Fusion of cellobiose phosphorylase and potato alpha-glucan phosphorylase facilitates substrate channeling for enzymatic conversion of cellobiose to starch.

We previously reported an in vitro enzymatic pathway for conversion of nonfood cellulose to starch (PNAS,110 (18): 7182-7187, 2013), in which the two sequential enzymes cellobiose phosphorylase (CBP) from Clostridium thermocellum and potato alpha-glucan phosphorylase (PGP) from Solanum tuberosum were the two key enzymes responsible for the whole conversion rate. In this work CBP and PGP were fused to form a large enzyme and it turned out that the fusion protein could exhibit a good bifunctionality when PGP moiety was put at the N-terminus and CBP moiety at the C-terminus (designated as PGP-CBP). Although the coupled reaction rate of PGP-CBP was decreased by 23.0% compared with the free enzymes, substrate channeling between the two active sites in PGP-CBP was formed, demonstrated by the introduction of the competing enzyme of PGP to the reaction system. The potential of PGP-CBP fusion enzyme being applied to the conversion of cellulose to amylose was discussed.

Effect of cellobiose supplementation and dietary soluble fibre content on in vitro caecal fermentation of carbohydrate-rich substrates in rabbits.

The in vitro caecal fermentation of five substrates low in starch and protein content [d-(+)-glucose (GLU), d-cellobiose (CEL), sugar beet pectin (PEC), sugar beet pulp (SBP) and wheat straw (WS)] was investigated using soft faeces from rabbits receiving different levels of cellobiose and soluble fibre as inoculum. A total of 24 rabbits were supplemented 3 levels of cellobiose in the drinking water (0.0, 7.5, 15.0 g/l) and fed two experimental diets containing either low soluble fibre (LSF) or high soluble fibre (HSF) levels (84.0 and 130 g/kg dry matter). All substrates were subjected to a two-step pepsin/pancreatin in vitro pre-digestion, and the whole residue was used as substrate for the in vitro incubations. Gas production was measured until 144 h, and volatile fatty acid (VFA) production was determined at 24 h incubation. Experimental treatments did not affect SBP fermentation and had only a subtle influence on fermentation of WS and GLU. In contrast, cellobiose supplementation × donors' diet interactions were detected for most gas production parameters for CEL. Both the fractional gas production (k) and maximal gas production rates were linearly increased (p ≤ 0.042) and the initial delay in the onset of gas production (Lag) linearly decreased (p < 0.001) by cellobiose supplementation with the HSF inoculum, with no differences between the 7.5 and 15.0 doses. In contrast, with the LSF inoculum cellobiose supplementation only affected k values, which were quadratically increased (p = 0.043) and had maximal values for the 7.5 dose. A quadratic effect (p ≤ 0.018) of cellobiose supplementation was observed for total VFA production at 24 h when CEL and PEC were fermented, obtaining the maximal VFA production for the 7.5 dose of cellobiose. Total VFA production for CEL was greater with LSF than with HSF inoculum (20.7 vs. 12.9 mmol/l; p = 0.014), but the opposite was found for WS (3.97 vs. 6.21 mmol/l; p = 0.005). The use of LSF inoculum for CEL fermentation sharply reduced acetate (p = 0.001) and increased butyrate proportions (p ≤ 0.001) compared with the HSF inoculum. A positive relationship between total VFA caecal concentrations in rabbits receiving the same experimental treatments and in vitro values was only observed when WS was used as substrate (r = 0.90; p = 0.015; n = 6). The results suggest that experimental factors influenced the fermentative activity of caecal digesta, but the observed response differed with the incubated substrate, being the CEL the most affected.

Cellulose-Derived Oligomers Act as Damage-Associated Molecular Patterns and Trigger Defense-Like Responses.

