Bacterial glycobiotechnology: A biosynthetic route for the production of biopharmaceutical glycans.

The recent advancement in the human glycome and progress in the development of an inclusive network of glycosylation pathways allow the incorporation of suitable machinery for protein modification in non-natural hosts and explore novel opportunities for constructing next-generation tailored glycans and glycoconjugates. Fortunately, the emerging field of bacterial metabolic engineering has enabled the production of tailored biopolymers by harnessing living microbial factories (prokaryotes) as whole-cell biocatalysts. Microbial catalysts offer sophisticated means to develop a variety of valuable polysaccharides in bulk quantities for practical clinical applications. Glycans production through this technique is highly efficient and cost-effective, as it does not involve expensive initial materials. Metabolic glycoengineering primarily focuses on utilizing small metabolite molecules to alter biosynthetic pathways, optimization of cellular processes for glycan and glycoconjugate production, characteristic to a specific organism to produce interest tailored glycans in microbes, using preferably cheap and simple substrate. However, metabolic engineering faces one of the unique challenges, such as the need for an enzyme to catalyze desired substrate conversion when natural native substrates are already present. So, in metabolic engineering, such challenges are evaluated, and different strategies have been developed to overcome them. The generation of glycans and glycoconjugates via metabolic intermediate pathways can still be supported by glycol modeling achieved through metabolic engineering. It is evident that modern glycans engineering requires adoption of improved strain engineering strategies for creating competent glycoprotein expression platforms in bacterial hosts, in the future. These strategies include logically designing and introducing orthogonal glycosylation pathways, identifying metabolic engineering targets at the genome level, and strategically improving pathway performance (for example, through genetic modification of pathway enzymes). Here, we highlight current strategies, applications, and recent progress in metabolic engineering for producing high-value tailored glycans and their applications in biotherapeutics and diagnostics.

Rapid and cost-efficient microplate assay for the accurate quantification of total phenolics in seaweeds.

Brown seaweeds (Phaeophyceae) are a rich source of polyphenols (up to 20% dry weight) with a structure based on phloroglucinol (1,3,5-trihydroxybenzene). To-date the determination of total phenolics content (TPC) involves a redox reaction with the Folin-Ciocalteu (FC) reagent. However, side reactions with other reducing substances preclude accurate, direct measurement of TPC. This research reports a novel microplate assay involving a coupling reaction between phloroglucinol with Fast Blue BB (FBBB) diazonium salt, at basic pH, to form a stable tri-azo complex with maximum absorbance at 450 nm. Linear regression correlation values (R2) were ≥0.99 with phloroglucinol as standard. Direct quantification of TPCs (phloroglucinol equivalents, PGEs) in crude aqueous and ethanolic extracts from A. nodosum demonstrated that the new FBBB assay is not subject to side-redox interference and provides a more accurate estimate of TPC (1.2-3.9-fold lower than with the FC assay) in a relatively rapid (30 min), cost-effective (0.24€/test) microplate format.

Valorisation of algal biomass to value-added metabolites: emerging trends and opportunities.

Algal biomass is a promising feedstock for sustainable production of a range of value-added compounds and products including food, feed, fuel. To further augment the commercial value of algal metabolites, efficient valorization methods and biorefining channels are essential. Algal extracts are ideal sources of biotechnologically viable compounds loaded with anti-microbial, anti-oxidative, anti-inflammatory, anti-cancerous and several therapeutic and restorative properties. Emerging technologies in biomass valorisation tend to reduce the significant cost burden in large scale operations precisely associated with the pre-treatment, downstream processing and waste management processes. In order to enhance the economic feasibility of algal products in the global market, comprehensive extraction of multi-algal product biorefinery is envisaged as an assuring strategy. Algal biorefinery has inspired the technologists with novel prospectives especially in waste recovery, carbon concentration/sequestration and complete utilisation of the value-added products in a sustainable closed-loop methodology. This review critically examines the latest trends in the algal biomass valorisation and the expansive feedstock potentials in a biorefinery perspective. The recent scope dynamics of algal biomass utilisation such as bio-surfactants, oleochemicals, bio-stimulants and carbon mitigation have also been discussed. The existing challenges in algal biomass valorisation, current knowledge gaps and bottlenecks towards commercialisation of algal technologies are discussed. This review is a comprehensive presentation of the road map of algal biomass valorisation techniques towards biorefinery technology. The global market view of the algal products, future research directions and emerging opportunities are reviewed.

