Holger Zorn

Fungal secretomes--nature's toolbox for white biotechnology.

Adapting their metabolism to varying carbon and nitrogen sources, saprophytic fungi produce an arsenal of extracellular enzymes, the secretome, which allows for an efficient degradation of lignocelluloses and further biopolymers. Based on fundamental advances in electrophoretic, chromatographic, and mass spectrometric techniques on the one hand and the availability of annotated fungal genomes and sophisticated bioinformatic software tools on the other hand, a detailed analysis of fungal secretomes has become feasible. While a number of reports on ascomycetous secretomes of, e.g., Aspergillus, Trichoderma, and Fusarium species are already available, studies on basidiomycetes have been mainly focused on the two model organisms Phanerochaete chrysosporium and Coprinopsis cinerea so far. Though an impressive number and diversity of fungal biocatalysts has been revealed by secretome analyses, the identity and function of many extracellular proteins still remains to be elucidated. A comprehensive understanding of the qualitative and quantitative composition of fungal secretomes, together with their synergistic actions and kinetic expression profiles, will allow for the development of optimized enzyme cocktails for white biotechnology.

Flavors and Fragrances

The awareness that fungi produce enticing flavors is not new. Presumably it was their pleasant aroma that prompted the notorious Roman emperor Nero to name mushrooms “cibus deorum,” food of the gods. Not many years ago fungi were accessible for consumption if one knew how to identify edible species and had access to sites where they grew. Modern cultivation techniques have made several edible fungi much more widely available. For field mushroom, shiitake, and oyster mushroom, the three most important edible fungi worldwide, the annual production exceeds 3,400 million tons (Hobbythek, 1999). In one sense, this is an enormous agrobiotechnological production of fungal flavors. The aroma of the field mushroom (Agaricus bisporus) and of the oyster mushroom (Pleurotus spp.) originates mainly from the enzymatic oxidative degradation of linoleic acid, with 1-octen-3-ol (Figure 13.1) being the most prominent flavor compound (Venkateshwarlu et al., 1999; Husson et al., 2001). Shiitake (Lentinus edodes), which is widely consumed in China and Japan, has a very intensive aroma due to 1,2,3,5,6-pentathiepane (lenthionine; Figure 13.1) as well as sulfur containing degradation products of S-alkyl cysteine sulfoxide (lentinic acid) (Belitz and Grosch, 1987). Beyond these well known representatives, the spectrum of fungi producing flavor compounds is immense. In many cases their capability is reflected in genus or species names that point to aroma characteristics: butyrace- (butter-like), delicat- (delicious), odor-/osm- (fragrant), olid- (ambrosial), suav- (sweet), nidoros- (pungent) and foetens-/foetid- (fetid). Furthermore, trivial names in many different languages often give hints to floral, spicy, or aromatic flavor impressions. Currently, elucidation of flavor profiles either focuses on edible wild mushrooms or is employed as a tool in chemotaxonomic approaches. The volatile compounds emitted by the fruiting bodies of basidiomycetes are usually analyzed by coupled gas chromatography-mass spectrometry (GC-MS) or GC-olfactometry. Olfactometry requires dynamic headspace concentration through cryotrapping, solvent extraction, simultaneous distillation and solvent extraction (SDE), or solid phase microextraction (SPME). In an investigation of 26 basidiomycetes, more than 140 volatiles were detected, among which were mono- and sesquiterpenes, aromatics, ketones, esters, alcohols, and sulfur-containing compounds (Talou et al., 2000). In a series of 80 wild mushroom species, 34 strains synthesized a total of 28 different monoterpenes (Breheret et al., 1997). Altogether 16 aromatic compounds, which were likely to be biogenically derived from lignin of the host tree, were identified in hot water extracts of the white-rot fungus Gloeophyllum odoratum (Rosecke and Konig, 2000).

Nootkatone—a biotechnological challenge

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.

Evidence for methane production by saprotrophic fungi

Methane in the biosphere is mainly produced by prokaryotic methanogenic archaea, biomass burning, coal and oil extraction, and to a lesser extent by eukaryotic plants. Here we demonstrate that saprotrophic fungi produce methane without the involvement of methanogenic archaea. Fluorescence in situ hybridization, confocal laser-scanning microscopy and quantitative real-time PCR confirm no contribution from microbial contamination or endosymbionts. Our results suggest a common methane formation pathway in fungal cells under aerobic conditions and thus identify fungi as another source of methane in the environment. Stable carbon isotope labelling experiments reveal methionine as a precursor of methane in fungi. These findings of an aerobic fungus-derived methane formation pathway open another avenue in methane research and will further assist with current efforts in the identification of the processes involved and their ecological implications.

