Expression of a fungal ferulic acid esterase increases cell wall digestibility of tall fescue (Festuca arundinacea)

In the cell walls of forage grasses, ferulic acid is esterified to arabinoxylans and participates with lignin monomers in oxidative coupling pathways to generate ferulate–polysaccharide–lignin complexes that cross-link the cell wall. Such cross-links hinder cell wall degradation by ruminant microbes, reducing plant digestibility. In this study, genetically modified Festuca arundinacea plants were produced expressing an Aspergillus niger ferulic acid esterase (FAEA) targeted to the vacuole. The rice actin promoter proved to be effective for FAEA expression, as did the cauliflower mosaic virus (CaMV) 35S and maize ubiquitin promoters. Higher levels of expression were, however, found with inducible heat-shock and senescence promoters. Following cell death and subsequent incubation, vacuole-targeted FAEA resulted in the release of both monomeric and dimeric ferulic acids from the cell walls, and this was enhanced several fold by the addition of exogenous endo-1,4-β-xylanase. Most of the FAEA-expressing plants showed increased digestibility and reduced levels of cell wall esterified phenolics relative to non-transformed plants. It is concluded that targeted FAEA expression is an effective strategy for improving wall digestibility in Festuca and, potentially, other grass species used for fodder or cellulosic ethanol production.     

Improvement of cell wall digestibility in tall fescue by <Emphasis Type="Italic">Oryza sativa</Emphasis> SECONDARY WALL NAC DOMAIN PROTEIN2 chimeric repressor

Forage digestibility is one of the most important factors in livestock performance. As grasses grow and mature, dry matter increases but they become fibrous with secondary cell wall deposition and lignification of sclerenchyma cells, and forage quality drops. In rice (Oryza sativa), the SECONDARY WALL NAC DOMAIN PROTEIN2 fused with the modified EAR-like motif repression domain (OsSWN2-SRDX) reduces secondary cell wall thickening in sclerenchyma cells. We introduced OsSWN2-SRDX under the control of the OsSWN1 promoter into tall fescue (Festuca arundinacea Schreb.) to increase cell wall digestibility. Of 23 transgenic plants expressing OsSWN2-SRDX, nine had brittle internodes that were easily broken by bending. Their secondary cell walls were significantly thinner than those of the wild type in interfascicular fibers of internodes and in cortical fiber cells between leaf epidermal cells and vascular bundles. The dry matter digestibility increased by 11.8% in stems and by 6.8% in leaves compared with the wild type, and therefore forage quality was improved. In stem interfascicular fibers, acid detergent fiber and acid insoluble lignin were greatly reduced. Thus, the reduction of indigestible fiber composed of cellulose and lignin increased the degradability of sclerenchyma cell walls. OsSWN2-SRDX plants offer great potential in the genetic improvement of forage digestibility.     

Modification of esterified cell wall phenolics increases vulnerability of tall fescue to herbivory by the fall armyworm

Feruloylation of arabinoxylan in grass cell walls leads to cross-linked xylans. Such cross-linking appears to play a role in plant resistance to pathogens and insect herbivores. In this study, we investigated the effect of ferulate cross-linking on resistance to herbivory by fall armyworm (Spodoptera frugiperda) making use of genetically modified tall fescue [Schedonorus arundinaceus (Festuca arundinacea)] expressing a ferulic acid esterase gene. Mature leaves of these plants have significant reduced levels of cell wall ferulates and diferulates but no change in acid detergent lignin. These reduced levels of esterified cell wall ferulates in transgenic plants had a positive effect on all measures of armyworm larval performance examined. More larvae survived (89 vs. 57?%) and grew faster (pupated 2.1?days sooner) when fed transgenic leaves with reduced levels of cell wall ferulates, than when fed control tall fescue leaves where levels of cell wall ferulates were not altered. Overall, mortality, growth and food utilization were negatively associated with level of esterified cell wall ferulates and diferulates in leaves they were fed. This study is the first to use transgenic plants with modified level of cell wall esterified ferulates to test the role of feruloylation in plant resistance to insects. It is concluded that the accumulation of ferulates and the cross-linking of arabinoxylans via diferulate esters in the leaves of tall fescue underlies the physical barrier to insect herbivory. Reducing ferulate cross-linking in grass cell walls could increase susceptibility of these plants to insect folivores.     

