Succession and potential role of bacterial communities during Pleurotus ostreatus production.

There is an increasing interest in studying bacterial-fungal interactions (BFIs), also the interactions of Pleurotus ostreatus, a model white-rot fungus and important cultivated mushroom. In Europe, P. ostreatus is produced on a wheat straw-based substrate with a characteristic bacterial community, where P. ostreatus is exposed to the microbiome during substrate colonisation. This study investigated how the bacterial community structure was affected by the introduction of P. ostreatus into the mature substrate. Based on the results obtained, the effect of the presence and absence of this microbiome on P. ostreatus production in an experimental cultivation setup was determined. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) and amplicon sequencing revealed a definite succession of the microbiome during substrate colonisation and fruiting body production: a sharp decrease in relative abundance of Thermus spp. and Actinobacteria, and the increasing dominance of Bacillales and Halomonas spp. The introduced experimental cultivation setup proved the protective role of the microbial community against competing fungi without affecting P. ostreatus growth. We could also demonstrate that this effect could be attributed to both living microbes and their secreted metabolites. These findings highlight the importance of bacterial-fungal interactions during mushroom production.

Assignment of Vibrational Circular Dichroism Cross-Referenced Electronic Circular Dichroism Spectra of Flexible Foldamer Building Blocks: Towards Assigning Pure Chiroptical Properties of Foldamers.

Assignment of the most established electronic circular dichroism (ECD) spectra of polypeptides and foldamers is either "evidence based" or relies on the 3D structures of longer oligomers of limited internal dynamics, which are derived from NMR spectroscopy (or X-ray) data. Critics warn that the use of NMR spectroscopy and ECD side by side has severe limitations for flexible molecules because explicit knowledge of conformational ensembles is a challenge. Herein, an old-new method of comparing ab initio computed and measured vibrational circular dichroism (VCD) data is presented to validate both the structures (conf(i)) and their relative weights (c(i)) that make up the conformational ensemble. Based on the array of {conf(i), c(i)}, the pure ECD spectra, g(i)conf(i) , can be ab initio calculated. The reconstructed spectrum Σc(i)g(i)conf(i) can thus help to assign any experimental ECD counterparts. Herein, such a protocol is successfully applied to flexible foldamer building blocks of sugar ß-amino acid diamides. The epimeric pair of the model system was selected because these molecules were conformationally tunable by simple chemical modification, and thus, the robustness of the current approach could be probed. The initial hydrogen bond (NH⋅⋅⋅O) eliminated by N-methylation reorients the amide plain, which influences the chiroptical properties of the foldamer building block; this structural change is successfully monitored by changes to the VCD and ECD transitions, which are now assigned to pure conformers. The current method seems to be general and effective without requiring extensive CPU and spectroscopic resources.

α/ß-Chimera peptide synthesis with cyclic ß-sugar amino acids: the efficient coupling protocol.

The synthesis of α/ß-chimeras comprises peptide bond formation from α- to ß-, from ß- to ß-, and from ß- to α-amino acid residues. The fine-tuned solid phase synthesis of -GXXG- chimera peptides containing the simplest achiral α-amino acid glycine and two cyclic SAAs of different ring size [X denoting cyclic ß-Sugar Amino Acids (ß-SAA)] is reported, variants containing Fmoc-RibAFU(ip)-OH a furanoid-, and Fmoc-GlcAPU(Me)-OH a pyranoid-type structural "Lego-element". Systematic search for the best coupling strategy with both H-ß-SAA-OHs is described, including the comparison of the different coupling reagents and conditions. Selecting the optimal reagent (from commonly used PyBOP, HATU and HOBt) was assisted by time-resolved 1H-NMR: formation and stability of the Fmoc protected active esters were compared. We found that PyBOP is the best choice for successfully coupling both H-ß-SAA-OH prototypes. The present comparative results open a reasonable route for building efficiently various -ß-SAA- containing homo- and heterooligomers.

Unwanted hydrolysis or α/ß-peptide bond formation: how long should the rate-limiting coupling step take?

Nowadays, in Solid Phase Peptide Synthesis (SPPS), being either manual, automated, continuous flow or microwave-assisted, the reaction with various coupling reagents takes place via in situ active ester formation. In this study, the formation and stability of these key active esters were investigated with time-resolved 1H NMR by using the common PyBOP/DIEA and HOBt/DIC coupling reagents for both α- and ß-amino acids. Parallel to the amide bond formation, the hydrolysis of the α/ß-active esters, a side reaction that is a considerable efficacy limiting factor, was studied. Based on the chemical nature/constitution of the active esters, three amino acid categories were determined: (i) the rapidly hydrolyzing ones (t < 6 h) with smaller (Ala) or even longer side chains (Arg) holding a large protecting group; (ii) branched amino acids (Ile, Thr) with slowly hydrolyzing (6 < t < 24 h) propensities, and (iii) non-hydrolyzing ones, such as the hard-to-couple ß-amino acids or ß-sugar amino acid derivatives, stable for longer times (t > 24 h) in solution. The current insight into the kinetics of this key hydrolysis side reaction serves as a guide to optimize the coupling conditions of α- and ß-amino acids, thereby saving time and minimizing the amounts of reagents and amino acids to be used - all key factors of more environmentally friendly chemistry.

