Characterisation of the enzyme transport path between shipworms and their bacterial symbionts.

BACKGROUND: Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal's gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. RESULTS: Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. CONCLUSION: Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm's mouth and digestive tract, where they aid in wood digestion.

Discovery, activity and characterisation of an AA10 lytic polysaccharide oxygenase from the shipworm symbiont Teredinibacter turnerae.

BACKGROUND: The quest for novel enzymes for cellulosic biomass-degradation has recently been focussed on lytic polysaccharide monooxygenases (LPMOs/PMOs), Cu-containing proteins that catalyse the oxidative degradation of otherwise recalcitrant polysaccharides using O2 or H2O2 as a co-substrate. RESULTS: Although classical saprotrophic fungi and bacteria have been a rich source of lytic polysaccharide monooxygenases (LPMOs), we were interested to see if LPMOs from less evident bio-environments could be discovered and assessed for their cellulolytic activity in a biofuel context. In this regard, the marine shipworm Lyrodus pedicellatus represents an interesting source of new enzymes, since it must digest wood particles ingested during its natural tunnel boring behaviour and plays host to a symbiotic bacterium, Teredinibacter turnerae, the genome of which has revealed a multitude of enzymes dedicated to biomass deconstruction. Here, we show that T. turnerae encodes a cellulose-active AA10 LPMO. The 3D structure, at 1.4 Å resolution, along with its EPR spectrum is distinct from other AA10 polysaccharide monooxygenases insofar as it displays a "histidine-brace" catalytic apparatus with changes to the surrounding coordination sphere of the copper. Furthermore, TtAA10A possesses a second, surface accessible, Cu site 14 Å from the classical catalytic centre. Activity measurements show that the LPMO oxidises cellulose and thereby significantly augments the rate of degradation of cellulosic biomass by classical glycoside hydrolases. CONCLUSION: Shipworms are wood-boring marine molluscs that can live on a diet of lignocellulose. Bacterial symbionts of shipworms provide many of the enzymes needed for wood digestion. The shipworm symbiont T. turnerae produces one of the few LPMOs yet described from the marine environment, notably adding to the capability of shipworms to digest recalcitrant polysaccharides.

Hemocyanin facilitates lignocellulose digestion by wood-boring marine crustaceans.

Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for innovations in lignocellulose biorefining.

Uncovering the molecular mechanisms of lignocellulose digestion in shipworms.

Lignocellulose forms the structural framework of woody plant biomass and represents the most abundant carbon source in the biosphere. Turnover of woody biomass is a critical component of the global carbon cycle, and the enzymes involved are of increasing industrial importance as industry moves away from fossil fuels to renewable carbon resources. Shipworms are marine bivalve molluscs that digest wood and play a key role in global carbon cycling by processing plant biomass in the oceans. Previous studies suggest that wood digestion in shipworms is dominated by enzymes produced by endosymbiotic bacteria found in the animal's gills, while little is known about the identity and function of endogenous enzymes produced by shipworms. Using a combination of meta-transcriptomic, proteomic, imaging and biochemical analyses, we reveal a complex digestive system dominated by uncharacterized enzymes that are secreted by a specialized digestive gland and that accumulate in the cecum, where wood digestion occurs. Using a combination of transcriptomics, proteomics, and microscopy, we show that the digestive proteome of the shipworm Lyrodus pedicellatus is mostly composed of enzymes produced by the animal itself, with a small but significant contribution from symbiotic bacteria. The digestive proteome is dominated by a novel 300 kDa multi-domain glycoside hydrolase that functions in the hydrolysis of ß-1,4-glucans, the most abundant polymers in wood. These studies allow an unprecedented level of insight into an unusual and ecologically important process for wood recycling in the marine environment, and open up new biotechnological opportunities in the mobilization of sugars from lignocellulosic biomass.

An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion.

Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia's LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.

