Acremonium sp. diglycosidase-aid chemical diversification: valorization of industry by-products.

The fungal diglycosidase α-rhamnosyl-ß-glucosidase I (αRßG I) from Acremonium sp. DSM 24697 catalyzes the glycosylation of various OH-acceptors using the citrus flavanone hesperidin. We successfully applied a one-pot biocatalysis process to synthesize 4-methylumbellipheryl rutinoside (4-MUR) and glyceryl rutinoside using a citrus peel residue as sugar donor. This residue, which contained 3.5 % [w/w] hesperidin, is the remaining of citrus processing after producing orange juice, essential oil, and peel-juice. The low-cost compound glycerol was utilized in the synthesis of glyceryl rutinoside. We implemented a simple method for the obtention of glyceryl rutinoside with 99 % yield, and its purification involving activated charcoal, which also facilitated the recovery of the by-product hesperetin through liquid-liquid extraction. This process presents a promising alternative for biorefinery operations, highlighting the valuable role of αRßG I in valorizing glycerol and agricultural by-products. KEYPOINTS: • αRßG I catalyzed the synthesis of rutinosides using a suspension of OPW as sugar donor. • The glycosylation of aliphatic polyalcohols by the αRßG I resulted in products bearing a single rutinose moiety. • αRßG I catalyzed the synthesis of glyceryl rutinoside with high glycosylation/hydrolysis selectivity (99 % yield).

Discovery of TCMs and derivatives against the main protease of SARS-CoV-2 via high throughput screening, ADMET analysis, and inhibition assay in vitro.

The rapidly evolving Coronavirus Disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide with thousands of deaths and infected cases. For the identification of effective treatments against this disease, the main protease (Mpro) of SARS­CoV­2 was found to be an attractive drug target, as it played a central role in viral replication and transcription. Here, we report the results of high-throughput molecular docking with 1,045,468 ligands' structures from 116 kinds of traditional Chinese medicine (TCM). Subsequently, 465 promising candidates were obtained, showing high binding affinities. The dynamic simulation, ADMET (absorption, distribution, metabolism, excretion and toxicity) and drug-likeness properties were further analyzed the screened docking results. Basing on these simulation results, 23 kinds of Chinese herbal extracts were employed to study their inhibitory activity for Mpro of SARS­CoV­2. Plants extracts from Forsythiae Fructus, Radix Puerariae, Radix astragali, Anemarrhenae Rhizoma showed acceptable inhibitory efficiencies, which were over 70%. The best candidate was Anemarrhenae Rhizoma, reaching 78.9%.

Production of an Anise- and Woodruff-like Aroma by Monokaryotic Strains of Pleurotus sapidus Grown on Citrus Side Streams.

The production of natural flavors by means of microorganisms is of great interest for the food and flavor industry, and by-products of the agro-industry are particularly suitable as substrates. In the present study, Citrus side streams were fermented using monokaryotic strains of the fungus Pleurotus sapidus. Some of the cultures exhibited a pleasant smell, reminiscent of woodruff and anise, as well as herbaceous notes. To evaluate the composition of the overall aroma, liquid/liquid extracts of submerged cultures of a selected monokaryon were prepared, and the volatiles were isolated via solvent-assisted flavor evaporation. Aroma extract dilution analyses revealed p-anisaldehyde (sweetish, anisic- and woodruff-like) with a flavor dilution factor of 218 as a character impact compound. The coconut-like, herbaceous, and sweetish smelling acyloin identified as (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone also contributed to the overall aroma and was described as an aroma-active substance with an odor threshold in air of 0.2 ng L-1 to 2.4 ng L-1 for the first time. Supplementation of the culture medium with isotopically substituted l-tyrosine elucidated this phenolic amino acid as precursor of p-anisaldehyde as well as of (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone. Chiral analysis via HPLC revealed an enantiomeric excess of 97% for the isolated product produced by P. sapidus.

Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression.

