Efficient and Economic Utilization of Cellobiose for Glycosylation Modification by Regulating Carbon Source Supply and Metabolic Pathway In Vivo.

Glycosylation, one of the most common and significant modifications in nature, has prompted the development of a cellobiose phosphorolysis route for glycosylation in vivo. However, the process of glycosylation is hampered by the notably low conversion rate of cellobiose. In this work, regulation of the carbon source supply by changing the ratio of glucose to cellobiose improved the conversion rate of cellobiose, resulting in enhancing the efficiency of glycosylation and the production of vitexin. Moreover, three genes (pgm, agp, and ushA) involved in the degradation of UDP-glucose were knocked out to relieve the degradation and diversion of the cellobiose phosphorolysis route. Finally, through the optimization of conversion conditions, we observed a continuous enhancement in cellobiose conversion rate and vitexin production in BL21ΔushAΔagp-TcCGT-CepA, corresponding to an increased concentration of added glucose. The maximum production of vitexin reached 2228 mg/L with the addition of 2 g/L cellobiose and 6 g/L glucose, which was 312% of that in BL21-TcCGT-CepA with the addition of 2 g/L cellobiose. The conversion rate of cellobiose in BL21ΔushAΔagp-TcCGT-CepA reached 88%, which was the highest conversion rate of cellobiose to date. Therefore, this study presents a cost-effective and efficient method to enhance the conversion rate of cellobiose during the glycosylation process.

Cost-effective simultaneous saccharification and fermentation of l-lactic acid from bagasse sulfite pulp by Bacillus coagulans CC17.

The main barriers to cost-effective lactic acid production from lignocellulose are the high cost of enzymes and the ineffective utilization of the xylose within the hydrolysate. In the present study, the thermophilic Bacillus coagulans strain CC17 was used for the simultaneous saccharification and fermentation (SSF) of bagasse sulfite pulp (BSP) to produce l-lactic acid. Unexpectedly, SSF by CC17 required approximately 33.33% less fungal cellulase than did separate hydrolysis and fermentation (SHF). More interestingly, CC17 can co-ferment cellobiose and xylose without any exogenous ß-glucosidase in SSF. Moreover, adding xylanase could increase the concentration of lactic acid produced via SSF. Up to 110g/L of l-lactic acid was obtained using fed-batch SSF, resulting in a lactic acid yield of 0.72g/g cellulose. These results suggest that SSF using CC17 has a remarkable advantage over SHF and that a potentially low-cost and highly-efficient fermentation process can be established using this protocol.

Heterologously expressed Aspergillus aculeatus ß-glucosidase in Saccharomyces cerevisiae is a cost-effective alternative to commercial supplementation of ß-glucosidase in industrial ethanol production using Trichoderma reesei cellulases.

Trichoderma reesei is a filamentous organism that secretes enzymes capable of degrading cellulose to cellobiose. The culture supernatant of T. reesei, however, lacks sufficient activity to convert cellobiose to glucose using ß-glucosidase (BGL1). In this study, we identified a BGL (Cel3B) from T. reesei (TrCel3B) and compared it with the active ß-glucosidases from Aspergillus aculeatus (AaBGL1). AaBGL1 showed higher stability and conversion of sugars to ethanol compared to TrCel3B, and therefore we chose to express this recombinant protein for use in fermentation processes. We expressed the recombinant protein in the yeast Saccharomyces cerevisiae, combined it with the superb T. reesei cellulase machinery and used the combination in a simultaneous saccharification and fermentation (SSF) process, with the hope that the recombinant would supplement the BGL activity. As the sugars were processed, the yeast immediately converted them to ethanol, thereby eliminating the problem posed by end product inhibition. Recombinant AaBGL1 activity was compared with Novozyme 188, a commercially available supplement for BGL activity. Our results show that the recombinant protein is as effective as the commercial supplement and can process sugars with equal efficiency. Expression of AaBGL1 in S. cerevisiae increased ethanol production effectively. Thus, heterologous expression of AaBGL1 in S. cerevisiae is a cost-effective and efficient process for the bioconversion of ethanol from lignocellulosic biomass.

Modular system for assessment of glycosyl hydrolase secretion in Geobacillus thermoglucosidasius.

