Trends in Systems Biology for the Analysis and Engineering of Clostridium acetobutylicum Metabolism.

Clostridium acetobutylicum has received renewed interest worldwide as a promising producer of biofuels and bulk chemicals such as n-butanol, 1,3-propanediol, 1,3-butanediol, isopropanol, and butyrate. To develop commercial processes for the production of bulk chemicals via a metabolic engineering approach it is necessary to better characterize both the primary metabolism and metabolic regulation of C. acetobutylicum. Here, we review the history of the development of omics studies of C. acetobutylicum, summarize the recent application of quantitative/integrated omics approaches to the physiological analysis and metabolic engineering of this bacterium, and provide directions for future studies to address current challenges.

An efficient method for markerless mutant generation by allelic exchange in Clostridium acetobutylicum and Clostridium saccharobutylicum using suicide vectors.

BACKGROUND: Clostridium acetobutylicum and Clostridium saccharobutylicum are Gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of their genomes has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals. RESULTS: The method used in this paper to knock-out, knock-in, or edit genes in C. acetobutylicum and C. saccharobutylicum combines an improved electroporation method with the use of (i) restrictionless Δupp (which encodes uracil phosphoribosyl-transferase) strains and (ii) very small suicide vectors containing a markerless deletion/insertion cassette, an antibiotic resistance gene (for the selection of the first crossing-over) and upp (from C. acetobutylicum) for subsequent use as a counterselectable marker with the aid of 5-fluorouracil (5-FU) to promote the second crossing-over. This method was successfully used to both delete genes and edit genes in both C. acetobutylicum and C. saccharobutylicum. Among the edited genes, a mutation in the spo0A gene that abolished solvent formation in C. acetobutylicum was introduced in C. saccharobutylicum and shown to produce the same effect. CONCLUSIONS: The method described in this study will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.

Reviving the Weizmann process for commercial n-butanol production.

Developing a commercial process for the biological production of n-butanol is challenging as it needs to combine high titer, yield, and productivities. Here we engineer Clostridium acetobutylicum to stably and continuously produce n-butanol on a mineral media with glucose as sole carbon source. We further design a continuous process for fermentation of high concentration glucose syrup using in situ extraction of alcohols by distillation under low pressure and high cell density cultures to increase the titer, yield, and productivity of n-butanol production to the level of 550 g/L, 0.35 g/g, and 14 g/L/hr, respectively. This process provides a mean to produce n-butanol at performance levels comparable to that of corn wet milling ethanol plants using yeast as a biocatalyst. It may hold the potential to be scaled-up at pilot and industrial levels for the commercial production of n-butanol.

Cap0037, a Novel Global Regulator of Clostridium acetobutylicum Metabolism.

An operon comprising two genes, CA_P0037 and CA_P0036, that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicum upstream of adhE2 A CA_P0037::int (189/190s) mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037 was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol) and alcohologenic (producing mainly butyrate, acetate, and lactate) chemostat cultures but not in solventogenic or batch cultures. Transcriptomic investigation of the CA_P0037::int (189/190s) mutant showed that inactivation of CA_P0037 significantly affected the expression of more than 258 genes under acidogenic conditions. Surprisingly, genes belonging to the Fur regulon, involved in iron transport (CA_C1029-CA_C1032), or coding for the main flavodoxin (CA_C0587) were the most significantly expressed genes under all conditions, whereas fur (coding for the ferric uptake regulator) gene expression remained unchanged. Furthermore, most of the genes of the Rex regulon, such as the adhE2 and ldhA genes, and of the PerR regulon, such as rbr3A-rbr3B and dfx, were overexpressed in the mutant. In addition, the whole CA_P0037-CA_P0036 operon was highly expressed under all conditions in the CA_P0037::int (189/190s) mutant, suggesting a self-regulated expression mechanism. Cap0037 was shown to bind to the CA_P0037-CA_P0036 operon, sol operon, and adc promoters, and the binding sites were determined by DNA footprinting. Finally, a putative Cap0037 regulon was generated using a bioinformatic approach. IMPORTANCE: Clostridium acetobutylicum is well-known for its ability to produce solvents, especially n-butanol. Understanding the regulatory network of C. acetobutylicum will be crucial for further engineering to obtain a strain capable of producing n-butanol at high yield and selectivity. This study has discovered that the Cap0037 protein is a novel regulator of C. acetobutylicum that drastically affects metabolism under both acidogenic and alcohologenic fermentation conditions. This is pioneering work for further determining the regulatory mechanism of Cap0037 in C. acetobutylicum and studying the role of proteins homologous to Cap0037 in other members of the phylum Firmicutes.

Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum.

BACKGROUND: Clostridium acetobutylicum is a gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of its genome has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals. RESULTS: The method used in this paper to knock-out or knock-in genes in C. acetobutylicum combines the use of an antibiotic-resistance gene for the deletion or replacement of the target gene, the subsequent elimination of the antibiotic-resistance gene with the flippase recombinase system from Saccharomyces cerevisiae, and a C. acetobutylicum strain that lacks upp, which encodes uracil phosphoribosyl-transferase, for subsequent use as a counter-selectable marker. A replicative vector containing (1) a pIMP13 origin of replication from Bacillus subtilis that is functional in Clostridia, (2) a replacement cassette consisting of an antibiotic resistance gene (MLS (R) ) flanked by two FRT sequences, and (3) two sequences homologous to selected regions around target DNA sequence was first constructed. This vector was successfully used to consecutively delete the Cac824I restriction endonuclease encoding gene (CA_C1502) and the upp gene (CA_C2879) in the C. acetobutylicum ATCC824 chromosome. The resulting C. acetobutylicum Δcac1502Δupp strain is marker-less, readily transformable without any previous plasmid methylation and can serve as the host for the "marker-less" genetic exchange system. The third gene, CA_C3535, shown in this study to encode for a type II restriction enzyme (Cac824II) that recognizes the CTGAAG sequence, was deleted using an upp/5-FU counter-selection strategy to improve the efficiency of the method. The restriction-less marker-less strain and the method was successfully used to delete two genes (ctfAB) on the pSOL1 megaplasmid and one gene (ldhA) on the chromosome to get strains no longer producing acetone or l-lactate. CONCLUSIONS: The restriction-less, marker-less strain described in this study, as well as the maker-less genetic exchange coupled with positive selection, will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.

