Targeted gene engineering in Clostridium cellulolyticum H10 without methylation

Genetic engineering of Clostridium cellulolyticum has been developed slowly compared with that of other clostridial species, and one of the major reasons might be the restriction and modification (RM) system which degrades foreign DNA. Here, a putative Mspl endonuclease gene, ccel2866, was inactivated by a ClosTron-based gene disruption method. The resulting C cellulolyticum mutant H10 Delta mspl lost the Mspl endonuclease activity and can accept unmethylated DNA efficiently. Following that, an oxygen-independent green fluorescence protein gene was introduced into H10 Delta mspl without methylation, generating a convenient reporter system to evaluate the expression of heterologous protein in C. cellulolyticum by green fluorescence. To further demonstrate the efficiency of the H10 Delta mspl, double mutants H10 Delta mspl Delta ldh and H10 Delta mspl Delta ack were constructed by disrupting lactate dehydrogenase gene ccel2485 and acetate kinase gene ccel2136 in H10 Delta mspl, respectively, without DNA methylation, and the stability of the double mutation was confirmed after the 100th generation. The mutant H10 Delta mspl constructed here can be used as a platform for further targeted gene manipulation conveniently and efficiently. It will greatly facilitate the metabolic engineering of C. cellulolyticum aiming at faster cellulose degradation and higher biofuel production at the molecular level. (c) 2012 Elsevier B.V. All rights reserved.