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Background   Clostridium cellulolyticum  can degrade lignocellulosic biomass, and ferment the soluble sugars to produce valuable chemicals such as lactate, acetate, ethanol and hydrogen. However, the cellulose utilization efficiency of  C. cellulolyticum  still remains very low, impeding its application in consolidated bioprocessing for biofuels production. In this study, two metabolic engineering strategies were exploited to improve cellulose utilization efficiency, including sporulation abolishment and carbon overload alleviation.    Results  The  spo0A  gene at locus  Ccel_1894 , which encodes a master sporulation regulator was inactivated. The  spo0A  mutant abolished the sporulation ability. In a high concentration of cellulose (50 g/l), the performance of the  spo0A  mutant increased dramatically in terms of maximum growth, final concentrations of three major metabolic products, and cellulose catabolism. The microarray and gas chromatography–mass spectrometry (GC-MS) analyses showed that the valine, leucine and isoleucine biosynthesis pathways were up-regulated in the  spo0A  mutant. Based on this information, a partial isobutanol producing pathway modified from valine biosynthesis was introduced into  C. cellulolyticum  strains to further increase cellulose consumption by alleviating excessive carbon load. The introduction of this synthetic pathway to the wild-type strain improved cellulose consumption from 17.6 g/l to 28.7 g/l with a production of 0.42 g/l isobutanol in the 50 g/l cellulose medium. However, the  spo0A  mutant strain did not appreciably benefit from introduction of this synthetic pathway and the cellulose utilization efficiency did not further increase. A technical highlight in this study was that an  in vivo  promoter strength evaluation protocol was developed using anaerobic fluorescent protein and flow cytometry for  C. cellulolyticum .    Conclusions  In this study, we inactivated the  spo0A  gene and introduced a heterologous synthetic pathway to manipulate the stress response to heavy carbon load and accumulation of metabolic products. These findings provide new perspectives to enhance the ability of cellulolytic bacteria to produce biofuels and biocommodities with high efficiency and at low cost directly from lignocellulosic biomass .

Improvement of cellulose catabolism in Clostridium cellulolyticum by sporulation abolishment and carbon alleviation