Abstract
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.
Highlights
The widely used gene deletion approach of the conventional sacB counter-selectable system in C. glutamicum ATCC13032 is based on two rounds of homologous recombination, during which only 2% of the events correspond to double-crossover events and which usually requires more than 10 days[16]
RecE/RecT (RecET), Orf47/Orf[48] and OrfB/OrfC, which have been identified to perform the function of linear double-strand DNA (dsDNA) recombineering in E.coli[29], were selected to test the recombineering activity in C. glutamicum ATCC14067
A 0.5 μg linear dsDNA cassette of CrtB/400-Kan was used for the verification (Supplementary Fig. S1)
Summary
The widely used gene deletion approach of the conventional sacB counter-selectable system in C. glutamicum ATCC13032 is based on two rounds of homologous recombination, during which only 2% of the events correspond to double-crossover events and which usually requires more than 10 days[16]. It does not work in C. glutamicum ATCC140676, 17, which may be due to specific or unclear genetic information among different Corynebacteria[12]. We designed a self-excisable linear dsDNA cassette combining the Cre/loxP system to perform markerless deletion via RecET recombineering system This strategy provides a new simple and efficient markerless deletion strategy for C. glutamicum ATCC14067
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