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Background  The thermostable serine protease pernisine originates from the hyperthermophilic  Archaeaon Aeropyrum pernix  and has valuable industrial applications. Due to its properties,  A. pernix  cannot be cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation.    Results  We achieved production of active extracellular pernisine in a  Streptomyces rimosus  host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the  srT  signal sequence from the  S. rimosus  protease was fused to pernisine. To promote correct processing and folding of pernisine, the  srT  functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by  S. rimosus  retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease.    Conclusion  Functional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a  S. rimosus  heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that  S. rimosus  can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.

Extracellular production of the engineered thermostable protease pernisine from Aeropyrum pernix K1 in Streptomyces rimosus