Discovering autoregulator systems in industrially useful basidiomycetes: a novel approach to improve fungal bioprocesses by endogenous extracellular signals

Autoregulators are endogenous extracellular metabolites signalling information on environmental conditions or on the status of cells within the mycelium, thus ensuring a coordinated colony function. This mechanism results in the regulation of transitions between alternative morphogenetic and/or functional programmes of the colony as a living unit. The white-rot fungus Pleurotus ostreatus belongs to a subclass of white-rot fungi producing many ligninolitic enzymes such as laccases, MnPs and AAO. Isoenzymes with interesting structural and functional properties are expressed by this fungus and their production is differentially regulated by culture conditions. However, nothing is known about properties of the fungus secondary metabolites and correlation between them and fungal morpho-physiological switch. Regulation of fungal development stages and expression of ligninolytic system through autoregulatory signals may allow controlling fungal phenotype and improving fungal application. Characterization of the endogenous exo-metabolome of P. ostreatus growing in submerged culture model is reported. 11 GC/MS-detected volatile compounds related to shikimic acid pathway (aryl compounds) had been differentially produced during submerged growth. Endogenous exo-metabolites extracted from spent media positively affected the general mechanisms of laccase expression in liquid culture. Other excreted eight-carbon aliphatic compounds had been previously detected during P. ostreatus mushroom fructification and reported in literature. Effects of some of these commercially available aryl compounds and eight carbon volatile molecules were evaluated in P. ostreatus cultures. Fungal pellet morphology, sporal germination, radial growth in agar medium and laccase production profiles in submerged cultures were monitored in presence and absence of each potential autoregulator. Morphological and physiological analysis showed at least 3 autoregulators involved in different processes. Benzaldehyde indicates a dramatic decrease of nutrient source (starvation) and induces increase of main hyphal length in order to enhance the probability of finding new nutrient sources. 2-hydroxy-4-nitrobenzoic acid is secreted during a long starvation stress. This chemical inhibits mycelium growth and induces, up to 10 times, laccase production in liquid culture. These two physiological reactions can be considered as defence mechanisms in natural behaviours. Finally, 1-octen-3one inhibited basidiospore germination. Analougus mechanism regulat ing similar sexual processes are reported for other fungal species. Moreover, the production of new P. ostreatus dikaryotic strains with improved efficiencies in laccase expression was performed through inter-crossing different variants. The effect of the 2-hydroxy 4-nitrobenzoic acid autoregulator on laccase production was also tested on the best performing strains. The signal molecule increased laccase production levels confirming that its signalling role is not strain - dependent. As a result this work allowed the knowledge of autoregulatory mechanisms in P. ostreatus in order to control physiological phenomena for industrial purposes such as protein production capability. Because a very efficient heterlogous expression system in P. ostreatus is still not available, this classical natural method, as well as classical breeding, represent valid and eco-compatible methods for bioproductions improvement in comparison with random mutagenesis. As parallel section a new laccase from P. ostreatus phenoloxidase multigene family was isolated from the mature fruiting body and enzymatically characterized for the first time.