Biochemical and structural characterisation of a family GH5 cellulase from endosymbiont of shipworm P. megotara.

BACKGROUND: Cellulases play a key role in the enzymatic conversion of plant cell-wall polysaccharides into simple and economically relevant sugars. Thus, the discovery of novel cellulases from exotic biological niches is of great interest as they may present properties that are valuable in the biorefining of lignocellulosic biomass. RESULTS: We have characterized a glycoside hydrolase 5 (GH5) domain of a bi-catalytic GH5-GH6 multi-domain enzyme from the unusual gill endosymbiont Teredinibacter waterburyi of the wood-digesting shipworm Psiloteredo megotara. The catalytic GH5 domain, was cloned and recombinantly produced with or without a C-terminal family 10 carbohydrate-binding module (CBM). Both variants showed hydrolytic endo-activity on soluble substrates such as ß-glucan, carboxymethylcellulose and konjac glucomannan, respectively. However, low activity was observed towards the crystalline form of cellulose. Interestingly, when co-incubated with a cellulose-active LPMO, a clear synergy was observed that boosted the overall hydrolysis of crystalline cellulose. The crystal structure of the GH5 catalytic domain was solved to 1.0 Å resolution and revealed a substrate binding cleft extension containing a putative + 3 subsite, which is uncommon in this enzyme family. The enzyme was active in a wide range of pH, temperatures and showed high tolerance for NaCl. CONCLUSIONS: This study provides significant knowledge in the discovery of new enzymes from shipworm gill endosymbionts and sheds new light on biochemical and structural characterization of cellulolytic cellulase. Study demonstrated a boost in the hydrolytic activity of cellulase on crystalline cellulose when co-incubated with cellulose-active LPMO. These findings will be relevant for the development of future enzyme cocktails that may be useful for the biotechnological conversion of lignocellulose.

Alginate Degradation: Insights Obtained through Characterization of a Thermophilic Exolytic Alginate Lyase.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to polysaccharide lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics data set. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.2.2.26), which can degrade alginate, poly-ß-d-mannuronate, and poly-α-l-guluronate within a broad range of pHs, temperatures, and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as a catalytic acid, indeed is essential for catalysis, whereas mutation of tyrosine Y446, previously proposed to act as a catalytic base, did not affect enzyme activity. The observed reaction products are protonated and deprotonated forms of the 4,5-unsaturated uronic acid monomer, Δ, two hydrates of DEH (4-deoxy-l-erythro-5-hexulosuronate), which are formed after ring opening, and, finally, two epimers of a 5-member hemiketal called 4-deoxy-d-manno-hexulofuranosidonate (DHF), formed through intramolecular cyclization of hydrated DEH. The detection and nuclear magnetic resonance (NMR) assignment of these hemiketals refine our current understanding of alginate degradation.IMPORTANCE The potential markets for seaweed-derived products and seaweed processing technologies are growing, yet commercial enzyme cocktails for complete conversion of seaweed to fermentable sugars are not available. Such an enzyme cocktail would require the catalytic properties of a variety of different enzymes, where fucoidanases, laminarinases, and cellulases together with endo- and exo-acting alginate lyases would be the key enzymes. Here, we present an exo-acting alginate lyase that efficiently produces monomeric sugars from alginate. Since it is only the second characterized exo-acting alginate lyase capable of degrading alginate at a high industrially relevant temperature (≥60°C), this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation revealed previously undescribed rearrangement products of the unsaturated monomeric sugars generated from exo-acting lyases. The insight provided by the NMR assignment of these products facilitates future assessment of product formation by alginate lyases.

