Biotechnology for Biofuels 2015-01-01

Combining free and aggregated cellulolytic systems in the cellulosome-producing bacterium Ruminiclostridium cellulolyticum.

Julie Ravachol, Romain Borne, Isabelle Meynial-Salles, Philippe Soucaille, Sandrine Pagès, Chantal Tardif, Henri-Pierre Fierobe

Index: Biotechnol. Biofuels 8 , 114, (2015)

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Abstract

Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans (formerly known as Clostridium cellulolyticum and Clostridium phytofermentans, respectively) are anaerobic bacteria that developed different strategies to depolymerize the cellulose and the related plant cell wall polysaccharides. Thus, R. cellulolyticum produces large extracellular multi-enzyme complexes termed cellulosomes, while L. phytofermentans secretes in the environment some cellulose-degrading enzymes as free enzymes. In the present study, the major cellulase from L. phytofermentans was introduced as a free enzyme or as a cellulosomal component in R. cellulolyticum to improve its cellulolytic capacities.The gene at locus Cphy_3367 encoding the major cellulase Cel9A from L. phytofermentans and an engineered gene coding for a modified enzyme harboring a R. cellulolyticum C-terminal dockerin were cloned in an expression vector. After electrotransformation of R. cellulolyticum, both forms of Cel9A were found to be secreted by the corresponding recombinant strains. On minimal medium containing microcrystalline cellulose as the sole source of carbon, the strain secreting the free Cel9A started to grow sooner and consumed cellulose faster than the strain producing the cellulosomal form of Cel9A, or the control strain carrying an empty expression vector. All strains reached the same final cell density but the strain producing the cellulosomal form of Cel9A was unable to completely consume the available cellulose even after an extended cultivation time, conversely to the two other strains. Analyses of their cellulosomes showed that the engineered form of Cel9A bearing a dockerin was successfully incorporated in the complexes, but its integration induced an important release of regular cellulosomal components such as the major cellulase Cel48F, which severely impaired the activity of the complexes on cellulose. In contrast, the cellulosomes synthesized by the control and the free Cel9A-secreting strains displayed similar composition and activity. Finally, the most cellulolytic strain secreting free Cel9A, was also characterized by an early production of lactate, acetate and ethanol as compared to the control strain.Our study shows that the cellulolytic capacity of R. cellulolyticum can be augmented by supplementing the cellulosomes with a free cellulase originating from L. phytofermentans, whereas integration of the heterologous enzyme in the cellulosomes is rather unfavorable.