The facultatively anaerobic, thermophilic bacterium Geobacillus thermoglucosidasius is being developed as an industrial micro-organism for cellulosic bioethanol production. Process improvement would be gained by enhanced secretion of glycosyl hydrolases. Here we report the construction of a modular system for combining promoters, signal peptide encoding regions and glycosyl hydrolase genes to facilitate selection of the optimal combination in G. thermoglucosidasius. Initially, a minimal three-part E. coli-Geobacillus sp. shuttle vector pUCG3.8 was constructed using Gibson isothermal DNA assembly. The three PCR amplicons contained the pMB1 E. coli origin of replication and multiple cloning site (MCS) of pUC18, the Geobacillus sp. origin of replication pBST1 and the thermostable kanamycin nucleotidyltransferase gene (knt), respectively. G. thermoglucosidasius could be transformed with pUCG3.8 at an increased efficiency [2.8×10(5) c.f.u. (µg DNA)(-1)] compared to a previously reported shuttle vector, pUCG18. A modular cassette for the inducible expression and secretion of proteins in G. thermoglucosidasius, designed to allow the simple interchange of parts, was demonstrated using the endoglucanase Cel5A from Thermotoga maritima as a secretion target. Expression of cel5A was placed under the control of a cellobiose-inducible promoter (Pßglu) together with a signal peptide encoding sequence from a G. thermoglucosidasius C56-YS93 endo-ß-1,4-xylanase. The interchange of parts was demonstrated by exchanging the cel5A gene with the 3' region of a gene with homology to celA from Caldicellulosiruptor saccharolyticus and substituting Pßglu for the synthetic, constitutive promoter PUp2n38, which increased Cel5A activity five-fold. Cel5A and CelA activities were detected in culture supernatants indicating successful expression and secretion. N-terminal protein sequencing of Cel5A carrying a C-terminal FLAG epitope confirmed processing of the signal peptide sequence.

Dekkera/Brettanomyces yeasts for ethanol production from renewable sources under oxygen-limited and low-pH conditions.

Industrial fermentation of lignocellulosic hydrolysates to ethanol requires microorganisms able to utilise a broad range of carbon sources and generate ethanol at high yield and productivity. D. bruxellensis has recently been reported to contaminate commercial ethanol processes, where it competes with Saccharomyces cerevisiae [4, 26]. In this work Brettanomyces/Dekkera yeasts were studied to explore their potential to produce ethanol from renewable sources under conditions suitable for industrial processes, such as oxygen-limited and low-pH conditions. Over 50 strains were analysed for their ability to utilise a variety of carbon sources, and some strains grew on cellobiose and pentoses. Two strains of D. bruxellensis were able to produce ethanol at high yield (0.44 g g(-1) glucose), comparable to those reported for S. cerevisiae. B. naardenensis was shown to be able to produce ethanol from xylose. To obtain ethanol from synthetic lignocellulosic hydrolysates we developed a two-step fermentation strategy: the first step under aerobic conditions for fast production of biomass from mixtures of hexoses and pentoses, followed by a second step under oxygen limitation to promote ethanol production. Under these conditions we obtained biomass and ethanol production on synthetic lignocellulosic hydrolysates, with ethanol yields ranging from 0.2 to 0.3 g g(-1) sugar. Hexoses, xylose and arabinose were consumed at the end of the process, resulting in 13 g l(-1) of ethanol, even in the presence of furfural. Our studies showed that Brettanomyces/Dekkera yeasts have clear potential for further development for industrial processes aimed at production of ethanol from renewable sources.

Assessment of saccharification efficacy in the cellulase system of the brown rot fungus Gloeophyllum trabeum.

Brown rot fungi uniquely degrade wood by creating modifications thought to aid in the selective removal of polysaccharides by an incomplete cellulase suite. This naturally successful mechanism offers potential for current bioprocessing applications. To test the efficacy of brown rot cellulases, southern yellow pine wood blocks were first degraded by the brown rot fungus Gloeophyllum trabeum for 0, 2, 4, and 6 weeks. Characterization of the pine constituents revealed brown rot decay patterns, with selective polysaccharide removal as lignin compositions increased. G. trabeum liquid and solid state cellulase extracts, as well as a commercial Trichoderma reesei extract (Celluclast 1.5 L), were used to saccharify this pretreated material, using beta-glucosidase amendment to remove limitation of cellobiose-to-glucose conversion. Conditions varied according to source and concentration of cellulase extract and to pH (3.0 vs. 4.8). Hydrolysis yields were maximized using solid state G. trabeum extracts at a pH of 4.8. However, the extent of glucose release was low and was not significantly altered when cellulase loading levels were increased threefold. Furthermore, Celluclast 1.5 L continually outperformed G. trabeum cellulase extracts, although extent of glucose release never exceeded 22.0%. Results suggest methodological advances for utilizing crude G. trabeum cellulases and imply that the suboptimal hydrolysis levels obtained with G. trabeum and Celluclast 1.5 L cellulases, even at high loading levels, may be due to brown rot modifications insufficiently distributed throughout the pretreated material.

[Industrial exploitation of renewable resources: from ethanol production to bioproducts development]. / Valorisation des ressources renouvelables : de la production d'éthanol au développement de nouveaux bioproduits.

