Due to its several industrial applications the recombinant p

Due to its several industrial applications, the recombinant production of collagen and all its derivatives as gelatins has been pursued for many years in different biological systems as mammalian 155 8 mg (Toman et al., 1999), tobacco plants (Ruggiero et al., 2000), silkworms (Tomita et al., 2003) and yeast cells (Vuorela et al., 1997). Among these systems, the yeast model proved to be the best performing in terms of yield/cost ratio (Olsen et al., 2003). With a few exceptions (De Bruin et al., 2002), the most commonly used species for this application, i.e. S. cerevisiae and P. pastoris, are devoid of endogenous P4H activity. Through a complex system of yeast cell triple transformation with both α and β P4H and collagenous genes, Vuorela and collaborators obtained a fully hydroxylated human procollagen type III in the methanotrophic yeast P. pastoris. Furthermore, in the same system it was observed that co-expression of type III procollagen was also able to enhance the enzymatic activity of the recombinant P4H itself (Vuorela et al., 1997). Marine sponges are organisms naturally rich in collagens whose features are peculiar if compared to their vertebrate counterparts (Garrone et al., 1975). Sponges are an interesting alternative to the classical bovine and porcine sources for the extraction of these molecules, and their exploitation is increasing with time due to the health risks arisen from the use of bovine collagen (i.e. bovine spongiform encephalopathy, Moon et al., 2014) as well as to ethical concerns for the use of porcine collagen (for Muslim religious). There are several fields of applications of collagen extracts derived from marine sponges related to biomedicine, drug preparation and cosmetics, as for example the use of marine spongin as a biomimetic scaffold for human osteoprogenitor cell attachment, growth and differentiation (Green et al., 2003). One promising candidate for applications in humans is the collagen extracted from the marine sponge Chondrosia reniformis, because of its significant biocompatibility with human skin (Swatschek et al., 2002). The use of collagen extracts from C. reniformis was also proposed in the form of nanoparticles as penetration enhancers for the transdermal delivery of 17 beta-estradiol-hemihydrate in hormone therapy (Nicklas et al., 2009a) and as enteric coating for gastro resistant delayed-release tablets (Nicklas et al., 2009b). It was also observed that C. reniformis collagen can be used as an organic template for silica polymerization (Ehrlich et al., 2010). Recently, a C. reniformis non fibrillar collagen type was described at the molecular level (Pozzolini et al., 2012) and the full length cDNA coding for the α and β subunits of C. reniformis P4H were cloned and characterized (Pozzolini et al., 2015). To date, all the cosmetic and pharmaceutical preparations that use sponge collagen extracts are not characterized at the molecular level. Thus, the optimization of a system for the recombinant production of marine sponge collagen would allow the possibility to take advantage of safe and well-defined molecular types and furthermore, to limit the expensive procedures of sample recovery and purification as well as to avoid environmental issues due to sea pauperization of marine sponges. In the present work, we produced a sponge collagen-specific proline-hydroxylating yeast expression system. Such system contains an active C. reniformis P4H enzyme by co-expression of its α and β subunits in Pichia pastoris. In particular, using four different Pichia strains and two different expression vectors for the αP4H we obtained eight different αP4H+ yeast strains. The strain with the best recombinant enzymatic activity was then selected and further transformed with a non fibrillar collagen gene from C. reniformis marine sponge in order to assess the recombinant P4H activity directly inside the yeast cells on their natural target protein by mass spectrometric analysis of proline-hydroxylated collagen-derived peptides.