Tricholoma matsutake is a representative mushroom species with a characteristic pleasant aroma. The characteristic aroma component is methyl cinnamate, which is also produced in many plants. In basil, cinnamic acid is produced from l-phenylalanine (l-Phe) by phenylalanine ammonia-lyase (PAL) and converted to methyl cinnamate by a cinnamate/p-coumarate carboxyl methyltransferase. Two PAL genes, Tmpal1 and Tmpal2, have been isolated from T. matsutake. In this study, we aimed to clarify the relationships between l-Phe, methyl cinnamate production, and PAL expression in the mycelium of T. matsutake strain NBRC 30605. For this purpose, methyl cinnamate content, PAL activity, and transcript levels of Tmpal1 and Tmpal2 were examined in the mycelia of T. matsutake supplemented with l-Phe. The mycelia were cultured in 20 mL of a liquid medium (2% glucose, 0.15% yeast extract, and 0.15% Bacto Soytone) at 20 °C for 45 d, supplemented with 0.5-6 mM l-Phe, and then grown for a further 15 d. Mycelia cultured without l-Phe supplementation for 60 d in the medium were used as a control. Crude extracts were prepared from the mycelia harvested for enzymatic, protein, and methyl cinnamate assays. Methyl cinnamate was measured using gas chromatography. PAL activity was assayed by measuring the rate of trans-cinnamic acid formation as the absorbance at 290 nm (ɛ290 = 10,000 M−1 cm−1). The transcript levels of Tmpal1 and Tmpal2 were examined by performing real-time reverse transcriptase-quantitative PCR on the total RNA. Methyl cinnamate was detected in very low levels in cultures without l-Phe supplementation, but its content per mg of protein increased markedly with increasing concentrations of l-Phe, especially at 4-6 mM. When 6 mM l-Phe was added to the culture medium, the methyl cinnamate content was approximately 55-fold higher than that of the control sample. The specific activity of PAL also increased in cultures supplemented with l-Phe, especially at 4-6 mM. When l-Phe was added to the culture medium, the methyl cinnamate content in the mycelia was relatively well correlated with PAL activity. These results indicated that supplementation with l-Phe, a precursor of methyl cinnamate, increases the specific activity of PAL, leading to an increase in methyl cinnamate production in the mycelia of T. matsutake. The transcript level of Tmpal1 did not change markedly with l-Phe supplementation. In contrast, the transcript level of Tmpal2 increased greatly in cultures supplemented with 4-6 mM l-Phe. These results suggested that the expression of Tmpal1 and Tmpal2 was controlled by different regulatory mechanisms and that they may have different biological functions in T. matsutake. In addition, the pattern of PAL activity in the presence of l-Phe was similar to that of the transcript level of Tmpal2, but not Tmpal1, suggesting that the increase in PAL activity was dependent on the increased transcription of Tmpal2.

The characteristic aroma of mushrooms is one of their attractive elements as food materials. The major aroma compound in most mushrooms is 1-octen-3-ol. The biosynthesis of 1-octen-3-ol starts with the oxidation of linoleic acid by lipoxygenase (LOX). The resultant intermediate hydroperoxide is then cleaved by hydroperoxide lyase. LOX is a non-heme iron-containing dioxygenase widely found in plants, animals, fungi, and bacteria. It catalyzes the insertion of molecular oxygen into polyunsaturated fatty acids containing Z,Z-1,4-pentadiene moieties, such as linoleic acid, linolenic acid, and arachidonic acid, yielding the corresponding hydroperoxides. Two LOX genes, Polox1 and Polox2, have been isolated from P. ostreatus, which has higher LOX activity than other edible mushrooms. Polox1 and Polox2 were found to show different expression patterns during the development of the fruiting body. However, the biochemical properties of PoLOX1 and PoLOX2, encoded by Polox1 and Polox2, respectively, have been not fully elucidated. In this study, we engineered these two LOX genes of P. ostreatus into a heterologous host, Escherichia coli, and characterized the recombinant proteins. The coding regions of Polox1 and Polox2 were amplified by RT-PCR from the total RNA of P. ostreatus PC15 mycelia. The RT-PCR products were digested with appropriate restriction enzymes and ligated into