The mesophilic cellulosome-producing Clostridium cellulovorans is a versatile strain for metabolic engineering towards consolidated n-butanol production from cellulose. However, the host restriction systems prevented the transformation of recombinant plasmids to express heterologous genes in C. cellulovorans. By analyzing the host restriction systems, possible restriction enzymes and their recognition sites were identified. In view of the failure of in vivo plasmid methylation, a new restriction site free shuttle plasmid was constructed by removing all unnecessary DNA fragments and mutating the E. coli replicon. The obtained plasmid was not digested by host cell lysate and was successfully transformed by electroporation. Subsequent transformation of different recombinant plasmids confirmed the applicability of the new shuttle plasmid and our strategy in overcoming transformation barrier, opening the door for efficient strain engineering of C. cellulovorans.

Overcoming the transformation hurdle of Clostridium cellulovorans for efficient metabolic engineering towards consolidated n-butanol production from cellulose

253rd National Meeting (2017)

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