Fruiting body formation is the most important developmental event in the edible mushroom life cycle; however, the genetic regulation of this process is not well understood. Pleurotus eryngii is a widely cultivated mushroom with high economic value. The mating of two monokaryons carrying compatible A and B mating-type genes is required for the development of fruiting bodies in P. eryngii. In this study, we showed that the monokaryons of P. eryngii transformed with compatible homeodomain (A mating type) and pheromone (B mating type) genes can complete fruiting body development but cannot form basidiospores. Transcriptional analyses revealed that expression of endogenous homeodomain and pheromone receptor genes and mating signaling pathways were activated by transferred homeodomain and pheromone genes in the transformants. Our findings provide a novel model for studying fruiting body development, which may accelerate the genetic breeding of edible mushrooms in the future. IMPORTANCE Fruiting bodies of edible mushrooms have high nutritional value. However, the fruiting body development of mushrooms is not well understood, and thus, many wild edible mushrooms of economic importance cannot be cultivated artificially. Moreover, variety among cultivatable mushrooms has improved marginally. Under natural conditions, fruiting body development can be initiated only in a dikaryon, the sexual mycelium obtained from mating two compatible monokaryons. The present work showed induction of fruiting body development in Pleurotus eryngii monokaryons by genetic manipulation. Gene expression analyses revealed key genes and signaling pathways involved in the fruiting body development of P. eryngii.

Lentinula edodes is a tetrapolar basidiomycete with two haploid nuclei in each cell during most of their life cycle. Understanding the two haploid nuclei genome structures and their interactions on growth and fruiting body development has significant practical implications, especially for commercial cultivars. In this study, we isolated and assembled the two haploid genomes from a commercial strain of L. edodes using Illumina, HiFi, and Hi-C technologies. The total genome lengths were 50.93 Mb and 49.80 Mb for the two monokaryons SP3 and SP30, respectively, with each assembled into 10 chromosomes with 99.63% and 98.91% anchoring rates, respectively, for contigs more than 100 Kb. Genome comparisons suggest that two haploid nuclei likely derived from distinct genetic ancestries, with ~30% of their genomes being unique or non-syntenic. Consistent with a tetrapolar mating system, the two mating-type loci A (matA) and B (matB) of L. edodes were found located on two different chromosomes. However, we identified a new but incomplete homeodomain (HD) sublocus at ~2.8 Mb from matA in both monokaryons. Our study provides a solid foundation for investigating the relationships among cultivars and between cultivars and wild strains and for studying how two genetically divergent nuclei coordinate to regulate fruiting body formation in L. edodes.

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.

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.