## 4 Discussion  
Several studies have confirmed that DOX's oxidize linoleic acid to 10-HPOD, and HPLs specifically cleave 10-HPOD to 1-octen-3-ol and 10-ODA in _A. bisporus_[3, 28, 32]. However, to date, the corresponding enzymes have not been identified. In this study, we validated the idea that the combination of heterologously expressed _Ab_LOX and _Ab_HPL catalyzed the oxidative cleavage of linoleic acid to 1-octen-3-ol with an optimum pH of 7.2, in agreement with the crude enzyme homogenate obtained from the mushrooms [21]. A reduced _AbLOX_ expression correlated with the decreased production of 1-octen-3-ol by _A. bisporus_ mycelia, suggesting that _Ab_LOX may be the only fatty acid oxidase that catalyzes the production of 1-octen-3-ol from linoleic acid in _A. bisporus_, most likely as DOX. In addition, the reaction catalyzed by _Ab_LOX was probably the rate-limiting step in the synthesis of 1-octen-3-ol from linoleic acid in _A. bisporus_. _Ab_LOX was highly homologous to the sesquiterpene synthase Cop4 of _C. cinerea._ Interestingly, a dioxygenase from _P. sapidus_ was able to convert the sesquiterpene (+)-valencene to (+)-nootkatone, as well as convert linoleic acid to 13-HPOD; since it is functional as a lipoxygenase, it was designated as LOXPsa1 [51]. In addition, LOXPsa1 can oxidize piperine to piperonal and 3_A_-methylenedioxycinnamaldehyde depending on the co-oxidation of linoleic acid [52]. The multisubstrate property of sesquiterpene synthase from fungi has not yet been reported. This study found that _Ab_LOX was capable of catalyzing the formation of fatty acid hydroperoxides from linoleic acid, possibly with multiple substrate properties. Plant HPLs are CYP74 proteins that catalyze the cleavage of 9- and/or 13-HPODs to form an aldehyde and \(\omega\)-oxo-acid [36]. The HPL from the cyanobacterium _Nostoc punctiforme_ is a heme catalase with 10-HPOD cleavage activity [37], whereas the other bacterial HPL from _Methylobacterium nodulans_ is a CYP74 protein with 13-HPOD cleavage activity (53). A small number of identified HPLs from animals were the CYP74 clan proteins [53, 54, 55]. An HPL cloned from matsutake was considered to be capable of cleaving HPOD to form 1-octen-3-ol [18], which was highly homologous to sterol C-22 desaturase encoded by CYP61 in _Saccharomyces cerevisiae_[56]. Whether sterol C-22 desaturase has multisubstrate properties was not recorded. In this study, _Ab_HPL, with a high homology compared to HPL from matsutake, can catalyze the products of _Ab_LOX oxidation of linoleic acid to generate 1-octen-3-ol, indicating that _Ab_HPL has different structures and functions to HPLs in plants, animals, and bacteria. A large quantity of 1-octen-3-ol was synthesized by _A. bisporus_ mycelia and its developed fruiting bodies [57, 58, 59, 32]. Similarly, _C. cinerea_ mycelia and its developed fruiting bodies also yielded high quantities of 1-octen-3-ol as well as sesquiterpenes, such as \(\beta\)-limachalene and cuparene [60]. The LOX and CYP74 protein genes were not retrieved from the genome of _C. cinerea_ okayama 7#130 [61], whereas six genes of terpen synthase [62]; three genes of LDS, all of which were DOX-(CYP), and one gene of sterol C-22 desaturase were retrieved. The deduced amino acid sequence of the sterol C-22 desaturase was 69.85% identical to _Ab_HPL. Furthermore, six genes encoding terpene synthases, including _Ab_LOX, were retrieved from the genome of _A. bisporus_ H97 [44]. Therefore, _A. bisporus_ and _C. cinerea_ should have a similar biosynthesis pathway for 1-octen-3-ol. Nonetheless, the enzymatic pathways for the biosynthesis of 1-octen-3-ol in mushroom fungi were probably quite diverse. A high amount of 1-octen-3-ol was synthesized in the growing mycelia and early developing fruiting bodies of _Cyclocyhe aegerita_. A total of two putative DOX-CYP genes, five putative LOX genes, and eleven putative HPL (CYP74 proteins) genes were found in the _C. aegerita_ transcriptome.