Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

<p>Abstract</p> <p>Background</p> <p>The model bacterium <it>Clostridium cellulolyticum </it>efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H₂and CO₂, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for <it>C. cellulolyticum</it>, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering.</p> <p>Results</p> <p>The first targeted gene inactivation system was developed for <it>C. cellulolyticum</it>, based on a mobile group II intron originating from the <it>Lactococcus lactis </it>L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous <smcaps>L</smcaps>-lactate dehydrogenase (<it>Ccel_2485; ldh</it>) and <smcaps>L</smcaps>-malate dehydrogenase (<it>Ccel_0137; mdh</it>) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant's tricarboxylic acid pathway.</p> <p>Conclusions</p> <p>The efficient intron-based gene inactivation system produced the first non-random, targeted mutations in <it>C. cellulolyticum</it>. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in <it>C</it>. <it>cellulolyticum </it>and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from cellobiose, cellulose and switchgrass.</p

Whole proteome analyses on Ruminiclostridium cellulolyticum show a modulation of the cellulolysis machinery in response to cellulosic materials with subtle differences in chemical and structural properties

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the xyl-doc cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics.This work was supported by a grant [R2DS 2010-08] from Conseil Regional d'Ile-de-France through DIM R2DS programs (http://www.r2ds-ile-de-france.com/). Irstea (www.irstea.fr/) contributed to the funding of a PhD grant for the first author. The funders provided support in the form of salaries for author [NB], funding for consumables and laboratory equipment, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Omics Services provided support in the form of salaries for authors [VS, MD], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors [NB, VS, MD] are articulated in the 'author contributions' section.info:eu-repo/semantics/publishedVersio

Regulation of cel genes of C. cellulolyticum: identification of GlyR2, a transcriptional regulator regulating cel5D gene expression

Transcription and expression regulation of some individual cel genes (cel5A, cel5I, cel5D and cel44O) of Clostridium cellulolyticum were investigated. Unlike the cip-cel operon, these genes are transcribed as monocistronic units of transcription, except cel5D. The location of the transcription initiation sites was determined using RT-PCR and the mRNA 5′-end extremities were detected using primer extension experiments. Similarly to the cip-cel operon, cel5A and cel5I expressions are regulated by a carbon catabolite repression mechanism, whereas cel44O and cel5D expressions do not seem to be submitted to this regulation. The role of the putative transcriptional regulator GlyR2 in the regulation of cel5D expression was investigated. The recombinant protein GlyR2 was produced and was shown to bind in vitro to the cel5D and glyR2 promoter regions, suggesting that besides regulating its own expression, GlyR2 may regulate cel5D expression. To test this hypothesis in vivo, an insertional glyR2 mutant was generated and the effect of this disruption on cel5D expression was evaluated. Levels of cel5D mRNAs in the mutant were 16 fold lower than that of the wild-type strain suggesting that GlyR2 acts as an activator of cel5D expression