Identification of volatile producing enzymes in higher fungi: Combining analytical and bioinformatic methods.

Filamentous fungi harbor the genetic potential for the biosynthesis of several secondary metabolites including various volatile organic compounds (VOCs). Nonetheless, under standard laboratory conditions, many of these VOCs are not formed. Furthermore, little is known about enzymes involved in the production of fungal VOCs. To tap these interesting topics, we developed an approach to identify enzymes putatively involved in the fungal VOC biosynthesis. In this chapter, we highlight different fungal cultivation methods and techniques for the extraction of VOCs, including a method that allows the noninvasive analysis of VOCs. In addition using terpene synthases as an example, it is depicted how enzymes putatively involved in VOC synthesis can be identified by means of bioinformatic approaches. Transcriptomic data of chosen genes combined with volatilome data obtained during different developmental stages is demonstrated as a powerful tool to identify enzymes putatively involved in fungal VOC biosynthesis. Especially with regard to subsequent enzyme characterization, this procedure is a target-oriented way to save time and efforts by considering only the most important enzymes.

Chromosome-level reference genome assembly provides insights into aroma biosynthesis in passion fruit (Passiflora edulis).

Passion fruit, native to tropical America, is an agriculturally, economically and ornamentally important fruit plant that is well known for its acid pulp, rich aroma and distinctive flavour. Here, we present a chromosome-level genome assembly of passion fruit by incorporating PacBio long HiFi reads and Hi-C technology. The assembled reference genome is 1.28 Gb size with a scaffold N50 of 126.4 Mb and 99.22% sequences anchored onto nine pseudochromosomes. This genome is highly repetitive, accounting for 86.61% of the assembled genome. A total of 39,309 protein-coding genes were predicted with 93.48% of those being functionally annotated in the public databases. Genome evolution analysis revealed a core eudicot-common γ whole-genome triplication event and a more recent whole-genome duplication event, possibly contributing to the expansion of certain gene families. The 33 rapidly expanded gene families were significantly enriched in the pathways of isoflavone biosynthesis, galactose metabolism, diterpene biosynthesis and fatty acid metabolism, which might be responsible for the formation of featured flavours in the passion fruit. Transcriptome analysis revealed that genes related to ester and ethylene biosynthesis were significantly upregulated in the mature fruit and the expression levels of those genes were consistent with the accumulation of volatile lipid compounds. The passion fruit genome analysis improves our understanding of the genome evolution of this species and sheds new lights into the molecular mechanism of aroma biosynthesis in passion fruit.

Biosynthetic Mechanism of Key Volatile Biomarkers of Harvested Lentinula edodes Triggered by Spore Release.

In this study, headspace solid-phase microextraction-gas chromatography-mass spectrometry, multivariate analyses, and transcriptomics were used to explore the biosynthesis of key volatiles and the formation of spores in Lentinula (L.) edodes. Among the 50 volatiles identified, 1-octen-3-ol, phenethyl alcohol, and several esters were considered key aromas because of their higher odor activity values. Eleven volatiles were screened as biomarkers by orthogonal partial least squares discriminant analysis, and hierarchical cluster analysis showed that these biomarkers could represent all volatiles to distinguish the spore release stage. The activities of lipoxygenase (LOX), hydroperoxide lyase, alcohol dehydrogenase, and alcohol acyltransferase were higher in L. edodes with spore release. Moreover, linolenic acid and phenylalanine metabolism were involved in aroma biosynthesis. One LOX-related gene and five aryl alcohol dehydrogenase-related genes could regulate the biosynthesis of 1-octen-3-ol, phenethyl alcohol, and phenylacetaldehyde. In addition, several key genes were involved in meiosis to regulate sporulation.