Crosstalk of Cellulose and Mannan Perception Pathways Leads to Inhibition of Cellulase Production in Several Filamentous Fungi.

It is essential for microbes to acquire information about their environment. Fungi use soluble degradation products of plant cell wall components to understand the substrate composition they grow on. Individual perception pathways have been well described. However, the interconnections between pathways remain poorly understood. In the present work, we provide evidence of crosstalk between the perception pathways for cellulose and the hemicellulose mannan being conserved in several filamentous fungi and leading to the inhibition of cellulase expression. We used the functional genomics tools available for Neurospora crassa to investigate this overlap at the molecular level. Crosstalk and competitive inhibition could be identified both during uptake by cellodextrin transporters and intracellularly. Importantly, the overlap is independent of CRE-1-mediated catabolite repression. These results provide novel insights into the regulatory networks of lignocellulolytic fungi and will contribute to the rational optimization of fungal enzyme production for efficient plant biomass depolymerization and utilization.IMPORTANCE In fungi, the production of enzymes for polysaccharide degradation is controlled by complex signaling networks. Previously, these networks were studied in response to simple sugars or single polysaccharides. Here, we tackled for the first time the molecular interplay between two seemingly unrelated perception pathways: those for cellulose and the hemicellulose (gluco)mannan. We identified a so far unknown competitive inhibition between the respective degradation products acting as signaling molecules. Competition was detected both at the level of the uptake and intracellularly, upstream of the main transcriptional regulator CLR-2. Our findings provide novel insights into the molecular communication between perception pathways. Also, they present possible targets for the improvement of industrial strains for higher cellulase production through the engineering of mannan insensitivity.

Cellulose Deficiency Is Enhanced on Hyper Accumulation of Sucrose by a H+-Coupled Sucrose Symporter.

In order to understand factors controlling the synthesis and deposition of cellulose, we have studied the Arabidopsis (Arabidopsis thaliana) double mutant shaven3 shaven3-like1 (shv3svl1), which was shown previously to exhibit a marked cellulose deficiency. We discovered that exogenous sucrose (Suc) in growth medium greatly enhances the reduction in hypocotyl elongation and cellulose content of shv3svl1 This effect was specific to Suc and was not observed with other sugars or osmoticum. Live-cell imaging of fluorescently labeled cellulose synthase complexes revealed a slowing of cellulose synthase complexes in shv3svl1 compared with the wild type that is enhanced in a Suc-conditional manner. Solid-state nuclear magnetic resonance confirmed a cellulose deficiency of shv3svl1 but indicated that cellulose crystallinity was unaffected in the mutant. A genetic suppressor screen identified mutants of the plasma membrane Suc/H(+) symporter SUC1, indicating that the accumulation of Suc underlies the Suc-dependent enhancement of shv3svl1 phenotypes. While other cellulose-deficient mutants were not specifically sensitive to exogenous Suc, the feronia (fer) receptor kinase mutant partially phenocopied shv3svl1 and exhibited a similar Suc-conditional cellulose defect. We demonstrate that shv3svl1, like fer, exhibits a hyperpolarized plasma membrane H(+) gradient that likely underlies the enhanced accumulation of Suc via Suc/H(+) symporters. Enhanced intracellular Suc abundance appears to favor the partitioning of carbon to starch rather than cellulose in both mutants. We conclude that SHV3-like proteins may be involved in signaling during cell expansion that coordinates proton pumping and cellulose synthesis.

PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomyces cerevisiae.

The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.

Identification of MEDIATOR16 as the Arabidopsis COBRA suppressor MONGOOSE1.

We performed a screen for genetic suppressors of cobra, an Arabidopsis mutant with defects in cellulose formation and an increased ratio of unesterified/esterified pectin. We identified a suppressor named mongoose1 (mon1) that suppressed the growth defects of cobra, partially restored cellulose levels, and restored the esterification ratio of pectin to wild-type levels. mon1 was mapped to the MEDIATOR16 (MED16) locus, a tail mediator subunit, also known as SENSITIVE TO FREEZING6 (SFR6). When separated from the cobra mutation, mutations in MED16 caused resistance to cellulose biosynthesis inhibitors, consistent with their ability to suppress the cobra cellulose deficiency. Transcriptome analysis revealed that a number of cell wall genes are misregulated in med16 mutants. Two of these genes encode pectin methylesterase inhibitors, which, when ectopically expressed, partially suppressed the cobra phenotype. This suggests that cellulose biosynthesis can be affected by the esterification levels of pectin, possibly through modifying cell wall integrity or the interaction of pectin and cellulose.

Evaluating endoglucanase Cel7B-lignin interaction mechanisms and kinetics using quartz crystal microgravimetry.

The kinetics and mechanisms of protein interactions with solid surfaces are important to fields as diverse as industrial biocatalysis, biomedical engineering, food science, and cell biology. The nonproductive adsorption of cellulase enzymes to lignin, a plant cell wall polymer, reduces their effectiveness in saccharifying biomass. Cellulase has been shown to interact with lignin, but the heterogeneity of lignin surfaces, challenges in measuring irreversible components of these interactions, and fast adsorption rates make quantifying the reaction kinetics difficult. This work employs quartz crystal microgravimetry with dissipation monitoring (QCM-D) for real-time measurement of adsorbed mass on a flat lignin surface. We have developed a method for casting homogeneous lignin films that are chemically similar to lignin found in pretreated biomass, and used QCM-D to compare three models of reversible-irreversible binding behavior: a single-site transition model, a transition model with changing adsorbate footprint, and a two-site transition model. Of the three models tested, the two-site transition model provides the only kinetic mechanism able to describe the behavior of Cel7B binding to lignin. While the direct implications of lignin-cellulase interactions may be limited to biomass deconstruction for renewable energy and green chemistry, the analytical and experimental methods demonstrated in this work are relevant to any system in which the kinetics and reaction mechanism of reversible and irreversible protein adsorption at a solid-liquid interface are important.

