Clostridium Cellulolyticum H10 Research Articles

Engineering the Metabolic Profile of Clostridium cellulolyticum with Genomic DNA Libraries

Clostridium cellulolyticum H10 (ATCC 35319) has the ability to ferment cellulosic substrates into ethanol and weak acids. The growth and alcohol production rates of the wild-type organism are low and, therefore, targets of metabolic engineering. A genomic DNA expression library was produced by a novel application of degenerate oligonucleotide primed PCR (DOP-PCR) and was serially enriched in C. cellulolyticum grown on cellobiose in effort to produce fast-growing and productive strains. The DNA library produced from DOP-PCR contained gene-sized DNA fragments from the C. cellulolyticum genome and from the metagenome of a stream bank soil sample. The resulting enrichment yielded a conserved phage structural protein fragment (part of Ccel_2823) from the C. cellulolyticum genome that, when overexpressed alone, enabled the organism to increase the ethanol yield by 250% compared to the plasmid control strain. The engineered strain showed a reduced production of lactate and a 250% increased yield of secreted pyruvate. Significant changes in growth rate were not seen in this engineered strain, and it is possible that the enriched protein fragment may be combined with the existing rational metabolic engineering strategies to yield further high-performing cellulolytic strains.

Engineering of Acid-Resistant d-Allulose 3-Epimerase for Functional Juice Production

d-Allulose, a rare sugar and functional sweetener, can be biosynthesized by d-allulose 3-isomerase (DAE). However, most of the reported DAEs exhibit poor resistance under acidic conditions, which severely limited their application. Here, surface charge engineering and random mutagenesis were used to construct a mutant library of CcDAE from Clostridium cellulolyticum H10, combined with high-throughput screening to identify mutants with high activity and resistance under acidic conditions. The mutant M3 (I114R/K123E/H209R) exhibited high activity (3.36-fold of wild-type) and acid resistance (10.6-fold of wild-type) at pH 4.5. The structure-function relationship was further analyzed by molecular dynamics (MD) simulations, which indicated that M3 had a higher number of hydrogen bonds and negative surface charges than the wild type. A multienzyme cascade system including M3 was used to convert high-calorie sugars in acidic juices, and functional juices containing 7.8-15.4 g/L d-allulose were obtained. Our study broadens the manufacture of functional foods containing d-allulose.

Optimization of Ultrahigh-Throughput Screening Assay for Protein Engineering of d-Allulose 3-Epimerase

d-Allulose is the corresponding epimer of d-fructose at the C-3 position, which exhibits a similar taste and sweetness to sucrose. As a low-calorie sweetener, d-allulose has broad application prospects in the fields of medicine, food, and so on. Currently, the production method of d-allulose is mainly the enzymatic conversion of d-fructose by d-allulose 3-epimerase (DAEase). However, the limited specific activity and thermal stability of DAEase restrict its industrial application. Herein, an ultrahigh-throughput screening assay based on the transcription factor PsiR was extensively optimized from the aspects of culture medium components, screening plasmid, and expression host, which enhanced the correction between the fluorescent readout and the enzyme activity. Then, the error-prone PCR (epPCR) library of Clostridium cellulolyticum H10 DAEase (CcDAEase) was screened through the above optimized method, and the variant I228V with improved specific activity and thermal stability was obtained. Moreover, after combining two beneficial substitutions, D281G and C289R, which were previously obtained by this optimized assay, the specific activity of the triple-mutation variant I228V/D281G/C289R reached up to 1.42-fold of the wild type (WT), while its half-life (T1/2) at 60 °C was prolonged by 62.97-fold. The results confirmed the feasibility of the optimized screening assay as a powerful tool for the directed evolution of DAEase.

Boosting the Heterologous Expression of d-Allulose 3-Epimerase in Bacillus subtilis through Protein Engineering and Catabolite-Responsive Element Box Engineering.

