Due to its pleasant grapefruit-like aroma and various further interesting molecular characteristics, (+)-nootkatone represents a highly sought-after specialty chemical. (+)-Nootkatone is accumulated in its producer plants in trace amounts only, and the demand of the food, cosmetics and pharmaceutical industry is currently predominantly met by chemical syntheses. These typically require environmentally critical reagents, catalysts and solvents, and the final product must not be marketed as a "natural flavour" compound. Both the market pull and the technological push have thus inspired biotechnologists to open up more attractive routes towards natural (+)-nootkatone. The multifaceted approaches for the de novo biosynthesis or the biotransformation of the precursor (+)-valencene to (+)-nootkatone are reviewed. Whole-cell systems of bacteria, filamentous fungi and plants, cell extracts or purified enzymes have been employed. A prominent biocatalytic route is the allylic oxidation of (+)-valencene. It allows the production of natural (+)-nootkatone in high yields under mild reaction conditions. The first sequence data of (+)-valencene-converting activities have just become known.

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Novel refreshments with pleasant flavors were developed by fermentation of wort with basidiomycetes. Among 31 screened fungi, shiitake (Lentinula edodes) produced the most pleasant flavor. It was perceived as fruity, slightly sour, and plum-like. Flavor compounds were isolated by liquid-liquid extraction (LLE) and by headspace solid phase microextraction (HS-SPME). The key odor-active compounds were analyzed by a gas chromatography system equipped with a tandem mass spectrometry detector and an olfactory detection port (GC-MS/MS-O) and aroma extract dilution analysis (AEDA). For HS-SPME, a revised method of increasing the GC inlet split ratio was used. Most of the key odor-active compounds (e.g., 2-acetylpyrrole, β-damascenone, (E)-2-nonenal, and 2-phenylethanol) were detected with both extraction techniques. However, distinct differences between these two methods were observed.

As a result of their pleasant odor qualities and low odor thresholds, iso-and anteiso-fatty aldehydes represent promising candidates for applications in flavoring preparations. A novel cyanobacterial α-dioxygenase from Crocosphaera subtropica was heterologously expressed in Escherichia coli and applied for the biotechnological production of C 12 −C 15 branched-chain fatty aldehydes. The enzyme has a sequence identity of less than 40% to well-investigated α-dioxygenase from rice. Contrary to the latter, it efficiently transformed short-chained fatty acids. The kinetic parameters of α-dioxygenase toward unbranched and iso-branchedchain substrates were studied by means of an oxygen-depletion assay. The transformation products (C 12 −C 15 iso-and anteisoaldehydes) were extensively characterized, including their sensory properties. The aldehydes exhibited green-soapy, sweety odors with partial citrus-like, metallic, peppery, and savory-tallowy nuances. Moreover, the two C 14 isomers showed particularly low odor threshold values of 0.2 and 0.3 ng/L in air as determined by means of gas chromatography−olfactometry.

The basidiomycete Pleurotus sapidus (PSA) was grown in submerged cultures with molasses as substrate for the production of mycelium as a protein source for food applications. The volatilomes of the substrate, the submerged culture, and the mycelia were analyzed by gas chromatography-tandem mass spectrometry-olfactometry. For compound identification, aroma dilution analyses by means of headspace solid phase microextraction and stir bar sorptive extraction were performed via variation of the split vent flow rate. Among the most potent odorants formed by PSA were arylic compounds (e.g., p-anisaldehyde), unsaturated carbonyls (e.g., 1-octen-3-one, ( E)-2-octenal, ( E, E)-2,4-decadienal), and cyclic monoterpenoids (e.g., 3,9-epoxy- p-menth-1-ene, 3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3 H)-one). Several compounds from the latter group were described for the first time in Pleurotus spp. After separation of the mycelia from the medium, the aroma compounds were mainly enriched in the culture supernatant. The sensory analysis of the mycelium correlated well with the instrumental results.

The enzymatic conversion of alpha-pinene to verbenols, verbenone, and minor volatile flavors was studied using submerged cultured cells, lyophilisate, and microsomal fractions of the edible basidiomycete Pleurotus sapidus . The similarity of the product range obtained by the bioconversions with the range of products found after autoxidation of alpha-pinene at 100 degrees C suggested similar initial pinene radicals. Extracts of the bioconversions were analyzed using thin layer chromatography with hydroperoxide staining and cool on-column capillary gas chromatography-mass spectrometry. Two isomer alpha-pinene hydroperoxides were identified as the key intermediates and their structures confirmed by comparison with synthesized reference samples and by microchemical reduction to (Z)- and (E)-verbenol. When the biocatalysts were supplemented with one of the verbenols, only the (Z)-isomer was oxidized, indicating the activity of a highly stereospecific monoterpenol dehydrogenase. The structural comparison of subunits shows that fungal oxifunctionalization reactions of some common terpene substrates, such as (+)-limonene or (+)-valencene, might likewise be catalyzed by dioxygenases rather than by CYP450 enzymes, as previously assumed.

Submerged cultures of the basidiomycota Cystostereum murrayi emit an intensive coconut-like, sweetish, and buttery smell. For identification of the key aroma compounds, an aroma dilution analysis using dynamic headspace was performed by adjusting the split ratio of the GC inlet system. Flavor dilution (FD) factors varied from 22 up to ≥218, whereby the largest class of compounds represented terpenoids, including two rare stereoisomers of 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran (dill ether, ee ≥ 99.9). By means of nuclear magnetic resonance spectroscopy, the substances with the highest FD factors (29, 212, and 218) were identified as diastereomers of 3,6-dimethyl-3a,4,5,6,7,7a-hexayhydro-3H-1-benzofuran-2-one (dihydromenthofurolactone) and as its corresponding C3-unsaturated lactone. The latter two compounds have not been described for Cystostereum murrayi or for any other basidiomycota previously. Supplementation studies using 2-13C-d-glucose indicated that these lactones as well as the two stereoisomers of dill ether and other terpenoids were formed de novo by the fungus.

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