crucial to develop a simple and efficient CRISPR genome editing method for C. glutamicum. Results In this study, we developed a RecombinAtion Prior to Induced Double-strand-break (RAPID) genome editing technology for C. glutamicum, as Cpf1 cleavage was found to disrupt RecET-mediated homologous recombination (HR) of the donor template into the genome. The RAPID toolbox enabled highly efficient gene deletion and insertion, and notably, a linear DNA template was sufficient for gene deletion. Due to the simplified procedure and iterative operation ability, this methodology could be widely applied in C. glutamicum genetic manipulations. As a proof of concept, a high-yield D-pantothenic acid (vitamin B5)-producing strain was constructed, which, to the best of our knowledge, achieved the highest reported titer of 18.62 g/L from glucose only. Conclusions We developed a RecET-assisted CRISPR–Cpf1 genome editing technology for C. glutamicum that harnessed CRISPR-induced DSBs as a counterselection. This method is of great importance to C. glutamicum genome editing in terms of its practical applications, which also guides the development of CRISPR genome editing tools for other microorganisms.

Expanding the CRISPR Toolbox for Engineering Lycopene Biosynthesis in Corynebacterium glutamicum

“…Corynebacterium glutamicum is an ideal and secure strain for the industrial production of numerous compounds [6], including amino acids, vitamins, terpenoids, and biofuels [7].…”

Optimized Linear DNA Recombineering for CRISPR-Cpf1 System in Corynebacterium glutamicum

“…The CRISPR/Cpf1-RecET system was used to delete 1 kb, 5 kb, and 20 kb DNA fragments from the genome with editing efficiencies of 79.6%, 91.3%, and 36.4%, respectively, thus achieving effective deletion of large segments of DNA in C. glutamicum [31]. Recently, our research team also developed a gene editing system for inducing double-strand breaks before recombination, which used an inducible promoter to express the FnCpf1 protein; the editing efficiency was improved by adding inducers and extending the electrotransformation recovery time (5 h) [33]. Currently, the all-in-one single-plasmid system (SpCas9/FnCpf1, sgRNA, and homologous sequences expressed in the same vector) suffers from plasmid size limitation.…”