Proteomic analysis of Viscozyme L and its major enzyme components for pectic substrate degradation.

Enzymatic degradation of plant biomass requires the coordinated action of various enzymes. In this study, the production of reducing sugars from pectic substrates and sugar beet pulp (SBP) was investigated and compared using commercial enzyme preparations, including M2, pectinase (E1), Viscozyme L (V-L) and L-40. V-L, a cellulolytic enzyme mix produced by Aspergillus sp. was further evaluated as the most robust enzyme cocktail with the strongest SBP degradation ability in terms of the release of monosaccharides, methanol, and acetate from SBP. Mass-spectrometry-based proteomics analysis of V-L revealed 156 individual proteins. Of these, 101 proteins were annotated as containing a carbohydrate-active enzyme module. Notably, of the 50 most abundant proteins, ca. 44 % were predicted to be involved in pectin degradation. To reveal the role of individual putative key enzymes in pectic substrate decomposition, two abundant galacturonases (PglA and PglB), were heterologously expressed in Pichia pastoris and further characterized. PglA and PglB demonstrated maximum activity at 57 °C and 68 °C, respectively, and exhibited endo-type cleavage patterns towards polygalacturonic acid. Further studies along this line may lead to a better understanding of efficient SBP degradation and may help to design improved artificial enzyme mixtures with lower complexity for future application in biotechnology.

N-substituted pyrrole carboxylic acid derivatives from 3,4-dihydroxyketons.

Since the chemical industry is largely dependent on petrol-based feedstocks, new sources are required for a sustainable industry. Conversion of biomass to high-value compounds provides an environmentally friendly and sustainable approach, which might be a potential solution to reduce petrol-based starting materials. This also applies for N-heterocycles, which are a common structural motif in natural products, pharmaceuticals and functional polymers. The synthesis of pyrroles is a well-studied and established process. Nevertheless, most routes described are not in line with the principles of green and sustainable chemistry and employ harsh reaction conditions and harmful solvents. In this study, 3,4-dihydroxyketons are used as excellent platform chemicals for the production of N-substituted pyrrole-2-carboxylic- and pyrrole-2,5-dicarboxylic acids, as they can be prepared from glucose through the intermediate d-glucarate and converted into pyrrolic acid derivatives under mild conditions in water. The scope of this so far unknown reaction was examined using a variety of primary amines and aqueous ammonium chloride leading to pyrrolic acid derivatives with N-substituents like alkane-, alkene-, phenyl- and alcohol-groups with yields up to 20 %. The combination of both, enzymatic conversion and chemical reaction opens up new possibilities for further process development. Therefore, a continuous chemo-enzymatic system was set up by first employing an immobilized enzyme to catalyze the conversion of d-glucarate to the 3,4-dihydroxyketone, which is further converted to the pyrrolic acid derivatives by a chemical step in continuous flow.

Investigation of exopolysaccharide formation and its impact on anaerobic succinate production with Vibrio natriegens.

Vibrio natriegens is an emerging host for biotechnology due to its high growth and substrate consumption rates. In industrial processes typically fed-batch processes are applied to obtain high space-time yields. In this study, we established an aerobic glucose-limited fed-batch fermentation with the wild type (wt) of V. natriegens which yielded biomass concentrations of up to 28.4 gX L-1 . However, we observed that the viscosity of the culture broth increased by a factor of 800 at the end of the cultivation due to the formation of 157 ± 20 mg exopolysaccharides (EPS) L-1 . Analysis of the genomic repertoire revealed several genes and gene clusters associated with EPS formation. Deletion of the transcriptional regulator cpsR in V. natriegens wt did not reduce EPS formation, however, it resulted in a constantly low viscosity of the culture broth and altered the carbohydrate content of the EPS. A mutant lacking the cps cluster secreted two-fold less EPS compared to the wt accompanied by an overall low viscosity and a changed EPS composition. When we cultivated the succinate producer V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pycCg (Succ1) under anaerobic conditions on glucose, we also observed an increased viscosity at the end of the cultivation. Deletion of cpsR and the cps cluster in V. natriegens Succ1 reduced the viscosity five- to six-fold which remained at the same level observed at the start of the cultivation. V. natriegens Succ1 ΔcpsR and V. natriegens Succ1 Δcps achieved final succinate concentrations of 51 and 46 g L-1 with a volumetric productivity of 8.5 and 7.7 gSuc L-1 h-1 , respectively. Both strains showed a product yield of about 1.4 molSuc molGlc -1 , which is 27% higher compared with that of V. natriegens Succ1 and corresponds to 81% of the theoretical maximum.