Valorization of sugar beet pulp to value-added products: A review.

The processing of sugar beet in the sugar production industry releases huge amounts of sugar beet pulp as waste which can be considered a valuable by-product as a source of cellulose, hemicellulose, and pectin. Valorization of sugar beet pulp into value added products occurs through acid hydrolysis, hydrothermal techniques, and enzymatic hydrolysis. Biochemical conversion of beet pulp into simple fermentable sugars for producing value added products occurs through enzymatic hydrolysis is a cost effective and eco-friendly process. While beet pulp has predominantly been used as a fodder for livestock, recent developments in its biotechnological valorization have unlocked its value as a feedstock in the production of biofuels, biohydrogen, biodegradable plastics, and platform chemicals such as lactic acid, citric acid, alcohols, microbial enzymes, single cell proteins, and pectic oligosaccharides. This review brings forward recent biotechnological developments made in the valorization of sugar beet pulp into valuable products.

Characterization and gelling properties of a bioactive extract from Ascophyllum nodosum obtained using a chemical-free approach.

The bioactivity and gelling properties of a carbohydrate-rich algal extract obtained from locally harvested Ascophyllum nodosum seaweed using a chemical-free approach were investigated for its potential interest in food applications. Physicochemical characterisation and compositional analysis of the extract, using FTIR, biochemical methods and monosaccharide analysis, confirmed the presence of alginates and fucoidans, although the main polysaccharide present in it was laminarin. Significant amounts of phenolic compounds (~9 â€‹mg phloroglucinol/100 â€‹mg sample) were also detected. As a result, the extract exhibited good antioxidant activity. It also showed promising prebiotic potential, promoting the growth of beneficial Lactobacillus sp. and Bifidobacteria sp. when compared with commercial prebiotics, but not that of pathogenic bacteria such as E. coli or P. aeruginosa. The gelling properties of the raw extract were explored to optimize hydrogel bead formation by external gelation in CaCl2 solutions. This was enhanced at neutral to alkaline pHs and high extract and CaCl2 concentrations. The mechanical strength, nano- and microstructure of the hydrogel beads prepared under optimised conditions were determined using compression tests, synchrotron small- and wide-angle X-ray scattering (SAXS/WAXS) and scanning electron microscopy (SEM). It was concluded that the raw algal extract at neutral pH had potential for use as a gelling agent, although further enrichment with alginate improved the mechanical properties of the obtained gels. The advantages and disadvantages of applying the non-purified algal extract in comparison with purified carbohydrates are discussed.

Technological advances for improving fungal cellulase production from fruit wastes for bioenergy application: A review.

Fruit wastes can be imperative to elevate economical biomass to biofuels production process at pilot scale. Because of the renewable features, huge availability, having low lignin content organic nature and low cost; these wastes can be of much interest for cellulase enzyme production. This review provides recent advances on the fungal cellulase production using fruit wastes as a potential substrate. Also, the availability of fruit wastes, generation and processing data and their potential applications for cellulase enzyme production have been discussed. Several aspects, including cellulase and its function, solid-state fermentation, process parameters, microbial source, and the application of enzyme in biofuels industries have also been discussed. Further, emphasis has been made on various bottlenecks and feasible approaches such as use of nanomaterials, co-culture, molecular techniques, genetic engineering, and cost economy analysis to develop a low-cost based comprehensive technology for viable production of cellulase and its application in biofuels production technology.

Fungi populate deep-sea coral gardens as well as marine sediments in the Irish Atlantic Ocean.