Anionic carbohydrate-containing cell wall polymers of <Emphasis Type="Italic">Streptomyces melanosporofaciens</Emphasis> and related species

The structures of cell wall anionic carbohydrate-containing polymers in Streptomyces melanosporofaciens VKM Ac-1864^T and phylogenetically close organisms—S. hygroscopicus subsp. hygroscopicus VKM Ac-831^T, S. violaceusniger VKM Ac-583^T, S. endus VKM Ac-1331^T, S. endus VKM Ac-129, and S. rutgersensis subsp. castelarensis VKM Ac-832^T—have been comparatively studied by chemical and NMR spectroscopic methods. The natural polymer of a new, previously unknown structure, Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid) with β-galactose residues at C-9, has been found in the cell walls of all the strains under study. The cell walls of all the studied organisms contain three teichoic acids (TA): a predominant TA (1,3-poly(glycerol phosphate) with N-acetylated α-glucosaminyl substitutes by C-2 of glycerol, and minor TAs, 1,3-and 2,3-poly(glycerol phosphate) polymers without substitution. Their chains have O-acetyl and O-lysyl groups. Microorganisms of the above-mentioned species differ in the number of α-glucosaminyl substitutes and in the degree of their acetylation in the predominant teichoic acid.     

Discovery of novel feruloyl esterase activity of BioH in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21(DE3)

Objectives

To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3.

Results

The gene encoding BioH was cloned and overexpressed in E. coli. The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p-coumarate substrates were: K _m values of 0.48, 6.3, and 1.9 mM, respectively, and k _cat /K _m values of 9.3, 3.8, and 3.8 mM^?1 s^?1, respectively.

Conclusions

Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.

     

Anionic polymers of the cell wall of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. VKM Ac-2534

The cell wall of Streptomyces sp. VKM Ac-2534, the causative agent of common scab in potato tubers, which does not synthesize thaxtomin and is phylogenetically close to phytopathogen Streptomyces setonii sp. ATCC 25497, contains two anionic carbohydrate-containing polymers. The major polymer is teichuronic acid, whose repeating unit is disaccharide → 4)-β-D-ManpNAc3NAcyA-(1 → 3)-α-D-GalpNAc-(1→, where Acy is a residue of acetic or L-glutamic acid. The polymer of such structure has been found in Gram-positive bacteria for the first time. The minor polymer is teichoic acid [1,5-poly(ribitol phosphate)], in which a part of the ribitol residues are glycosylated at C4 with β-D-Glcp and, probably, with β-D-GlcpNAc and some residues are O-acylated with Lys residues. The structures were proved by chemical and NMR spectroscopic methods. It is likely that the presence of acidic polysaccharides on the surface of the phytopathogenic streptomycete is necessary for its attachment to the host plant.     

Ion-exchange properties of the cell walls isolated from suspension-cultured plant cells

Ion-exchange capacity of the cell walls isolated from suspension-cultured Panax japonicus, Polyscias filicifolia and Dioscorea deltoidea cells was analyzed at pH 2.8–12 and constant ionic strength (100 mM). The cell walls of all cultures contain three types of ion-exchange groups: primary amino groups (pK _a < 3), carboxyl groups of polygalacturonic acid (pK _a 3.71), and carboxyl groups of hydroxycinnamic acids (pK _a 7.62). Amount of primary amino groups ranges from 500 (D. deltoidea) to 710 (P. japonicus) µmol/g cell wall dry weight, carboxyl groups with pK _a 3.71—from 570 (D. deltoidea) to 670 (P. filicifolia), carboxyl groups with pK _a 7.62—from 270 (P. filicifolia) to 370 (P. japonicus) µmol/g cell wall dry weight. The comparison of the data obtained by elemental and functional analyses demonstrated that the cell walls of all cultures are characterized by high content of pectins (~40% by weight) and structural proteins (~17–30% by weight), but do not contain phenolic OH–groups, which presumably signifies the absence of lignin in them.     