The basidiomycete Pleurotus sapidus produced a dye-decolorizing peroxidase (PsaPOX) with alkene cleavage activity, implying potential as a biocatalyst for the fragrance and flavor industry. To increase the activity, a daughter-generation of 101 basidiospore-derived monokaryons (MK) was used. After a pre-selection according to the growth rate, the activity analysis revealed a stable intraspecific variability of the strains regarding peroxidase and alkene cleavage activity of PsaPOX. Ten monokaryons reached activities up to 2.6-fold higher than the dikaryon, with MK16 showing the highest activity. Analysis of the PsaPOX gene identified three different enzyme variants. These were co-responsible for the observed differences in activities between strains as verified by heterologous expression in Komagataella phaffii. The mutation S371H in enzyme variant PsaPOX_high caused an activity increase alongside a higher protein stability, while the eleven mutations in variant PsaPOX_low resulted in an activity decrease, which was partially based on a shift of the pH optimum from 3.5 to 3.0. Transcriptional analysis revealed the increased expression of PsaPOX in MK16 as reason for the higher PsaPOX activity in comparison to other strains producing the same PsaPOX variant. Thus, different expression profiles, as well as enzyme variants, were identified as crucial factors for the intraspecific variability of the PsaPOX activity in the monokaryons.

Virtual screening for functional foods against the main protease of SARS-CoV-2.

The special attention was paid on the interaction between functional foods and the main protease of severe acute respiratory syndrome coronavirus (SARS-CoV-2). Here, 10,870 ligands were employed and screened by the molecular docking, which involved 12 kinds of functional foods (carbohydrates, fatty acids, phospholipids, vitamin, ß-sitosterol, flavonoids, nordihydroguaiaretic acid, curcumin, nootkatone, ß-pinene, tincturoid, betulinic acid, and their isomers/analogs/derivatives). Then, 60 ligands were obtained with the good docking affinity. Most of them belong to quercetrin and its isomers/analogs/derivatives, which also showed the highest affinity for the main protease of SARS-CoV-2. The dynamic simulation indicated that quercetrin-protease and quercetrin-analog-protease showed the excellent stability. Compared with reported docking results, quercetrin should be the best inhibitor for the main protease of SARS-CoV-2. Considering the green and white tea are rich in quercetrin and its isomers/analogs/derivatives, tea and relative beverages may become a good option to regulate our metabolism and help us to overcome this special time. PRACTICAL APPLICATIONS: The docking and molecular dynamics technology were combined to screen the functional foods, which would be the potential candidate of the inhibitor for SARS-CoV-2. Many functional foods screened in this work belong to necessary nutrients for body. Thus, SARS-CoV-2 would consume some necessary nutrients, and thus, damage our body. It should be further consideration whether exogenous nutrients should be provided to slow, halt, or reverse biochemical alterations and structural deterioration in our body. On the contrary, this work also provided a new possibility to design a functional food or drug to help us overcome this special time.

Biotransformation of rice and sunflower side-streams by dikaryotic and monokaryotic strains of Pleurotus sapidus: Impact on phenolic profiles and bioactive properties.

Fungi are known to modify the properties of lignocellulosic materials during solid-state fermentation (SSF). In this study, agricultural side-streams (sunflower seed hulls, rice husks and rice straw) were used as substrates for SSF with dikaryotic and monokaryotic strains of Pleurotus sapidus. The phenolic profiles of the mentioned substrates were characterized by LC-DAD/ESI-MSn pre- and post- fermentation. Moreover, antioxidant, cytotoxic and antimicrobial activities were screened against oxidizable cellular substrates, tumour and primary cell lines, and different bacteria and fungi, respectively. The concentration of phenolic compounds in the crop side-streams was reduced after fermentation with both strains of the fungus. The fermented extracts also displayed lower antioxidant and cytotoxic activities and had no hepatotoxicity. The antimicrobial activity depended upon the crop side-stream and/or SSF conditions. These results indicate that P. sapidus represent a good candidate to modify the phenolic fraction presents in crop side-streams with a consequent decrease in its bioactivities. However, the SSF with P. sapidus strains play an interesting role in the detoxification of plant materials which can be used for different applications according to the "reduce - reuse - recycle" concept contributing with the sustainable land use and circular economy.

Biorefining via solid-state fermentation of rice and sunflower by-products employing novel monosporic strains from Pleurotus sapidus.