As a natural sweetener with low calories and various physiological activities, d-allulose has drawn worldwide attention. Currently, d-allulose 3-epimerase (DAEase) is mainly used to catalyze the epimerization of d-fructose to d-allulose. Therefore, it is quite necessary to enhance the food-grade expression of DAEase to meet the surging market demand for d-allulose. In this study, initially, the promising variant H207L/D281G/C289R of Clostridium cellulolyticum H10 DAEase (CcDAEase) was generated by protein engineering, the specific activity and the T1/2 of which were 2.24-fold and 13.45-fold those of the CcDAEase wild type at 60 °C, respectively. After that, PamyE was determined as the optimal promoter for the recombinant expression of CcDAEase in Bacillus subtilis, and catabolite-responsive element (CRE) box engineering was further performed to eliminate the carbon catabolite repression (CCR) effect. Lastly, high-density fermentation was carried out and the final activity peaked at 4971.5 U mL-1, which is the highest expression level and could effectively promote the industrial production of DAEase. This research provides a theoretical basis and technical support for the molecular modification of DAEase and its efficient fermentation preparation.

Enhancing the thermostability of D-allulose 3-epimerase from Clostridium cellulolyticum H10 via a dual-enzyme screening system

D-Allulose 3-epimerase (DAE) is promising to be used for the production of the rare sugar D-allulose in industry. However, the poor thermostability and low catalytic efficiency limited its large-scale industrial applications. A dual-enzyme screening method was developed to measure the activity of the D-allulose 3-epimerase from Clostridium cellulolyticum H10 by employing a xylose isomerase, enabling high-throughput screening of mutants with higher thermostability. After two rounds of directed evolution, the H56R, Q277R, H56R/Q277R and H56R/Q277R/S293R variants were obtained with 1.9, 1.8, 3.5 and 7.1 °C improvement in T505, the temperature at which the enzyme activity becomes half of the original after the 5 min treatment and 3.1-, 4.2-, 4.4- and 9.47- fold improvement in the half life at 60 °C, respectively, compared with the wild-type enzyme. Among them, triple mutant H56R/Q277R/S293R showed significant improvement in kcat/Km compared to the wild type enzyme. Molecular dynamics simulations provided the insights into improving the thermostability by three arginine mutations. The research will aid the development of industrial biocatalysts for the production of D‑allulose.

Efficient D-allulose synthesis under acidic conditions by auto-inducing expression of the tandem D-allulose 3-epimerase genes in Bacillus subtilis

BackgroundD-allulose, a hexulose monosaccharide with low calorie content and high sweetness, is commonly used as a functional sugar in food and nutrition. However, enzyme preparation of D-allulose from D-frutose was severely hindered by the non-enzymatic browning under alkaline and high-temperature, and the unnecessary by-products further increased the difficulties in separation and extraction for industrial applications. Here, to address the above issue during the production process, a tandem D-allulose 3-epimerase (DPEases) isomerase synergistic expression strategy and an auto-inducible promoter engineering were levered in Bacillus subtilis 168 (Bs168) for efficient synthesis of D-allulose under the acidic conditions without browning.ResultsFirst, based on the dicistron expression system, two DPEases with complementary functional characteristics from Dorea sp. CAG:317 (DSdpe) and Clostridium cellulolyticum H10 (RCdpe) were expressed in tandem under the promoter HpaII in one cell. A better potential strain Bs168/pMA5-DSdpe-RCdpe increases enzyme activity to 18.9 U/mL at acidic conditions (pH 6.5), much higher than 17.2 and 16.7 U/mL of Bs168/pMA5-DSdpe and Bs168/pMA5-RCdpe, respectively. Subsequently, six recombinant strains based on four constitutive promoters were constructed in variable expression cassettes for improving the expression level of protein. Among those engineered strains, Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe exhibited the highest enzyme activity with 480.1 U/mL on fed-batch fermentation process in a 5 L fermenter at pH 6.5, about 2.1-times higher than the 228.5 U/mL of flask fermentation. Finally, the maximum yield of D-allulose reached as high as 163.5 g/L at the fructose concentration (50% w/v) by whole-cell biocatalyst.ConclusionIn this work, the engineered recombinant strain Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe was demonstrated as an effective microbial cell factory for the high-efficient synthesis of D-allulose without browning under acidic conditions. Based on the perspectives from this research, this strategy presented here also made it possible to meet the requirements of the industrial hyper-production of other rare sugars under more acidic conditions in theory.