C-3 epimers of sugar amino acids as foldameric building blocks: improved synthesis, useful derivatives, coupling strategies.

To obtain key sugar derivatives for making homooligomeric foldamers or α/ß-chimera peptides, economic and multigram scale synthetic methods were to be developed. Though described in the literature, the cost-effective making of both 3-amino-3-deoxy-ribofuranuronic acid (H-t X-OH) and its C-3 epimeric stereoisomer, the 3-amino-3-deoxy-xylofuranuronic acid (H-c X-OH) from D-glucose is described here. The present synthetic route elaborated is (1) appropriate for large-scale synthesis; (2) reagent costs reduced (e.g. by a factor of 400); (3) yields optimized are ~80% or higher for all six consecutive steps concluding -t X- or -c X- and (4) reaction times shortened. Thus, a new synthetic route step-by-step optimized for yield, cost, time and purification is given both for D-xylo and D-ribo-amino-furanuronic acids using sustainable chemistry (e.g. less chromatography with organic solvents; using continuous-flow reactor). Our study encompasses necessary building blocks (e.g. -X-OMe, -X-OiPr, -X-NHMe, Fmoc-X-OH) and key coupling reactions making -Aaa-t X-Aaa- or -Aaa-t X-t X-Aaa- type "inserts". Completed for both stereoisomers of X, including the newly synthesized Fmoc-c X-OH, producing longer oligomers for drug design and discovery is more of a reality than a wish.

Origin of problems related to Staudinger reduction in carbopeptoid syntheses.

We report the solid phase synthesis of -GG-X-GG- type α/ß-carbopeptoids incorporating RibAFU(ip) (1a, tX) or XylAFU(ip) (2a, cX) sugar amino acids. Though coupling efficacy is moderate, both the lengthier synthetic route using Fmoc derivative (e.g., Fmoc-RibAFU(ip)-OH) and the azido derivative (e.g., N3-RibAFU(ip)-OH) via Staudinger reaction with nBu3P can be successfully applied. Both X-ray diffraction, 1H- and 31P-NMR, and theoretical (QM) data support and explain why the application of Ph3P as Staudinger reagent is "ineffective" in the case of a cis stereoisomer, if cX is attached to the preceding residue with a peptide (-CONH-) bond. The failure of the polypeptide chain elongation with N3-cX originates from the "coincidence" of a steric crowdedness and an electronic effect disabling the mandatory nucleophilic attack during the hydrolysis of a quasi penta-coordinated triphenylphosphinimine. Nevertheless, the synthesis of the above α/ß-chimera peptides as completed now by a new pathway via 1,2-O-isopropylidene-3-azido-3-deoxy-ribo- and -xylo-furanuronic acid (H-RibAFU(ip)-OH 1a and H-XylAFU(ip)-OH 2a) coupled with N-protected α-amino acids on solid phase could serve as useful examples and starting points of further synthetic efforts.

Characterisation of the large-scale production process of oyster mushroom (Pleurotus ostreatus) with the analysis of succession and spatial heterogeneity of lignocellulolytic enzyme activities.

Oyster mushroom (Pleurotus ostreatus) lignocellulolytic enzyme activity pattern and variation was investigated in a large-scale facility from spawning until the end of the second flush. In the first cultivation cycle laccase production reached its peak during vegetative growth stage, while manganese-peroxidase showed the highest activity during fruiting body induction. Cellulose and hemicellulose degrading enzymes had maximal activity at the beginning of flush and harvest stage. The enzyme activities showed similar tendencies among five different mushroom substrate blocks representing a production house. The spatial variability analysis of enzyme activities pointed out the within substrate block heterogeneity as the main source if variation. This result was confirmed by Combined Cluster and Discriminant Analysis (CCDA) method showing minimal among block heterogeneity considering the whole investigation period; furthermore in the first cultivation cycle all blocks were grouped into one cluster.

An improved sequence-aided T-RFLP analysis of bacterial succession during oyster mushroom substrate preparation.