Pleurotus sapidus monokaryotic strains (Mk) were screened as a novel source of mycelia to valorize rice straw (RS), rice husks (RH) and sunflower seed hulls (SSH) into value-added products through solid-state fermentation (SSF). P. sapidus Dk3174 basidiospores were cultured in the presence of Remazol Brillant Blue R for strain selection, revealing the ligninolytic ability of emerging colonies. Further screening demonstrated the intraspecific variability in dye degradation and enzyme production of 63 strains. Growth rate, biomass and enzyme production in plates containing RS, RH or SSH pointed at MkP6 as a suitable strain for pilot-scale SSF. MkP6 presented a similar laccase profile as the parental Dk3174, being greater in pasteurized substrates (300-1200 U/Kg) than in sterilized substrates (30-250 U/Kg). Peroxidase represented 25% of the total ligninolytic activity measured. The SSH fermented biomass with MkP6 obtained good yields of nanocellulose (67%) and the saccharide release for ethanol production increased by 3-4 times.

Long-Term Monokaryotic Cultures of Pleurotus ostreatus var. florida Produce High and Stable Laccase Activity Capable to Degrade ß-Carotene.

An extracellular laccase (Lacc10) was discovered in submerged cultures of Pleurotus ostreatus var. florida bleaching ß-carotene effectively without the addition of a mediator (650 mU/L, pH 4). Heterologous expression in P. pastoris confirmed the activity and structural analyses revealed a carotenoid-binding domain, which formed the substrate-binding pocket and is reported here for the first time. In order to increase activity, 106 basidiospore-derived monokaryons and crosses of compatible progenies were generated. These showed high intraspecific variability in growth rate and enzyme formation. Seventy-two homokaryons exhibited a higher activity-to-growth-rate-relation than the parental dikaryon, and one isolate produced a very high activity (1800 mU/L), while most of the dikaryotic hybrids showed lower activity. The analysis of the laccase gene of the monokaryons revealed two sequences differing in three amino acids, but the primary sequences gave no clue for the diversity of activity. The enzyme production in submerged cultures of monokaryons was stable over seven sub-cultivation cycles.

Low Phenotypic Penetrance and Technological Impact of Yeast [GAR +] Prion-Like Elements on Winemaking.

[GAR +] prion-like elements partially relieve carbon catabolite repression in Saccharomyces cerevisiae. They have been hypothesized to contribute to wine yeast survival and alcohol level reduction, as well as communication with bacteria and stuck fermentation. In this work, we selected [GAR +] derivatives from several genetic backgrounds. They were characterized for phenotypic penetrance, heritability and confirmed as prion-like through curing by desiccation. In terms of fermentation kinetics, the impact of the prion on anaerobic wine fermentation (natural grape juice) was either neutral or negative, depending on the genetic background. Likewise, residual sugars were higher or similar for [GAR +] as compared to the cognate [gar -] strains. The prions had little or no impact on glycerol and ethanol yields; while acetic acid yields experienced the highest variations between [GAR +] and [gar -] strains. Strains analyzed under aerobic conditions followed the same pattern, with either little or no impact on fermentation kinetics, ethanol or glycerol yield; and a clearer influence on volatile acidity. Although no clear winemaking advantages were found for [GAR +] strains in this work, they might eventually show interest for some combinations of genetic background or winemaking conditions, e.g., for reducing acetic acid yield under aerated fermentation.

Biotransformation of 1,8-cineole by solid-state fermentation of Eucalyptus waste from the essential oil industry using Pleurotus ostreatus and Favolus tenuiculus.

Biotechnological conversion of low-cost agro-industrial by-products, such as industrial waste or terpenes from the distillation of essential oils from plants into more valuable oxygenated derivatives, can be achieved by using microbial cells or enzymes. In Argentina, the essential oil industry produces several tons of waste each year that could be used as raw materials in the production of industrially relevant and value-added compounds. In this study, 1,8-cineole, one of the components remaining in the spent leaves of the Eucalyptus cinerea waste, was transformed by solid-state fermentation (SSF) using the two edible mushrooms Pleurotus ostreatus and Favolus tenuiculus. As a result, two new oxygenated derivatives of 1,8-cineole were identified: 1,3,3-trimethyl-2-oxabicyclo [2.2.2]octan-6-ol and 1,3,3-trimethyl-2-oxabicyclo [2.2.2]octan-6-one. Additionally, changes in the relative percentages of other aroma compounds present in the substrate were observed during SSF. Both fungal strains have the ability to produce aroma compounds with potential applications in the food and pharmaceutical industries.

