Biosynthesis of (R)-(-)-1-Octen-3-ol in Recombinant Saccharomyces cerevisiae with Lipoxygenase-1 and Hydroperoxide Lyase Genes from Tricholoma matsutake

Abstract

Tricholoma matsutake is an ectomycorrhizal fungus, related with the host of Pinus densiflora. Most of studies on T. matsutake have focused on mycelial growth, genes and genomics, phylogenetics, symbiosis, and immune activity of this strain. T. matsutake is known for its unique fragrance in Eastern Asia. The most major component of its scent is (R)-(-)-1-octen-3-ol and is biosynthesized from the substrate linoleic acid by the sequential reaction of lipoxygenase and peroxide lyase. Here, we report for the first time the biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake using the yeast Saccharomyces cerevisiae as a host. In this study, cDNA genes correlated with these reactions were cloned from T. matsutake, and expression studies of theses genes were carried out in the yeast Saccharomyces cerevisiae. The product of these genes expression study was carried out with Western blotting. The biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake in recombinant Saccharomyces cerevisiae was subsequently identified with GC-MS chromatography analysis. The biosynthesis of (R)-(-)-1-octen-3-ol with S. cerevisiae represents a significant step forward.

Keywords: (R)-(-)-1-octen-3-ol; Saccharomyces cerevisiae; Tricholoma matsutake; hydroperoxide lyase; lipoxygenase.

Fig. 1

Pathway for the bioconversion of linoleic acid to (R)-1- octen-3-ol by lipoxygenase and hydroperoxide lyase. Lipoxygenase participate in transforming linoleic acid into (S)-10- hydroperoxy-(8E,12Z)-8, 12, octadecadienoic acid (10-HPODE) and Hydroperoxide lyase participate in producing (R)-1-octen-3-ol.

Fig. 2

Western blot analysis of lipoxygenase-1, lipoxygenase- 2, lipoxygenase-3 and hydroperoxide lyase proteins. (A) The reaction of antibody was conducted. Lane M is the prestained Protein Marker (ELPIS), lane 1 is crud protein of INVSc1 without vector, lane 2 is pYES3/CT without inserted gene, lane 3 is pYES2/CT without inserted gene, lane 4 is lipoxygenase-1, lane 5 is lipoxygenase-2, lane 6 is lipoxygenase-3 and lane 7 is hydroperoxide lyase. (B) Western blot analysis of lipoxygenase-1 and hydroperoxide lyase proteins from the co-transformants. Lane M is the pre-stained Protein Marker (ELPIS), lane 1 is crud protein of INVSc1 without vector, lane 2 is lipoxygenase-1 and hydroperoxide lyase from cotransformants induced for 0 h, lane 3 is proteins from cotransformants induced for 8 h, lane 4 is proteins from cotransformants induced for 16 h, lane 5 is proteins from cotransformants induced for 28 h, lane 6 is proteins from cotransformants induced for 32 h. (C) Values are ratios of band density to band density of GAPDH at each condition and are mean ± SD.

Fig. 3

GC-MS chromatograms of bioconversion in transformants with lipoxygenase-1 and hydroperoxide lyase. (A) cell lysates incubated without linoleic acid, (B) incubated supernatant without linoleic acid, (C) cell lysates incubated with linoleic acid, (D) incubated supernatant with linoleic acid. 1: ethyl alcohol, 2: 1, 3, 5-undecatriene 3: 1-octen-3-ol.

Fig. 4

(R)-(-)-1-octen-3-ol biosynthesis in transgenic yeast with lipoxygense-1 and hydroperoxide lyase genes. (A) Effect of linoleic acid concentration on (R)-(-)-1-octen-3-ol biosynthesis at 30°C for 20 h, (B) effect of incubation time and temperature on (R)-(-)-1-octen-3-ol biosynthesis at 15°C and 30°C for 12, 24, 36 and 48 h.