Expediting Genome Synthesis of Corynebacterium glutamicum with An Artificial Chromosome Vector

Recent advances in genome synthesis have relied on scalable DNA assembly and delivery, and efficient recombination techniques. While these methods have enabled rapid progress for E. coli and yeast, they are often inadequate for other microorganisms. Here, we devise a Corynebacterium glutamicum Artificial Chromosome (CAC), which combines a replicating system from a closely related strain with an innate partitioning system. This CAC vector can efficiently deliver DNA fragments up to 56 kb and maintain stability in C. glutamicum. Leveraging the CAC vector, we developed CACEXER (CAC EXcision Enhanced Recombination), a streamlined strategy for iterative genome replacements for C. glutamicum. Using this approach, we integrated 361 kb (11%) of synthetic DNA into the genome, creating semi-synCG-A. This strain exhibited stability with minimal growth defects. This paves the way to establish C. glutamicum as the third industrial microorganism, alongside E. coli and S. cerevisiae, to undergo large-scale genome synthesis.