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Optimizing recombineering in Corynebacterium glutamicum
Name
10.1002-bit.27737.pdf
Size
591.72 KB
Format
Adobe PDF
Checksum (MD5)
3fd5ac6043262bba601f6001f28c11bf
Author(s)
Li, Cheng
Swofford, Charles A.
Rückert, Christian
Sinskey, Anthony J.
Date Issued
March 11, 2021
Journal
Biotechnology and Bioengineering
Publisher
Wiley
Citation
Li, Cheng, Swofford, Charles A., Rückert, Christian and Sinskey, Anthony J. 2021. "Optimizing recombineering in Corynebacterium glutamicum." Biotechnology and Bioengineering, 118 (6).
Version
Author's final manuscript
Abstract
Owing to the increasing demand for amino acids and valuable commodities that can be produced by Corynebacterium glutamicum, there is a pressing need for new rapid genome engineering tools that improve the speed and efficiency of genomic insertions, deletions, and mutations. Recombineering using the λ Red system in Escherichia coli has proven very successful at genetically modifying this organism in a quick and efficient manner, suggesting that optimizing a recombineering system for C. glutamicum will also improve the speed for genomic modifications. Here, we maximized the recombineering efficiency in C. glutamicum by testing the efficacy of seven different recombinase/exonuclease pairs for integrating single-stranded DNA and double-stranded DNA (dsDNA) into the genome. By optimizing the homologous arm length and the amount of dsDNA transformed, as well as eliminating codon bias, a dsDNA recombineering efficiency of 13,250 transformed colonies/10 viable cells was achieved, the highest efficiency currently reported in the literature. Using this optimized system, over 40,000 bp could be deleted in one transformation step. This recombineering strategy will greatly improve the speed of genetic modifications in C. glutamicum and assist other systems, such as clustered regularly interspaced short palindromic repeats and multiplexed automated genome engineering, in improving targeted genome editing.
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Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
http://dx.doi.org/10.1002/bit.27737
