The pyrroloquinoline-quinone (PQQ)-dependent quinohemoprotein pyranose dehydrogenase from Coprinopsis cinerea (CcPDH), belonging to the AA12 family, drives lytic polysaccharide monooxygenase (LPMO) action

Fungi secrete a set of glycoside hydrolases and oxidoreductases, including lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant polysaccharides. LPMOs accelerate the decomposition of cellulose by cellulases by catalyzing the oxidative cleavage of glycosidic bonds after activation by an external electron donor (1-3). LPMOs procure electrons from non-enzymatic electron donors, such as ascorbic acid, lignin and other plant biomass-derived phenols (1-3), or they can be activated by flavin-dependent oxidoreductases, directly or through plant-derived diphenols and quinones acting as redox mediators (3-7). Cellobiose dehydrogenase, in particular, efficiently transfers electrons from its AA3_1 dehydrogenase domain to LPMOs via an appended AA8 cytochrome domain (7). Here we show that LPMOs can be activated by a quinohemoprotein, namely the pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase CcPDH from Coprinopsis cinerea, the founding member of the recently discovered AA12 family (8). CcPDH has a domain composition similar to that of cellobiose dehydrogenases (CDHs) but contains a central catalytic AA12 dehydrogenase domain, rather than an AA3_1 domain. We have studied the ability of full length CcPDH and its truncated variants to drive catalysis by two Neurospora crassa LPMOs, NcLPMO9F and NcLPMO9C. Our study shows that both the AA8 and CBM1 domains of CcPDH have a positive effect on the CcPDH-NcLPMO system. The interplay between the PDH and LPMOs seemed also to depend on whether the LPMO contained a CBM. Unlike the single dehydrogenase domain of MtCDH from Myriococcum thermophylum, the AA12 dehydrogenase domain of CcPDH could drive the LPMO reaction, which is due to the non-covalently bound PQQ co-factor acting as a diphenol/quinone redox mediator. CcPDH does not oxidize cello-oligosaccharides, which makes this enzyme a useful tool in future studies of LPMOs and redox enzyme systems involved in cellulose degradation. References: [1] Vaaje-Kolstad, G. et al. (2010) Science 330, 219-222. [2] Hemsworth, G. R., et al. (2015) Trends Biotechnol 33:747-761. [3] Kracher, D. et al. (2016) Science 352, 1098-1101. [4] Westereng, B. et al. (2015) Sci Rep 5:18561. [5] Langston, J.A. et al. (2011) Appl Environ Microbiol 77:7007-7015. [6] Garajova, S. et al. (2016) Sci Rep 6:28276. [7] Tan, T.C. et al. (2015) Nat Commun 6:7542. [8] Takeda, K. et al. (2015) PLoS One 10:e0115722

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guerin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum. The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo. The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. IMPORTANCE This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guerin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigenspecific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice

Specific xylan activity revealed for AA9 Lytic Polysaccharide Monooxygenases of the thermophilic fungus Malbranchea cinnamomea by functional characterization

The thermophilic biomass-degrader\ua0Malbranchea cinnamomea\ua0exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea\ua0AA9s)\ua0McAA9s, namely,\ua0McAA9A,\ua0McAA9B,\ua0McAA9F, and\ua0McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized\ua0McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the\ua0McAA9s,\ua0McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends,\ua0McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO.\ua0McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers’ physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease of Bacillus stearothermophilus were analyzed using a three-dimensional model that was inferred from the crystal structure of thermolysin, the highly homologous neutral protease of B.thermoproteolyticus (85% sequence identity). Site-directed mutagenesis was used to fill some of these cavities, thereby improving hydrophobic packing in the protein interior. The mutations had small effects on the thermostability, even after drastic changes, such as Leu284 --> Trp and Met168 --> Trp. The effects on T50, the temperature at which 50% of the enzyme is irreversibly inactivated in 30 min, ranged from 0.0 to +0.4-degrees-C. These results can be explained by assuming that the mutations have positive and negative structural effects of approximately the same magnitude. Alternatively, it could be envisaged that the local unfolding steps, which render the enzyme susceptible towards autolysis and which are rate limiting in the process of thermal inactivation, are only slightly affected by alterations in the hydrophobic core

Glycan processing in gut microbiomes

Microbiomes and their enzymes process many of the nutrients accessible in the gastrointestinal tract of bilaterians and play an essential role in host health and nutrition. In this review, we describe recent insights into nutrient processing in microbiomes across three exemplary yet contrasting gastrointestinal ecosystems (humans, ruminants and insects), with focus on bacterial mechanisms for the utilization of common and atypical dietary glycans as well as host-derived mucus glycans. In parallel, we discuss findings from multi-omic studies that have provided new perspectives on understanding glycan-dependent interactions and the complex food-webs of microbial populations in their natural habitat. Using key examples, we emphasize how increasing understanding of glycan processing by gut microbiomes can provide critical insights to assist ‘microbiome reprogramming’, a growing field that seeks to leverage diet to improve animal growth and host health

Discovery, characterization and engineering of bacterial thermostable cellulose- degrading enzymes

