초록
<P><B>Background</B></P><P><I>Clostridium cellulolyticum</I> 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 <I>C. cellulolyticum</I> 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.</P><P><B>Results</B></P><P>The <I>spo0A</I> gene at locus <I>Ccel_1894</I>, which encodes a master sporulation regulator was inactivated. The <I>spo0A</I> mutant abolished the sporulation ability. In a high concentration of cellulose (50 g/l), the performance of the <I>spo0A</I> 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 <I>spo0A</I> mutant. Based on this information, a partial isobutanol producing pathway modified from valine biosynthesis was introduced into <I>C. cellulolyticum</I> 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 <I>spo0A</I> 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 <I>in vivo</I> promoter strength evaluation protocol was developed using anaerobic fluorescent protein and flow cytometry for <I>C. cellulolyticum</I>.</P><P><B>Conclusions</B></P><P>In this study, we inactivated the <I>spo0A</I> 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<I>.</I></P>