At the genome-wide level, the metabolic network of Corynebacterium glutamicum can be regulated and modified using synthetic biological approaches. However, Cre recombinase expression requires a tightly controlled system. To achieve this, a Cumate-ATc AND-gate inducible system was constructed, exhibiting a markedly lower uninduced relative fluorescence expression (14.28) compared to the traditional IPTG-inducible system (4 238.54). Cre recombinase was used to perform SCRaMbLE on the semi-synthetic C. glutamicum strain semi-syn CG-A1-A2 (containing 40 loxPsym sites within a 111.35 kb synthetic genome) under the control of an inducible system with additional structural optimization. Thirty-two SCRaMbLEd strains with specific site deletion preferences were verified using PCRTags. Genome sequencing revealed that the synthetic genome length of these SCRaMbLEd strains ranged from 64.50 kb to 165.31 kb. Among them, the SCRaMbLEd strain S-sCG-H underwent 15 simultaneous rearrangement events. The correlation between the genotype and phenotype of the SCRaMbLEd strains was analyzed in conjunction with growth curve and resistance phenotype assessments. The findings highlight that SCRaMbLE was successfully executed in a semi-synthetic C. glutamicum strain containing 40 loxPsym sites using the proposed system, providing modification targets for the precise breeding of C. glutamicum and serving as a reference for evolutionary studies in other strains.