DEVELOPMENT OF RAPID GENOME ENGINEERING TOOLS FOR CORYNEBACTERIUM GLUTAMICUM

Background: Over the past 60 years, Corynebacterium glutamicum has been the predominant producer of ʟ-amino acids. Due to the increasing worldwide demand of amino acids and other novel compounds, there is a pressing need for rapid improvement in strain engineering and production yields of chemical compounds in C. glutamicum. Current technology for strain engineering and genome modification in C. glutamicumis notoriously slow. Single mutations are deleted by a double-crossover homologous recombination event that takes at least one week to confirm. This work aims to improve the speed of strain engineering by developing rapid genome editing tools using recombinase-based systems that introduce mutations at high transformation efficiencies. Methods: Seven different recombinase-exonuclease (R-E) pairs from different phages were tested for recombination efficiency in C. glutamicum. Each pair was cloned into an expression vector and induced for four hours prior to making cells electrocompetent. Point mutation efficiency was determined by transforming ssDNA oligos that introduced an A to C transversion in the rpsL gene that confers streptomycin resistance. To test large-scale genomic insertions and deletions, dsDNA encoding a kanamycin resistance cassette was transformed that subsequently deleted various sizes of the genome. Each pair was also tested in a previously developed C. glutamicum strain that eliminates codon bias and improves translation efficiency of transgenes. Results: The RecT/E recombinase-exonuclease pair from the Rac prophage of E. coli demonstrated the highest efficiency for both ssDNA and dsDNA recombination. Max efficiency occurred with 1µg of DNA oligos that contained homologous arms of 1000bp in length. With these parameters, we could delete upwards of 40,000bp of the C. glutamicum genome in one step. Genomic modification using a codon-unbiased strain improved recombination efficiency for all pairs. Using this strain, we have the highest reported recombination efficiency for C. glutamicumin the literature. Conclusion: Recombineering in C. glutamicum using the RecT/E recombinase/exonuclease pair is a powerful tool that reduces the time it takes to make large scale genomic modifications from one week to one day. In addition, our results show that codon optimization is important for improving the expression of transgenes in a foreign organism. This tool will vastly improve strain engineering in C. glutamicum and eliminate barriers for synthesizing novel compounds through fermentation processes.