A thermostable bacterial lytic polysaccharide monooxygenase with high operational stability in a wide temperature range.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are oxidative, copper-dependent enzymes that function as powerful tools in the turnover of various biomasses, including lignocellulosic plant biomass. While LPMOs are considered to be of great importance for biorefineries, little is known about industrial relevant properties such as the ability to operate at high temperatures. Here, we describe a thermostable, cellulose-active LPMO from a high-temperature compost metagenome (called mgLPMO10). RESULTS: MgLPMO10 was found to have the highest apparent melting temperature (83 °C) reported for an LPMO to date, and is catalytically active up to temperatures of at least 80 °C. Generally, mgLPMO10 showed good activity and operational stability over a wide temperature range. The LPMO boosted cellulose saccharification by recombinantly produced GH48 and GH6 cellobiohydrolases derived from the same metagenome, albeit to a minor extent. Cellulose saccharification studies with a commercial cellulase cocktail (Celluclast®) showed that the performance of this thermostable bacterial LPMO is comparable with that of a frequently utilized fungal LPMO from Thermoascus aurantiacus (TaLPMO9A). CONCLUSIONS: The high activity and operational stability of mgLPMO10 are of both fundamental and applied interest. The ability of mgLPMO10 to perform oxidative cleavage of cellulose at 80 °C and the clear synergy with Celluclast® make this enzyme an interesting candidate in the development of thermostable enzyme cocktails for use in lignocellulosic biorefineries.

Identification and characterization of a hyperthermophilic GH9 cellulase from the Arctic Mid-Ocean Ridge vent field.

A novel GH9 cellulase (AMOR_GH9A) was discovered by sequence-based mining of a unique metagenomic dataset collected at the Jan Mayen hydrothermal vent field. AMOR_GH9A comprises a signal peptide, a catalytic domain and a CBM3 cellulose-binding module. AMOR_GH9A is an exceptionally stable enzyme with a temperature optimum around 100°C and an apparent melting temperature of 105°C. The novel cellulase retains 64% of its activity after 4 hours of incubation at 95°C. The closest characterized homolog of AMOR_GH9A is TfCel9A, a processive endocellulase from the model thermophilic bacterium Thermobifida fusca (64.2% sequence identity). Direct comparison of AMOR_GH9A and TfCel9A revealed that AMOR_GH9A possesses higher activity on soluble and amorphous substrates (phosphoric acid swollen cellulose, konjac glucomannan) and has an ability to hydrolyse xylan that is lacking in TfCel9A.

Production, Characterization, and Application of an Alginate Lyase, AMOR_PL7A, from Hot Vents in the Arctic Mid-Ocean Ridge.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. We describe a thermostable alginate lyase belonging to Polysaccharide Lyase family 7 (PL7), which can be used to degrade brown seaweed, Saccharina latissima, at conditions also suitable for a commercial cellulase cocktail (Cellic CTec2). This enzyme, AMOR_PL7A, is a ß-d-mannuronate specific (EC 4.2.2.3) endoacting alginate lyase, which degrades alginate and poly mannuronate within a broad range of pH, temperature and salinity. At 65 °C and pH 6.0, its Km and kcat values for sodium alginate are 0.51 ± 0.09 mg/mL and 7.8 ± 0.3 s-1 respectively. Degradation of seaweed with blends of Cellic CTec2 and AMOR_PL7A at 55 °C in seawater showed that the lyase efficiently reduces viscosity and increases glucose solublization. Thus, AMOR_PL7A may be useful in development of efficient protocols for enzymatic seaweed processing.

Discovery and characterization of a thermostable two-domain GH6 endoglucanase from a compost metagenome.

Enzymatic depolymerization of recalcitrant polysaccharides plays a key role in accessing the renewable energy stored within lignocellulosic biomass, and natural biodiversities may be explored to discover microbial enzymes that have evolved to conquer this task in various environments. Here, a metagenome from a thermophilic microbial community was mined to yield a novel, thermostable cellulase, named mgCel6A, with activity on an industrial cellulosic substrate (sulfite-pulped Norway spruce) and a glucomannanase side activity. The enzyme consists of a glycoside hydrolase family 6 catalytic domain (GH6) and a family 2 carbohydrate binding module (CBM2) that are connected by a linker rich in prolines and threonines. MgCel6A exhibited maximum activity at 85°C and pH 5.0 on carboxymethyl cellulose (CMC), but in prolonged incubations with the industrial substrate, the highest yields were obtained at 60°C, pH 6.0. Differential scanning calorimetry (DSC) indicated a Tm(app) of 76°C. Both functional data and the crystal structure, solved at 1.88 Å resolution, indicate that mgCel6A is an endoglucanase. Comparative studies with a truncated variant of the enzyme showed that the CBM increases substrate binding, while not affecting thermal stability. Importantly, at higher substrate concentrations the full-length enzyme was outperformed by the catalytic domain alone, underpinning previous suggestions that CBMs may be less useful in high-consistency bioprocessing.