Enhancing the thermostability of d-allulose 3-epimerase from Clostridium cellulolyticum H10 via directed evolution

d-allulose, the epimer at C-3 position of d-fructose, is a low-calorie functional rare sugar, which is regarded as one of the most potential sweeteners. At present, the main production method of d-allulose is epimerization of d-fructose by d-allulose 3-epimerase (DAE). However, industrial applications of DAE are still limited by its poor thermostability. Herein, directed evolution was applied to improve the thermostability of DAE from Clostridium cellulolyticum H10 (CcDAE). Two optimal mutants D281G and C289R, exhibiting 13.80-fold and 13.88-fold t1/2 values as that of wild type at 65 ℃, respectively, were obtained. To further enhance the thermostability, the triple mutant A107P/D281G/C289R was constructed after combination of mutants D281G, C289R, and previously identified thermostability-enhanced mutant A107P. The Tm and optimal temperature of triple mutant were increased by 14.39 ℃ and 5 ℃, respectively, compared to the wild type, meanwhile, the half-life of triple mutant was 58.85-fold as that of wild type at 65 ℃. Furthermore, the conversion rate of triple mutant was increased from 24.76% of wild type to 27.53% using 300 g/L d-fructose as substrate at 70 ℃. The effectiveness of directed evolution was verified and the triple mutant with enhanced thermostability had great application value in the large-scale production of d-allulose.

DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 Type II-C system.

Type II CRISPR–Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

Highly efficient production of Clostridium cellulolyticum H10 d-psicose 3-epimerase in Bacillus subtilis and use of these cells to produce d-psicose

Backgroundd-Psicose 3-epimerase (DPEase) catalyzes the isomerization of d-fructose to the rare sugar d-psicose, which may help prevent obesity, reduce blood sugar and blood fat, and inhibit intra-abdominal fat accumulation.ResultsIn this study, the DPEase of Clostridium cellulolyticum H10 was expressed in the food-grade host Bacillus subtilis. Optimization of the culture medium during shake-flask experiments yielded a DPEase activity of 314 U/mL. The optimal medium included 20 g/L peptone, 15 g/L corn steep powder, 5 g/L glycerol, and 1 mM Ca2+. Controlling the carbon source concentration was important because elevated concentrations can result in catabolite metabolic suppression (CCR). To avoid CCR and increase DPEase expression, we developed a fed-batch strategy in a 3.6-L fermenter. We altered the ratio of carbon source to nitrogen source (C/N) in the feeding medium and employed a constant feeding rate (6 g/L/h). This strategy improved the DPEase activity to 2246 U/mL (7.8 g/L), which is almost 15 times higher than that observed in the original shake-flask cultures. Finally, we used the DPEase-expressing B. subtilis cells to produce d-psicose from d-fructose, and a 28% conversion yield was achieved with these cells, demonstrating their potential use in d-psicose production.ConclusionsThis is the first report to enhance recombinant DPEase production in B. subtilis using efficient and convenient fermentation strategy, and the DPEase yield is three times higher than the highest yield reported to date. The recombinant B. subtilis cells were further used in the efficient synthesis of d-psicose. This study provides a basis for the industrial production of d-psicose.

