Metaprofiling of the Bacterial Community in Colonized Compost Extracts by Agaricus subrufescens.

It is well-known that bacteria and fungi play important roles in the relationships between mycelium growth and the formation of fruiting bodies. The sun mushroom, Agaricus subrufescens, was discovered in Brazil ca. 1960 and it has become known worldwide due to its medicinal and nutritional properties. This work evaluated the bacterial community present in mushroom-colonized compost extract (MCCE) prepared from cultivation of A. subrufescens, its dynamics with two different soaking times and the influence of the application of those extracts on the casing layer of a new compost block for A. subrufescens cultivation. MCCEs were prepared through initial submersion of the colonized compost for 1 h or 24 h in water followed by application on casing under semi-controlled conditions. Full-length 16S rRNA genes of 1 h and 24 h soaked MCCE were amplified and sequenced using nanopore technology. Proteobacteria, followed by Firmicutes and Planctomycetes, were found to be the most abundant phyla in both the 1 h and 24 h soaked MCCE. A total of 275 different bacterial species were classified from 1 h soaked MCCE samples and 166 species from 24 h soaked MCCE, indicating a decrease in the bacterial diversity with longer soaking time during the preparation of MCCE. The application of 24 h soaked MCCE provided increases of 25% in biological efficiency, 16% in precociousness, 53% in the number of mushrooms and 40% in mushroom weight compared to control. Further investigation is required to determine strategies to enhance the yield and quality of the agronomic traits in commercial mushroom cultivation.

Influence of strains and environmental cultivation conditions on the bioconversion of ergosterol and vitamin D2 in the sun mushroom.

BACKGROUND: The fungus Agaricus subrufescens is grown commercially in China, the USA, Brazil, Taiwan and Japan, among others. However, each country adopts a cultivation system that significantly influences the agronomical parameters and chemical composition of the harvested mushrooms. In this study, the influence of the cultivation process on the content of ergosterol and vitamin D2 was evaluated. RESULTS: Four commercial strains of A. subrufescens (ABL 04/49, ABL CS7, ABL 18/01 and ABL 19/01) and two environmental cultivation conditions (in the field and a controlled chamber with the absence of sunlight) were used. Infield cultivation, ABL CS7 and ABL 19/01 strains presented better agronomic parameters, whereas in a protected environment ABL 19/01, ABL 04/49 and ABL 18/01 demonstrated better performance, respectively. The highest biological efficiency value (64%) was provided by ABL 19/01 strain in a controlled environment. CONCLUSION: The highest content in ergosterol (990 mg kg-1 ) and vitamin D2 (36.8 mg kg-1 ) were observed in mushrooms obtained in the field from strain ABL 04/49, which presents reasonable agronomic parameters for cultivation. © 2021 Society of Chemical Industry.

L-DOPA extradiol dioxigenase de amanita muscaria: revisão da sequência codificadora, expressão heteróloga, caracterização funcional e contextualização filogenética / Amanita muscaria L-DOPA extradiol dioxygenase: coding sequence review, heterologous expression, functional characterization, and phylogenetic context