Feruloyl and p-coumaroyl esterase from anaerobic fungi in relation to plant cell wall degradation

Summary Trans-feruloyl and trans-p-coumaroyl esterases were found in the culture filtrates of two monocentric (Piromyces MC-1, Neocallimastix MC-2) and three polycentric (Orpinomyces PC-2, Orpinomyces PC-3, and PC-1, an unnamed genus with uniflagellated zoospores) isolates of anaerobic rumen fungi. Treatment of cell walls of Coastal bermudagrass shoots with the filtrates released the trans isomers of ferulic and p-coumaric acids; results of microscopic observations indicated that fungal isolates degraded primarily unlignified cell walls in leaf blades and stems. A greater proportion of ferulic than p-coumaric acid was released by this treatment when compared with the amounts of the acids released by saponification of the walls with 1 M NaOH. The filtrates also showed esterase activities against the trans isomers of methyl ferulate and methyl p-coumarate, with ferulic acid being released at a faster rate than p-coumaric acid. Assays for other cell-wall-degrading enzymes (xylanase, -xylosidase, -l-arabinosidase, cellulase, -glucosidase) indicated that only -xylosidase correlated with ferulate and p-coumarate esterase activities. The monocentric isolate MC-2 had the highest esterase activity against both the plant cell wall and methyl ester substrates and the highest specific activities of acetyl esterase, -xylosidase, -l-arabinosidase, cellulase and -glucosidase. Isolate MC-2 produced substantially greater amounts of feruloyl and p-coumaroyl esterase when the growth substrate contained higher levels of saponifiable ferulic and p-coumaric acids. Offprint requests to: W. S. Borneman     

Cell wall glycopolymers of type strains from three species of the genus <Emphasis Type="Italic">Actinoplanes</Emphasis>

The structures of cell wall glycopolymers from the type strains of three Actinoplanes species were investigated using chemical methods, NMR spectroscopy, and mass spectrometry. Actinoplanes digitatis VKM Ac-649^T contains two phosphate-containing glycopolymers: poly(diglycosyl-1-phosphate) →6)-α-D-GlcpNAc-(1-P-6)-α-D-GlcpN-(1→ and teichoic acid →1)-sn-Gro-(3-P-3)-β-[β-D-GlcpNAc-(1→2]-D-Galp-(1→. Two glycopolymers were identified in A. auranticolor VKM Ac-648^T and A. cyaneus VKM Ac-1095^T: minor polymer–unsubstituted 2,3-poly(glycerol phosphate), widely abundant in actinobacteria (Ac-648^T), and mannan with trisaccharide repeating unit →2)-α-D-Manp-(1→2)-α-D-Manp(1→6)-α-D-Manp-(1→(Ac-1095^T). In addition, both microorganisms contain a teichuronic acid of unique structure containing a pentasaccharide repeating unit with two residues of glucopyranose and three residues of diaminouronic acids in D-manno- and/or D-gluco-configuration. Each of the strains demonstrates peculiarities in the structure of teichuronic acid with respect to the ratio of diaminouronic acids and availability and location of O-methyl groups in glucopyranose residues. All investigated strains contain a unique set of glycopolymers in their cell walls with structures not described earlier for prokaryotes.     

