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Destructuring plant biomass: focus on fungal and extremophilic cell wall hydrolases.

ABSTRACT: The use of plant biomass as feedstock for biomaterial and biofuel production is relevant in the current bio-based economy scenario of valorizing renewable resources. Fungi, which degrade complex and recalcitrant plant polymers, secrete different enzymes that hydrolyze plant cell wall polysaccharides. The present review discusses the current research trends on fungal, as well as extremophilic cell wall hydrolases that can withstand extreme physico-chemical conditions required in efficient industrial processes. Secretomes of fungi from the phyla Ascomycota, Basidiomycota, Zygomycota and Neocallimastigomycota are presented along with metabolic cues (nutrient sensing, coordination of carbon and nitrogen metabolism) affecting their composition. We conclude the review by suggesting further research avenues focused on the one hand on a comprehensive analysis of the physiology and epigenetics underlying cell wall degrading enzyme production in fungi and on the other hand on the analysis of proteins with unknown function and metagenomics of extremophilic consortia. The current advances in consolidated bioprocessing, altered secretory pathways and creation of designer plants are also examined. Furthermore, recent developments in enhancing the activity, stability and reusability of enzymes based on synergistic, proximity and entropic effects, fusion enzymes, structure-guided recombination between homologous enzymes and magnetic enzymes are considered with a view to improving saccharification.

SUBMITTER: Guerriero G

PROVIDER: S-EPMC4937988 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Destructuring plant biomass: focus on fungal and extremophilic cell wall hydrolases.

Guerriero Gea G Hausman Jean-Francois JF Strauss Joseph J Ertan Haluk H Siddiqui Khawar Sohail KS

Plant science : an international journal of experimental plant biology 20150225

The use of plant biomass as feedstock for biomaterial and biofuel production is relevant in the current bio-based economy scenario of valorizing renewable resources. Fungi, which degrade complex and recalcitrant plant polymers, secrete different enzymes that hydrolyze plant cell wall polysaccharides. The present review discusses the current research trends on fungal, as well as extremophilic cell wall hydrolases that can withstand extreme physico-chemical conditions required in efficient industr ...[more]

PMID: 25804821

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Project description:BackgroundBiofuel use is one of many means of addressing global change caused by anthropogenic release of fossil fuel carbon dioxide into Earth's atmosphere. To make a meaningful reduction in fossil fuel use, bioethanol must be produced from the entire plant rather than only its starch or sugars. Enzymes produced by fungi constitute a significant percentage of the cost of bioethanol production from non-starch (i.e., lignocellulosic) components of energy crops and agricultural residues. We, and others, have reasoned that fungi that naturally deconstruct plant walls may provide the best enzymes for bioconversion of energy crops.ResultsPreviously, we have reported on the isolation of 106 fungi from decaying leaves of Miscanthus and sugarcane (Appl Environ Microbiol 77:5490-504, 2011). Here, we thoroughly analyze 30 of these fungi including those most often found on decaying leaves and stems of these plants, as well as four fungi chosen because they are well-studied for their plant cell wall deconstructing enzymes, for wood decay, or for genetic regulation of plant cell wall deconstruction. We extend our analysis to assess not only their ability over an 8-week period to bioconvert Miscanthus cell walls but also their ability to secrete total protein, to secrete enzymes with the activities of xylanases, exocellulases, endocellulases, and beta-glucosidases, and to remove specific parts of Miscanthus cell walls, that is, glucan, xylan, arabinan, and lignin.ConclusionThis study of fungi that bioconvert energy crops is significant because 30 fungi were studied, because the fungi were isolated from decaying energy grasses, because enzyme activity and removal of plant cell wall components were recorded in addition to biomass conversion, and because the study period was 2 months. Each of these factors make our study the most thorough to date, and we discovered fungi that are significantly superior on all counts to the most widely used, industrial bioconversion fungus, Trichoderma reesei. Many of the best fungi that we found are in taxonomic groups that have not been exploited for industrial bioconversion and the cultures are available from the Centraalbureau voor Schimmelcultures in Utrecht, Netherlands, for all to use.

Project description:This study was a proteomic and secretomic survey of the Gram-negative saprophytic bacterium Cellvibio japonicus during growth using fungal polysaccharides or authentic fungal biomass as a sole nutrient source. The purified polysaccharides were beta-glucan (derived from yeast) and alpha chitin (derived from shrimp shells). The authentic fungal biomass came from Saccharomyces cerevisiae and Aspergillus nidulans. The proteome and secretome samples were taken during mid-exponential growth in biological triplicate. Bacterial cells were homogenized via probe sonication and reduced and alkylated followed by trypsin digestion using a suspension trap (S-trap). Supernatants were reduced and alkylated and followed by trypsin digestion via MStern Blotting. An SPQC was made by combining equal volumes of each sample for both sample types. After lyophilization and reconstitution, 2 ul of cell digests and 15 ul of supernatant digests were loaded onto Evotips. Quantitative LC/MS/MS was performed using an Evosep One LC coupled to a Thermo Orbitrap Astral via a Nanospray Flex ionization source. Samples were block randomized and interspersed with the SPQC replicates. The Evosep used a 60 sample-per-day method with a Pepsep 8 cm x 150 um column (1.5 um particle size) with a PepSep Sprayer and stainless steel (30 um) emitter. The MS analysis used a 240,000 resolution precursor ion (MS1) scan from 380-980 m/z, AGC target of 500% and maximum injection time (IT) of 50 ms, collected every 0.6 s in centroid mode. MS/MS was performed using a DIA method with default charge state = 3, precursor mass range of 380-980, 10 m/z isolation windows, AGC target of 500% and maximum IT of 20 ms, and a NCE of 28. An RF lens of 40% was used for MS1 and DIA scans. Raw MS data was converted to .htrms format using HTRMS converter and processed in Spectronaut 19 (Biognosys). A spectral library was built using direct-DIA searches of all DIA analyses using a Cellvibrio japonicus database, downloaded from Uniprot on 10/29/2024 and appended contaminant sequences using FragPipeSearch settings included N-terminal trypsin/P specificity up to 2 missed cleavages; peptide length from 7-52 amino acids with the following modifications: acetyl (N-term) and carbamidomethyl(Cys). For DIA analysis, default extraction, calibration, identification, and protein inference settings were used.