Extradiol dioxigenases são enzimas que catalisam a clivagem oxidativa de ligações C-C entre grupos hidroxila fenólicos adjacentes utilizando catecóis como substratos. Esta classe de enzimas é bem caracterizada em bactérias, onde catalisam a degradação de compostos aromáticos. Na maioria das plantas Caryophyllales, como a beterraba, primavera e a maravilha, L-3,4-diidroxifenilalanina (L-DOPA) extradiol dioxigenases (DODAs) catalisam a clivagem oxidativa de L-DOPA na posição 4,5 gerando o ácido betalâmico, aldeído precursor das betalaínas, uma classe de pigmentos naturais que substitui as antocianinas na pigmentação dessas espécies. Alguns fungos basidiomicetos também produzem betalaínas, como o agário-das-moscas (Amanita muscaria). Nesse organismo, DODA é capaz de catalisar uma clivagem adicional na posição 2,3 da L-DOPA, formando muscaflavina, um isômero do ácido betalâmico que dá origem a uma outra classe de pigmentos naturais: as higroaurinas. Desde a caracterização do gene dodA, o qual codifica para a DODA de A. muscaria (AMAMU), não existem relatos na literatura que explorem a promiscuidade catalítica desta enzima, sua relação com outras linhagens de DODAs e a síntese quimioenzimática de betalaínas a partir desta enzima. Dessa forma, buscamos contextualizar as relações filogenéticas e funcionais entre AMAMU e diferentes linhagens de DODAs, bem como estabelecer um método que viabilize a clonagem, expressão heteróloga e caracterização funcional destaenzima. As análises filogenéticas revelaram que AMAMU possui uma evolução convergente com DODAs de plantas e bactérias e que, apesar de AMAMU ser funcionalmente homóloga à DODA da bactéria Escherichia coli, esta última apresenta homologia com DODAs de plantas. Logo, não há uma relação direta entre a sequência primária de DODAs e sua função. Nós também demonstramos que não há uma relação entre a expressão de transcritos de BvDODA1, e de seu parálogo BvDODA2, e a diferença de pigmentação entre variedades de beterrabas amarelas e vermelhas. A clonagem da sequência codificadora (CDS) publicada para o gene dodA de A. muscaria resultou na retenção do primeiro íntron, o que impedia a sua expressão. Então, uma nova CDS de 558 nucleotídeos foi proposta para este gene, a qual inclui um códon de início da tradução que se mantém na fase de leitura e codifica para uma proteína de 185 resíduos, 43 a menos que AMAMU. A expressão desta CDS resultou na proteína recombinante AmDODA, capaz de catalisar a síntese de ácido betalâmico e muscaflavina a partir de L-DOPA e D-DOPA. AmDODA possui um tamanho aproximado de 22 kDa, com um pH ótimo de atividade de 8,5 e uma constante de Michaelis (KM) de 3,7 ± 0,9 mmol L-1 e de velocidade máxima (Vmax) de 3,3 ± 0,4 µ mol min-1 mg-1. Sua utilização foi demonstrada na síntese quimioenzimática de betalaínas-modelo com potencial aplicação como sondas para microscopia confocal de fluorescência de dois fótons. Neste contexto, esta Tese explora os aspectos moleculares, bioquímicos e biológicos da DODA do fungo A. muscaria e traz importantes contribuições acerca da pigmentação por betalaínas na natureza

Extradiol dioxigenases are enzymes that catalyze the oxidative cleavage of C-C bonds between adjacent phenolic hydroxyl groups using catechols as substrates. This class of enzymes is well characterized in bacteria, where they catalyze the degradation of aromatic compounds. In most plants of the Order Caryophyllales, such as beet, paperflower and four o'clock flower, L-3,4-dihydroxyphenylalanine (L-DOPA) extradiol dioxygenases (DODAs) catalyze the oxidative 4,5-cleavage of L-DOPA generating the betalamic acid, an aldehyde precursor of the betalains, a class of natural pigments that replaces anthocyanins in the pigmentation of these species. Some basidiomycete fungi also produce betalains, such as the fly agaric (Amanita muscaria). In this organism, DODA is able to catalyze an additional 2,3-cleavage of L-DOPA, yielding muscaflavine, an isomer of betalamic acid that gives rise to another class of natural pigments: the hygroaurins. Since the characterization of the dodA gene, which encodes the A. muscaria DODA (AMAMU), there are no reports in the literature that explore the catalytic promiscuity of this enzyme, its relation to other DODAs and the chemoenzymatic synthesis of betalains from this enzyme. Thus, we seek to contextualize the phylogenetic and functional relationships between AMAMU and different DODA lineages, as well as to establish a method that enable the cloning, heterologous expression and functional characterization of this enzyme. Phylogenetic analysis revealed that AMAMU has a convergent evolutionwith plant and bacterial DODAs and that although AMAMU is functionally homologous to the DODA of the Escherichia coli bacteria, this latter is homologous to the plant DODAs. Therefore, there is no direct relationship between the primary sequence of DODAs and their function. We have also shown that there is no relationship between the expression of BvDODA1 transcripts, and its BvDODA2 paralogue, and the pigment difference between yellow and red beet varieties. Cloning of the published coding sequence (CDS) for the dodA gene of A. muscaria resulted in the retention of the first intron, which prevented its expression. Then, a new CDS of 558 nucleotides was proposed for this gene, which includes a translation start codon that remains in the open reading frame and encodes for a protein 185 residues long, 43 less than AMAMU. Expression of this CDS resulted in the recombinant AmDODA protein, able to catalyze the synthesis of betalamic acid and muscaflavine from L-DOPA and D-DOPA. AmDODA has an approximate size of 22 kDa, with an optimum activity pH of 8.5 and a Michaelis constant (KM) of 3.7 ± 0.9 mmol L-1 and a maximum velocity (Vmax) of 3.3 ± 0.4 µmol min-1 mg-1. Its use was demonstrated in the chemoenzymatic synthesis of betalains-model with potential application as probes for confocal microscopy of two-photon fluorescence. In this context, this thesis explores the molecular, biochemical and biological aspects of the DODA of the fungus A. muscaria and brings important contributions about the pigmentation by betalains in nature