Characterization of the cellulolytic secretome of Trichoderma harzianum during growth on sugarcane bagasse and analysis of the activity boosting effects of swollenin.

This study demonstrates the production of an active enzyme cocktail produced by growing Trichoderma harzianum on sugarcane bagasse. The component enzymes were identified by LCMS-MS. Glycosyl hydrolases were the most abundant class of proteins, representing 67% of total secreted protein. Other carbohydrate active enzymes involved in cell wall deconstruction included lytic polysaccharide mono-oxygenases (AA9), carbohydrate-binding modules, carbohydrate esterases and swollenin, all present at levels of 1%. In total, proteases and lipases represented 5 and 1% of the total secretome, respectively, with the rest of the secretome being made up of proteins of unknown or putative function. This enzyme cocktail was efficient in catalysing the hydrolysis of sugarcane bagasse cellulolignin to fermentable sugars for potential use in ethanol production. Apart from mapping the secretome of T. harzianum, which is a very important tool to understand the catalytic performance of enzyme cocktails, the gene coding for T. harzianum swollenin was expressed in Aspergillus niger. This novel aspect in this work, allowed increasing the swollenin concentration by 95 fold. This is the first report about the heterologous expression of swollenin from T. harzianum, and the findings are of interest in enriching enzyme cocktail with this important accessory protein which takes part in the cellulose amorphogenesis. Despite lacking detectable glycoside activity, the addition of swollenin of T. harzianum increased by two-fold the hydrolysis efficiency of a commercial cellulase cocktail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:327-336, 2016.

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance.

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.

Overexpression of the UGT73C6 alters brassinosteroid glucoside formation in Arabidopsis thaliana.

BACKGROUND: Brassinosteroids (BRs) are signaling molecules that play essential roles in the spatial regulation of plant growth and development. In contrast to other plant hormones BRs act locally, close to the sites of their synthesis, and thus homeostatic mechanisms must operate at the cellular level to equilibrate BR concentrations. Whilst it is recognized that levels of bioactive BRs are likely adjusted by controlling the relative rates of biosynthesis and by catabolism, few factors, which participate in these regulatory events, have as yet been identified. Previously we have shown that the UDP-glycosyltransferase UGT73C5 of Arabidopsis thaliana catalyzes 23-O-glucosylation of BRs and that glucosylation renders BRs inactive. This study identifies the closest homologue of UGT73C5, UGT73C6, as an enzyme that is also able to glucosylate BRs in planta. RESULTS: In a candidate gene approach, in which homologues of UGT73C5 were screened for their potential to induce BR deficiency when over-expressed in plants, UGT73C6 was identified as an enzyme that can glucosylate the BRs CS and BL at their 23-O-positions in planta. GUS reporter analysis indicates that UGT73C6 shows over-lapping, but also distinct expression patterns with UGT73C5 and YFP reporter data suggests that at the cellular level, both UGTs localize to the cytoplasm and to the nucleus. A liquid chromatography high-resolution mass spectrometry method for BR metabolite analysis was developed and applied to determine the kinetics of formation and the catabolic fate of BR-23-O-glucosides in wild type and UGT73C5 and UGT73C6 over-expression lines. This approach identified novel BR catabolites, which are considered to be BR-malonylglucosides, and provided first evidence indicating that glucosylation protects BRs from cellular removal. The physiological significance of BR glucosylation, and the possible role of UGT73C6 as a regulatory factor in this process are discussed in light of the results presented. CONCLUSION: The present study generates essential knowledge and molecular and biochemical tools, that will allow for the verification of a potential physiological role of UGT73C6 in BR glucosylation and will facilitate the investigation of the functional significance of BR glucoside formation in plants.

Suppression of microRNA accumulation via RNA interference in Arabidopsis thaliana.