The targeting of starch binding domains from starch synthase III to the cell wall alters cell wall composition and properties

Key message

Starch binding domains of starch synthase III from Arabidopsis thaliana (SBD123) binds preferentially to cell wall polysaccharides rather than to starch in vitro. Transgenic plants overexpressing SBD123 in the cell wall are larger than wild type. Cell wall components are altered in transgenic plants. Transgenic plants are more susceptible to digestion than wild type and present higher released glucose content. Our results suggest that the transgenic plants have an advantage for the production of bioethanol in terms of saccharification of essential substrates.

Abstract

The plant cell wall, which represents a major source of biomass for biofuel production, is composed of cellulose, hemicelluloses, pectins and lignin. A potential biotechnological target for improving the production of biofuels is the modification of plant cell walls. This modification is achieved via several strategies, including, among others, altering biosynthetic pathways and modifying the associations and structures of various cell wall components. In this study, we modified the cell wall of A. thaliana by targeting the starch-binding domains of A. thaliana starch synthase III to this structure. The resulting transgenic plants (E8-SDB123) showed an increased biomass, higher levels of both fermentable sugars and hydrolyzed cellulose and altered cell wall properties such as higher laxity and degradability, which are valuable characteristics for the second-generation biofuels industry. The increased biomass and degradability phenotype of E8-SBD123 plants could be explained by the putative cell-wall loosening effect of the in tandem starch binding domains. Based on these results, our approach represents a promising biotechnological tool for reducing of biomass recalcitrance and therefore, the need for pretreatments.

     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.     

Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

Background

Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB). In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine.

Results

Multiple sequence alignment revealed that the C-terminal region (LcsB) of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains S_A and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP) or beta-galactosidase (Gal) was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor.

Conclusion

The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological application.

     

Feruloyl esterase from <Emphasis Type="Italic">Alternaria tenuissima</Emphasis> that hydrolyses lignocellulosic material to release hydroxycinnamic acids

An extracellular feruloyl esterase from the culture filtrates of the isolated fungus Alternaria tenuissima was successfully purified to apparent homogeneity by anion-exchange and size-exclusion chromatography. Peptide fragments of purified enzyme (designated as AltFAE; molecular weight of 30.3 kDa determined by SDS-PAGE) were identified by mass spectrometry using a NanoLC-ESI-MS/MS system. Michaelis-Menten constants (K_M) and catalytic efficiencies (k_cat/K_M) were determined for typical substrates of feruloyl esterase, and the lowest K_M of 50.6 μM (i.e., the highest affinity) and the highest k_cat/K_M (3.1 × 10⁵ s^—1 M^–1) were observed for methyl ^p-coumarate and methyl ferulate, respectively. Not least, AltFAE catalyzed conversion of lignocellulosic material (e.g. wood meal) to release hydroxycinnamic products, i.e. ferulic- and ^p-coumaric acids.     

The increase of O-acetylation and N-deacetylation in cell wall promotes acid resistance and nisin production through improving cell wall integrity in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Cell wall is closely related to bacterial robustness and adsorption capacity, playing crucial roles in nisin production in Lactococcus lactis. Peptidoglycan (PG), the essential component of cell wall, is usually modified with MurNAc O-acetylation and GlcNAc N-deacetylation, catalyzed by YvhB and XynD, respectively. In this study, increasing the two modifications in L. lactis F44 improved autolysis resistance by decreasing the susceptibility to PG hydrolases. Furthermore, both modifications were positively associated with overall cross-linkage, contributing to cell wall integrity. The robust cell wall rendered the yvhB/xynD-overexpression strains more acid resistant, leading to the increase of nisin production in fed-batch fermentations by 63.7 and 62.9%, respectively. Importantly, the structural alterations also reduced nisin adsorption capacity, resulting in reduction of nisin loss. More strikingly, the co-overexpression strain displayed the highest nisin production (76.3% higher than F44). Our work provides a novel approach for achieving nisin overproduction via extensive cell wall remodeling.     