Fungi populate deep Oceans in extreme habitats characterized by high hydrostatic pressure, low temperature and absence of sunlight. Marine fungi are potential major contributors to biogeochemical events, critical for marine communities and food web equilibrium under climate change conditions and a valuable source of novel extremozymes and small molecules. Despite their ecophysiological and biotechnological relevance, fungal deep-sea biodiversity has not yet been thoroughly characterized. In this study, we describe the culturable mycobiota associated with the deepest margin of the European Western Continental Shelf: sediments sampled at the Porcupine Bank and deep-water corals and sponges sampled in the Whittard Canyon. Eighty-seven strains were isolated, belonging to 43 taxa and mainly Ascomycota. Ten species and four genera were detected for the first time in the marine environment and a possible new species of Arachnomyces was isolated from sediments. The genera Cladosporium and Penicillium were the most frequent and detected on both substrates, followed by Candida and Emericellopsis. Our results showed two different fungal communities: sediment-associated taxa which were predominantly saprotrophic and animal-associated taxa which were predominantly symbiotic. This survey supports selective fungal biodiversity in the deep North Atlantic, encouraging further mycological studies on cold water coral gardens, often overexploited marine habitats.

A Novel High-Throughput Screening Platform Identifies Itaconate Derivatives from Marine Penicillium antarcticum as Inhibitors of Mesenchymal Stem Cell Differentiation.

Worldwide diffused diseases such as osteoarthritis, atherosclerosis or chronic kidney disease are associated with a tissue calcification process which may involve unexpected local stem cell differentiation. Current pharmacological treatments for such musculoskeletal conditions are weakly effective, sometimes extremely expensive and often absent. The potential to develop new therapies is represented by the discovery of small molecules modulating resident progenitor cell differentiation to prevent aberrant tissue calcification. The marine environment is a rich reserve of compounds with pharmaceutical potential and many novel molecules are isolated from macro and microorganisms annually. The potential of small molecules synthetized by marine filamentous fungi to influence the osteogenic and chondrogenic differentiation of human mesenchymal stem/stromal cells (hMSCs) was investigated using a novel, high-throughput automated screening platform. Metabolites synthetized by the marine-derived fungus Penicillium antarcticum were evaluated on the platform. Itaconic acid derivatives were identified as inhibitors of calcium elaboration into the matrix of osteogenically differentiated hMSCs and also inhibited hMSC chondrogenic differentiation, highlighting their capacity to impair ectopic calcification. Bioactive small molecule discovery is critical to address ectopic tissue calcification and the use of biologically relevant assays to identify naturally occurring metabolites from marine sources represents a strategy that can contribute to this effort.

Production of a recombinant swollenin from Trichoderma harzianum in Escherichia coli and its potential synergistic role in biomass degradation.

BACKGROUND: Fungal swollenins (SWOs) constitute a class of accessory proteins that are homologous to canonical plant expansins. Expansins and expansin-related proteins are well known for acting in the deagglomeration of cellulose structure by loosening macrofibrils. Consequently, SWOs can increase the accessibility and efficiency of the other enzymes involved in the saccharification of cellulosic substrates. Thus, SWOs are promising targets for improving the hydrolysis of plant biomass and for use as an additive to enhance the efficiency of an enzyme cocktail designed for the production of biofuels. RESULTS: Here, we report the initial characterization of an SWO from Trichoderma harzianum (ThSwo) that was successfully produced using Escherichia coli as a host. Initially, transcriptome and secretome data were used to compare swo gene expression and the amount of secreted ThSwo. The results from structural modeling and phylogenetic analysis of the ThSwo protein showed that ThSwo does preserve some structural features of the plant expansins and family-45 glycosyl hydrolase enzymes, but it evolutionarily diverges from both of these protein classes. Recombinant ThSwo was purified at a high yield and with high purity and showed secondary folding similar to that of a native fungal SWO. Bioactivity assays revealed that the purified recombinant ThSwo created a rough and amorphous surface on Avicel and displayed a high synergistic effect with a commercial xylanase from T. viride, enhancing its hydrolytic performance up to 147 ± 7%. CONCLUSIONS: Many aspects of the structure and mechanism of action of fungal SWOs remain unknown. In the present study, we produced a recombinant, active SWO from T. harzianum using a prokaryotic host and confirmed its potential synergistic role in biomass degradation. Our work paves the way for further studies evaluating the structure and function of this protein, especially regarding its use in biotechnology.