Lignocellulose is the most abundant biomass on Earth, and thus our largest organic carbon reservoir. Enzymatic depolymerization of recalcitrant polysaccharides, notably cellulose, is a major cost driver in accessing the renewable energy stored within lignocellulosic biomass. Natural biodiversities may be explored to discover microbial enzymes that have evolved to conquer this task in various environments. We are studying novel enzymes from various biodiversities for the conversion of lignocellulosic materials, using (meta)genome mining and functional screening of fosmid libraries. Targeted biodiversities include deep-sea hot vents of the Arctic mid-ocean ridge (AMOR), the microbiome of the wood-eating Arctic shipworm, thermophilic enrichment cultures from biogas reactors, the Svalbard reindeer gut microbiome, and publicly available metagenomic data from various hot environments. Bioprospecting of the different biodiversities has so far resulted in the discovery of approximately 20 novel enzymes active on lignocellulosic substrates. The significant differences in the origin of the enzymes is reflected in their properties, both beneficial and challenging, and provide us with interesting engineering targets for improved performance in industrial settings. We will present case studies, including work on a novel thermostable cellulase named mgCel6A, with good activity on sulfite-pulped Norway spruce. This enzyme consists of a glycoside hydrolase family 6 catalytic domain (GH6) connected to a family 2 carbohydrate binding module (CBM2) and both the activity profile and predicted structural similarities to known cellulases suggest that mgCel6A is an endo-acting cellulase. Comparison of the full-length enzyme with the catalytic domain showed that the CBM strongly increases substrate binding, while not affecting thermal stability. However, importantly, in reactions with 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. This enzyme is currently being targeted for rational engineering in an effort to decrease the pH optimum and improve the pH stability. Other case studies include GH48 cellulases and lytic polysaccharide monooxygenases (LPMOs). One important aspect of this work concerns the possible assembly of novel enzyme cocktails for lignocellulose processing that can compete with exiting commercial cocktails, which are primarily composed of fungal enzymes. Thus, comparative studies of our most promising bacterial enzymes with their well-known fungal counterparts are also being conducted

Metagenomics of the Svalbard Reindeer Rumen Microbiome Reveals Abundance of Polysaccharide Utilization Loci

Lignocellulosic biomass remains a largely untapped source of renewable energy predominantly due to its recalcitrance and an incomplete understanding of how this is overcome in nature. We present here a compositional and comparative analysis of metagenomic data pertaining to a natural biomass-converting ecosystem adapted to austere arctic nutritional conditions, namely the rumen microbiome of Svalbard reindeer (Rangifer tarandus platyrhynchus). Community analysis showed that deeply-branched cellulolytic lineages affiliated to the Bacteroidetes and Firmicutes are dominant, whilst sequence binning methods facilitated the assemblage of metagenomic sequence for a dominant and novel Bacteroidales clade (SRM-1). Analysis of unassembled metagenomic sequence as well as metabolic reconstruction of SRM-1 revealed the presence of multiple polysaccharide utilization loci-like systems (PULs) as well as members of more than 20 glycoside hydrolase and other carbohydrate-active enzyme families targeting various polysaccharides including cellulose, xylan and pectin. Functional screening of cloned metagenome fragments revealed high cellulolytic activity and an abundance of PULs that are rich in endoglucanases (GH5) but devoid of other common enzymes thought to be involved in cellulose degradation. Combining these results with known and partly re-evaluated metagenomic data strongly indicates that much like the human distal gut, the digestive system of herbivores harbours high numbers of deeply branched and as-yet uncultured members of the Bacteroidetes that depend on PUL-like systems for plant biomass degradation

Methane Potential and Enzymatic Saccharification of Steam-exploded Bagasse

To evaluate the biofuel potential of bagasse, an abundant co-product in sugarcane-based industries, the effect of steam explosion on the efficiency of enzymatic saccharification and anaerobic digestion was studied. Bagasse was steam exploded at four different severity levels, and the impact of pretreatment was evaluated by analyzing the release of glucose after enzymatic saccharification with Cellic CTec2 and by analyzing methane production during anaerobic batch digestions. Increasing the severity of pretreatment led to degradation of xylan and the formation of pseudo-lignin. The severity of pretreatment was correlated with the enzymatic release of glucose; at optimal conditions, > 90% of the glucan was released. The highest methane yield (216 mL/gVS) was 1.3 times higher than the yield from untreated bagasse. More importantly, the pretreatment dramatically increased the rate of methane production; after 10 days, methane production from pretreated material was approximately twice that of the untreated material. To assess the possibility of developing combined processes, steam-exploded bagasse was enzymatically pre-hydrolyzed and, after the removal of released sugars, the remaining solid was subjected to anaerobic digestion. The results indicated that, in terms of total heating value, combined ethanol and biogas production is as beneficial as producing only biogas

In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose

Background: Biochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation. Results: Here, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation–reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt. Conclusion: Our results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation–reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics

Can we make Chitosan by Enzymatic Deacetylation of Chitin?

Chitin, an insoluble linear polymer of β-1,4-N-acetyl-d-glucosamine (GlcNAc; A), can be converted to chitosan, a soluble heteropolymer of GlcNAc and d-glucosamine (GlcN; D) residues, by partial deacetylation. In nature, deacetylation of chitin is catalyzed by enzymes called chitin deacetylases (CDA) and it has been proposed that CDAs could be used to produce chitosan. In this work, we show that CDAs can remove up to approximately 10% of N-acetyl groups from two different (α and β) chitin nanofibers, but cannot produce chitosan