Cloning and expression of ferulic acid esterase gene and its effect on wort filterability

Objectives

To optimize the expression of type A ferulic acid esterase (FaeA) from Aspergillus niger in Pichia pastoris X-33 using codon optimization.

Results

Recombinant FaeA was purified from the fermentation broth, with the maximum specific activity of 48.4 ± 0.1 U mg^?1. Adding it during mashing process for beer brewing raised the filtration rate by 14.5% while the turbidity and viscosity declined by 22 and 6.9%, respectively. Addition of FaeA increased the concentrations of free ferulic acid (FA) and arabinoxylan (AX) in the wort, while the polymeric arabinoxylans content declined significantly.

Conclusions

Recombinant FaeA was capable to prevent the oxidative gelation of PAX formation by breaking the cross-linking of FA among AX chains and improve the filtration performance of wort.

     

A cinnamoyl esterase from Aspergillus niger can break plant cell wall cross-links without release of free diferulic acids.

A cinnamoyl esterase, ferulic acid esterase A, from Aspergillus niger releases ferulic acid and 5-5- and 8-O-4-dehydrodiferulic acids from plant cell walls. The breakage of one or both ester bonds from dehydrodimer cross-links between plant cell wall polymers is essential for optimal action of carbohydrases on these substrates, but it is not known if cinnamoyl esterases can break these cross-links by cleaving one of the ester linkages which would not release the free dimer. It is difficult to determine the mechanism of the reaction on complex substrates, and so we have examined the catalytic properties of ferulic acid esterase A from Aspergillus niger using a range of synthetic ethyl esterified dehydrodimers (5-5-, 8-5-benzofuran and 8-O-4-) and two 5-5-diferulate oligosaccharides. Our results show that the esterase is able to cleave the three major dehydrodiferulate cross-links present in plant cell walls. The enzyme is highly specific at hydrolysing the 5-5- and the 8-5-benzofuran diferulates but the 8-O-4-is a poorer substrate. The hydrolysis of dehydrodiferulates to free acids occurs in two discrete steps, one involving dissociation of a monoesterified intermediate which is negatively charged at the pH of the reaction. Although ferulic acid esterase A was able to release monoesters as products of reactions with all three forms of diesters, only the 5-5- and the 8-O-4-monoesters were substrates for the enzyme, forming the corresponding free diferulic acids. The esterase cannot hydrolyse the second ester bond from the 8-5-benzofuran monoester and therefore, ferulic acid esterase A does not form 8-5-benzofuran diferulic acid. Therefore, ferulic acid esterase A from Aspergillus niger contributes to total plant cell wall degradation by cleaving at least one ester bond from the diferulate cross-links that exist between wall polymers but does not always release the free acid product.     

Effect of nitrogen deficiency on the ion-exchange properties of cell wall polymers from wheat roots

The ion-exchange properties of cell wall polymers isolated from the roots of wheat (Triticum aestivum L.) plants grown on either nitrate-free (N-deficient) or nitrate-containing (+N) hydroponic nutrient medium have been investigated. Irrespective of the nitrogen nutrition regimen, the studied cell walls contained four types of ion-exchange groups: primary amino groups of structural proteins (pK_a < 3), carboxyl groups of polygalacturonic acid in pectin (pK _a ~4.7), carboxyl groups of hydroxycinnamic acids (pK _a ~7.3), and phenolic OH-groups of lignin (pK_a ~10.2). The quantitative ratio between these types of ion-exchange groups, the mass fraction of cell walls in the dry weight of roots, and the swelling coefficient of cell walls depended on the nitrate presence in the growing medium. Compared to the +N variant, the N-deficient variant was characterized by a 2.4 times higher content of phenolic OH-groups in cell walls and 1.24 times higher mass fraction of cell walls; at the same time, the swelling coefficient for this variant was lower by 10%. The obtained data indicate that nitrogen deficiency results in a formation of thicker root cell walls with a higher degree of polymer cross-linking that may be caused by the increased lignin content.