A rational design to enhance the resistance of Escherichia coli phytase AppA to trypsin

D-Allulose 3-epimerase (DAE) is promising to be used for the production of the rare sugar D-allulose in industry. However, the poor thermostability and low catalytic efficiency limited its large-scale industrial applications. A dual-enzyme screening method was developed to measure the activity of the D-allulose 3-epimerase from Clostridium cellulolyticum H10 by employing a xylose isomerase, enabling high-throughput screening of mutants with higher thermostability. After two rounds of directed evolution, the H56R, Q277R, H56R/Q277R and H56R/Q277R/S293R variants were obtained with 1.9, 1.8, 3.5 and 7.1 °C improvement in T505, the temperature at which the enzyme activity becomes half of the original after the 5 min treatment and 3.1-, 4.2-, 4.4- and 9.47- fold improvement in the half life at 60 °C, respectively, compared with the wild-type enzyme. Among them, triple mutant H56R/Q277R/S293R showed significant improvement in kcat/Km compared to the wild type enzyme. Molecular dynamics simulations provided the insights into improving the thermostability by three arginine mutations. The research will aid the development of industrial biocatalysts for the production of D‑allulose.

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 (R2=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.

Dockerin-containing protease inhibitor protects key cellulosomal cellulases from proteolysis inClostridium cellulolyticum

Cellulosomes are key for lignocellulosic biomass degradation in cellulolytic Clostridia. Better understanding of the mechanism of cellulosome regulation would allow us to improve lignocellulose hydrolysis. It is hypothesized that cellulosomal protease inhibitors would regulate cellulosome architecture and then lignocellulose hydrolysis. Here, a dockerin-containing protease inhibitor gene (dpi) in Clostridium cellulolyticum H10 was characterized by mutagenesis and physiological analyses. The dpi mutant had a decreased cell yield on glucose, cellulose and xylan, lower cellulose utilization efficiency, and a 70% and 52% decrease of the key cellulosomal components, Cel48F and Cel9E respectively. The decreased cellulolysis is caused by the proteolysis of major cellulosomal components, such as Cel48F and Cel9E. Disruption of cel9E severely impaired cell growth on cellulose while loss of cel48F completely abolished cellulolytic activity. These observations are due to the combinational results of gene inactivation and polar effects caused by intron insertion. Purified recombinant Dpi showed inhibitory activity against cysteine protease. Taken together, Dpi protects key cellulosomal cellulases from proteolysis in H10. This study identified the physiological importance of cellulosome-localized protease inhibitors in Clostridia.

Correction to Cloning, Expression, and Characterization of a d-Psicose 3-Epimerase from Clostridium cellulolyticum H10

Correction to Cloning, Expression, and Characterization of a <scp>d</scp>-Psicose 3-Epimerase from Clostridium cellulolyticum H10

Targeted gene engineering in Clostridium cellulolyticum H10 without methylation

Genetic engineering of Clostridium cellulolyticum has been developed slowly compared with that of other clostridial species, and one of the major reasons might be the restriction and modification (RM) system which degrades foreign DNA. Here, a putative MspI endonuclease gene, ccel2866, was inactivated by a ClosTron-based gene disruption method. The resulting C. cellulolyticum mutant H10ΔmspI lost the MspI endonuclease activity and can accept unmethylated DNA efficiently. Following that, an oxygen-independent green fluorescence protein gene was introduced into H10ΔmspI without methylation, generating a convenient reporter system to evaluate the expression of heterologous protein in C. cellulolyticum by green fluorescence. To further demonstrate the efficiency of the H10ΔmspI, double mutants H10ΔmspIΔldh and H10ΔmspIΔack were constructed by disrupting lactate dehydrogenase gene ccel2485 and acetate kinase gene ccel2136 in H10ΔmspI, respectively, without DNA methylation, and the stability of the double mutation was confirmed after the 100th generation. The mutant H10ΔmspI constructed here can be used as a platform for further targeted gene manipulation conveniently and efficiently. It will greatly facilitate the metabolic engineering of C. cellulolyticum aiming at faster cellulose degradation and higher biofuel production at the molecular level.

Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars.