The plant cell wall, often the site of initial encounters between plants and their microbial pathogens, is composed of a complex mixture of cellulose, hemicellulose, and pectin polysaccharides as well as proteins. The concept of damage-associated molecular patterns (DAMPs) was proposed to describe plant elicitors like oligogalacturonides (OGs), which can be derived by the breakdown of the pectin homogalacturon by pectinases. OGs act via many of the same signaling steps as pathogen- or microbe-associated molecular patterns (PAMPs) to elicit defenses and provide protection against pathogens. Given both the complexity of the plant cell wall and the fact that many pathogens secrete a wide range of cell wall-degrading enzymes, we reasoned that the breakdown products of other cell wall polymers may be similarly biologically active as elicitors and may help to reinforce the perception of danger by plant cells. Our results indicate that oligomers derived from cellulose are perceived as signal molecules in Arabidopsis (Arabidopsis thaliana), triggering a signaling cascade that shares some similarities to responses to well-known elicitors such as chitooligomers and OGs. However, in contrast to other known PAMPs/DAMPs, cellobiose stimulates neither detectable reactive oxygen species production nor callose deposition. Confirming our idea that both PAMPs and DAMPs are likely to cooccur at infection sites, cotreatments of cellobiose with flg22 or chitooligomers led to synergistic increases in gene expression. Thus, the perception of cellulose-derived oligomers may participate in cell wall integrity surveillance and represents an additional layer of signaling following plant cell wall breakdown during cell wall remodeling or pathogen attack.

Citrobacter freundii as a test platform for recombinant cellulose degradation systems.

Cellulosic biomass represents a huge reservoir of renewable carbon, but converting it into useful products is challenging. Attempts to transfer cellulose degradation capability to industrially useful micro-organisms have met with limited success, possibly due to poorly understood synergy between multiple cellulases. This is best studied by co-expression of many combinations of cellulases and associated proteins. Here, we describe the development of a test platform based on Citrobacter freundii, a cellobiose-assimilating organism closely related to Escherichia coli. Standard E. coli cloning vectors worked well in Cit. freundii. Expression of cellulases CenA and Cex of Cellulomonas fimi in Cit. freundii gave recombinant strains which were able to grow at the expense of cellulosic filter paper or microcrystalline cellulose (Avicel) in a mineral medium supplemented with a small amount of yeast extract. Periodic physical agitation of the cultures was highly beneficial for growth at the expense of filter paper. This provides a test platform for the expression of combinations of genes encoding biomass-degrading enzymes to develop effective genetic cassettes for degradation of different biomass streams. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofuels have been shown to be the best sustainable and alternative source of fuel to replace fossil fuels. Of the different types of feedstocks used for producing biofuels, lignocellulosic biomass is the most abundant. Converting this biomass to useful products has met with little success. Different approaches are being used and microbial platforms are the most promising and sustainable method. This study shows that Citrobacter freundii is a better test platform than Escherichia coli for testing various combinations of cellulases for the development of microbial systems for biomass conversion.

Overexpression and characterization of a glucose-tolerant ß-glucosidase from T. aotearoense with high specific activity for cellobiose.

Thermoanaerobacterium aotearoense P8G3#4 produced ß-glucosidase (BGL) intracellularly when grown in liquid culture on cellobiose. The gene bgl, encoding ß-glucosidase, was cloned and sequenced. Analysis revealed that the bgl contained an open reading frame of 1314 bp encoding a protein of 446 amino acid residues, and the product belonged to the glycoside hydrolase family 1 with the canonical glycoside hydrolase family 1 (GH1) (ß/α)8 TIM barrel fold. Expression of pET-bgl together with a chaperone gene cloned in vector pGro7 in Escherichia coli dramatically enhanced the crude enzyme activity to a specific activity of 256.3 U/mg wet cells, which resulted in a 9.2-fold increase of that obtained from the expression without any chaperones. The purified BGL exhibited relatively high thermostability and pH stability with its highest activity at 60 °C and pH 6.0. In addition, the activities of BGL were remarkably stimulated by the addition of 5 mM Na(+) or K(+). The enzyme showed strong ability to hydrolyze cellobiose with a K m and V max of 25.45 mM and 740.5 U/mg, respectively. The BGL was activated by glucose at concentration varying from 50 to 250 mM and tolerant to glucose inhibition with a K i of 800 mM glucose. The supplement of the purified BGL to the sugarcane bagasse hydrolysis mixture containing a commercial cellulase resulted in about 20 % enhancement of the released reducing sugars. These properties of the purified BGL should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.