Plants, which are one of major groups of life forms, are constituted of an amazing number of molecules such as sugars, proteins, phenolic compounds etc. These molecules display multiple and complementary properties involved in various compartments of plants (structure, storage, biological activity etc.). The first uses of plants in industry were for food and feed, paper manufacturing or combustion. In the coming decades, these renewable biological materials will be the basis of a new concept: the "biorefiner" i.e. the chemical conversion of the whole plant to various products and uses. This concept, born in the 90ies, is analogous to today's petroleum refinery, which produces multiple fuels and derivative products from petroleum. Agriculture generates lots of co-products which were most often wasted. The rational use of these wasted products, which can be considered as valuable renewable materials, is now economically interesting and will contribute to the reduction of greenhouse has emissions by partially substituting for fossil fuels. Such substructures from biological waste products and transforming them into biofuels and new industrial products named "bioproducts". These compounds, such as bioplastics or biosurfactants, can replace equivalent petroleum derivatives. Towards that goal, lots of filamentous fungi, growing on a broad range of vegetable species, are able to produce enzymes adapted to the modification of these type of substrates. The best example, at least the more industrially developed to date, is the second generation biofuel technology using cellulose as a raw material. The process includes an enzymatic hydrolysis step which requires cellulases secreted from Trichoderma fungal species. This industrial development of a renewable energy will contribute to the diversification of energy sources used to transport and to the development of green chemistry which will partially substitute petrochemicals.

Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme.

The crystal structure of the catalytic domain of alkaline cellulase K was determined at 1.9 A resolution. Because of the most alkaliphilic nature and it's highest activity at pH 9.5, it is used commercially in laundry detergents. An analysis of the structural bases of the alkaliphilic character of the enzyme suggested a mechanism similar to that previously proposed for alkaline proteases, that is, an increase in the number of Arg, His, and Gln residues, and a decrease in Asp and Lys residues. Some ion pairs were formed by the gained Arg residues, which is similar to what has been found in the alkaline proteases. Lys-Asp ion pairs are disfavored and partly replaced with Arg-Asp ion pairs. The alkaline adaptation appeared to be a remodeling of ion pairs so that the charge balance is kept in the high pH range.

Analysis of glycosidase-catalyzed transglycosylation reaction using probabilistic model.

General mechanism of transglycosylation reaction by glycosidases contains branched paths to form and destroy the glycosylated intermediate. The probabilistic model was applied for the simulation and analysis of the transglycosylation mechanism. The model is composed of a single enzyme molecule and finite amounts of substrates and water molecules mimicking the possible smallest enzyme-catalyzed reaction system in a microcompartment. Using random numbers and probabilities, progress of distribution of reactants and products can be simulated and predicted with minimum adjustable parameters. Experimental data of beta-xylosidase and beta-glucosidase reactions were quantitatively analyzed with the simple scheme. Since the algorithm and simulation procedures are simple, the model is applicable to related complicated enzyme mechanisms containing many branched reaction paths.

Assessment of small intestinal damage in patients treated with pelvic radiotherapy.

Pelvic radiotherapy almost always induces intestinal symptoms. We investigated the radiation-induced damage to the small intestinal mucosa and evaluated its relationship with symptoms, using cellobiose/mannitol permeability test (CE/MA) and plasma postheparin diamine oxidase test (PHD) in 20 patients treated with pelvic radiotherapy. The symptoms developed during radiotherapy were noted. Intestinal permeability significantly (p=0.013) increased from 0.021 +/- 0.026 to 0.047 +/- 0.055 (mean +/- SD) after 15 days of radiotherapy, while it returned to normal values (0.010 0.015) at the end of radiotherapy. PHD values did not change. All patients developed intestinal symptoms. These findings indicate that pelvic radiotherapy induces an early small bowel mucosa damage followed by mucosal adaptation. Acute intestinal symptoms during pelvic radiotherapy may not depend only on small intestinal mucosal damage.

Assessment of intestinal permeability in the experimental rat with [3H]cellobiotol and [14C]mannitol.

1. The intestinal absorption of a mixture of [3H]cellobiotol and [14C]mannitol was determined by measuring the 3H/14C ratio in urine after oral administration of the labelled sugars to rats. This index of intestinal permeability was used to identify cellular dysfunction in experimental enteropathy in rats. 2. Rats with mucosal damage induced with ethanol showed an increased uptake of [3H]cellobiotol and a decreased uptake of [14C]mannitol compared with normal controls. The increased 3H/14C ratio in urine reflected the abnormal cell function known to be caused by ethanol. 3. Methotrexate treatment reduced the absorption of the two sugars whereas cetrimide treatment enhanced their absorption. However, in both methotrexate-induced and cetrimide-induced enteropathy the 3H/14C ratio in urine was unaffected by the mucosal damage. Here the permeability change was not related to absorptive dysfunction of the mucosal cells. 4. It is concluded that this labelled sugar absorption test enables the rapid and accurate identification of malfunction of intestinal mucosal cells in the rat. Moreover, the test distinguishes between changes in permeability caused by abnormal cell function and changes caused by gross disturbance of the structure of the mucosal surface.