Characterization of the Trichoderma reesei hydrophobins HFBI and HFBII: Dissertation

Hydrophobins are surface active proteins produced by filamentous fungi. They fulfil a wide variety of functions in fungal growth and development. These proteins for example render fungal aerial structures hydrophobic and affect the attachment of fungi to solid supports. The ability of hydrophobins to modify surface properties by interfacial self-assembly and their high surface activity provide a potential for several applications. In this work, properties and biological roles of the HFBI and HFBII hydrophobins produced by Trichoderma reesei were studied. In addition, the hydrophobins were crystallized for structure determination. Moreover, effects of these hydrophobins on the cultivation of T. reesei in bioreactors were evaluated. High production levels of HFBI and HFBII were obtained in T. reesei by introducing additional copies of the hfb1 or hfb2 genes into the genome. The T. reesei HFBI-overproducing strain (VTT D-98692) produced up to 1.4 grams HFBI per liter on glucose-containing culture medium, the highest production level of hydrophobin hitherto reported. The HFBII-overproducing strain (VTT D-99745) and its parent strain secreted 0.24 and 0.03 grams HFBII per liter, respectively, into the culture medium. HFBI and HFBII were purified from fungal cell walls and liquid culture medium, respectively. The purified hydrophobins were crystallized by vapour diffusion in hanging drops for structural analysis. Behaviour of the class II hydrophobins HFBI and HFBII of T. reesei and the class I hydrophobin SC3 of Schizophyllum commune was studied at various interfaces. All the hydrophobins were surface active. HFBI and HFBII reduced the surface tension of water faster than SC3. Self-assembly of HFBI and HFBII at water-air interfaces induced by mixing with air was not accompanied by a change in the circular dichroism spectra, in contrast to SC3. No clear ultrastructure of the dried class II hydrophobin film was observed in electron microscopy. However, the circular dichroism spectra of HFBI and HFBII changed when they were in contact with Teflon surface, indicating formation of helix structure, although not to the same extent as with SC3. Both HFBI and SC3 strongly interacted with hydrophobic Teflon surface, rendering it completely wettable. HFBI, HFBII and SC3 all stabilized oil emulsions but HFBI and SC3 appeared to be more effective than HFBII. The presence of HFBI or HFBII affected the solubility of SC3 aggregates, indicating interaction between these hydrophobins. Differences between the behaviour of class I and class II hydrophobins at the water-air interface are proposed to be due to the divergent size and shape of the hydrophobic parts of these proteins. In order to study the biological roles of HFBI and HFBII, the hfb1 and hfb2 genes were deleted in T. reesei. The hfb1 strain formed no aerial hyphae in static liquid cultures. Addition of purified HFBI to the medium restored the aerial hyphae formation. The aerial growth was also restored by expressing the gene encoding the SC3 hydrophobin of S. commune in the hfb1 strain. Colonies of hfb1 had a wettable and fluffy phenotype when grown on a solid medium. In shaken liquid cultivation, biomass formation of hfb1 was slower compared with the parent strain. Sporulating colonies of hfb2 were wettable and sporulation was only 50% of that of the parent strain. These results indicate that HFBI facilitates aerial growth of T. reesei, whereas HFBII is involved in sporulation. Process technological effects of HFBI and HFBII were studied by cultivating the hfb1, hfb2, HFBI-overproducing and HFBII-overproducing strains in laboratory bioreactors. Vegetative growth properties of the hydrophobin deletion and over-producing strains were similar to those of their parent strains. The strains overproducing the hydrophobins foamed extensively, especially the HFBII-overproducing strain. Foaming of the hfb2 strain (but not hfb1) was lower compared with the parent strain on lactose- and cellulose-containing media. This shows that the main cause of foaming in bioreactor cultivations is the HFBII hydrophobin.