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Based on its structural role and compatibility within the human body, collagen is a commonly used biomaterial in medical applications, such as cosmetic surgery, wound treatment and tissue engineering. Gelatin is in essence denatured and partly degraded collagen and is, as a result of its unique functional and chemical properties, also used in many medical and pharmaceutical products. Collagen and gelatin are traditionally extracted from animal tissues. The quality and the characteristics of the proteins are not very reproducible in today's batch-to-batch production processes and recently, potential contamination of collagen and gelatin with viruses and prions (causing BSE) became a matter of concern. BSE is thought to cause a new variety of the brain- wasting Creutzfeldt-Jacob disease in humans.Recombinant DNA technology may provide safe collagen and gelatins from which the quality and characteristics can precisely be controlled and reproduced and, in addition, opens up possibilities for novel functional "tailor-made" proteins.For the heterologous production of animal proteins yeasts are frequently used. Since yeasts are eukaryotes, most translational modification, needed for functionality and stability of recombinant animal proteins, normally occur. However prolyl 4-hydroxylation, essential for gelling properties of recombinant gelatin and thermal stability of recombinant collagen, is generally considered to be absent in yeast systems.In this study we explored the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris for their use as recombinant production systems of natural and "tailor-made" gelatins and human collagen.We found that both yeasts are well able to cope with the repetitive gene sequences encoding animal gelatin and human collagen and showed that P. pastoris can produce synthetic gelatins with highly hydrophilic properties at high levels. Furthermore, it was discovered that H. polymorpha unexpectedly produced endogenous collagen-like proteins with 4-hydroxyproline amino acid residues. This finding indicated that the yeast H. polymorpha , in contract to what was generally believed, must contain intrinsic proly 4-hydroxyalse activity. Indeed, expression of murine gelatin in H. polymorpha yielded a secreted and hydroxylated product. We also investigated if H. polymorpha could be used for the production of recombinant human collagen. Intract human collagen trimers were obtained but they were not stable at temperatures higher than 15 °C, indicating that hydroxylation in the product was poor.In the course of this study we found putative prolyl 4-hydroxylase genes in different eukaryotic microbial systems. In the future these genes may be used to further develop yeasts into cell factories for the production of animal gelatins and thermally stable human collagen.

Several yeast systems have recently been developed for the recombinant production of gelatin and collagen. Amino acid sequence-specific prolyl 4-hydroxylation is essential for the gel-forming capacity of gelatin and for the proper folding of (pro)collagen. This post-translational modification is generally considered to be absent in microbial eukaryotic systems and therefore co-expression of heterologous (human or animal) prolyl 4-hydroxylase would be required. However, we found that the well-known protein expression host Hansenula polymorpha unexpectedly does have the endogenous capacity for prolyl 4-hydroxylation. Without co-expression of a heterologous prolyl 4-hydroxylase, both an endogenous collagen-like protein and a heterologously expressed collagen fragment were found to be sequence-specifically hydroxylated.