Acetylesterase Activity Research Articles

Identification of pectin acetylesterase genes in moso bamboo (Phyllostachys edulis) reveals PePAE6 involved in pectin accumulation of leaves

As an indispensable polysaccharide, pectin profoundly affects various aspects of plant growth and development. Pectin acetylesterase (PAE) adjusts the acetylation level of pectin, thereby affecting the properties of pectin and cell walls, particularly in tissues such as leaves, where primary cell walls are predominant and rich in pectin. However, the PAE family in bamboo remains unknown. In the present study, ten PAE genes (PePAE1-PePAE10) were identified and characterized in moso bamboo (Phyllostachys edulis). All PePAEs possessed unique conserved domains and motifs of plant PAEs. The expression patterns of PePAEs varied across different tissues and developmental stages, indicating the sub-functionalization of these genes. Notably, the expression level of PePAE6 displayed the most remarkable change during leaf growth and was co-expressed with multiple transcription factors. Using various experimental approaches, we found that PeWRKY-1 and PeWRKY-2 negatively regulated PePAE6 by binding to its promoter. Knockdown of PeWRKY-1 or PeWRKY-2 inversely increased expression of PePAE6. While knockdown of PePAE6 resulted in increased acetylation levels and reduced galacturonic acid (GalA) content in moso bamboo leaves. Furthermore, overexpression of PePAE6 in rice indirectly enhanced vegetative growth and photosynthetic capacity, with a reduction in pectin acetylation levels and an increase in GalA. Additionally, an in vitro pectin acetylesterase activity assay confirmed that PePAE6 catalyzed pectin deacetylation, releasing acetic acid as a byproduct. These findings provide valuable insights into the basic characteristics of PAE in bamboo and reveal that PePAE6 is a functional gene involved in pectin accumulation, which could be helpful in genetic engineering in bamboo breeding.

Biochemical characterization of an esterase from Thermobifida fusca YX with acetyl xylan esterase activity.

Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58s-1mM-1, 1.09s-1mM-1, and 0.062s-1mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20min at 70°C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other esterases. The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.

TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells

Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana, we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity invitro. These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.

Oomycete pathogen pectin acetylesterase targets host lipid transfer protein to reduce salicylic acid signaling.

During initial stages of microbial invasion, the extracellular space (apoplast) of plant cells is a vital battleground between plants and pathogens. The oomycete plant pathogens secrete an array of apoplastic carbohydrate active enzymes, which are central molecules for understanding the complex plant-oomycete interactions. Among them, pectin acetylesterase (PAE) plays a critical role in the pathogenesis of plant pathogens including bacteria, fungi, and oomycetes. Here, we demonstrated that Peronophythora litchii (syn. Phytophthora litchii) PlPAE5 suppresses litchi (Litchi chinensis) plant immunity by interacting with litchi lipid transfer protein 1 (LcLTP1). The LcLTP1-binding activity and virulence function of PlPAE5 depend on its PAE domain but not on its PAE activity. The high expression of LcLTP1 enhances plant resistance to oomycete and fungal pathogens, and this disease resistance depends on BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and Suppressor of BIR1 (SOBIR1) in Nicotiana benthamiana. LcLTP1 activates the plant salicylic acid (SA) signaling pathway, while PlPAE5 subverts the LcLTP1-mediated SA signaling pathway by destabilizing LcLTP1. Conclusively, this study reports a virulence mechanism of oomycete PAE suppressing plant LTP-mediated SA immune signaling and will be instrumental for boosting plant resistance breeding.

Pseudomonas aeruginosa AlgF is a protein–protein interaction mediator required for acetylation of the alginate exopolysaccharide

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P.aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX invitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P.aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate invitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.