an expression vector (pET-16b). The resultant plasmids were introduced into E. coli BL21 (DE3) via transformation. Polox1 and Polox2 were then expressed by induction at 15°C with 0.4 mM IPTG for 18 h. The cells were harvested by centrifugation and resuspended in 10 mM potassium phosphate buffer (pH 7.0). The cell suspension was sonicated and again centrifuged at 15,000 ×g for 20 min at 4 °C. The resultant cell-free extract was used for subsequent experiments. Recombinant PoLOX1 and PoLOX2 were confirmed by SDS-PAGE analysis of the cell-free extract. PoLOX1 and PoLOX2 were estimated to have molecular weights of approximately 76,000 Da and 78,000 Da, respectively. The LOX activity was determined with linoleic acid as a substrate by a spectrophotometric procedure based on the formation of conjugated dienes. To characterize the biochemical properties of PoLOX1 and PoLOX2, in vitro enzymatic assays were performed using the total cell protein from E. coli expressing the two Polox genes, with linoleic acid as a substrate. The optimum pH of recombinant PoLOX1 and PoLOX2 was 7.5 and 5.5, respectively; the optimum temperatures of recombinant PoLOX1 and PoLOX2 were 55 °C and 30 °C, respectively. Recombinant PoLOX1 and PoLOX2 were stable at pH 5.0-9.0 and 6.0-8.0, respectively; recombinant PoLOX1 and PoLOX2 were relatively stable below 50 °C and 40 °C, respectively. Thus, PoLOX1 had higher thermal and pH stability than PoLOX2. The calculated Km values of PoLOX1 and PoLOX2 were 121 μM and 249 μM, respectively. The calculated Vmax values of PoLOX1 and PoLOX2 were 17.2 μmol/mg･min and 17.5 μmol/mg･min, respectively. These results indicated that PoLOX1 had a higher affinity for linoleic acid than PoLOX2. Collectively, our findings suggested that there were some differences between the biochemical properties of PoLOX1 and PoLOX2.

Lipoxygenase (LOX) is considered to be a key enzyme in the biosynthetic pathways of the most important mushroom aroma, 1-octen-3-ol. In previous work, we purified and characterized a LOX from Pleurotus ostreatus (probably H1 strain) fruit bodies [1] and also determined its partial amino acid sequence. In this study, to clarify the biosynthetic mechanism of 1-octen-3-ol, we isolated cDNA and genomic DNA corresponding to a LOX (Polox1) gene of P. ostreatus H1, and analyzed the expression of the gene in the fruit bodies. A commercial P. ostreatus H1 strain (Onuki kinjin, Utsunomiya, Japan) was used in this study. To isolate the Polox1 cDNA, RT-PCR was done using degenerate primers designed from the partial amino acid sequence. This approach generated a single DNA band of approximately 1.1 kbp, which was cloned and sequenced. The deduced amino acid sequence showed high similarity to LOXs of some ascomycetes fungi. To obtain the full-length cDNA of Polox1, clones corresponding to the Polox1 gene were isolated by plaque hybridization from a cDNA library of the P. ostreatus H1 fruit body. DNA sequences of all clones were determined. The 5’ end of the Polox1 cDNA was amplified by the 5’ RACE method and cloned. The full-length cDNA of Polox1 is 2,031 bp long and contains 640 amino acid residues. The deduced amino acid sequence contains LOX iron-binding catalytic domain signature sequences. Next, to determine the genomic DNA sequence of the Polox1 gene, inverse PCR and PCR was done with P. ostreatus H1 genomic DNA. After inverse PCR and PCR, 3.3 and 1.9 kbp DNA fragments, respectively, were amplified and sequenced. Sequence comparison between cDNA and genomic DNA showed that Polox1 gene contained one intron. To investigate expression of the Polox1 gene, northern blot analysis and measurement of LOX activity were performed. P. ostreatus fruit bodies were produced in a sawdust medium containing beech sawdust and rice bran and separated into pileus and stipe. Two transcripts were detected by northern blot analysis in both pileus and stipe. The band intensities were relatively higher in the stipe than in the pileus. The level of LOX activity in the stipe was 3.8 times higher than that in the pileus. By Southern blot analysis, several major bands were detected after the digestion of 4 restriction enzymes. These blot analyses suggest that the Polox1 gene is probably a member of a small gene family. [1] T. Kuribayashi et al., J. Agric. Food Chem., 50, 1247 (2002).