Rheological characterization of artificial paenan compositions produced by Paenibacillus polymyxa DSM 365.

Microbial exopolysaccharides offer a sustainable alternative to petroleum-based rheological modifiers. Recent studies revealed that the heteroexopolysaccharide produced by Paenibacillus polymyxa is composed of three distinct biopolymers, referred to as paenan I, II and III. Using CRISPR-Cas9 mediated knock-out variants of glycosyltransferases, defined polysaccharide compositions were produced and rheologically characterized in detail. The high viscosity and gel-like character of the wildtype polymer is proposed to originate from the non-covalent interaction between a pyruvate residue of paenan I and the glucuronic acid found in the backbone of paenan III. Paenan II conveys thermostable properties to the exopolysaccharide mixture. In contrast to the wildtype polymer mixture, knock-out variants demonstrated significantly altered rheological behavior. Using the rheological characterization performed in this study, tailor-made paenan variants and mixtures can be generated to be utilized in a wide range of applications including thickening agents, coatings, or high-value biomedical materials.

CRISPR-Cas9 driven structural elucidation of the heteroexopolysaccharides from Paenibacillus polymyxa DSM 365.

Paenibacillus polymyxa is a Gram-positive soil bacterium known for producing a wide range of exopolysaccharides. However, due to the biopolymer's complexity, structural elucidation has so far been inconclusive. Combinatorial knock-outs of glycosyltransferases were generated in order to separate distinct polysaccharides produced by P. polymyxa. Using a complementary analytical approach consisting of carbohydrate fingerprints, sequence analysis, methylation analysis as well as NMR spectroscopy, the structure of the repeating units of two additional heteroexopolysaccharides termed paenan I and paenan III were elucidated. Results for paenan I identified a trisaccharide backbone consisting of 1➔4-ß-d-Glc, 1➔4-ß-d-Man and a 1,3,4-branching ß-d-Gal residue with a sidechain comprising of a terminal ß-d-Gal3,4-Pyr and 1➔3-ß-d-Glc. For paenan III, results indicated a backbone consisting of 1➔3-ß-d-Glc, 1,3,4-linked α-d-Man and 1,3,4-linked α-d-GlcA. NMR analysis indicated monomeric ß-d-Glc and α-d-Man sidechains for the branching Man and GlcA residues respectively.

Structural elucidation of the fucose containing polysaccharide of Paenibacillus polymyxa DSM 365.

Paenibacillus polymyxa is an avid producer of exopolysaccharides of industrial interest. However, due to the complexity of the polymer composition, structural elucidation of the polysaccharide remained unfeasible for a long time. By using a CRISPR-Cas9 mediated knock-out strategy, all single glycosyltransferases as well as the Wzy polymerases were individually deleted in the corresponding gene cluster for the first time. Thereby, it was observed that the main polymer fraction was completely suppressed (or deleted) and a pure minor fucose containing polysaccharide could be isolated, which was named paenan II. Applying this combinatorial approach, the monosaccharide composition, sequence and linkage pattern of this novel polymer was determined via HPLC-MS, GC-MS and NMR. Furthermore, we demonstrated that the knock-out of the glycosyltransferases PepQ, PepT, PepU and PepV as well as of the Wzy polymerase PepG led to the absence of paenan II, attributing those enzymes to the assembly of the repeating unit.

Systematic optimization of exopolysaccharide production by Gluconacetobacter sp. and use of (crude) glycerol as carbon source.

The usage of polysaccharides as biodegradable polymers is of growing interest in the context of a sustainable and ecofriendly economy. For this, the production of exopolysaccharides (EPS) by Gluconacetobacter sp. was investigated. Glycerol as carbon source revealed to be beneficial compared to glucose. In addition, pure glycerol could be substituted by a crude glycerol waste stream from biodiesel production. Systematic analysis of the peptone and phosphate concentrations in glycerol-based media indicated a strong effect of peptone. Optimized parameters resulted in a titer of 25.4 ± 2.4 g/L EPS with a productivity of 0.46 ± 0.04 g*(L*h)-1. With decreasing peptone, a variation in the monomer ratios was observed. An accompanying change in molecular size distribution indicated the production of two different polysaccharides. Intensified analysis revealed the main polysaccharide to be composed of glucose (Glc), galactose (Gal), mannose (Man) and glucuronic acid (GlcA), and the minor polysaccharide of Gal, Man, ribose (Rib).

Correction: Shelat, K.J.; et al. Overall Nutritional and Sensory Profile of Different Species of Australian Wattle Seeds (Acacia spp.): Potential Food Sources in the Arid and Semi-Arid Regions. Foods 2019, 8, 482.