Genomic, proteomic, and biochemical analyses of oleaginous Mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production.

Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 ß-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one ß-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and ß-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

Nitrogen and sulfur requirements for Clostridium thermocellum and Caldicellulosiruptor bescii on cellulosic substrates in minimal nutrient media.

Growth media for cellulolytic Clostridium thermocellum ATCC 27405 and Caldicellulosiruptor bescii bacteria usually contain excess nutrients that would increase costs for consolidated bioprocessing for biofuel production and create a waste stream with nitrogen, sulfur and phosphate. C. thermocellum was grown on crystalline cellulose with varying concentrations of nitrogen and sulfur compounds, and growth rate and ethanol production response curves were determined. Both bacteria assimilated sulfate in the presence of ascorbate reductant, increasing the ratio of oxidized to reduced fermentation products. From these results, a low ionic strength, defined minimal nutrient medium with decreased nitrogen, sulfur, phosphate and vitamin supplements was developed for the fermentation of cellobiose, cellulose and acid-pretreated Populus. Carbon and electron balance calculations indicate the unidentified residual fermentation products must include highly reduced molecules. Both bacterial populations were maintained in co-cultures with substrates containing cellulose and xylan in defined medium with sulfate and basal vitamin supplements.

Impairment of cellulose- and cellobiose-degrading soil Bacteria by two acidic herbicides.

Herbicides have the potential to impair the metabolism of soil microorganisms. The current study addressed the toxic effect of bentazon and 4-chloro-2-methylphenoxyacetic acid on aerobic and anaerobic Bacteria that are involved in cellulose and cellobiose degradation in an agricultural soil. Aerobic saccharide degradation was reduced at concentrations of herbicides above environmental values. Microbial processes (e.g. fermentations, ferric iron reduction) that were linked to anaerobic cellulose and cellobiose degradation were reduced in the presence of both herbicides at concentrations above and at those that occur in crop field soil. 16S rRNA gene transcript numbers of total Bacteria, and selected bacterial taxa (Clostridia [Group I], Planctomycetaceae, and two uncultivated taxa of Bacteroidetes) decreased more in anoxic than in oxic cellulose-supplemented soil microcosms in the presence of both herbicides. Collectively, the results suggested that the metabolism of anaerobic cellulose-degrading Bacteria was impaired by typical in situ herbicide concentrations, whereas in situ concentrations did not impair metabolism of aerobic cellulose- and cellobiose-degrading soil Bacteria.

Enzymatic hydrolysis and simultaneous saccharification and fermentation of alkali/peracetic acid-pretreated sugarcane bagasse for ethanol and 2,3-butanediol production.

The enzymatic digestibility of alkali/peracetic acid (PAA)-pretreated bagasse was systematically investigated. The effects of initial solid consistency, cellulase loading and addition of supplemental ß-glucosidase on the enzymatic conversion of glycan were studied. It was found the alkali-PAA pulp showed excellent enzymatic digestibility. The enzymatic glycan conversion could reach about 80% after 24 h incubation when enzyme loading was 10 FPU/g solid. Simultaneous saccharification and fermentation (SSF) results indicated that the pulp could be well converted to ethanol. Compared with dilute acid pretreated bagasse (DAPB), alkali-PAA pulp could obtain much higher ethanol and xylose concentrations. The fermentation broth still showed some cellulase activity so that the fed pulp could be further converted to sugars and ethanol. After the second batch SSF, the fermentation broth of alkali-PAA pulp still kept about 50% of initial cellulase activity. However, only 21% of initial cellulase activity was kept in the fermentation broth of DAPB. The xylose syrup obtained in SSF of alkali-PAA pulp could be well converted to 2,3-butanediol by Klebsiella pneumoniae CGMCC 1.9131.