WssI from the Gram-negative bacterial cellulose synthase is an O-acetyltransferase that acts on cello-oligomers with several acetyl donor substrates

In microbial biofilms, bacterial cells are encased in a self-produced matrix of polymers (e.g., exopolysaccharides) that enable surface adherence and protect against environmental stressors. For example, the wrinkly spreader phenotype of Pseudomonas fluorescens colonizes food/water sources and human tissue to form robust biofilms that can spread across surfaces. This biofilm largely consists of bacterial cellulose produced by the cellulose synthase proteins encoded by the wss (WS structural) operon, which also occurs in other species, including pathogenic Achromobacter species. Although phenotypic mutant analysis of the wssFGHI genes has previously shown that they are responsible for acetylation of bacterial cellulose, their specific roles remain unknown and distinct from the recently identified cellulose phosphoethanolamine modification found in other species. Here, we have purified the C-terminal soluble form of WssI from P.fluorescens and Achromobacter insuavis and demonstrated acetylesterase activity with chromogenic substrates. The kinetic parameters (kcat/KM values of 13 and 8.0 M-1 s-1, respectively) indicate that these enzymes are up to four times more catalytically efficient than the closest characterized homolog, AlgJ from the alginate synthase. Unlike AlgJ and its cognate alginate polymer, WssI also demonstrated acetyltransferase activity onto cellulose oligomers (e.g., cellotetraose to cellohexaose) with multiple acetyl donor substrates (p-nitrophenyl acetate, 4-methylumbelliferyl acetate, and acetyl-CoA). Finally, a high-throughput screen identified three low micromolar WssI inhibitors that may be useful for chemically interrogating cellulose acetylation and biofilm formation.

Implications of phyto-feed additives supplementation in buffalo calves on rumen fermentation pattern and microbial population

Natural phyto-feed additives have been identified as a potential rumen fermentation modifier by in vitro studies and by few short-term in vivo trials. However, information on impact on animal performance by their long-term administration is still inadequate. In light of this, the present study was undertaken to examine the rumen fermentation pattern, rumen microbial enzymes and microbial profiles as influenced by long term supplementation of phyto-feed additives to buffalo calves. A six months feeding trial was conducted on 20 male buffaloes (165±4 kg body weight), divided into four groups and fed on diet supplemented with no additive (T0, control), with feed additive FAI @ 1% of dry matter intake (DMI) (T1), with FAII @ 1 ml/kg DMI (T2) and with FAI and FAII switched alternatively after every 15 days (T3). No significant effect was observed on rumen fermentation pattern as well as carboxymethylcellulase, avicelase, xylanase, acetyl esterase, and protease activities in the rumen of buffalo calves. The population density of methanogens, fungi, Ruminococcus flavefaciens, and R. albus decreased significantly in T3 where FAI and FAII were fed alternately, but Fibrobacter succinogenes decreased significantly in T2 where FAII was fed. When compared to the control, the microscopic count of protozoa decreased in all the three supplemented groups. It can be concluded that rumen fermentation, including rumen metabolites and microbial enzymes, were unaffected; however, phyto-feed additives exhibited changes in rumen microbes.

Pectin acetylesterase 8 influences pectin acetylation in the seed coat, seed imbibition, and dormancy in common bean (Phaseolus vulgaris L.)

AbstractThe results of a prior transcript profiling study identified a large difference in transcript accumulation of a pectin acetylesterase gene, designated as PAE8, in two genetically related lines of common bean (Phaseolus vulgaris). The results of reverse transcription‐quantitative PCR experiments confirmed this difference and revealed that the gene is expressed specifically in the seed coat of developing seeds. Genomic sequence data identified a non‐functional allele, due to a five‐base pair insertion resulting in a frameshift and premature stop codon. The non‐functional pae8 allele was associated with a lack of detectable protein as determined by Western blot. PAE8 accounted for approximately 65% of total pectin acetylesterase activity in the developing seed coat. Lack of functional PAE8 resulted in an approximately 2.5‐fold increase in acetylation of soluble pectin in the mature seed coat. The presence of a non‐functional pae8 allele was associated with an increased rate of water absorption by the seed and increased percentage of germination in aged seeds. The data suggest that the decreased acetylation of pectin leads to enhanced interaction with Ca2+, contributing to water impermeability.

Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans

BackgroundNowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized ‘treasure troves’ in the era of exponentially growing numbers of sequenced genomes.ResultsWe determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/β-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs.ConclusionsThe bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.

