Abstract
BackgroundClostridium 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.ResultsThe 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.ConclusionsIn 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.
Highlights
Clostridium cellulolyticum can degrade lignocellulosic biomass, and ferment the soluble sugars to produce valuable chemicals such as lactate, acetate, ethanol and hydrogen
Group II intron-mediated spo0A inactivation C. cellulolyticum cells transformed with pLyc1217Erbased vectors yielded erythromycin-resistant colonies on agar plates
One possible explanation is that a connection between sporulation rate and the availability of amino acids in B. subtilis was reported [28], and our omics analyses showed that valine, leucine and isoleucine biosynthesis pathways were altered in the spo0A mutant, indicating sporulation abolishment could probably affect amino acid biosynethsis
Summary
Clostridium cellulolyticum can degrade lignocellulosic biomass, and ferment the soluble sugars to produce valuable chemicals such as lactate, acetate, ethanol and hydrogen. The cellulose utilization efficiency of C. cellulolyticum still remains very low, impeding its application in consolidated bioprocessing for biofuels production. Two metabolic engineering strategies were exploited to improve cellulose utilization efficiency, including sporulation abolishment and carbon overload alleviation. Consolidated bioprocessing (CBP), which integrates saccharolytic enzymes production, cellulose fiber degradation, and fermentation of resulting sugars into a single step, is considered a promising technology for significantly reducing the processing cost [3]. Clostridium cellulolyticum is a mesophilic gram-positive bacterium capable of degrading cellulose via an extracellular enzymatic complex called the cellulosome and fermenting the sugars from cellulose degradation to lactate, acetate, ethanol, hydrogen and CO2 [4]. Other metabolic engineering strategies need to be exploited to improve the cellulose utilization efficiency
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