Systems analysis in Cellvibrio japonicus resolves predicted redundancy of ß-glucosidases and determines essential physiological functions.

Degradation of polysaccharides forms an essential arc in the carbon cycle, provides a percentage of our daily caloric intake, and is a major driver in the renewable chemical industry. Microorganisms proficient at degrading insoluble polysaccharides possess large numbers of carbohydrate active enzymes (CAZymes), many of which have been categorized as functionally redundant. Here we present data that suggests that CAZymes that have overlapping enzymatic activities can have unique, non-overlapping biological functions in the cell. Our comprehensive study to understand cellodextrin utilization in the soil saprophyte Cellvibrio japonicus found that only one of four predicted ß-glucosidases is required in a physiological context. Gene deletion analysis indicated that only the cel3B gene product is essential for efficient cellodextrin utilization in C. japonicus and is constitutively expressed at high levels. Interestingly, expression of individual ß-glucosidases in Escherichia coli K-12 enabled this non-cellulolytic bacterium to be fully capable of using cellobiose as a sole carbon source. Furthermore, enzyme kinetic studies indicated that the Cel3A enzyme is significantly more active than the Cel3B enzyme on the oligosaccharides but not disaccharides. Our approach for parsing related CAZymes to determine actual physiological roles in the cell can be applied to other polysaccharide-degradation systems.

Lotus japonicus flowers are defended by a cyanogenic ß-glucosidase with highly restricted expression to essential reproductive organs.

Flowers and leaves of Lotus japonicus contain α-, ß-, and γ-hydroxynitrile glucoside (HNG) defense compounds, which are bioactivated by ß-glucosidase enzymes (BGDs). The α-HNGs are referred to as cyanogenic glucosides because their hydrolysis upon tissue disruption leads to release of toxic hydrogen cyanide gas, which can deter herbivore feeding. BGD2 and BGD4 are HNG metabolizing BGD enzymes expressed in leaves. Only BGD2 is able to hydrolyse the α-HNGs. Loss of function mutants of BGD2 are acyanogenic in leaves but fully retain cyanogenesis in flowers pointing to the existence of an alternative cyanogenic BGD in flowers. This enzyme, named BGD3, is identified and characterized in this study. Whereas all floral tissues contain α-HNGs, only those tissues in which BGD3 is expressed, the keel and the enclosed reproductive organs, are cyanogenic. Biochemical analysis, active site architecture molecular modelling, and the observation that L. japonicus accessions lacking cyanogenic flowers contain a non-functional BGD3 gene, all support the key role of BGD3 in floral cyanogenesis. The nectar of L. japonicus flowers was also found to contain HNGs and additionally their diglycosides. The observed specialisation in HNG based defence in L. japonicus flowers is discussed in the context of balancing the attraction of pollinators with the protection of reproductive structures against herbivores.

Carbohydrate-binding modules of fungal cellulases: occurrence in nature, function, and relevance in industrial biomass conversion.

In this review, the present knowledge on the occurrence of cellulases, with a special emphasis on the presence of carbohydrate-binding modules (CBMs) in various fungal strains, has been summarized. The importance of efficient fungal cellulases is growing due to their potential uses in biorefinery processes where lignocellulosic biomasses are converted to platform sugars and further to biofuels and chemicals. Most secreted cellulases studied in detail have a bimodular structure containing an active core domain attached to a CBM. CBMs are traditionally been considered as essential parts in cellulases, especially in cellobiohydrolases. However, presently available genome data indicate that many cellulases lack the binding domains in cellulose-degrading organisms. Recent data also demonstrate that CBMs are not necessary for the action of cellulases and they solely increase the concentration of enzymes on the substrate surfaces. On the other hand, in practical industrial processes where high substrate concentrations with low amounts of water are employed, the enzymes have been shown to act equally efficiently with and without CBM. Furthermore, available kinetic data show that enzymes without CBMs can desorb more readily from the often lignaceous substrates, that is, they are not stuck on the substrates and are thus available for new actions. In this review, the available data on the natural habitats of different wood-degrading organisms (with emphasis on the amount of water present during wood degradation) and occurrence of cellulose-binding domains in their genome have been assessed in order to identify evolutionary advantages for the development of CBM-less cellulases in nature.

Engineering microbial surfaces to degrade lignocellulosic biomass.

Renewable lignocellulosic plant biomass is a promising feedstock from which to produce biofuels, chemicals, and materials. One approach to cost-effectively exploit this resource is to use consolidating bioprocessing (CBP) microbes that directly convert lignocellulose into valuable end products. Because many promising CBP-enabling microbes are non-cellulolytic, recent work has sought to engineer them to display multi-cellulase containing minicellulosomes that hydrolyze biomass more efficiently than isolated enzymes. In this review, we discuss progress in engineering the surfaces of the model microorganisms: Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. We compare the distinct approaches used to display cellulases and minicellulosomes, as well as their surface enzyme densities and cellulolytic activities. Thus far, minicellulosomes have only been grafted onto the surfaces of B. subtilis and S. cerevisiae, suggesting that the absence of an outer membrane in fungi and Gram-positive bacteria may make their surfaces better suited for displaying the elaborate multi-enzyme complexes needed to efficiently degrade lignocellulose.