Use of a mannitol rich ensiled grass press juice (EGPJ) as a sole carbon source for polyhydroxyalkanoates (PHAs) production through high cell density cultivation.

This study demonstrates the use of a mannitol rich ensiled grass press juice (EGPJ) as a renewable carbon substrate for polyhydroxyalkanoates (PHA) production in shaking flask experiments and fed-batch stirred tank reactor cultivations. Fed-batch cultivations of Burkholderia sacchari IPT101 using EGPJ as sole carbon source produced 44.5 g/L CDW containing 33% polyhydroxybutyrate (PHB) in 36 h, while Pseudomonas chlororaphis IMD555 produced a CDW of 37 g/L containing 10% of medium chain length polyhydroxyalkanoates (mcl-PHA) in 34 h. PHB and mcl-PHA extracted from B. sacchari IPT101 and P. chlororaphis IMD555, grown on EGPJ, had a molecular weight of 548 kg/mol and 115.4 kg/mol, respectively. While mcl-PHA can be produced from EGPJ, PHB production is more interesting as there is a 4-fold higher volumetric productivity compared to mcl-PHA.

Conversion of grass biomass into fermentable sugars and its utilization for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas strains.

This study investigated the potential of grass biomass as a feedstock for mcl-PHA production. Pretreatments (2% NaOH at 120°C or hot water at 120°C) of perennial ryegrass were employed alone or in combination with sodium chlorite/acetic acid (SC/AA) delignification to evaluate the enzymatic digestibility and subsequent utilization of resultant sugars by Pseudomonas strains. NaOH pretreated sample had better digestibility than raw and hot water treated samples and this hydrolysate supported good growth of all tested strains with limited mcl-PHA (6-17% of cell dry mass (CDM)) accumulation. Digestibility of both untreated and pretreated samples was improved after SC/AA delignification and produced glucose (74-77%) rich hydrolysates. Tested strains accumulated 20-34% of CDM as PHA when these hydrolysates were used as sole carbon and energy source. CDM and PHA yields obtained for these strains when tested with laboratory grade sugars was similar to that achieved with grass derived sugars.

Cloning, overexpression in Escherichia coli, and characterization of a thermostable fungal acetylxylan esterase from Talaromyces emersonii.

The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels.

Influence of unit operations on the levels of polyacetylenes in minimally processed carrots and parsnips: An industrial trial.

Carrots and parsnips are often consumed as minimally processed ready-to-eat convenient foods and contain in minor quantities, bioactive aliphatic C17-polyacetylenes (falcarinol, falcarindiol, falcarindiol-3-acetate). Their retention during minimal processing in an industrial trial was evaluated. Carrot and parsnips were prepared in four different forms (disc cutting, baton cutting, cubing and shredding) and samples were taken in every point of their processing line. The unit operations were: peeling, cutting and washing with chlorinated water and also retention during 7days storage was evaluated. The results showed that the initial unit operations (mainly peeling) influence the polyacetylene retention. This was attributed to the high polyacetylene content of their peels. In most cases, when washing was performed after cutting, less retention was observed possibly due to leakage during tissue damage occurred in the cutting step. The relatively high retention during storage indicates high plant matrix stability. Comparing the behaviour of polyacetylenes in the two vegetables during storage, the results showed that they were slightly more retained in parsnips than in carrots. Unit operations and especially abrasive peeling might need further optimisation to make them gentler and minimise bioactive losses.

Characterisation of a Talaromyces emersonii thermostable enzyme cocktail with applications in wheat dough rheology.

