Pichia pastoris production of a prolyl 4-hydroxylase derived from Chondrosia reniformis sponge: A new biotechnological tool for the recombinant production of marine collagen

Prolyl 4-hydroxylase (P4H) is a alpha-2 beta-2 tetramer catalyzing the post-translational hydroxylation of prolinesin collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to gener-ate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applicationsis the collagen extracted from the marine spongeChondrosia reniformis, because of its biocompatibilityand because is devoid of the health risks associated with bovine and porcine collagens.Here we report on the production and selection, by enzymatic and biomolecular analyses, of a tripletransformedPichia pastorisstrain expressing a stable P4H tetramer derived fromC. reniformissponge anda hydroxylated non fibrillar procollagen polypeptide from the same animal. The percentage of recom-binant procollagen hydroxylated prolines inside the transformed yeast was of 36.3% analyzed by massspectrometry indicating that the recombinant enzyme is active on its natural substrate inside the yeastcell host. Furthermore, the recombinant sponge P4H has the ability to hydroxylate its natural substratein both X and Y positions in the Xaa-Yaa-Gly collagenous triplets.In conclusion thisPichiasystem seems ideal for high-level production of hydroxylated sponge- ormarine-derived collagen polypeptides as well as of conotoxins or other marine proteins of high pharma-cological interest needing this particular post-translational modification