Chenggang Xu

Factors influencing cellulosome activity in Consolidated Bioprocessing of cellulosic ethanol

The cellulosome, a multi-subunit protein complex catalyzing cellulose degradation in cellulolytic Clostridium thermocellum, plays a crucial role in Consolidated Bioprocessing (CBP) of lignocellulose into ethanol. Here, activity of cellulosome was tested under varying concentrations of chemical compounds derived from lignocellulose pretreatment and fermentation. We found that, firstly, the cellulolytic activity of cellulosome was actually promoted by formate, acetate and lactate; secondly, cellulosome was tolerant up to 5mM furfural, 50mM p-hydroxybenzoic acid and 1mM catechol. Furthermore, the cellulosome exhibited higher ethanol tolerance and thermostability than commercialized fungal (Trichoderma reesei) cellulase. To probe the implication of these unique enzyme-features, C. thermocellum JYT01 was cultured under conditions optimal for cellulosome activity. This CBP system yielded 491 mM ethanol, the highest level reported thus far for C. thermocellum monocultures. These findings demonstrate the potential advantages of bacterial cellulosome, and provide a novel strategy for design, selection and optimization of the cellulosome-ethanologen partnership.

Structure and regulation of the cellulose degradome in Clostridium cellulolyticum

BackgroundMany bacteria efficiently degrade lignocellulose yet the underpinning genome-wide metabolic and regulatory networks remain elusive. Here we revealed the “cellulose degradome” for the model mesophilic cellulolytic bacterium Clostridium cellulolyticum ATCC 35319, via an integrated analysis of its complete genome, its transcriptomes under glucose, xylose, cellobiose, cellulose, xylan or corn stover and its extracellular proteomes under glucose, cellobiose or cellulose.ResultsProteins for core metabolic functions, environment sensing, gene regulation and polysaccharide metabolism were enriched in the cellulose degradome. Analysis of differentially expressed genes revealed a “core” set of 48 CAZymes required for degrading cellulose-containing substrates as well as an “accessory” set of 76 CAZymes required for specific non-cellulose substrates. Gene co-expression analysis suggested that Carbon Catabolite Repression (CCR) related regulators sense intracellular glycolytic intermediates and control the core CAZymes that mainly include cellulosomal components, whereas 11 sets of Two-Component Systems (TCSs) respond to availability of extracellular soluble sugars and respectively regulate most of the accessory CAZymes and associated transporters. Surprisingly, under glucose alone, the core cellulases were highly expressed at both transcript and protein levels. Furthermore, glucose enhanced cellulolysis in a dose-dependent manner, via inducing cellulase transcription at low concentrations.ConclusionA molecular model of cellulose degradome in C. cellulolyticum (Ccel) was proposed, which revealed the substrate-specificity of CAZymes and the transcriptional regulation of core cellulases by CCR where the glucose acts as a CCR inhibitor instead of a trigger. These features represent a distinct environment-sensing strategy for competing while collaborating for cellulose utilization, which can be exploited for process and genetic engineering of microbial cellulolysis.

Effect of location of the His-tag on the production of soluble and functional Buthus martensii Karsch insect toxin.

The low yield and poor folding efficiency in vivo of soluble and active recombinant cysteine-rich proteins expressed in Escherichia coli are a major challenge for large-scale protein production and purification. Expression vectors containing Buthus martensii Karsch insect toxin (BmK IT) fused to the C terminus of the intein Ssp DnaB were constructed in an attempt to overcome this problem. Following purification and intein self-cleavage, the fusion protein His(6)-intein-IT produced insoluble BmK IT, while intein-IT-His(6) generated soluble and properly folded BmK IT. This result indicated that the positioning of the His(6) tag has a key role in the production of soluble and functional BmK IT.

Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization

The mechanism, physiological relevance and evolutionary implication of selective RNA processing and stabilization (SRPS) remain elusive. Here we report the genome-wide maps of transcriptional start sites (TSs) and post-transcriptional processed sites (PSs) for Clostridium cellulolyticum. The PS-associated genes are preferably associated with subunits of heteromultimeric protein complexes, and the intergenic PSs (iPSs) are enriched in operons exhibiting highly skewed transcript-abundance landscape. Stem-loop structures associated with those iPSs located at 3′ termini of highly transcribed genes exhibit folding free energy negatively correlated with transcript-abundance ratio of flanking genes. In the cellulosome-encoding cip-cel operon, iPSs and stem-loops precisely regulate structure and abundance of the subunit-encoding transcripts processed from a primary polycistronic RNA, quantitatively specifying cellulosome stoichiometry. Moreover, cellulosome evolution is shaped by the number, position and biophysical nature of TSs, iPSs and stem-loops. Our findings unveil a genome-wide RNA-encoded strategy controlling in vivo stoichiometry of protein complexes.

Flavin mononucleotide (FMN)-based fluorescent protein (FbFP) as reporter for promoter screening in Clostridium cellulolyticum

Conventional methods for screening promoters in anaerobic bacteria are generally based on detection of enzymatic reactions and thus usually complicated or strain specific. Therefore a more efficient and universal method will be valuable. Here, using cellulolytic bacteria Clostridium cellulolyticum H10 as a model, we employed an oxygen-independent flavin-based fluorescent protein (FbFP) derived from Pseudomonas putida as a quantitative reporter for the screening of promoter via monitoring fluorescence intensity. The stability and reliability of FbFP fluorescence were proven by the high correlation (R(2)=0.87) between fluorescence intensity and abundance of FbFP. Moreover, two endogenous promoters with exceptional performance were identified and characterized, including a constitutive promoter p3398 and an inducible promoter p1133. Compared to the existing reporter systems widely used in clostridia, this FbFP-based method is more rapid, intuitive and versatile, and the endogenous promoters reported here should enrich the synthetic biology toolbox for this and related organisms.