Crosses between monokaryons of Pleurotus sapidus or Pleurotus florida show an improved biotransformation of (+)-valencene to (+)-nootkatone.

Several hundred monokaryotic and new dikaryotic strains derived thereof were established from (+)-valencene tolerant Pleurotus species. When grouped according to their growth rate on agar plates and compared to the parental of Pleurotus sapidus 69, the slowly growing monokaryons converted (+)-valencene more efficiently to the grapefruit flavour compound (+)-nootkatone. The fast growing monokaryons and the slow×slow and the fast×fast dikaryotic crosses showed similar or inferior yields. Some slow×fast dikaryons, however, exceeded the biotransformation capability of the parental dikaryon significantly. The activity of the responsible enzyme, lipoxygenase, showed a weak correlation with the yields of (+)-nootkatone indicating that the determination of enzyme activity using the primary substrate linoleic acid may be misleading in predicting the biotransformation efficiency. This exploratory study indicated that a classical genetics approach resulted in altered and partly improved terpene transformation capability (plus 60%) and lipoxygenase activity of the strains.

Non-additive transcriptional profiles underlie dikaryotic superiority in Pleurotus ostreatus laccase activity.

BACKGROUND: The basidiomycete Pleurotus ostreatus is an efficient producer of laccases, a group of enzymes appreciated for their use in multiple industrial processes. The aim of this study was to reveal the molecular basis of the superiority of laccase production by dikaryotic strains compared to their parental monokaryons. METHODOLOGY/PRINCIPAL FINDINGS: We bred and studied a set of dikaryotic strains starting from a meiotic population of monokaryons. We then completely characterised the laccase allelic composition, the laccase gene expression and activity profiles in the dikaryotic strain N001, in two of its meiotic full-sib monokaryons and in the dikaryon formed from their mating. CONCLUSIONS/SIGNIFICANCE: Our results suggested that the dikaryotic superiority observed in laccase activity was due to non-additive transcriptional increases in lacc6 and lacc10 genes. Furthermore, the expression of these genes was divergent in glucose- vs. lignocellulose-supplemented media and was highly correlated to the detected extracellular laccase activity. Moreover, the expression profile of lacc2 in the dikaryotic strains was affected by its allelic composition, indicating a putative single locus heterozygous advantage.

Induction of laccase activity in the white rot fungus Pleurotus ostreatus using water polluted with wheat straw extracts.

The purpose of this work was to explore the use of polluted water effluents from wheat straw using industries as inducers of lignocellulolytic enzymatic activities in cultures of white rot basidiomycetes. For this purpose, we studied the effect of a wheat straw water extract on the evolution of the laccase activity recovered from submerged cultures of Pleurotus ostreatus made in different media and under various culture conditions. Our results demonstrated an accumulative induction effect in all the cultures and conditions tested. This induction is parallel to changes in the laccase electrophoretic profiles recovered from the culture supernatants. The isoenzyme that appeared to be mainly responsible for the laccase activity under these conditions was laccase 10, as confirmed by sequencing the induced protein. These results support the idea of using wheat straw effluents as inducers in liquid cultures of P. ostreatus mycelia for the production of ligninolytic enzymatic cocktails.

Transcriptional and enzymatic profiling of Pleurotus ostreatus laccase genes in submerged and solid-state fermentation cultures.

The genome of the white rot basidiomycete Pleurotus ostreatus includes 12 phenol oxidase (laccase) genes. In this study, we examined their expression profiles in different fungal strains under different culture conditions (submerged and solid cultures) and in the presence of a wheat straw extract, which was used as an inducer of the laccase gene family. We used a reverse transcription-quantitative PCR (RT-qPCR)-based approach and focused on determining the reaction parameters (in particular, the reference gene set for the normalization and reaction efficiency determinations) used to achieve an accurate estimation of the relative gene expression values. The results suggested that (i) laccase gene transcription is upregulated in the induced submerged fermentation (iSmF) cultures but downregulated in the solid fermentation (SSF) cultures, (ii) the Lacc2 and Lacc10 genes are the main sources of laccase activity in the iSmF cultures upon induction with water-soluble wheat straw extracts, and (iii) an additional, as-yet-uncharacterized activity (Unk1) is specifically induced in SSF cultures that complements the activity of Lacc2 and Lacc10. Moreover, both the enzymatic laccase activities and the Lacc gene family transcription profiles greatly differ between closely related strains. These differences can be targeted for biotechnological breeding programs for enzyme production in submerged fermentation reactors.