Project description:Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay and pathogenesis as well as symbiotic associations. In many of these processes, fungi secrete carbohydrate-active enzymes (CAZymes) to modify and/or degrade carbohydrates. Ten years ago, while evaluating the potential of a secretome from the maize pathogen Ustilago maydis to supplement lignocellulolytic cocktails, we noticed it contained many unknown or poorly characterized CAZymes. Here, and after reannotation of this data set and detailed phylogenetic analyses, we observed that several CAZymes (including glycoside hydrolases and carbohydrate oxidases) are predicted to act on the fungal cell wall (FCW), notably on β-1,3-glucans. We heterologously produced and biochemically characterized two new CAZymes, called UmGH16_1-A and UmAA3_2-A. We show that UmGH16_1-A displays β-1,3-glucanase activity, with a preference for β-1,3-glucans with short β-1,6 substitutions, and UmAA3_2-A is a dehydrogenase catalyzing the oxidation of β-1,3- and β-1,6-gluco-oligosaccharides into the corresponding aldonic acids. Working on model β-1,3-glucans, we show that the linear oligosaccharide products released by UmGH16_1-A are further oxidized by UmAA3_2-A, bringing to light a putative biocatalytic cascade. Interestingly, analysis of available transcriptomics data indicates that both UmGH16_1-A and UmAA3_2-A are coexpressed, only during early stages of U. maydis infection cycle. Altogether, our results suggest that both enzymes are connected and that additional accessory activities still need to be uncovered to fully understand the biocatalytic cascade at play and its physiological role. IMPORTANCE Filamentous fungi play a central regulatory role on Earth, notably in the global carbon cycle. Regardless of their lifestyle, filamentous fungi need to remodel their own cell wall (mostly composed of polysaccharides) to grow and proliferate. To do so, they must secrete a large arsenal of enzymes, most notably carbohydrate-active enzymes (CAZymes). However, research on fungal CAZymes over past decades has mainly focused on finding efficient plant biomass conversion processes while CAZymes directed at the fungus itself have remained little explored. In the present study, using the maize pathogen Ustilago maydis as model, we set off to evaluate the prevalence of CAZymes directed toward the fungal cell wall during growth of the fungus on plant biomass and characterized two new CAZymes active on fungal cell wall components. Our results suggest the existence of a biocatalytic cascade that remains to be fully understood.

Project description:BackgroundPlant lignocellulosic biomass is an abundant, renewable feedstock for the production of biobased fuels and chemicals. Previously, we showed that iron can act as a co-catalyst to improve the deconstruction of lignocellulosic biomass. However, directly adding iron catalysts into biomass prior to pretreatment is diffusion limited, and increases the cost of biorefinery operations. Recently, we developed a new strategy for expressing iron-storage protein ferritin intracellularly to accumulate iron as a catalyst for the downstream deconstruction of lignocellulosic biomass. In this study, we extend this approach by fusing the heterologous ferritin gene with a signal peptide for secretion into Arabidopsis cell walls (referred to here as FerEX).ResultsThe transgenic Arabidopsis plants. FerEX. accumulated iron under both normal and iron-fertilized growth conditions; under the latter (iron-fertilized) condition, FerEX transgenic plants showed an increase in plant height and dry weight by 12 and 18 %, respectively, compared with the empty vector control plants. The SDS- and native-PAGE separation of cell-wall protein extracts followed by Western blot analyses confirmed the extracellular expression of ferritin in FerEX plants. Meanwhile, Perls' Prussian blue staining and X-ray fluorescence microscopy (XFM) maps revealed iron depositions in both the secondary and compound middle lamellae cell-wall layers, as well as in some of the corner compound middle lamella in FerEX. Remarkably, their harvested biomasses showed enhanced pretreatability and digestibility, releasing, respectively, 21 % more glucose and 34 % more xylose than the empty vector control plants. These values are significantly higher than those of our recently obtained ferritin intracellularly expressed plants.ConclusionsThis study demonstrated that extracellular expression of ferritin in Arabidopsis can produce plants with increased growth and iron accumulation, and reduced thermal and enzymatic recalcitrance. The results are attributed to the intimate colocation of the iron co-catalyst and the cellulose and hemicellulose within the plant cell-wall region, supporting the genetic modification strategy for incorporating conversion catalysts into energy crops prior to harvesting or processing at the biorefinery.

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Destructuring plant biomass: focus on fungal and extremophilic cell wall hydrolases.

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Destructuring plant biomass: focus on fungal and extremophilic cell wall hydrolases.

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