While oyster mushroom (Pleurotus spp.) is one of the most popular cultivated edible mushrooms, there is scanty information about the microbial community taking part in mushroom substrate production. In this study, an improved sequence-aided terminal restriction fragment length polymorphism (T-RFLP) was used to identify and (semi-)quantify the dominant bacteria of oyster mushroom substrate preparation. The main features of the improved T-RFLP data analysis were the alignment of chromatograms with variable clustering thresholds, the visualization of data matrix with principal component analysis ordination superimposed with cluster analysis, and the search for stage-specific peaks (bacterial taxa) with similarity percentage (analysis of similarity) analysis, followed by identification with clone libraries. By applying this method, the dominance of the following bacterial genera was revealed during oyster mushroom substrate preparation: Pseudomonas and Sphingomonas at startup, Bacillus, Geobacillus, Ureibacillus, Pseudoxanthomonas, and Thermobispora at the end of partial composting, and finally several genera of Actinobacteria, Thermus, Bacillus, Geobacillus, Thermobacillus, and Ureibacillus in the mature substrate. As the proportion of uncultured bacteria increased during the process, it is worth establishing strain collections from partial composting and from mature substrate for searching new species.

Characterization of pseudomonads isolated from decaying sporocarps of oyster mushroom.

Pleurotus ostreatus is one of the most extensively cultivated mushrooms in the world; however, the success of cultivation often depends on the proliferation of different bacterial pathogens. Pseudomonas tolaasii is thought as the major cause of brown blotch disease of Agaricus bisporus and yellowing of Pleurotus ostreatus. In this study we examined the pathogenicity and assessed the industrial damage causing effect of 41 Pseudomonas strains isolated from deformed, yellowing oyster mushroom (P. ostreatus) sporocarps. Identification of the isolates at species level by the partial sequence analysis of the hypervariable region of the rpoB gene revealed nine Pseudomonas sps. We analyzed the presence of the tolaasin gene-cluster, the production of fluorescent pigments, the oxidase- and nitrite reductase activities, the growth at restrictive temperatures and the carbon source utilizing abilities of each strain. Complex lipopeptide production (including tolaasin) was examined with thin layer chromatography and a novel in vitro necrosis-test was developed and evaluated for the investigation of the pathogenic effect of Pseudomonas strains. Our results underline the importance of extracellular enzyme production in the sporocarp decaying process. Strong correlations were found between the secretion of trypsin-like proteases and lipases and the necrotic effect of these bacteria. All the results clearly established that besides Ps. tolaasii, Ps. fluorescens biovar V strains were pathogenic to P. ostreatus and cause serious losses during mushroom production. Our results underline the importance of extracellular enzyme production in the sporocarp decaying process, especially the trypsin-like proteases and lipases.

Microbial community structure changes during oyster mushroom substrate preparation.

Although oyster mushroom (Pleurotus spp.) is a valuable food, cultivated worldwide on an industrial scale, still very little is known about the microbial dynamics during oyster mushroom substrate preparation. Therefore, the characterization of the microbial dynamics by chemical and biological tools was the objective of this study. During substrate preparation, enzymatic digestibility of the substrate improved by 77%, whereas the cellulose and hemicellulose to lignin ratios decreased by 9% and 19%, respectively. Fluorescein diacetate hydrolysis reached its minimum value at the temperature maximum of the process during the composting phase and exceeded the initial level at the end of the process. Fungal species played part in the initial mesophilic phase of the substrate preparation process, but they disappeared after pasteurization in tunnels at constant elevated temperatures. Changes in the microbiota showed a marked bacterial community succession during substrate preparation investigated by 16S ribosomal deoxyribonucleic acid-based terminal restriction fragment length polymorphism (T-RFLP). Mature samples represented the least variance, which indicated the effect of the standardized preparation protocol. The relation between mushroom yield and the bacterial community T-RFLP fingerprints was investigated, but the uniformity of mushroom yields did not support any significant correlation.

Molecular identification of Trichoderma species associated with Pleurotus ostreatus and natural substrates of the oyster mushroom.

Green mold of Pleurotus ostreatus, caused by Trichoderma species, has recently resulted in crop losses worldwide. Therefore, there is an emerging need for rapid means of diagnosing the causal agents. A PCR assay was developed for rapid detection of Trichoderma pleurotum and Trichoderma pleuroticola, the two pathogens causing green mold of P. ostreatus. Three oligonucleotide primers were designed for identifying these species in a multiplex PCR assay based on DNA sequences within the fourth and fifth introns in the translation elongation factor 1alpha gene. The primers detected the presence of T. pleurotum and/or T. pleuroticola directly in the growing substrates of oyster mushrooms, without the need for isolating the pathogens. The assay was used to assess the presence of the two species in natural environments in which P. ostreatus can be found in Hungary, and demonstrated that T. pleuroticola was present in the growing substrates and on the surface of the basidiomes of wild oyster mushrooms. Other Trichoderma species detected in these substrates and habitats were Trichoderma harzianum, Trichoderma longibrachiatum and Trichoderma atroviride. Trichoderma pleurotum was not found in any of the samples from the forested areas tested in this study.