BACKGROUND Corynebacterium glutamicum is an important microorganism for the industrial production of high value-added compounds. Therefore, powerful genetic engineering tools are required for metabolic engineering of C. glutamicum. RESULTS In this study, the application of ssDNA recombineering was extended to realize efficient and rapid genetic engineering of C. glutamicum. The efficiency of one-step ssDNA recombineering reached 60% without the need for an additional selective marker. Multi-nucleotide modifications and strain iterative engineering were also achieved. As evidence of its potential in metabolic engineering, a base substitution mutation of aspartokinase T311I was introduced to relieve the feedback inhibition involved in L-lysine biosynthesis while simultaneously investigating the combinatory effect of adjacent residues in one step. Iterative engineering enabled rapid mutation of 11 nucleotides in the RBS/initiation codon region to improve lysine production 100-fold compared with the wild-type organism. CONCLUSIONS CRISPR-Cas9-assisted ssDNA recombineering was developed for genome editing of C. glutamicum with high efficiency. This strategy contributes to the toolbox available for genetic engineering of C. glutamicum and boosts the strain engineering for more industrial applications. (c) 2018 Society of Chemical Industry