Recombinant Lactobacillus plantarum induces immune responses to cancer testis antigen NY-ESO-1 and maturation of dendritic cells.

Given their safe use in humans and inherent adjuvanticity, Lactic Acid Bacteria may offer several advantages over other mucosal delivery strategies for cancer vaccines. The objective of this study is to evaluate the immune responses in mice after oral immunization with Lactobacillus (L) plantarum WCFS1 expressing a cell-wall anchored tumor antigen NY-ESO-1. And to investigate the immunostimulatory potency of this new candidate vaccine on human dendritic cells (DCs). L. plantarum displaying NY-ESO-1 induced NY-ESO-1 specific antibodies and T-cell responses in mice. By contrast, L. plantarum displaying conserved proteins such as heat shock protein-27 and galectin-1, did not induce immunity, suggesting that immune tolerance to self-proteins cannot be broken by oral administration of L. plantarum. With respect to immunomodulation, immature DCs incubated with wild type or L. plantarum-NY-ESO-1 upregulated the expression of co-stimulatory molecules and secreted a large amount of interleukin (IL)-12, TNF-α, but not IL-4. Moreover, they upregulated the expression of immunosuppressive factors such as IL-10 and indoleamine 2,3-dioxygenase. Although L. plantarum-matured DCs expressed inhibitory molecules, they stimulated allogeneic T cells in-vitro. Collectively, the data indicate that L. plantarum-NY-ESO-1 can evoke antigen-specific immunity upon oral administration and induce DC maturation, raising the potential of its use in cancer immunotherapies.

Lactobacillus plantarum WCFS1 O-linked protein glycosylation: an extended spectrum of target proteins and modification sites detected by mass spectrometry.

It has recently been shown that the major autolysin Acm2 from Lactobacillus plantarum WCFS1 undergoes intracellular O-GlcNAcylation [Fredriksen L, Mathiesen G, Moen A, Bron PA, Kleerebezem M, Eijsink VG, Egge-Jacobsen W. 2012. The major autolysin Acm2 from Lactobacillus plantarum undergoes cytoplasmic O-glycosylation. J Bacteriol. 194(2):325-333]. To gain more insight into the occurrence of this protein modification, methods based on the higher energy collisional fragmentation of the Orbitrap XL mass spectrometer to generate both diagnostic oxonium (glycan) ions and significant peptide sequencing information were used to detect and identify novel glycoproteins. This led to the identification of 10 novel glycoproteins, including four proteins with well-known functions in the cytoplasm, a compartment not previously recognized to contain glycosylated proteins in bacteria: the molecular chaperone DnaK, the E2 subunit of the pyruvate dehydrogenase complex PdhC, the signal recognition particle receptor FtsY and the DNA translocase FtsK1. Among the other, glycosylated proteins were two extracellular peptidoglycan hydrolases and a mucus-binding protein. In total, 49 glycosylation sites for N-acetylhexosamine (HexNAc) were detected in the 11 Lactobacillus glycoproteins found so far. Most of the attached glycans consisted of a single HexNAc per site, whereas hexose moieties were also found in a few cases (in both of the peptidoglycan hydrolases and in DnaK).

Surface display of N-terminally anchored invasin by Lactobacillus plantarum activates NF-κB in monocytes.

The probiotic lactic acid bacterium Lactobacillus plantarum is a potential delivery vehicle for mucosal vaccines because of its generally regarded as safe (GRAS) status and ability to persist at the mucosal surfaces of the human intestine. However, the inherent immunogenicity of vaccine antigens is in many cases insufficient to elicit an efficient immune response, implying that additional adjuvants are needed to enhance the antigen immunogenicity. The goal of the present study was to increase the proinflammatory properties of L. plantarum by expressing a long (D1 to D5 [D1-D5]) and a short (D4-D5) version of the extracellular domain of invasin from the human pathogen Yersinia pseudotuberculosis. To display these proteins on the bacterial surface, four different N-terminal anchoring motifs from L. plantarum were used, comprising two different lipoprotein anchors, a transmembrane signal peptide anchor, and a LysM-type anchor. All these anchors mediated surface display of invasin, and several of the engineered strains were potent activators of NF-κB when interacting with monocytes in cell culture. The most distinct NF-κB responses were obtained with constructs in which the complete invasin extracellular domain was fused to a lipoanchor. The proinflammatory L. plantarum strains constructed here represent promising mucosal delivery vehicles for vaccine antigens.