Characterization of a novel strain of Aspergillus aculeatinus: From rhamnogalacturonan type I pectin degradation to improvement of fruit juice filtration

Aspergillus spp. are well-known producers of pectinases commonly used in the industry. Aspergillus aculeatinus is a recently identified species but poorly characterized. This study aimed at giving a comprehensive characterization of the enzymatic potential of the O822 strain to produce Rhamnogalacturonan type I (RGI)-degrading enzymes. Proteomic analysis identified cell wall degrading enzymes (cellulases, hemicellulases, and pectinases) that accounted for 92 % of total secreted proteins. Twelve out of fifty proteins were identified as RGI-degrading enzymes. NMR and enzymatic assays revealed high levels of arabinofuranosidase, arabinanase, galactanase, rhamnogalacturonan hydrolases and rhamnogalacturonan acetylesterase activities in aqueous extracts. Viscosity assays carried out with RGI-rich camelina mucilage confirmed the efficiency of enzymes secreted by O822 to hydrolyze RGI, by decreasing viscosity by 70 %. Apple juice trials carried out at laboratory and pilot scale showed an increase in filtration flow rate and yield, paving the way for an industrial use of enzymes derived from A. aculeatinus.

Multimodular fused acetyl\u2013feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass

BackgroundPlant biomass is an abundant and renewable carbon source that is recalcitrant towards both chemical and biochemical degradation. Xylan is the second most abundant polysaccharide in biomass after cellulose, and it possesses a variety of carbohydrate substitutions and non-carbohydrate decorations which can impede enzymatic degradation by glycoside hydrolases. Carbohydrate esterases are able to cleave the ester-linked decorations and thereby improve the accessibility of the xylan backbone to glycoside hydrolases, thus improving the degradation process. Enzymes comprising multiple catalytic glycoside hydrolase domains on the same polypeptide have previously been shown to exhibit intramolecular synergism during degradation of biomass. Similarly, natively fused carbohydrate esterase domains are encoded by certain bacteria, but whether these enzymes can result in similar synergistic boosts in biomass degradation has not previously been evaluated.ResultsTwo carbohydrate esterases with similar architectures, each comprising two distinct physically linked catalytic domains from families 1 (CE1) and 6 (CE6), were selected from xylan-targeting polysaccharide utilization loci (PULs) encoded by the Bacteroidetes species Bacteroides ovatus and Flavobacterium johnsoniae. The full-length enzymes as well as the individual catalytic domains showed activity on a range of synthetic model substrates, corn cob biomass, and Japanese beechwood biomass, with predominant acetyl esterase activity for the N-terminal CE6 domains and feruloyl esterase activity for the C-terminal CE1 domains. Moreover, several of the enzyme constructs were able to substantially boost the performance of a commercially available xylanase on corn cob biomass (close to twofold) and Japanese beechwood biomass (up to 20-fold). Interestingly, a significant improvement in xylanase biomass degradation was observed following addition of the full-length multidomain enzyme from B. ovatus versus the addition of its two separated single domains, indicating an intramolecular synergy between the esterase domains. Despite high sequence similarities between the esterase domains from B. ovatus and F. johnsoniae, their addition to the xylanolytic reaction led to different degradation patterns.ConclusionWe demonstrated that multidomain carbohydrate esterases, targeting the non-carbohydrate decorations on different xylan polysaccharides, can considerably facilitate glycoside hydrolase-mediated hydrolysis of xylan and xylan-rich biomass. Moreover, we demonstrated for the first time a synergistic effect between the two fused catalytic domains of a multidomain carbohydrate esterase.

Functional investigation and applications of the acetylesterase activity of the Citrus sinensis (L.) Osbeck peel

The hydrolysis of acetyl moieties on a set of commercially relevant substrates was performed by employing the whole tissue of Citrus sinensis (L.) Osbeck peel as an efficient biocatalyst in mild reaction conditions with high degree of regioselectivity. The reaction is done in aqueous media and the product is easily recovered. Optimal reaction conditions were deduced and two practical applications were investigated: the elaboration of acetylglicerols and the preparation of vitamin K1 precursor. Peel waste (flavedo and albedo) from orange juice manufacturing was successfully employed as a biocatalyst.

Functional exploration of Pseudoalteromonas atlantica as a source of hemicellulose-active enzymes: Evidence for a GH8 xylanase with unusual mode of action

To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a β-xylosidase, a α-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end.

