Triple deletion of clpC, porB, and mepA enhances production of small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide in Corynebacterium glutamicum.

In our previous work, a two-plasmid CRISPR/Cas9 system was constructed for genome editing in Corynebacterium glutamicum. To increase the transformation efficiency and simplify the plasmid curing steps, an all-in-one CRISPR/Cas9 system was constructed for efficient genome editing. In addition, to research proteolysis during the production of recombinant proteins and generate a host for enhanced expression of recombinant proteins, the system was used to delete three genes, clpC, porB, and mepA in C. glutamicum CGMCC1.15647, which encoded the Clp protease subunit ClpC, anion selective channel protein B, and metallopeptidase A, respectively. After the evaluation of different plasmids and hosts, small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide (SUMO-NT-proBNP), an important protein used for the diagnosis of mild heart failure was successfully expressed in the triple mutant ΔclpCΔporBΔmepA, which exhibit threefold higher levels of protein expression compared with the wild-type. In conclusion, we created a simplified CRISPR tool for genome editing in C. glutamicum, provided a method to generate a host for enhanced expression of recombinant proteins and successfully expressed SUMO-NT-proBNP in C. glutamicum. This tool and method will greatly facilitate genetic engineering and metabolic optimization of this important platform organism.