Elucidation of the structure and biosynthetic pathways of dihydromenthofurolactones as highly potent aroma compounds of basidiomycetes

Abstract

Increasing consumer rejection towards non-natural food ingredients is driving the global demand for natural food ingredients, including natural flavors. Many biocatalytic production processes are used in order to meet this demand, offering the advantage that, unlike many chemical syntheses, they often adhere to the principles of green chemistry. As fungi are known to be excellent biocatalysts harboring a broad enzymatic portfolio, research on their potential as natural flavor source is increasingly conducted. The present work aimed to investigate the structure, biogenesis, and olfactory properties of a highly interesting group of aroma compounds, the bicyclic benzofuran derivatives. Cystostereum murrayi, a rarely occurring species found in the Black Forest National Park, was identified to de novo form derivatives of the above-mentioned compounds. Volatile compounds produced by the fungus in submerged culture were identified after aroma dilution analysis (ADA) using dynamic headspace extraction (DHS) after sensory evaluation of the culture supernatant. Among the perceived substances were two diastereoisomers of 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran (dill ether). The identification of the three substances with the highest flavor dilution (FD) values was achieved after isolation using preparative HPLC by means of nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HR-MS). Hereby, the compounds were identified as two diastereoisomers of 3,6-dimethyl-3a,4,5,6,7,7a-hexahydro-3H-1-benzofuran-2-one, known as dihydromenthofurolactones (dml), as well as the C3-unsaturated analogue (Δ3-dml). Semiquantitative analyses revealed comparatively high concentrations of 8 mg L-1 (dml a), 22 mg L-1 (Δ3-dml), and 86 mg L-1 (dml c). As the benzofuran derivatives studied within this work are chiral aroma compounds, their olfactory properties such as odor impression and threshold may differ depending on the investigated enantiomer. Employing chiral analyses by means of enantioselective multidimensional gas chromatography (MDGC) in comparison to authentic standards, either of natural origin or from chemical synthesis, revealed a stereospecific formation (enantiomeric ratio (er) >99.9:0.1) of the respective stereoisomer. Thereby, dill ether and dml differed in the orientation of the methyl group at the C3* position, at which Δ3-dml contains a double-bond. Noteworthy, the dill ether and lactones’ stereochemistry differs significantly at two positions within the tetrahydrofuran ring system, suggesting independent biosynthetic pathways. Indeed, supplementations studies using labeled and non-labeled potential terpenoid precursors suggested dill ether formation via p-menth-1-en-9-ol. The latter may thereby be formed as a biotransformation product of limonene, which, however, does not necessarily act as a precursor within the fungus. Analogous precursors could be excluded for the (Δ3)-dml. The high FD value determined for dml a at comparatively low concentrations indicated a low odor threshold. For the determination, a novel approach by means of direct analysis via gas chromatography-olfactory (GC–O) in conjunction with a flame ionization detector (FID) was established. The determined odor threshold of 1.9 x 10-6 ng L-1 air represents one of the lowest thresholds known to date. Additionally, the combination of aroma analysis data and bioinformatics allowed for the identification of an O-methyltransferase in Pleurotus sapidus, a close relative to the well-known oyster mushroom, involved in aroma biogenesis. The enzyme was heterologously expressed in Escherichia coli, crystallized, and enabled the transmethylation of both hydroxylated compounds and a thiol-nucleophile.