The major autolysin Acm2 from Lactobacillus plantarum undergoes cytoplasmic O-glycosylation.

The major autolysin Acm2 from the probiotic strain Lactobacillus plantarum WCFS1 contains high proportions of alanine, serine, and threonine in its N-terminal so-called AST domain. It has been suggested that this extracellular protein might be glycosylated, but this has not been experimentally verified. We used high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the possible occurrence of glycans on peptides generated from lactobacillary surface proteins by protease treatment. This approach yielded five glycopeptides in various glycoforms, all derived from the AST domain of Acm2. All five glycopeptides contained the hydroxy-amino acids serine and threonine, suggesting that Acm2 is O-glycosylated. By using lectin blotting with succinylated wheat germ agglutinin, and by comparing the wild-type strain with an Acm2-negative derivative (NZ3557), we found that the attached N-acetylhexosamines are most likely N-acetylglucosamines (GlcNAc). NZ3557 was further used as a genetic background to express an Acm2 variant lacking its secretion signal, resulting in intracellular expression of Acm2. We show that this intracellular version of Acm2 is also glycosylated, indicating that the GlcNAc modification is an intracellular process.

A food-grade system for inducible gene expression in Lactobacillus plantarum using an alanine racemase-encoding selection marker.

Food-grade gene expression systems for lactic acid bacteria are useful for applications in the food industry. We describe a new food-grade host/vector system for Lactobacillus plantarum based on pSIP expression vectors and the use of the homologous alanine racemase gene (alr) as selection marker. A new series of expression vectors were constructed by exchanging the erythromycin resistance gene (erm) in pSIP vectors by the L. plantarum WCFS1 alr gene. The vectors were applied for the overexpression of ß-galactosidase genes from L. reuteri L103 and L. plantarum WCFS1 in an alr deletion mutant of L. plantarum WCFS1. The expression levels obtained in this way, i.e. without the use of antibiotics, were comparable to the levels obtained with the conventional system based on selection for erythromycin resistance. The new system is suitable for the production of ingredients and additives for the food industry.

Cell wall anchoring of the 37-kilodalton oncofetal antigen by Lactobacillus plantarum for mucosal cancer vaccine delivery.

The 37-kDa oncofetal antigen (OFA), a tumor immunogen expressed on all mammalian cancers examined to date, was secreted and anchored to the cell wall of Lactobacillus plantarum using homologous signal peptides and LPxTG anchors. Orally administered L. plantarum expressing anchored OFA induced a specific immune response against OFA in mice.

Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1.

BACKGROUND: Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality. RESULTS: We have constructed a library of 76 Sec-type signal peptides from L. plantarum WCFS1 that were predicted to be cleaved by signal peptidase I. SP functionality was studied using staphylococcal nuclease (NucA) as a reporter protein. 82% of the SPs gave significant extracellular NucA activity. Levels of secreted NucA varied by a dramatic 1800-fold and this variation was shown not to be the result of different mRNA levels. For the best-performing SPs all produced NucA was detected in the culture supernatant, but the secretion efficiency decreased for the less well performing SPs. Sequence analyses of the SPs and their cognate proteins revealed four properties that correlated positively with SP performance for NucA: high hydrophobicity, the presence of a transmembrane helix predicted by TMHMM, the absence of an anchoring motif in the cognate protein, and the length of the H+C domain. Analysis of a subset of SPs with a lactobacillal amylase (AmyA) showed large variation in production levels and secretion efficiencies. Importantly, there was no correlation between SP performance with NucA and the performance with AmyA. CONCLUSION: This is the first comprehensive experimental study showing that predicted SPs in the L. plantarum genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in L. plantarum. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.