D-psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of D-psicose, a rare hexose sugar, from D-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticum DPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)(8) TIM barrel fold with a Mn(2+) metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/products (D-psicose, D-fructose, D-tagatose and D-sorbose) were also determined. From the complex structures of C. cellulolyticum DPEase with D-psicose and D-fructose, the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexose-bound complex structures, a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.

Cloning, Expression, and Characterization of a d-Psicose 3-Epimerase from Clostridium cellulolyticum H10

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.

The Family 1 Glycoside Hydrolase from Clostridium cellulolyticum H10 is a Cellodextrin Glucohydrolase

The only family 1 glycoside hydrolase in Clostridium cellulolyticum H10 (CcGH1) is annotated as a beta-galactosidase but has high sequence homology with many beta-glucosidases. Given the possible importance of beta-glucosidase in cellulose utilization by C. cellulolyticum, the encoding open reading frame Ccel_0374 was cloned and expressed in E. coli as a soluble fusion protein with thioredoxin. After tag cleavage, the purified enzyme had a molecular mass of 52 kDa and was active in dimeric form on a broad range of substrates, including cellobiose, cellotriose, cellotetraose, p-nitrophenyl-beta-glucopyranoside, lactose, and o-nitrophenyl-beta-galactopyranoside. The enzyme showed lower K(m) and higher catalytic efficiency (k (cat)/K(m)) on cellodextrins with degree of polymerization from 2 to 4 than on lactose, and the k (cat)/K (m) values on cellodextrins increased in the order of cellobiose < cellotriose < cellotetraose, suggesting that CcGH1 was a cellodextrin glucohydrolase (EC 3.2.1.74). The high K(m) (69 mM) on cellobiose implies that CcGH1 likely has a minimal role in the intracellular hydrolysis of cellobiose in C. cellulolyticum. The three-dimensional structure model of CcGH1 generated by homology modeling showed a typical (alpha/beta)(8) barrel topology characteristic of family 1 glycoside hydrolases.

Adhesion and growth rate of Clostridium cellulolyticum ATCC 35319 on crystalline cellulose.

The rate of tritiated-thymidine incorporation into DNA was used to estimate Clostridium cellulolyticum H10 growth rates on Avicel cellulose, taking into consideration both the unattached cells and the cells adhered to the substrate. The generation time on cellobiose calculated from the data on cell density (4.5 h) agreed well with the generation time calculated by tritiated-thymidine incorporation (3.8 h). Growth on Avicel cellulose occurred when bacteria were adhered to their substrate; 80% of the biomass was detected on the cellulose. Taking into consideration attached and free bacteria, the generation time as determined by thymidine incorporation was about 8 h, whereas by bacterial-protein estimation it was about 13 h. In addition to the growth rate of the bacteria on the cellulose, the release of adhered cells constituted an important factor in the efficiency of the cellulolysis. The stage of growth influenced adhesion of C. cellulolyticum; maximum adhesion was found during the exponential phase. Under the conditions used, the end of growth was characterized by an acute release of biomass and cellulase activity from the cellulose. An exhaustion of the accessible cellulose could be responsible for this release.

Partial purification and characterization of the cellulases from clostridium cellulolyticum h10

AbstractThe extracellular cellulolytic complex from Clostridium cellulolyticum H10 has been isolated and partially characterized. Through application of gel filtration and ion exchange chromatography techniques it has been established that in its native state this complex essentially consists of highly stable macromolecular structures (Mr c. 20 000 kDa) (similar to those described for different cellulolytic bacteria and currently termed cellulosomes) exhibiting avicelase, carboxymethylcellulase (CMCase) and xylanase activities. Furthermore, immunoaffinity assays show that this extracellular cellutosomal form of the complex is immunochemically similar to the cell‐associated form.Another feature of the C. cellulolyticum H10 complex is that the Avicel‐adsorbed fraction consists essentially of three major components, two of them (those of 160–170 and 72 kDa) having no detectable activity and one (that of 95 kDa) being associated with strong CMCase activity.