In this paper, we report new sequence data for secreted thermostable fungal enzymes from the un-sequenced xylanolytic filamentous fungus Talaromyces emersonii and reveal novel insights on the potential role of enzymes relevant as wheat dough improvers. The presence of known and de novo enzyme sequences were confirmed through NanoLC-ESI-MS/MS and resultant peptide sequences were identified using SWISS PROT databases. The de novo protein sequences were assigned identity based on homology to known fungal proteins. Other proteins were assigned function based on the limited T. emersonii genome coverage. This approach allowed the identification of enzymes with relevance as wheat dough improvers. Rheological examination of wheat dough and wheat flour components treated with the thermostable fungal enzyme cocktail revealed structural alterations that can be extrapolated to the baking process. Thermoactive amylolytic, xylanolytic, glucanolytic, proteolytic and lipolytic enzyme activities were observed. Previously characterized T. emersonii enzymes present included; ß-glucosidase, xylan-1,4-ß-xyloxidase, acetylxylan esterase, acid trehalase, avenacinase, cellobiohydrolase and endo-glucanase. De novo sequence analysis confirmed peptides as being; α-glucosidase, endo-1,4-ß-xylanase, endo-arabinase, endo-glucanase, exo-ß-1,3-glucanase, glucanase/cellulase, endopeptidase and lipase/acylhydrolase. Rheology tests using wheat dough and fractioned wheat flour components in conjunction with T. emersonii enzymes show the role of these novel biocatalysts in altering properties of wheat substrates. Enzyme treated wheat flour fractions showed the effects of particular enzymes on appropriate substrates. This proteomic approach combined with rheological characterization is the first such report to the authors' knowledge.

An unfractionated fucoidan from Ascophyllum nodosum: extraction, characterization, and apoptotic effects in vitro.

An unfractionated fucoidan was extracted from the brown alga Ascophyllum nodosum. Extraction of fucoidan from seaweed was carried out using an innovative low-chemical process. A combinational approach involving compositional analysis, HPAEC, IR analysis, GPC, and NMR was employed to elucidate the composition and structure of an unfractionated fucoidan from A. nodosum. This fucoidan is composed mainly of fucose (52.1%), and also galactose (6.1%), glucose (21.3%), and xylose (16.5%). Sulfate content was determined to be 19%. GPC data indicated a polydisperse fucoidan containing two main size fractions (47 and 420 kDa). NMR analyses revealed a fucoidan displaying broad, complex signals as expected for such a high molecular weight and heterogeneous polymer with resonances consistent with a fucoidan isolated previously from A. nodosum. The effects of fucoidan on the apoptosis of human colon carcinoma cells and fucoidan-mediated signaling pathways were also investigated. Fucoidan decreased cell viability and induced apoptosis of HCT116 colon carcinoma cells. Fucoidan treatment of HCT116 cells induced activation of caspases-9 and -3 and the cleavage of PARP, led to apoptotic morphological changes, and altered mitochondrial membrane permeability. These results detail the structure and biological activity of an unfractionated fucoidan from A. nodosum.

Evolution and diversity of plant cell walls: from algae to flowering plants.

All photosynthetic multicellular Eukaryotes, including land plants and algae, have cells that are surrounded by a dynamic, complex, carbohydrate-rich cell wall. The cell wall exerts considerable biological and biomechanical control over individual cells and organisms, thus playing a key role in their environmental interactions. This has resulted in compositional variation that is dependent on developmental stage, cell type, and season. Further variation is evident that has a phylogenetic basis. Plants and algae have a complex phylogenetic history, including acquisition of genes responsible for carbohydrate synthesis and modification through a series of primary (leading to red algae, green algae, and land plants) and secondary (generating brown algae, diatoms, and dinoflagellates) endosymbiotic events. Therefore, organisms that have the shared features of photosynthesis and possession of a cell wall do not form a monophyletic group. Yet they contain some common wall components that can be explained increasingly by genetic and biochemical evidence.

Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii.

