Microbial production of synthetic spider silk proteins
Mikola, Saara (2018-09-05)
Microbial production of synthetic spider silk proteins
Mikola, Saara
(05.09.2018)
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Turun yliopisto
Tiivistelmä
Spider dragline silks are polymers composed mainly of proteins called spidroins. The outstanding strength and elasticity of dragline silks is due to multiple repeats of crystalline and amorphous regions in the spidroins. These properties make spider silk an attractive candidate for developing novel functional materials. Because of the territorial nature of spiders, industrial applications of spider silk require the production of recombinant spidroins. Two organisms widely used in industrial production of recombinant proteins are Pichia pastoris, a methylotrophic yeast, and Trichoderma reesei, a cellulase-producing filamentous fungus. Spidroin production in P. pastoris has been previously studied, but with varying results.
The aim of this study was to generate new spidroin-producing strains of both P. pastoris and T. reesei. Three variants of a synthetic spidroin called AQ12 were used: either AQ12 by itself, or flanked by bacterial or fungal carbohydrate-binding modules. A P. pastoris strain with AQ12 under a constitutive promoter was cloned. Growth condition optimization for P. pastoris was carried out using a model strain where AQ12 is under the methanol-inducible alcohol oxidase 1 promoter. Protease inhibitors were shown to prevent degradation of the product in both of these strains. The P. pastoris AQ12 strains were cultured in bioreactors to get material for protein purification, and a production level of 600 mg/l was achieved. Expression levels of AQ12 constructs both under a strong native promoter and under a synthetic expression system were tested in T. reesei. AQ12 produced from the synthetic expression system was free of contaminating native proteins. The AQ12 proteins were purified from culture supernatants by affinity chromatography, and by heating the supernatant to precipitate auxiliary proteins. Ability of the produced proteins to bind to cellulose was demonstrated.
This study indicates that T. reesei is a more promising host for spidroin production than P. pastoris. The generated strains and the developed purification methods form a foundation for future industrial collaborations in spider silk production at VTT Ltd.
The aim of this study was to generate new spidroin-producing strains of both P. pastoris and T. reesei. Three variants of a synthetic spidroin called AQ12 were used: either AQ12 by itself, or flanked by bacterial or fungal carbohydrate-binding modules. A P. pastoris strain with AQ12 under a constitutive promoter was cloned. Growth condition optimization for P. pastoris was carried out using a model strain where AQ12 is under the methanol-inducible alcohol oxidase 1 promoter. Protease inhibitors were shown to prevent degradation of the product in both of these strains. The P. pastoris AQ12 strains were cultured in bioreactors to get material for protein purification, and a production level of 600 mg/l was achieved. Expression levels of AQ12 constructs both under a strong native promoter and under a synthetic expression system were tested in T. reesei. AQ12 produced from the synthetic expression system was free of contaminating native proteins. The AQ12 proteins were purified from culture supernatants by affinity chromatography, and by heating the supernatant to precipitate auxiliary proteins. Ability of the produced proteins to bind to cellulose was demonstrated.
This study indicates that T. reesei is a more promising host for spidroin production than P. pastoris. The generated strains and the developed purification methods form a foundation for future industrial collaborations in spider silk production at VTT Ltd.