High-yield production of hydrophobins RodA and RodB from Aspergillus fumigatus in Pichia pastoris

Abstract

Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins in Pichia pastoris and present fed-batch fermentation yields of 200-300 mg/l fermentation broth. Protein bands of expected sizes were detected by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy and fed-batch production using P. pastoris may be transferred to hydrophobins from other species.

Effect of gene copy number and chaperone coexpression on recombinant hydrophobin HFBI biosurfactant production in Pichia pastoris

“…These differences are exacerbated in the case of the 3‐copy‐HFBI group, where PDI1 coexpression resulted in 7.8±0.2 fold increase, and ERO1 coexpression resulted in 30±4.0 fold increase relative to the 1‐copy‐HFBI/no chaperone strain, with each 3‐copy‐HFBI/chaperone coexpressing strain being statistically significant compared with the 3‐copy‐HFBI/no chaperone parent strain. The approximately 30‐fold increase in HFBI secretion for the 3‐copy‐HFBI/Ero1p strain corresponded to a production level of approximately 330 mg/L in the 5 ml scale used in this study, which is in the same range as previously reported production levels for class I hydrophobins RodA and RodB produced in a bioreactor under optimized conditions (Pedersen et al, ), and for purified HFBI produced in shake flasks (Lohrasbi‐Nejad et al, ). Statistical comparison for the effect of HFBI gene copy number on HFBI expression across groups is presented in Table .…”

“…Eukaryotic expression hosts such as yeasts have produced overall higher hydrophobin yields in a folded state. In particular, the methylotropic yeast Pichia pastoris as a heterologous host for recombinant hydrophobin production has been relatively successful in producing recombinant hydrophobins, with yields on the order of hundreds of milligrams per liter (Lohrasbi‐Nejad, Torkzadeh‐Mahani, & Hosseinkhani, ; Niu, Wang, Yang, Xu, & Qiao, ; Pedersen et al, ; Wang, Feng, Huang, Li, et al, ). However, this stands to be further improved to make use of hydrophobins in large‐scale applications feasible.…”

Heterologous expression and characterization of the hydrophobin HFBI in Pichia pastoris and evaluation of its contribution to the food industry

“…The hydrophobic patch on the surface of rHFBI comprises only a small part of the total surface area and the major part is hydrophilic. In addition, it was speculated that the measurement parameters for WCA data including the hydrophobin amount, the incubation temperature and time might affect the changes in WCA after modification of mica surfaces with rHFBI (Askolin et al 2006;Pedersen et al 2011;Qin et al 2007). Thus, the WCA data demonstrated that similar to the native HFBI, the rHFBI performed a unique amphiphile character that allowed them to self-assemble on siliconized glass and mica surfaces leading to the change of the wettability of rHFBI-coated solid substrates.…”

A visualized fusion protein based on self-assembly hydrophobin HGFI

“…Class I hydrophobins including HGFI possess lower solubility in aqueous solution, therefore Class I hydrophobins have not been used as a tag for purification by now. Compared with Class II hydrophobins, Class I hydrophobin HGFI come from edible mushroom is considered safe and available because of its self-assembly and biocompatibility [6,[25][26][27] . However, there had been no study about fusing a bio-macromolecule with Class I hydrophobin before.…”