The moderately thermophilic aerobic ascomycete Talaromyces emersonii secretes, under selected growth conditions, several ß-glucan hydrolases including an exo-1,3-ß-glucanase. This enzyme was purified to apparent homogeneity in order to characterise its biochemical properties and investigate hydrolysis of different ß-glucans, including laminaran, a 1,3-ß-glucan from brown algae. The native enzyme is monomeric with a molecular mass of ~40 kDa and a pI value of 4.3, and is active over broad ranges of pH and temperature, with optimum activity observed at pH 5.4 and 65 °C. At pH 5.0, the enzyme displays strict specificity for laminaran (apparent K(m) 1.66 mg mL⁻¹; V(max) 7.69 IU mL⁻¹) and laminari-oligosaccharides and did not yield activity against 1,4-ß-glucans, 1,3;1,4-ß-glucans or 4-nitrophenyl- and methylumbelliferyl-ß-D: -glucopyranosides. Analysis of hydrolysis products formed during time-course hydrolysis of laminaran by high-performance anion exchange chromatography with pulsed amperometric detection revealed a strict exo mode of action, with glucose being the sole reaction product even at the initial stages of hydrolysis. The T. emersonii exo-1,3-ß-glucanase was inhibited by glucono-δ-lactone (K(i) 1.25 mM) but at significantly higher concentrations than typically inhibitory for exo-glycosidases such as ß-glucosidase. 'De novo' sequence analysis of the purified enzyme suggests that it belongs to family GH5 of the glycosyl hydrolase superfamily. The results clearly show that the exo-1,3-ß-glucanase is yet another novel enzyme present in the ß-glucanolytic enzyme system of T. emersonii.

A multi-step chromatographic strategy to purify three fungal endo-ß-glucanases.

Fungi and fungal enzymes have traditionally occupied a central role in biotechnology. Understanding the biochemical properties of the variety of enzymes produced by these eukaryotes has been an area of research interest for decades and again more recently due to global interest in greener bio-production technologies. Purification of an individual enzyme allows its unique biochemical and functional properties to be determined, can provide key information as to the role of individual biocatalysts within a complex enzyme system, and can inform both protein engineering and enzyme production strategies in the development of novel green technologies based on fungal biocatalysts. Many enzymes of current biotechnological interest are secreted by fungi into the extracellular culture medium. These crude enzyme mixtures are typically complex, multi-component, and generally also contain other non-enzymatic proteins and secondary metabolites. In this chapter, we describe a multi-step chromatographic strategy required to isolate three new endo-ß-glucanases (denoted EG V, EG VI, and EG VII) with activity against cereal mixed-linkage ß-glucans from the thermophilic fungus Talaromyces emersonii. This work also illustrates the challenges frequently involved in isolating individual extracellular fungal proteins in general.

Influence of Sous Vide and water immersion processing on polyacetylene content and instrumental color of parsnip (Pastinaca sativa) disks.

The effect of blanching (95 +/- 3 degrees C) followed by sous vide (SV) processing (90 degrees C for 10 min) on levels of two polyacetylenes in parsnip disks immediately after processing and during chill storage was studied and compared with the effect of water immersion (WI) processing (70 degrees C for 2 min.). Blanching had the greatest influence on the retention of polyacetylenes in sous vide processed parsnip disks resulting in significant decreases of 24.5 and 24% of falcarinol (1) and falcarindiol (2) respectively (p < 0.05). Subsequent SV processing did not result in additional significant losses in polyacetylenes compared to blanched samples. Subsequent anaerobic storage of SV processed samples resulted in a significant decrease in 1 levels (p < 0.05) although no change in 2 levels was observed (p > 0.05). 1 levels in WI processed samples were significantly higher than in SV samples (p

Talaromyces emersonii thermostable enzyme systems and their applications in wheat baking systems.

In this study, novel extracellular thermozymes were produced by the thermophilic fungus Talaromyces emersonii (IMI 392299) on low-cost carbon inducers. This paper reports the cocktail characterization, substrate hydrolysis studies, and their application in baking. Relevant enzymes were optimally active at pH 4.5-5.0 and 70 degrees C. Model studies confirmed production of significant levels of yeast monosaccharide sugars during cereal flour hydrolysis. The "thermozyme cocktails" are thermostable secreted T. emersonii enzyme blends. In baking trials, these thermozyme cocktails showed significant improvements in bread quality with respect to hardness, staling, and loaf volume (p < 0.5). Thermozyme cocktail B- treated loaf volume was 23.2% greater than the control and 49.5% softer. Staling analysis showed that bread treated with cocktail B was 41.7% softer than the control. This is the first report of T. emersonii thermozymes positively influencing bread quality.