Plant growth–promoting rhizobacteria (PGPR) and fungi-bacterial biofilms are both important biofertilizer inoculants for sustainable agriculture. However, the strongest chemoattractant for bacteria to colonize the rhizosphere and mycelia is not clear. Coincidentally, almost all the PGPRs possess 1-aminocyclopropane-1-carboxylate (ACC) deaminase (AcdS) and can utilize ACC as the sole nitrogen source. Here, we found that ACC was a novel, metabolic dependent and methyl-accepting chemoreceptor–involved chemoattractant for Pseudomonas putida UW4. The chemotactic response of UW4 to ACC is significantly greater than that to the amino acids and organic acids identified in the plant root and fungal hyphal exudates. The colonization counts of the UW4 acdS or cheR deletion mutants in the wheat rhizosphere and on Agaricus bisporus mycelia were reduced one magnitude compared with those of UW4. The colonization counts of UW4 on A. bisporus antisense ACC oxidase mycelia with a high ACC production significantly increased compared with A. bisporus, followed by the UW4 cheR complementary strain and the ethylene chemoreceptor gene–deletion mutant. The colonization counts of the UW4 strains on A. bisporus acdS⁺ mycelia with a low ACC production decreased significantly compared with A. bisporus wild type. These results suggested that ACC and not ethylene should be the strongest chemoattractant for the PGPR that contain AcdS.

The biosynthetic pathway from linoleic acid to 1-octen-3-ol in Agaricus bisporus has long been established, in which linoleic acid is converted to 10-hydroperoxide (10-HPOD) by deoxygenation, and 10-HPOD is subsequently cleaved to yield 1-octene-3-ol and 10-oxodecanoic acid. However, the corresponding enzymes have not been identified and cloned. In the present study, four putative genes involved in oxylipid biosynthesis, including one lipoxygenase gene named AbLOX, two linoleate diol synthase genes named AbLDS1 and AbLDS2, and one hydroperoxide lyase gene named AbHPL were retrieved from the A. bisporus genome by a homology search and cloned and expressed prokaryotically. AbLOX, AbLDS1, and AbLDS2 all exhibited fatty acid dioxygenase activity, catalyzing the conversion of linoleic acid to generate hydroperoxide, and AbHPL showed a cleaving hydroperoxide activity, as was determined by the KI-starch method. AbLOX and AbHPL catalyzed linoleic acid to 1-octen-3-ol with an optimum temperature of 35 °C and an optimum pH of 7.2, whereas AbLDS1, AbLDS2, and AbHPL catalyzed linoleic acid without 1-octen-3-ol. Reduced AbLOX expression in antisense AbLOX transformants was correlated with a decrease in the yield of 1-octen-3-ol. AbLOX and AbHPL were highly homologous to the sesquiterpene synthase Cop4 of Coprinus cinerea and the yeast sterol C-22 desaturase, respectively. These results reveal that the enzymes for the oxidative cleavage of linoleic acid to synthesize 1-octen-3-ol in A. bisporus are the multifunctional fatty acid dioxygenase AbLOX and hydroperoxide lyase AbHPL.

Mushroom β-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for β-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl₂-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of β-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of β-glucans. This is the first report of successful swapping of promoters in filamentous fungi.

Cultivating oyster mushrooms (Pleurotus ostreatus), a typical primary decomposer of lignocellulose, on a short composting substrate is a novel procedure which possesses energy conserves, reduced the chance of infection by competitive species, shorter production duration and achieved high production efficiency. However, the microbiome and microbial metabolic functions in the composting substrate during the mushroom cropping is unknown. In the present study, the contents of hemicellulose, cellulose and lignin and the activities of protease, laccase and cellulase were evaluated in the corncob short composting substrate from before oyster mushroom spawning to first flush fructification; meanwhile the changes in the microbiome and microbial metabolic functions were surveyed by using metagenomic sequencing. Results showed that the hemicellulose, cellulose and lignin in the short composting substrate were decomposed of 42.76, 34.01, and 30.18%, respectively, during the oyster mushroom cropping process. In addition, the contents of hemicellulose, cellulose and lignin in the composting substrate were reduced rapidly and negatively correlated with the abundance of the Actinobacteria phylum. The activities of protease, laccase and cellulase fastly increased in the period of before oyster mushroom spawning to full colonization and were positively correlated to the abundance of Actinobacteria phylum. The total abundance of bacteria domain gradually decreased by only approximately 15%, while the abundance of Actinobacteria phylum increased by 68% and was positively correlated with that of oyster mushroom. The abundance of oyster mushroom increased by 50 times from spawning to first flush fructification. The dominant genera, all in the order of Actinomycetales, were Cellulosimicrobium, Mycobacterium, Streptomyces and Saccharomonospora. The total abundance of genes with functions annotated in the Clusters of Orthologous Groups of proteins (COG) for Bacteria and Archaea and Kyoto Encyclopedia of Genes and Genomes (KEGG) database for all three life domains was positively correlated.The three metabolic pathways for carbohydrates, amino acids and energy were the primary enrichment pathways in KEGG pathway, accounting for more than 30% of all pathways, during the mushroom cropping in which the glycine metabolic pathway, carbon fixation pathways in prokaryotes and methane metabolism were all dominated by bacteria. The genes of cellulolytic enzymes, hemicellulolytic enzymes, laccase, chitinolytic enzymes, peptidoglycanlytic enzymes and ammonia assimilation enzymes with abundances from 0.28 to 0.24%, 0.05 to 0.02%, 0.02 to 0.01%, 0.14 to 0.08%, 0.39 to 0.16%, and 0.13 to 0.12% during the mushroom cropping identified in the Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (eggNOG) database for all three life domains were all aligned to COG database. These results indicated that bacteria, especially Actinomycetales, were the main metabolism participants in the short composting substrate during the oyster mushroom cropping. The relationship between oyster mushrooms and bacteria was cooperative, Actinomycetales were oyster mushroom growth promoting bacteria (OMGPB).