Brettanomyces anomalus, a double drawback for cider aroma

Sixty-six French ciders were collected and characterized to build up a data base of eighty-eight variables, including sensory descriptors, aroma description and physicochemical variables. A classification in four main sensory categories was made on the basis of sensorial data. Fruity-Floral ciders were rich in acetate esters and low in volatile phenols, in opposition with phenol-like or off-flavour ciders having an inverse ratio. In order to explain this observation, an essential result was the demonstration of Brettanomyces anomalus implication, not only in volatile phenol synthesis, but also in acetate ester degradation. Brettanomyces anomalus exhibited an intracellular acetyl esterase activity. At last, both mechanisms, acetate ester degradation and phenol synthesis, contribute to the decrease of cider fruitiness.

Bacterial Fermentation of Water-Soluble Cellulose Acetate Raises Large-Bowel Acetate and Propionate and Decreases Plasma Cholesterol Concentrations in Rats.

We hypothesized that water-soluble cellulose acetate (WSCA) could be useful tool for the delivery of short-chain fatty acids to the large intestine. Rats were fed a control diet or a diet containing graded levels of WSCA for up to 21 days. Consuming WSCA dose-dependently increased large-bowel acetate and propionate concentrations through the bacterial fermentation. When WSCA was used as substrate, acetyl esterase activity in the cecal bacteria was detected solely in rats fed WSCA, in which the activity increased over time accompanied by an increased number of Bacteroides xylanisolvens. Consuming WSCA at a 4% level increased the goblet cell numbers and mucin contents in the cecum and lowered plasma cholesterol concentrations, which tended to correlate with the portal plasma concentrations of propionate. The results suggest that bacterial fermentation of WSCA is characterized by the greater production of acetate and propionate, which may contribute to the physiologic alterations.

Xyloglucan O-acetyltransferases from Arabidopsis thaliana and Populus trichocarpa catalyze acetylation of fucosylated galactose residues on xyloglucan side chains.

AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are O-acetyltransferases acetylating fucosylated galactose residues on xyloglucan and AXY9 does not directly catalyze O-acetylation of xyloglucan but exhibits weak acetylesterase activity. Xyloglucan is a major hemicellulose that cross-links cellulose in the primary walls of dicot plants and the galactose (Gal) residues on its side chains can be mono- and di-O-acetylated. In Arabidopsis thaliana, mutations of three AXY (altered xyloglucan) genes, AXY4, AXY4L and AXY9, have previously been shown to cause a reduction in xyloglucan acetylation, but their biochemical functions remain to be investigated. In this report, we demonstrated that recombinant proteins of AXY4/XGOAT1 (xyloglucan O-acetyltransferase1), AXY4L/XGOAT2 and their close homologs from Populus trichocarpa, PtrXGOATs, displayed O-acetyltransferase activities transferring acetyl groups from acetyl CoA onto xyloglucan oligomers. Structural analysis of XGOAT-catalyzed reaction products revealed that XGOATs mediated predominantly 6-O-monoacetylation and a much lesser degree of 3-O and 4-O-monoacetylation and 4,6-di-O-acetylation of Gal residues on xyloglucan side chains. XGOATs appeared to preferentially acetylate fucosylated Gal residues with little activity toward non-fucosylated Gal residues. Mutations of the conserved amino acid residues in the GDS and DXXH motifs in AXY4/XGOAT1 resulted in a drastic reduction in its ability to transfer acetyl groups onto xyloglucan oligomers. In addition, although recombinant AXY9 was unable to transfer acetyl groups from acetyl CoA onto xyloglucan oligomers, it was catalytically active as demonstrated by its weak acetylesterase activity that was also exhibited by AXY4/XGOAT1 and AXY4L/XGOAT2. Furthermore, we showed that the AXY8 fucosidase was able to hydrolyze fucosyl residues from both non-acetylated and acetylated xyloglucan oligomers. These findings provide biochemical evidence that AXY4/XGOAT1, AXY4L/XGOAT2 and PtrXGOATs are xyloglucan O-acetyltransferases catalyzing acetyl transfer onto fucosylated Gal residues on xyloglucan side chains and the defucosylation of these acetylated side chains by apoplastic AXY8 generates side chains with acetylated, non-fucosylated Gal residues.

