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Gene manipulation is essential for metabolic engineering and synthetic biology, but the current general gene manipulation methods are not applicable to the non-model strain  Corynebacterium glutamicum  ( C .  glutamicum ) ATCC14067, which is used for amino acid production. Here, we report an effective and sequential deletion method for  C .  glutamicum  ATCC14067 using the exonuclease-recombinase pair RecE + RecT (RecET) for recombineering via a designed self-excisable linear double-strand DNA (dsDNA) cassette, which contains the Cre /loxP  system, to accomplish markerless deletion. To the best of our knowledge, this is the first effective and simple strategy for recombination with markerless deletion in  C .  glutamicum  ATCC14067. This strategy provides a simple markerless deletion strategy for  C .  glutamicum  and builds a solid basis for producer construction.

Recombineering using RecET in Corynebacterium glutamicum ATCC14067 via a self-excisable cassette