MicroRNAs (miRNAs) are key regulatory molecules in plants. These small RNAs are processed in the nucleus from longer precursor transcripts that form distinct secondary structures. The miRNAs target specific messenger RNAs (mRNAs) and consequently down-regulate gene expression. The importance of these regulatory molecules is wide-ranging, however, few loss-of-function mutants have been identified in miRNA genes and understanding the biology of miRNA-target pairings has largely depended upon creating alterations in the sequences of the target genes. Here we demonstrate using Arabidopsis thaliana, that it is possible to use RNA interference (RNAi) to suppress accumulation of miRNAs. Significantly reduced accumulation of miR163 and miR171a was achieved using hairpin RNAi constructs that were designed to target both the primary miRNA transcripts and their promoters. The presence of DNA methylation in the targeted promoter regions suggests that inhibition of transcription of the miRNA precursors is responsible. Reduction of miRNA accumulation resulted in an increase in accumulation of the mRNA targets of these miRNAs. This demonstrates that knock-down of miRNA expression can be achieved, thereby providing a straightforward approach for disrupting miRNA-target pairings and studying miRNA functions.

The genetic map of Artemisia annua L. identifies loci affecting yield of the antimalarial drug artemisinin.

Artemisinin is a plant natural product produced by Artemisia annua and the active ingredient in the most effective treatment for malaria. Efforts to eradicate malaria are increasing demand for an affordable, high-quality, robust supply of artemisinin. We performed deep sequencing on the transcriptome of A. annua to identify genes and markers for fast-track breeding. Extensive genetic variation enabled us to build a detailed genetic map with nine linkage groups. Replicated field trials resulted in a quantitative trait loci (QTL) map that accounts for a significant amount of the variation in key traits controlling artemisinin yield. Enrichment for positive QTLs in parents of new high-yielding hybrids confirms that the knowledge and tools to convert A. annua into a robust crop are now available.

The glucosyltransferase UGT72E2 is responsible for monolignol 4-O-glucoside production in Arabidopsis thaliana.

The phenylpropanoid pathway in plants leads to the synthesis of a wide range of soluble secondary metabolites, many of which accumulate as glycosides. In Arabidopsis, a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases shown to glucosylate several phenylpropanoids in vitro, including monolignols, hydroxycinnamic acids and hydroxycinnamic aldehydes. The role of these genes in planta has now been investigated through genetically downregulating the expression of individual genes or silencing the entire cluster. Analysis of these transgenic Arabidopsis plants showed that the levels of coniferyl and sinapyl alcohol 4-O-glucosides that accumulate in light-grown roots were significantly reduced. A 50% reduction in both glucosides was observed in plants in which UGT72E2 was downregulated, whereas silencing the three genes led to a 90% reduction, suggesting some redundancy of function within the cluster. The gene encoding UGT72E2 was constitutively overexpressed in transgenic Arabidopsis to determine whether increased glucosylation of monolignols could influence flux through the soluble phenylpropanoid pathway. Elevated expression of UGT72E2 led to increased accumulation of monolignol glucosides in root tissues and also the appearance of these glucosides in leaves. In particular, coniferyl alcohol 4-O-glucoside accumulated to massive amounts (10 micromol g(-1) FW) in root tissues of these plants. Increased glucosylation of other phenylpropanoids also occurred in plants overexpressing this glycosyltransferase. Significantly changing the pattern of glycosides in the leaves also led to a pronounced change in accumulation of the hydroxycinnamic ester sinapoyl malate. The data demonstrate the plasticity of phenylpropanoid metabolism and the important role that glucosylation of secondary metabolites can play in cellular homeostasis.

Use of the glucosyltransferase UGT71B6 to disturb abscisic acid homeostasis in Arabidopsis thaliana.