The Arabidopsis COBRA protein facilitates cellulose crystallization at the plasma membrane.

Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual ß1-4-linked glucan chains with a KD of 3.2 µm. Competition assays suggests that COBRA binds individual ß1-4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging ß1-4-glucan chains by acting as a "polysaccharide chaperone."

Solution-state 2D NMR spectroscopy of plant cell walls enabled by a dimethylsulfoxide-d6/1-ethyl-3-methylimidazolium acetate solvent.

Lignocellulosic biomass is composed of the polysaccharides cellulose and hemicellulose and the polyphenol lignin. Many current methods for analyzing the structure of lignocelluloses involve a sequential extraction of the material and subsequent analysis of the resulting fractions, which is labor-intensive and time-consuming. The work presented here assesses the dissolution of whole lignocellulosic material, focusing on biomass derived from the perennial bioenergy grass Miscanthus. The solvent dimethylsulfoxide (DMSO)-d6 containing 1-ethyl-3-methylimidazolium acetate ([Emim]OAc) was able to dissolve lignocellulosic material completely and gave high-resolution 2D heteronuclear single quantum coherence (HSQC) NMR spectra of the entire array of wall polymers. Extrapolated time-zero HSQC was applied using DMSO-d6/[Emim]OAc-d14 and enabled quantitative analysis of structural traits of lignocellulose components.

Characterization of Miscanthus giganteus lignin isolated by ethanol organosolv process under reflux condition.

Miscanthus giganteus lignin was extracted by an organosolv process under reflux conditions (4 h) with varying concentrations of ethanol (65%, 75%, 85%, 95%) and 0.2 M hydrochloric acid as catalyst. The resulting lignin was extensively characterized by size exclusion chromatography (SEC), Fourier-transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC/MS), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR), and chemical analysis (residual sugars, Klason lignin, ash). The predominant linkage units present were ß-O-4' (82-84%), resinol (6-7%), and phenylcoumaran (10-11%). The 65% ethanol solvent system gave the lowest lignin yield (14% of starting biomass) compared to 29-32% of the other systems. Increasing ethanol concentration resulted in decreasing carbohydrate content of the lignins (3.6-1.1%), a higher solubility in tetrahydrofuran (THF), a slight reduction of the molecular weight (M(w) 2.72-2.25 KDa), an increasing α-ethoxylation, and an increase in ethoxylated phenylpropenoic compounds (p-coumaric and ferulic acid), but the S/G ratio of the monolignols (0.63, GC/MS) and Klason lignin content (86-88%) were unaffected. An extraction method for these ethyl-esterified phenylpropenoids and smaller molecular weight lignin compounds was developed. The effect of reaction time (2, 4, and 8 h) was investigated for the 95% ethanol solvent system. Besides increased lignin yield (13-43%), a slight increase in M(w) (2.21-2.38 kDa) and S/G ratio (0.53-0.68, GC-MS) was observed. Consecutive extractions suggested that these changes were not from lignin modifications (e.g., condensations) but rather from extraction of lignin of different composition. The results were compared to similar solvent systems with 95% acetone and 95% dioxane.

Isohalitulin and haliclorensins B and C, three marine alkaloids from Haliclona tulearensis.

Three new alkaloids, designated isohalitulin (4), haliclorensin B (5), and haliclorensin C (6), were isolated from two specimens of the Madagascan sponge Haliclona tulearensis, collected at two locations in Salary Bay, north of Tulear. Their structures were elucidated by extensive spectroscopic means. Alkaloids 4-6 exhibited mild toxicity in the brine shrimp test.

Saldedines A and B, dibromo proaporphine alkaloids from a Madagascan tunicate.

Two new dibromo proaporphine alkaloids, designated saldedines A (1) and B (2), were isolated from an unidentified tunicate collected at Salary Bay, Madagascar. Saldedines A and B are the first marine proaporphine alkaloids. Their structures were elucidated by extensive spectroscopic means, and the structure of 1 was confirmed by single-crystal X-ray diffraction analysis. Both saldedines A and B were tested for toxicity to brine shrimp and showed moderate activity.

Nuttingins A-F and malonganenones D-H, tetraprenylated alkaloids from the Tanzanian gorgonian Euplexaura nuttingi.

Six new tetraprenylated purine alkaloids, designated nuttingins A-F (1-6), were isolated together with three known malonganenones, A-C (12-14), and five new closely related malonganenones, D-H (7-11), from the gorgonian Euplexaura nuttingi collected in Pemba Island, Tanzania. The structures of the compounds were elucidated by interpretation of 1D and 2D NMR data including 15N chemical shifts obtained from 1H-15N HMBC spectra. Nuttingins A-E (1-5) and malonganenones D-G (7-10) displayed inhibitory activity against both K562 and UT7 tumor cell lines, compounds 3-5 being the most active ones, approximately 3-fold more potent than the others. Compounds 1-5 and 7-11 also induce apoptosis in transformed mammalian cells at a concentration of 1.25 microg/mL.