Abstract
BackgroundThe 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.ResultsWe 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.ConclusionFunctional 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.
Highlights
The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications
We initially evaluated the possible production of a wild-type version of pernisine in the S. rimosus background through the construction of expression plasmids that contained the DNA sequences for the native pernisine and the native codon-optimised pernisine (Table 1)
Engineering of the native and codon‐optimised prepropernisine gene and analysis of protein production The wild-type pernisine gene, prepropernisineWT, was PCR amplified with specific primers using genomic DNA of A. pernix as the template, which was subcloned into the pVF vector to generate pVFPER1, as shown in Fig. 1 and Table 1
Summary
The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. A. pernix cannot be cultivated in standard industrial fermentation facilities. Standard industrial fermentation facilities are not suitable for large-scale production of pernisine, due to its extreme cultivation conditions. Recombinant propernisine from Escherichia coli has similar properties to the wild-type enzyme, after an additional thermal activation step [7]. Since the recombinant enzyme is located in the intracellular region or in the periplasm, additional downstream operations are required for its isolation and effective temperature pre-treatment. These isolation procedures are very difficult to scale up to the industrial volume while still ensuring sufficient quantities of fully active pernisine
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