Overexpression of Escherichia coli Acetyl Esterase Using a Strategy of Multi-copy Promoters

Acetyl esterase (AE) plays an important role in bioconversion of lignocellulosic biomass. However, the expression level of AE from wild type strains is relatively low. The aim of the study was to enhance the activity of AE using the strategy of multi-copy promoters. A commercial strain E. coli JM110 was used as the host of expression plasmid. pKK223-3-ybaC (pK-ybaC) is a recombinant plasmid, and it carries ybaC gene (encoding AE) that was derived from E. coli RB3. E. coli JM110 (pK-ybaC; EcKy) was a recombinant strain that contains pK-ybaC. Fragment P3 that contains three copies of the core-tac-promoter and fragment P5 that contains five copies of the core-tac-promoter were synthesized. Plasmid pK-ybaC was extracted from EcKy and was linearized by EcoR I and BamH I to remove its wild promoter. Then fragment P3 was subcloned into linearized pK-ybaC to generate pKTH-ybaC and fragment P5 was inserted into pK-ybaC to generate pKFI-ybaC. In-Fusion kit (Takara) was used to construct recombinant plasmids according to the instruction of the manufacturer. The two plasmids were then transformed into E. coli JM110 to get the recombinant strain E. coli JM110 (pKTH-ybaC; EcTH) and E. coli JM110 (pKFI-ybaC; EcFI). The highest activity of AE from EcTH was 3.04 U mL−1. It was 7.8 times of that from the parental strain E. coli RB3. The peak value of AE activity from EcFI was 35.66 U mL−1, which was 91.4 times of that from E. coli RB3. EcFI showed the highest transcription level of ybaC gene, and its value was 4239 times of that in E. coli RB3. The K m value was 3.49 mmol L−1 and the V max value was 43.29 µmol s−1 mg−1 for AE from EcFI. The K cat/K m value was around fivefold of that from the parental strain E.coli RB3. Two engineered bacterial strains that contain three copies and five copies of the core-tac-promoter were constructed. The activity of AE from the two engineered strains was enhanced significantly compared to that from the parental strain. The results indicated the feasibility of enhancing AE activity using the strategy of multi-copy promoters.

Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni. Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro. The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target.

Isolation, identification and fibrolytic characteristics of rumen fungi grown with indigenous methanogen from yaks (Bos grunniens) grazing on the Qinghai-Tibetan Plateau.

To obtain co-cultures of anaerobic fungi and their indigenously associated methanogens from the rumen of yaks grazing on the Qinghai-Tibetan Plateau and investigate their morphology features and ability to degrade lignocellulose. Twenty fungus-methanogen co-cultures were obtained by Hungate roll-tube technique. The fungi were identified as Orpinomyces, Neocallimastix and Piromyces genera based on the morphological characteristics and internal transcribed spacer 1 sequences analysis. All methanogens were identified as Methanobrevibacter sp. by 16S rRNA gene sequencing. There were four types of co-cultures: Neocallimastix with Methanobrevibacter ruminantium, Orpinomyces with M.ruminantium, Orpinomyces with Methanobrevibacter millerae and Piromyces with M.ruminantium among 20 co-cultures. In vitro studies with wheat straw as substrate showed that the Neocallimastix with M.ruminantium co-cultures and Piromyces with M.ruminantium co-cultures exhibited higher xylanase, filter paper cellulase (FPase), ferulic acid esterase, acetyl esterase activities, invitro dry matter digestibility, gas, CH4 , acetate production, ferulic acid and p-coumaric acid releases. The Neocallimastix frontalis Yak16 with M.ruminantium co-culture presented the strongest lignocellulose degradation ability among 20 co-cultures. Twenty fungus-methanogen co-cultures were obtained from the rumen of grazing yaks. The N.frontalis with M.ruminantium co-cultures were highly effective combination for developing a fermentative system that bioconverts lignocellulose to high activity fibre-degrading enzyme, CH4 and acetate. The N.frontalis with M.ruminantium co-cultures from yaks grazing on the Qinghai-Tibetan Plateau present great potential in lignocellulose biodegradation industry.

Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose.

Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1-expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.