A glucosyltransferase (GT) of Arabidopsis, UGT71B6, recognizing the naturally occurring enantiomer of abscisic acid (ABA) in vitro, has been used to disturb ABA homeostasis in planta. Transgenic plants constitutively overexpressing UGT71B6 (71B6-OE) have been analysed for changes in ABA and the related ABA metabolites abscisic acid glucose ester (ABA-GE), phaseic acid (PA), dihydrophaseic acid (DPA), 7'-hydroxyABA and neo-phaseic acid. Overexpression of the GT led to massive accumulation of ABA-GE and reduced levels of the oxidative metabolites PA and DPA, but had marginal effect on levels of free ABA. The control of ABA homeostasis, as reflected in levels of the different metabolites, differed in the 71B6-OEs whether the plants were grown under standard conditions or subjected to wilt stress. The impact of increased glucosylation of ABA on ABA-related phenotypes has also been assessed. Increased glucosylation of ABA led to phenotypic changes in post-germinative growth. The use of two structural analogues of ABA, known to have biological activity but to differ in their capacity to act as substrates for 71B6 in vitro, confirmed that the phenotypic changes arose specifically from the increased glucosylation caused by overexpression of 71B6. The phenotype and profile of ABA and related metabolites in a knockout line of 71B6, relative to wild type, has been assessed during Arabidopsis development and following stress treatments. The lack of major changes in these parameters is discussed in the context of functional redundancy of the multigene family of GTs in Arabidopsis.

Evolution of substrate recognition across a multigene family of glycosyltransferases in Arabidopsis.

The complete sequence of the Arabidopsis genome enables definitive characterization of multigene families and analysis of their phylogenetic relationships. Using a consensus sequence previously defined for glycosyltransferases that use small-molecular-weight acceptors, 107 gene sequences were identified in the Arabidopsis genome and used to construct a phylogenetic tree. Screening recombinant proteins for their catalytic activities in vitro has revealed enzymes active toward physiologically important substrates, including hormones and secondary metabolites. The aim of this study has been to use the phylogenetic relationships across the entire family to explore the evolution of substrate recognition and regioselectivity of glucosylation. Hydroxycoumarins have been used as the model substrates for the analysis in which 90 sequences have been assayed and 48 sequences shown to recognize these compounds. The study has revealed activity in 6 of the 14 phylogenetic groups of the multigene family, suggesting that basic features of substrate recognition are retained across substantial evolutionary periods.

The activity of Arabidopsis glycosyltransferases toward salicylic acid, 4-hydroxybenzoic acid, and other benzoates.

Benzoates are a class of natural products containing compounds of industrial and strategic importance. In plants, the compounds exist in free form and as conjugates to a wide range of other metabolites such as glucose, which can be attached to the carboxyl group or to specific hydroxyl groups on the benzene ring. These glucosylation reactions have been studied for many years, but to date only one gene encoding a benzoate glucosyltransferase has been cloned. A phylogenetic analysis of sequences in the Arabidopsis genome revealed a large multigene family of putative glycosyltransferases containing a consensus sequence typically found in enzymes transferring glucose to small molecular weight compounds such as secondary metabolites. Ninety of these sequences have now been expressed as recombinant proteins in Escherichia coli, and their in vitro catalytic activities toward benzoates have been analyzed. The data show that only 14 proteins display activity toward 2-hydroxybenzoic acid, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid. Of these, only two enzymes are active toward 2-hydroxybenzoic acid, suggesting they are the Arabidopsis salicylic acid glucosyltransferases. All of the enzymes forming glucose esters with the metabolites were located in Group L of the phylogenetic tree, whereas those forming O-glucosides were dispersed among five different groups. Catalytic activities were observed toward glucosylation of the 2-, 3-, or 4-hydroxyl group on the ring. To further explore their regioselectivity, the 14 enzymes were analyzed against benzoic acid, 3-hydroxybenzoic acid, 2,3-, 2,4-, 2,5-, and 2,6-dihydroxybenzoic acid. The data showed that glycosylation of specific sites could be positively or negatively influenced by the presence of additional hydroxyl groups on the ring. This study provides new tools for biotransformation reactions in vitro and a basis for engineering benzoate metabolism in plants.