Characterization of extracellular cellulose-degrading enzymes from bacillus thuringiensis strains

The gram-positive spore-forming bacteria, Bacillus thuringiensis (Bt) strains produced novel cellulases which could liberate glucose from soluble cellulose, carboxymethyl cellulose (CMC), and insoluble crystalline cellulose. The maximal cellulase activities were obtained after 60 hrs incubation at 28ºC in a LB broth medium with 1 percent CMC. Maximum CMCase activities were got at 40ºC and pH 4.0, respectively, and more than 50 percent of its maximal activity was retained at 40-60ºC for 1 hr, while approximately 40 percent of its maximal activity was also retained after incubating at 70ºC for 1 hr. Most metal ions and reagents such as Ca2+, Mg2+, Cd2+, Pb2+, Zn2+, Cu2+, EDTA, and SDS inhibited the enzyme activities, but K+ and Mn2+ activated the activities. The enzymes from Bacillus thuringiensis strains could be applied in bioconversion of lignocellulosic biomass into fermentable sugars.

Evolution of herbivory in a carnivorous clade of minnows (teleostei: cyprinidae): effects on gut size and digestive physiology.

We constructed a phylogeny for 10 minnow species (family Cyprinidae) previously revealed to be members of sister genera with different dietary affinities and used the phylogeny to examine whether the evolution of digestive tract size and physiology is correlated with the evolution of diet in these fishes. We studied a total of 11 taxa: four herbivorous species in the genus Campostoma and six largely carnivorous species in the genus Nocomis, including two populations of Nocomis leptocephalus, the carnivorous Chattahoochee River drainage population and the herbivorous Altamaha River drainage population. Thus, we were able to compare digestive tract size and physiology among sister genera (Campostoma and Nocomis) and among sister taxa (N. leptocephalus Chattahoochee and N. leptocephalus Altamaha) in dietary and phylogenetic contexts. The herbivorous taxa had longer digestive tracts and higher activity of the carbohydrases amylase and laminarinase in their guts, whereas the carnivorous species had higher chitinase activity. Phylogenetic independent-contrast analysis suggested that the evolution of amylase and chitinase activities was correlated with the evolution of diet in these species, whereas trypsin and lipase activities showed no pattern associated with diet or phylogenetic history. Concentrations of short-chain fatty acids were low in all taxa, indicating that these fishes rely largely on endogenous digestive mechanisms to subsist on their respective diets. Subtle differences in tooth shape were observed between species in the two genera. Overall, our results suggest that dietary specialization can be observed on the level of anatomy and physiology of the digestive tracts of fishes but that such differences are most appropriately viewed in comparisons of closely related species with different diets.

Expression of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco.

An inexpensive source of active cellulases is critical to efficient and cost-effective conversion of lignocellulosic biomass to ethanol. Transgenic plants expressing foreign cellulases are potential sources of cellulases for biomass conversion. A number of foreign proteins have been reported to accumulate to high levels when the transgene is incorporated into the chloroplast genome rather than into the nuclear genome. We developed plastid transformation vectors carrying two Thermobifida fusca thermostable cellulases, Cel6A and Cel6B, and expressed them in nicotine-free or nicotine-containing tobacco varieties following chloroplast transformation. We obtained homoplasmic tobacco plants expressing Cel6A or Cel6B. Maximum estimates of expression levels ranged from 2 to 4% of total soluble protein. Enzyme assays indicated that both Cel6A and Cel6B expressed in transplastomic tobacco were active in hydrolyzing crystalline cellulose. With further optimization, it may be feasible to produce bacterial cellulases in tobacco chloroplasts in large quantities.

Mycobacterium tuberculosis strains possess functional cellulases.

The genomes of various Mycobacterium tuberculosis strains encode proteins that do not appear to play a role in the growth or survival of the bacterium in its mammalian host, including some implicated in plant cell wall breakdown. Here we show that M. tuberculosis H37Rv does indeed possess a functional cellulase. The x-ray crystal structure of this enzyme, in ligand complex forms, from 1.9 to 1.1A resolution, reveals a highly conserved substrate-binding cleft, which affords similar, and unusual, distortion of the substrate at the catalytic center. The endoglucanase activity, together with the existence of a putative membrane-associated crystalline polysaccharide-binding protein, may reflect the ancestral soil origin of the Mycobacterium or hint at a previously unconsidered environmental niche.

Action pattern, specificity, lytic activities, and physiological role of five digestive beta-glucanases isolated from Periplaneta americana.

Three laminarinases (LAM, LIC 1, and LIC 2) and two cellulases (CEL 1 and CEL 2) were purified to homogeneity from Periplaneta americana midguts. These beta-glucanases are secreted by salivary glands, stabilized by calcium ions, and have pH optima around 6. LAM (46 kDa) is active only on laminarin, native or with oxidized ends, and so it is an endo-beta-1,3-glucanase (EC 3.2.1.39). It processively releases mainly glucose from laminarin and shows lytic activity on fungal cells. LIC 1 (25 kDa) is an endo-beta-1,3(4)-glucanase (EC 3.2.1.6.), because it cleaves internal bonds on both laminarin and lichenin. LIC 1 lyses fungal cells and apparently have high affinity for sequences of cellotetraoses linked by beta-1,3 links, releasing cellotetraose from lichenin. The reaction catalyzed by LIC 1 is not in rapid equilibrium, as suggested by activity-pH data. These data also showed that a group in LIC 1 with pK=4.9 is necessary for substrate binding. LIC 2 (23 kDa) seems to be similar to LIC 1. The laminarinases are inactivated by carbodiimide, suggesting the presence of a carboxyl group involved in catalysis. LAM and LIC 2 are inhibited by excess laminarin as substrate. CEL 1 (72 kDa) and CEL 2 (73 kDa) quickly decrease the molecular weight of lichenin used as substrate. Therefore, they are endo-beta-1,4-glucanases (EC 3.2.1.4). Both CEL 1 and CEL 2 are also active on crystalline cellulose. The specificities of P. americana beta-glucanases agree with the omnivorous detritus-feeding habit of this insect, as they are able to attack plant (CEL 1, CEL 2, LIC 1 and LIC 2) and fungal (LIC 1 and LAM) cell walls.