[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.

Thaxtomin biosynthesis: the path to plant pathogenicity in the genus Streptomyces.

Streptomyces species are best known for their ability to produce a wide array of medically and agriculturally important secondary metabolites. However, there is a growing number of species which, like Streptomyces scabies, can function as plant pathogens and cause scab disease on economically important crops such as potato. All of these species produce the phytotoxin thaxtomin, a nitrated dipeptide which inhibits cellulose synthesis in expanding plant tissue. The biosynthesis of thaxtomin involves conserved non-ribosomal peptide synthetases, P450 monooxygenases, and a nitric oxide synthase, the latter being required for nitration of the toxin. This nitric oxide synthase is also responsible for the production of diffusible nitric oxide by scab-causing streptomycetes at the host-pathogen interface, suggesting that nitric oxide production might play an additional role during the infection process. The thaxtomin biosynthetic genes are transcriptionally regulated by an AraC/XylS family regulator, TxtR, which is conserved in pathogenic streptomycetes and is encoded within the thaxtomin biosynthetic gene cluster. The TxtR protein specifically binds cellobiose, a known inducer of thaxtomin biosynthesis, and cellobiose is required for expression of the biosynthetic genes. A second virulence gene in pathogenic Streptomyces species, nec1, encodes a novel secreted protein that may suppress plant defence responses. The thaxtomin biosynthetic genes and nec1 are contained on a large mobilizable pathogenicity island; the transfer of this island to recipient streptomycetes likely explains the rapid emergence of new pathogenic species. The newly available genome sequence of S. scabies will provide further insight into the mechanisms utilized by pathogenic streptomycetes during plant-microbe interactions.

Stimulatory and inhibitory effects of protein amino acids on growth rate and efficiency of mixed ruminal bacteria.

Mixed ruminal bacteria were incubated in vitro with glucose, xylose, cellobiose, and various protein amino acids replaced isonitrogenously with 25% (i.e., 25 mg of N/L) of ammonia-N, to determine the growth rate and the amount of sugar consumed in the exponential growth phase. The growth rate and efficiency (grams of bacteria per gram of sugars) increased by 46 and 15%, respectively, when a mixture of 20 amino acids was added. On the other hand, neither growth rate nor efficiency increased when any one of these amino acids was added singly, except for Glu and Gln, each of which produced significant but small improvements. The stimulatory effect of the combined amino acids on bacterial growth declined when each of Leu, Trp, Tyr, Glu, Met, Phe, and Val was removed from the original group of 20. When a mixture of only these seven amino acids was used as a supplement, their stimulatory effects on growth rate and efficiency were only 21 and 25%, respectively, of the effects that the mixture of 20 amino acids showed. The effects increased to 76 and 72% on growth rate and efficiency, respectively, when Gly, Cys, and His were supplied in addition to the seven amino acids. The growth rate and efficiency of the ruminal bacteria were inhibited by an addition of each of Ile, Thr, Cys, Phe, Leu, Lys, or Val to ammonia-N, and the effects of the first five of these amino acids were highly significant. Isoleucine, threonine, and phenylalanine were each inhibitory even at a low concentration (1 mg of NL), while cysteine and leucine showed inhibitory effects at higher concentrations (more than 10 mg of N/L). A higher growth rate of the ruminal bacteria when supplemented with amino acid mixtures was accompanied with a higher growth efficiency, which was attributable to a relatively smaller proportion of energy expended on maintenance according to the Pirt derivation.

Demethylation of [14C]-labelled veratric acid and oxidation of methanol and formaldehyde by the white-rot fungus Phlebia radiata.