Enzymatic hydrolysis of carotenoid esters of marigold flowers (Tagetes erecta L.) and red paprika (Capsicum annuum L.) by commercial lipases and Pleurotus sapidus extracellular lipase

The carotenoids lutein and capsanthin, widely used as colorants, mainly occur as esters of fatty acids in their producer plants marigold and paprika. The enzymatic hydrolysis of these esters is reported here. Out of 26 commercial lipases, fastest ester hydrolysis under conditions was obtained using an enzyme from Candida antarctica (69% release of capsanthin, 44% of lutein from the respective oleoresins). Bile salts were essential for the activity of all commercial enzymes. A novel hydrolase was discovered in the mycelium-free supernatant of submerged cultures of the edible basidiomycete Pleurotus sapidus. The fungal enzyme cleaved several carotenoid esters almost completely and in the absence of bile salts.

Molecular and phenotypic characterization of Sebacina vermifera strains associated with orchids, and the description of Piriformospora williamsii sp. nov.

Sebacinales was described in 2004 and is currently recognized as the earliest diverging lineage of mycorrhizal Basidiomycota. In addition, recent research has demonstrated that no other known fungal order harbours a broader spectrum of mycorrhizal types. Yet because of the character poor morphology of these inconspicuous fungi, a reliable systematic framework for Sebacinales is still out of reach. In order to increase the body of comparative data on Sebacinales, we followed a polyphasic approach using a sampling of seven diverse Sebacinales strains, including several isolates of Australian orchid mycorrhizae, Piriformospora indica, and a multinucleate rhizoctonia isolated from a pot culture of Glomus fasciculatum (Williams 1985) with clover. We performed molecular phylogenetic analyses from candidate barcoding regions [rDNA: internal transcribed spacer (ITS)1-5.8-ITS2, 28S; translation elongation factor 1-α (TEF)], enzymatic profiling, genome size estimation by quantitative polymerase chain reaction (PCR), and karyotype analysis using pulsed field gel electrophoresis. Here, we report significant differences in the physiological and molecular parameters inferred from these morphologically very similar strains. Particularly, our results indicate that intron sequences of the TEF gene are useful markers for Sebacinales at the species level. As a first taxonomic consequence, we describe Piriformospora williamsii as a new member of the so far monotypic genus Piriformospora and show that this genus contains still undescribed species that were recently discovered as endophytes of field-collected specimens of Anthyllis, Medicago, and Lolium in Germany.

Quantification and fatty acid profiles of sulfolipids in two halophytes and a glycophyte grown under different salt concentrations

This study was aimed at understanding the role of sulfolipids in salt tolerance mechanisms of the halophytes Aster tripolium L., Compositae, and Sesuvium portulacastrum L., Aizoaceae, and of the glycophyte Arabidopsis thaliana (L.) Heynh., Brassicaceae. In Aster and Sesuvium the sulfolipid contents increased significantly under salt stress conditions (517 mᴍ or 864 mᴍ). In Arabidopsis, changes in sulfolipid contents were not observed (NaCl up to 100 mᴍ). The fatty acid profile of sulfoquinovosyldiacylglycerol (SQDG) in Aster was modified with increasing NaCl concentrations. LC-MS analyses of sulfolipids from Aster and Sesuvium revealed the presence of 18:3/18:3 and 16:0/18:3 molecules. Obviously, the function of sulfolipids during salt stress differs between halophytic species and between halophytes and glycophytes where sulfolipid accumulation was not observed.

Foam fractionation of exo-lipases from a growing fungus (Pleurotus sapidus).

Active lipases were isolated from the culture supernatant of the basidiomycetous fungus Pleurotus sapidus by foam fractionation. The pH value, the gas flow, and the foaming period were systematically varied to optimize the transport of the enzymes into the foam phase. On a 70-mL scale, a maximum recovery of hydrolytic activity of 95% was obtained at pH 7.0 and 60 mL N2 min−1 after 50 min. The procedure, with minor modifications, was also applicable to native pellet cultures of P. sapidus. The same recovery of 95% was achieved, with purification and enrichment factors of 11.6 and 28.0, respectively.

Autoxidation versus Biotransformation of α-Pinene to Flavors with Pleurotus sapidus: Regioselective Hydroperoxidation of α-Pinene and Stereoselective Dehydrogenation of Verbenol

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.

Marasmius scorodonius extracellular dimeric peroxidase — Exploring its temperature and pressure stability

The temperature and pressure dependent stability and function of MsP1, an uncommon peroxidase from the basidiomycetous fungus Marasmius scorodonius were investigated. To this end, a series of biophysical techniques (DSC, fluorescence and FTIR spectroscopy, small-angle X-ray scattering) were combined with enzymatic studies of the enzyme. The dimeric MsP1 turned out to be not only rather thermostable, but also highly resistant to pressure, i.e., up to temperatures of about 65 degrees C and pressures as high as 8-10 kbar at ambient temperatures. Remarkably, the activity of MsP1 increased by a factor of two until approximately 500 bar. At about 2 kbar, the enzymatic activity was still as high as under ambient pressure conditions. As revealed by the fluorescence and SAXS data, the increased activity of MsP1 at pressures around 500 bar may result from slight structural changes, which might stabilize the transition state of the enzymatic reaction. Owing to this marked high pressure stability of MsP1, it may represent a valuable tool for industrial high pressure applications.