Genomics and transcriptomics characterization of genes expressed during postharvest at 4°C by the edible basidiomycete Pleurotus ostreatus.

Pleurotus ostreatus is an industrially cultivated basidiomycete with nutritional and environmental applications. Its genome, which was sequenced by the Joint Genome Institute, has become a model for lignin degradation and for fungal genomics and transcriptomics studies. The complete P. ostreatus genome contains 35 Mbp organized in 11 chromosomes, and two different haploid genomes have been individually sequenced. In this work, genomics and transcriptomics approaches were employed in the study of P. ostreatus under different physiological conditions. Specifically, we analyzed a collection of expressed sequence tags (EST) obtained from cut fruit bodies that had been stored at 4°C for 7 days (postharvest conditions). Studies of the 253 expressed clones that had been automatically and manually annotated provided a detailed picture of the life characteristics of the self-sustained fruit bodies. The results suggested a complex metabolism in which autophagy, RNA metabolism, and protein and carbohydrate turnover are increased. Genes involved in environment sensing and morphogenesis were expressed under these conditions. The data improve our understanding of the decay process in postharvest mushrooms and highlight the use of high-throughput techniques to construct models of living organisms subjected to different environmental conditions.

Genomics and transcriptomics characterization of genes expressed during postharvest at 4°C by the edible basidiomycete Pleurotus ostreatus

Pleurotus ostreatus is an industrially cultivated basidiomycete with nutritional and environmental applications. Its genome, which was sequenced by the Joint Genome Institute, has become a model for lignin degradation and for fungal genomics and transcriptomics studies. The complete P. ostreatus genome contains 35 Mbp organized in 11 chromosomes, and two different haploid genomes have been individually sequenced. In this work, genomics and transcriptomics approaches were employed in the study of P. ostreatus under different physiological conditions. Specifically, we analyzed a collection of expressed sequence tags (EST) obtained from cut fruit bodies that had been stored at 4°C for 7 days (postharvest conditions). Studies of the 253 expressed clones that had been automatically and manually annotated provided a detailed picture of the life characteristics of the self-sustained fruit bodies. The results suggested a complex metabolism in which autophagy, RNA metabolism, and protein and carbohydrate turnover are increased. Genes involved in environment sensing and morphogenesis were expressed under these conditions. The data improve our understanding of the decay process in postharvest mushrooms and highlight the use of high-throughput techniques to construct models of living organisms subjected to different environmental conditions (AU)

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Polyporus tenuiculus: a new naturally occurring mushroom that can be industrially cultivated on agricultural waste.

Polyporus tenuiculus is a naturally occurring species from Central and South America that is consumed by different ethnic groups in the region. To determine the optimal conditions for fruiting body production, two strains were assayed on wheat straw and sawdust with or without supplements. Sixty days of incubation at 25 degrees C were needed to produce a solid block. The highest yield was obtained with strain ICFC 383/00 grown on supplemented willow sawdust. In a second experiment the strain ICFC 383/00 and different supplements were used to improve the biological efficiency (BE) and to determine the quality traits and its biodegradation capacity. The highest yields were obtained on sawdust with 25% of supplements reaching 82.7% of BE. Supplements raised the number of flushes, generally from four to five, contributing to increased yields. The type of substrate had a significant effect on fruiting body diameters of P. tenuiculus, and the largest mushrooms were harvested on supplemented substrate with the highest BE coinciding with the highest dry matter loss in substrates. P. tenuiculus showed a capacity to degrade sawdust, causing a decrease of 67.2-74.5% in cellulose, 80.4-85.7% in hemicellulose, and 60.6-66.2% in lignin content at the end of the cultivation cycle. The decrease in hemicellulose was relatively greater than that of cellulose and lignin on supplemented substrates. This is the first report of the cultivation of P. tenuiculus on lignocellulosic waste, and it is a promising species both for commercial production and for its potential use in the degradation of other biowastes.