Veratric acids 14C-labelled in carboxyl group, 3-OCH3, 4-OCH3, or aromatic ring together with unlabelled veratric acid were supplemented in the cultures of the white-rot fungus Phlebia radiata. The effect of various carbon sources on the release of 14CO2 was studied. Veratric acid was readily decarboxylated, maximally already on day 1 from the addition of [14COOH]-veratric acid. High amounts (4%) of glucose slightly repressed the decarboxylation. In medium supplemented with cellulose the methoxyl group in position 4 was much more readily mineralized to CO2 than the group in position 3. The maximum evolution was achieved on day 5, two days from the addition. Cellulose did not repress methanol oxidation but repression of methanol oxidation by glucose was detected in media supplemented with [O14CH3]-veratric acids and 14CH3OH. However, glucose did not repress oxidation of H14CHO. The apparent uptake of 14C by fungal mycelium, especially from methoxyl groups, but also from the aromatic ring, may partially be due to the strong slime formation observed in cellobiose medium. Also in cellobiose medium apparent uptake of 14C from 14C-labelled methoxyl groups was observed.

Microbial reduction of Fe(III) in acidic sediments: isolation of Acidiphilium cryptum JF-5 capable of coupling the reduction of Fe(III) to the oxidation of glucose.

To evaluate the microbial populations involved in the reduction of Fe(III) in an acidic, iron-rich sediment, the anaerobic flow of supplemental carbon and reductant was evaluated in sediment microcosms at the in situ temperature of 12 degrees C. Supplemental glucose and cellobiose stimulated the formation of Fe(II); 42 and 21% of the reducing equivalents that were theoretically obtained from glucose and cellobiose, respectively, were recovered in Fe(II). Likewise, supplemental H(2) was consumed by acidic sediments and yielded additional amounts of Fe(II) in a ratio of approximately 1:2. In contrast, supplemental lactate did not stimulate the formation of Fe(II). Supplemental acetate was not consumed and inhibited the formation of Fe(II). Most-probable-number estimates demonstrated that glucose-utilizing acidophilic Fe(III)-reducing bacteria approximated to 1% of the total direct counts of 4', 6-diamidino-2-phenylindole-stained bacteria. From the highest growth-positive dilution of the most-probable-number series at pH 2. 3 supplemented with glucose, an isolate, JF-5, that could dissimilate Fe(III) was obtained. JF-5 was an acidophilic, gram-negative, facultative anaerobe that completely oxidized the following substrates via the dissimilation of Fe(III): glucose, fructose, xylose, ethanol, glycerol, malate, glutamate, fumarate, citrate, succinate, and H(2). Growth and the reduction of Fe(III) did not occur in the presence of acetate. Cells of JF-5 grown under Fe(III)-reducing conditions formed blebs, i.e., protrusions that were still in contact with the cytoplasmic membrane. Analysis of the 16S rRNA gene sequence of JF-5 demonstrated that it was closely related to an Australian isolate of Acidiphilium cryptum (99.6% sequence similarity), an organism not previously shown to couple the complete oxidation of sugars to the reduction of Fe(III). These collective results indicate that the in situ reduction of Fe(III) in acidic sediments can be mediated by heterotrophic Acidiphilium species that are capable of coupling the reduction of Fe(III) to the complete oxidation of a large variety of substrates including glucose and H(2).

Incorporation of soya oil hydrolysate in the diet of defaunated or refaunated sheep: effect on rumen fermentation in vitro.

The effects of incorporation in the diet of 7% soya oil hydrolysate (SOH) on in vitro incubations of cellobiose + maltose, maize starch and casein by rumen microbes were studied using defaunated and refaunated sheep as rumen fluid donors. Feeding refaunated sheep the SOH supplemented diet lowered the protozoal numbers in the rumen from 1.61 10(6)/ml to 6.1 10(5)/ml. SOH addition reduced in vitro methane production, rather by a depletion of methanogens is than by a simple inhibition of their activity. This reduction seemed to be independent of protozoa depletion. With cellobiose-maltose and maize starch incubations, SOH supplementation increased molar proportion of propionate while acetate decreased. Both variations could be linked to the inhibition of methanogenesis. Volatile fatty acid production from casein was strongly reduced by SOH supplementation with or without protozoa in the rumen of the donors animals.