The authors wish to make the following correction to the paper [1]:The title of Table 3 "Amino acid (g/100 g dry weight) profile of four different species of wattle seeds" should be changed to "Amino acid (mol% dry weight) profile of four different species of wattle seeds" [...].

Rheological characterization of Porphyridium sordidum and Porphyridium purpureum exopolysaccharides.

Porphyridium exopolysaccharides (EPSs), which contain sulfate and methyl groups, have a similar potential for use in multiple industrial applications as macroalgae counterparts but lack detailed characterization. For this reason, we produced 0.21 g L-1 of P. sordidum EPS and 0.17 g L-1P. purpureum EPS, followed by a thorough rheological characterization in respect to their differences in monomer composition, sulfate concentrations and methyl patterns. Furthermore, the effect of NaCl and CaCl2 was evaluated, and the effect of high salinity media on the rheological properties of the biopolymers was analyzed. Both Porphyridium EPSs show a remarkable stability at high temperature and under the effect of mono- and divalent cations, and high salinity cultivation medium, which was evidenced by the rheological properties of the EPS. This feature is not displayed by many carbohydrate polymers, making it possible to enrich current applications in which EPS are used.

Overall Nutritional and Sensory Profile of Different Species of Australian Wattle Seeds (Acacia spp.): Potential Food Sources in the Arid Semi-Arid Regions.

Wattle seed (Acacia spp.) is a well-known staple food within indigenous communities in Australia. A detailed investigation of the overall nutritional and sensory profile of four abundant and underutilized Acacia species-A. coriacea, A. cowleana, A. retinodes and A. sophorae-were performed. Additionally, molecular weight of protein extracts from the wattle seeds (WS) was determined. The seeds are rich in protein (23-27%) and dietary fibre (33-41%). Relatively high fat content was found in A. cowleana (19.3%), A. sophorae (14.8%) and A. retinodes (16.4%) with oleic acid being the predominant fatty acid. The seeds contained high amounts of essential amino acids (histidine, lysine, valine, isoleucine and leucine). A. coriacea is rich in iron (43 mg/kg), potassium (10 g/kg) and magnesium (1.7 g/kg). Pentose (xylose/arabinose), glucose, galactose and galacturonic acids were the major sugars found in the four species. Raw seeds from A. sophorae, A. retinodes and A. coriacea have the highest protein molecular weight, between 50-90 kDa, 80 kDa and 50-55 kDa, respectively. There was variation in the sensory profile of the WS species. This study showed that the four WS species have good nutritional value and could be included in human diet or used in food formulations.

Colorimetric Determination of Sulfate via an Enzyme Cascade for High-Throughput Detection of Sulfatase Activity.

High-throughput screening (HTS) methods have become decisive for the discovery and development of new biocatalysts and their application in numerous fields. Sulfatases, a broad class of biocatalysts that hydrolyze sulfate esters, are involved in diverse relevant cellular functions (e.g., signaling and hormonal regulation) and are therefore gaining importance, particularly in the medical field. Additionally, various technical applications have been recently devised. One of the major challenges in the field of enzyme development is the sensitive and high-throughput detection of the actual product of the biocatalyst of interest without the need for chromophore analogues. Addressing this issue, a colorimetric assay for sulfatases was developed and validated for detecting sulfate through a two-step enzymatic cascade, with a linear detection range of 3.3 (limit of detection) up to 250 µM. The procedure is compatible with relevant compounds employed in sulfatase reactions, including cosolvents, cations, and buffers. The assay was optimized and performed as part of a 96-well screening workflow that included bacterial growth, heterologous sulfatase expression, cell lysis, sulfate ester hydrolysis, inactivation of cell lysate, and colorimetric sulfate determination. With this procedure, the activity of an aryl and an alkyl sulfatase could be confirmed and validated. Overall, this assay provides a simple and fast alternative for screening and engineering sulfatases from DNA libraries (e.g., using metagenomics) with medical or synthetic relevance.

Screening of c-di-GMP-Regulated Exopolysaccharides in Host Interacting Bacteria.

Bacterial exopolysaccharides (EPS) often confer a survival advantage by protecting the cell against abiotic and biotic stresses, including host defensive factors. They are also main components of the extracellular matrix involved in cell-cell recognition, surface adhesion and biofilm formation. Biosynthesis of a growing number of EPS has been reported to be regulated by the ubiquitous second messenger c-di-GMP, which promotes the transition to a biofilm mode of growth in an intimate association with the eukaryotic host. Here we describe a strategy based on the combination of an approach to artificially increase the intracellular level of c-di-GMP in virtually any gram-negative bacteria with a high throughput screening (HTS) for the identification of monosaccharide composition and carbohydrate fingerprinting of novel EPS, or modified variants, that can be involved in host-bacteria interactions.