Optimization of ß-glucosidase production by a strain of Stereum hirsutum and its application in enzymatic saccharification.

A high beta-glucosidase (BGL)-producing strain, Stereum hirsutum, was identified and isolated and showed a maximum BGL activity (10.4 U/ml) when cultured with Avicel and tryptone as the carbon and nitrogen sources, respectively. In comparison with other BGLs, BGL obtained from S. hirsutum showed a higher level of activity to cellobiose (V(max) = 172 U/mg, and k(cat) = 281/s). Under the optimum conditions (600 rpm, 30°C, and pH 6.0), the maximum BGL activity of 10.4 U/ml with the overall productivity of 74.5 U/l/h was observed. BGL production was scaled up from a laboratory scale (7-L fermenter) to a pilot scale (70-L fermenter). When S. hirsutum was cultured in fed-batch culture with rice straw as the carbon source in a 70-L fermenter, a comparable productivity of 78.6 U/l/h was obtained. Furthermore, S. hirsutum showed high levels of activity of other lignocellulases (cellobiohydrolase, endoglucanase, xylanase, and laccase) that are involved in the saccharification of biomasses. Application of S. hirsutum lignocellulases in the hydrolysis of Pinus densiflora and Catalpa ovata showed saccharification yields of 49.7% and 43.0%, respectively, which were higher than the yield obtained using commercial enzymes.

Covalent immobilization of ß-1,4-glucosidase from Agaricus arvensis onto functionalized silicon oxide nanoparticles.

An efficient ß-1,4-glucosidase (BGL) secreting strain, Agaricus arvensis, was isolated and identified. The relative molecular weight of the purified A. arvensis BGL was 98 kDa, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. Using a crude enzyme preparation, A. arvensis BGL was covalently immobilized onto functionalized silicon oxide nanoparticles with an immobilization efficiency of 158%. The apparent V (max) (k (cat)) values of free and immobilized BGL under standard assay conditions were 3,028 U mg protein(-1) (4,945 s(-1)) and 3,347 U mg protein(-1) (5,466 s(-1)), respectively. The immobilized BGL showed a higher optimum temperature and improved thermostability as compared to the free enzyme. The half-life at 65 °C showed a 288-fold improvement over the free BGL. After 25 cycles, the immobilized enzyme still retained 95% of the original activity, thus demonstrating its prospects for commercial applications. High specific activity, high immobilization efficiency, improved stability, and reusability of A. arvensis BGL make this enzyme of potential interest in a number of industrial applications.

Conversion of woody biomass into fermentable sugars by cellulase from Agaricus arvensis.

Agaricus arvensis, a newly isolated basidiomycetous fungus, was found to secrete efficient cellulases. The strain produced the highest endoglucanase (EG), cellobiohydrolase (CBH) and beta-glucosidase (BGL) activities of 0.3, 3.2 and 8U/mg-protein, respectively, with rice straw as the carbon source. Saccharification of the woody biomass with A. arvensis cellulase as the enzyme source released a high level of fermentable sugars. Enzymatic hydrolysis of the poplar biomass was optimized using the response surface methodology in order to study the influence of the variables (pH, temperature, cellulases concentration and substrate concentration). The enzyme and substrate concentrations were identified as the limiting factors for the saccharification of poplar wood biomass. A total reducing sugar level of 29g/L (293mg/g-substrate) was obtained at an enzyme concentration of 65FPU/g-substrate after optimization of the hydrolysis parameters. The model validation showed a good agreement between the experimental results and the predicted responses. A. arvensis could be a good candidate for the production of reducing sugars from a cellulosic biomass.

One-step purification and characterization of a beta-1,4-glucosidase from a newly isolated strain of Stereum hirsutum.

A highly efficient beta-1,4-glucosidase (BGL) secreting strain, Stereum hirsutum SKU512, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. A BGL containing a carbohydrate moiety was purified to homogeneity from S. hirsutum culture supernatants using only a single chromatography step on a gel filtration column. The relative molecular weight of S. hirsutum BGL was determined as 98 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. S. hirsutum BGL showed the highest activity toward p-nitrophenyl-beta-D-glucopyranoside (V (max) = 3,028 U mg-protein(-1), k (cat) = 4,945 s(-1)) ever reported. The enzyme also showed good stability at an acidic pH ranging from 3.0 to 5.5. The BGL was able to promote transglycosylation with an activity of 42.9 U mg-protein(-1) using methanol as an acceptor and glucose as a donor. The internal amino acid sequences of the isolated enzyme showed significant homology with hydrolases from glycoside hydrolase family 1 (GH1), indicating that the S. hirsutum BGL is a member of GH1 family. The characteristics of S. hirsutum BGL could prove to be of interest in several potential applications, especially in enhancing flavor release during the wine fermentation process.