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

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.

Streptomycetes: Attractive Hosts for Recombinant Protein Production

“…The homologous host – the hyperthermophilic Aeropyrum pernix – was uncultivable in industrial fermentation facilities. The protein productivity (10 mg/L) in this case was comparable to that achieved in E. coli , but with the advantage of simplified downstream processes due to protein secretion in the streptomycete ( Šnajder et al, 2019 ). Similarly, the Streptomyces halstedii phospholipase expression was approximately 60 and 30 times higher in S. lividans TK24 than in E. coli and Pichia pastoris , respectively ( Tao et al, 2019 ).…”

“…lividans TK24 as host; the same protein could not be produced in E. coli. Very recently, Šnajder et al (2019) reported the first and so far the only case of expression of an archaeal thermozyme (pernisine) in Streptomyces rimosus . The homologous host – the hyperthermophilic Aeropyrum pernix – was uncultivable in industrial fermentation facilities.…”

“…( Figure 1B ). The thermostable cellulase from the bacteroidetes Rhodotermus marinus ( Hamed et al, 2017 ) and the archaeal thermozyme (pernisine) ( Šnajder et al, 2019 ), described above, complete the list of the prokaryote proteins.…”

Insights into the Maturation of Pernisine, a Subtilisin-Like Protease from the Hyperthermophilic Archaeon Aeropyrum pernix

“…This archaeon was isolated from a coastal solfataric vent and grows at 98°C to 103°C in situ (20). Pernisine is of interest for industrial purposes due to its extraordinary stability and its potential use for degradation of prion proteins (21)(22)(23). In this study, we show that maturation of pro-pernisine (pro-Per) is coupled to the Ca 2ϩ -induced transition from a disordered Ca 2ϩ -free state into the folded Ca 2ϩ -bound state.…”

Streptomyces as Microbial Chassis for Heterologous Protein Expression

“…Enhancing protein secretion efficiency is another important approach that drastically increases the protein yield. The native signal peptides are generally functional for protein secretion in the Streptomyces host; however, optimization of signal peptides improves protein production and secretion in terms of both the proportion of secretion and product yield (Snajder et al, 2019). For example, various signal peptides were tested for the production of XylE, the catechol 2,3-dioxygenase, from Pseudomonas putida in S. rimosus, and extracellular XylE activity was highest when the lipase signal peptide of S. rimosus was utilized, while the xysA signal peptide of S. halstedii resulted in the highest secretion efficiency (Carrillo Rincon et al, 2018).…”