Quantitative assay of ß-(1,3)-ß-(1,6)-glucans from fermentation broth using aniline blue.

An aniline blue fluorescence assay was constructed to quantify two fungal ß-(1,3)-glucans with ß-(1,6)-sidechains: scleroglucan and schizophyllan. Calibration curves were linear in the range from 30mg/l to 6g/l with R2>99.8%. The assay was robust against d-glucose (50 g/l), oxalic acid (22.5g/l), protein (0.667g/l), and KCl (13.3g/l). Sclerotium rolfsii and Schizophyllum commune cultures were sampled, and the test results of the assay and alcohol precipitation were compared. The correlation between assay results of samples at different stages of the purification with the values from the precipitation of the polymers was the best in the supernatant for S. rolfsii and after blending for S. commune. Using the precipitation data, correction factors of 2.46 and 3.83 for S. rolfsii and S. commune, respectively, were found. A range of other polymers were tested for their measurability with this assay. However, except for laminarin and microcrystalline cellulose no robust correlation between concentration and fluorescence intensity was found.

Tailor-made exopolysaccharides-CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa.

Application of state-of-the-art genome editing tools like CRISPR-Cas9 drastically increase the number of undomesticated micro-organisms amenable to highly efficient and rapid genetic engineering. Adaptation of these tools to new bacterial families can open up entirely new possibilities for these organisms to accelerate as biotechnologically relevant microbial factories, also making new products economically competitive. Here, we report the implementation of a CRISPR-Cas9 based vector system in Paenibacillus polymyxa, enabling fast and reliable genome editing in this host. Homology directed repair allows for highly efficient deletions of single genes and large regions as well as insertions. We used the system to investigate the yet undescribed biosynthesis machinery for exopolysaccharide (EPS) production in P. polymyxa DSM 365, enabling assignment of putative roles to several genes involved in EPS biosynthesis. Using this simple gene deletion strategy, we generated EPS variants that differ from the wild-type polymer not only in terms of monomer composition, but also in terms of their rheological behavior. The developed CRISPR-Cas9 mediated engineering approach will significantly contribute to the understanding and utilization of socially and economically relevant Paenibacillus species and extend the polymer portfolio.

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis.

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

Controlled production of polysaccharides-exploiting nutrient supply for levan and heteropolysaccharide formation in Paenibacillus sp.

Bacterial exopolysaccharides (EPSs) are promising sustainable alternatives to synthetic polymers. Here we describe the production and characterization of different EPSs produced by the recently isolated Paenibacillus sp. 2H2. A final EPS titer of 4.54gL(-1) was recovered after a 17-h fermentation, corresponding to a volumetric productivity of 0.27gL(-1)h(-1). Remarkably, supplying the fermentation with specific carbon and nitrogen sources could be exploited for the production of different polymers. A pure heteropolysaccharide composed of glucose, mannose, galactose, and glucuronic acid (3.5:2:1:0.1) was obtained when using glucose/glycerol and peptone as substrates. A pure levan-type polymer or mixture of both polymers was observed with sucrose and NaNO3 or peptone. To our knowledge, this is the first report that nutrients, particularly nitrogen sources, can be used to fine-tune EPS production in Paenibacillaceae. Rheological characterization of the heteropolysaccharide revealed impressive thickening properties, suggesting its potential application in commodity materials.

Methods to identify the unexplored diversity of microbial exopolysaccharides.

Microbial exopolysaccharides (EPS) are a structurally very diverse class of molecules. A number of them have found their application in rather diverging fields that extend from medicine, food, and cosmetics on the one side to construction, drilling, and chemical industry on the other side. The analysis of microbial strains for their competence in polysaccharide production has therefore been a major issue in the past, especially in the search for new polysaccharide variants among natural strain isolates. Concerning the fact that nearly all microbes carry the genetic equipment for the production of polysaccharides under specific conditions, the naturally provided EPS portfolio seems to be still massively underexplored. Therefore, there is a need for high throughput screening techniques capable of identifying novel variants of bacterial EPS with properties superior to the already described ones, or even totally new ones. A great variety of different techniques has been used in screening approaches for identifying microorganisms that are producing EPS in substantial amounts. Mucoid growth is often the method of choice for visual identification of EPS producing strains. Depending on the thickening characteristics of the polysaccharide, observation of viscosity in culture broth can also be an option to evaluate EPS production. Precipitation with different alcohols represents a common detection, isolation, and purification method for many EPS. A more quantitative approach is found in the total carbohydrate content analysis, normally determined, e.g., by phenol-sulfuric-acid-method. In addition, recently a new and reliable method for the detailed analysis of the monomeric composition and the presence of rare sugars and sugar substitutions has become available, which could give a first hint of the polymer structure of unknown EPS. This minireview will compare available methods and novel techniques and discuss their benefits and disadvantages.

