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The use of genetically engineered microorganisms is a cost-effective, scalable technology for the production of recombinant human collagen (rhC) and recombinant gelatin (rG). This review will discuss the use of yeast (Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha) and of bacteria (Escherichia coli, Bacillus brevis) genetically engineered for the production of rhC and rG. P. pastoris is the preferred production system for rhC and rG. Recombinant strains of P. pastoris accumulate properly hydroxylated triple helical rhC intracellularly at levels up to 1.5 g/l. Coexpression of recombinant collagen with recombinant prolyl hydroxylase results in the synthesis of hydroxylated collagen with thermal stability similar to native collagens. The purified hydroxylated rhC forms fibrils that are structurally similar to fibrils assembled from native collagen. These qualities make rhC attractive for use in many medical applications. P. pastoris can also be engineered to secrete high levels (3 to 14 g/l ) of collagen fragments with defined length, composition, and physiochemical properties that serve as substitutes for animal-derived gelatins. The replacement of animal-derived collagen and gelatin with rhC and rG will result in products with improved safety, traceability, reproducibility, and quality. In addition, the rhC and rG can be engineered to improve the performance of products containing these biomaterials.

The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heatstable heterotrimers in Saccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.Collagen is the single most abundant protein found in animals. It is found in all animals, including sponges. It is not expressed in yeast. In mammals, it is expressed in most tissues and plays both a structural as well as a signaling role in the development, maintenance, and repair of tissues and organs. More than 30 gene products compose the collagen family of molecules (1). Procollagens have several features and require numerous steps for production of functional molecules, including post-translational modifications (2). Key features in the collagen family are the formation of a triple helix composed of three polypeptide chains and the post-translational modification of proline residues to hydroxyproline, which provides stability of the triple helix against thermal denaturation and unfolding (T m ) 1 at the animal's body temperature (3). The content of proline and hydroxyproline is correlated with the temperature of an animal's environment (4). The triple helical domain of procollagen consists of -(GXY) n -repeats, where X and/or Y is frequently proline or hydroxyproline in the mature molecule. Prolyl 4-hydroxylase, an ␣ 2 ␤ 2 tetrameric enzyme composed of the prolyl hydroxylase ␣-subunit (␣PH) and the protein-disulfide isomerase (PDI) subunit in higher eucaryotes, is the enzyme that modifies proline residues to hydroxyproline. Additional steps for procollagen production include carbohydrate attachment, folding into a triple helix, secretion into the extracellular matrix, and cleavage by specific proteases to remove the propeptide domains to form mature collagen helices. A C-terminal non-helical propeptide facilitates the assembly of trimeric collagen molecules, leading to helix formation (5); the N-terminal propeptide may limit fiber diameter (6). The association and folding steps of three polypeptide chains that compose the triple helix potentially require chaperone functions in the endoplasmic reticulum, with PDI (7) and Hsp47 (8) as two proteins that have been implicated in the assembly of a procollagen trimer.A fundam...

The extracellular connective tissue matrix of the skin is a complex aggregate of distinct collagenous and non-collagenous components. Optimal quantities and delicate interactions of these components are necessary to maintain normal physiologic properties of skin. This overview summarizes the progress made in understanding the normal biology and biochemistry of the extracellular matrix, and will highlight cutaneous diseases with underlying molecular defects in the structure and expression of extracellular matrix components.