High throughput exopolysaccharide screening platform: from strain cultivation to monosaccharide composition and carbohydrate fingerprinting in one day.

Microbial exopolysaccharides (EPS) are multifunctional biogenic polymers, which exist in highly diverse chemical structures. To facilitate a fast determination of the carbohydrate composition of novel isolated strains or modified EPS variants a fast screening and analytical method is required. The platform as realized and described in this article is based on the fast carbohydrate analysis via liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection in 96-well format to detect different sugars, sugar derivatives and substituents such as pyruvate. Monosaccharide analysis from hydrolyzed polysaccharides was validated successfully by 16 commercially available polymers with known structure. The method is sensitive enough to distinguish various types of sphingans which solely differ in small alterations in the monomer composition. Even a quantitative detection of single monomers as present in complex plant polysaccharides like karaya gum, with the lowest recovery, was in accordance with literature. Furthermore, 94 bacterial strains for the validation of the screening platform were completely analyzed and 41 EPS producing strains were efficiently identified. Using the method a carbohydrate-fingerprint of the strains was obtained even allowing a very fast differentiation between strains belonging to the same species. This method can become a valuable tool not only in the fast analysis of strain isolates but also in the targeted screening for polysaccharides containing special rare sugars as well in the screening of strain libraries from genetic engineering for altered structures.

Fast carbohydrate analysis via liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection in 96-well format.

A fast carbohydrate screening platform processible in 96-well format is described. The method is suitable for the determination of various carbohydrates out of complex mixtures as obtained by acidic hydrolysis of carbohydrates polymers. The chromatographic conditions for an efficient separation (12min) and the derivatization process with 1-phenyl-3-methyl-5-pyrazolone (PMP) were optimized for high resolution separation and simultaneous determination of deoxy-, amino-, anhydro-sugars as well as hexoses, pentoses, dimers, uronic acids and degradation products like furfural and hydroxymethylfurfural (HMF). The potential to quantify with UV- and MS-detector in the same range has been demonstrated for 20 different compounds. Finally, the matrix effects of the hydrolysis were positively evaluated. The micro scale hydrolysis and PMP-derivatization without any extraction or drying steps, both in 96-well format, result in a fast and intuitive sample preparation. In combination with a fast liquid chromatography coupled to UV and electrospray ionization ion trap detection (LC-UV-ESI-MS/MS) for the qualification and quantification of various sugars, dimers and degradation products, this method shows great performance in carbohydrate analysis.

Novel CAD-like enzymes from Escherichia coli K-12 as additional tools in chemical production.

In analyzing the reductive power of Escherichia coli K-12 for metabolic engineering approaches, we identified YahK and YjgB, two medium-chain dehydrogenases/reductases subgrouped to the cinnamyl alcohol dehydrogenase family, as being important. Identification was achieved using a stepwise purification protocol starting with crude extract. For exact characterization, the genes were cloned into pET28a vector and expressed with N-terminal His tag. Substrate specificity studies revealed that a large variety of aldehydes but no ketones are converted by both enzymes. YahK and and YjgB strongly preferred NADPH as cofactor. The structure of YjgB was modeled using YahK as template for a comparison of the active center giving a first insight to the different substrate preferences. The enzyme activity for YahK, YjgB, and YqhD was determined on the basis of the temperature. YahK showed a constant increase in activity until 60 °C, whereas YjgB was most active between 37 and 50 °C. YqhD achieved the highest activity at 50 °C. Comparing YjgB and Yahk referring to the catalytic efficiency, YjgB achieved for almost all substrates higher rates (butyraldehyde 221 s⁻¹ mM⁻¹, benzaldehyde 1,305 s⁻¹ mM⁻¹). Exceptions are the two substrates glyceraldehydes (no activity for YjgB) and isobutyraldehyde (YjgB 0.26 s⁻¹ mM⁻¹) which are more efficiently converted by YahK (glyceraldehyde 2.8 s⁻¹ mM⁻¹, isobutyraldehyde 14.6 s⁻¹ mM⁻¹). YahK and even more so YjgB are good candidates for the reduction of aldehydes in metabolic engineering approaches and could replace the currently used YqhD.