Team:TU Munich/project/applications


Collagen production in E. coli

Collagen is the basic component of connective tissue and therefore present in skin, bone and cartilage. Bone receives its properties from the enclosure of minerals into the collagen. As an application for our project we originally imagined a 3D-bone printer, that produces custom-fit implants. E. coli could be induced to produce human collagen in a desired form which then will be hardened in a subsequent mineralization process.

As the structure of collagen is rather complex, the production in E. coli faces several problems. Three α polypeptide chains are connected via hydrogen bonds between the amino acid side chains and form a triple helix, the collagen molecule. The collagen type that is present in human bones, type I, consists of two different α chains: α1 and α2, that are left-handed helices. The collagen molecules organize themselves by self-assembly to fibrils with a diameter of 200 to 500 Å. The amino acid sequence of the α chains contains glycine in every third position (X-Y-G). The stability of the triple helix is thought to be determined by the amount and distribution of proline and hydroxyproline in the α chain. Therefore the enzyme prolyl-4-hydroxylase, which catalyzes the hydroxylation of proline to hydroxyproline in eukaryotes, plays a critical role in the maturation of collagen. This enzyme is an L-ascorbate-dependent oxygenase [1]. For producing correct human-like collagen in E. coli it is therefore necessary to engineer E. coli to produce hydroxyproline.

We considered two different approaches to realize the synthesis of human collagen in bacteria.

  1. One approach is to co-express the α peptide chain with the prolyl-4-hydroxylase (P4H) and the enzyme D-arabinono-1,4-lactone oxidase (ALO) while cultivating E. coli with sufficient oxygen supply on 1,4-lactones as a nutrient source. The expression of ALO is necessary as E. coli normally does not synthesize L-ascorbates and has no ascorbate transporter system [2]. This method seems to be suboptimal for our purpose, as the bacteria will grow under anaerobic conditions which is not suitable for P4H activity (see solid matrix experiments), because oxygen is obligatory.
  2. The second approach is to modify the cultivation conditions of E. coli for co-translational hydroxyproline insertion [3]. Hydroxyproline is aminoacylated by the native prolyl-tRNA synthetase at a low rate under natural conditions and is therefore inserted in polypeptide chains instead of proline on rare occasions. A higher hydroxyproline insertion rate can be achieved by high intracellular concentrations of hydroxyproline. E. coli accumulates proline and its analogues upon hyperosmotic shock. By cultivating E. coli under hyperosmolaric conditions (more than 500 mM NaCl in media) with a huge excess of hydroxyproline over proline in the media, hydroxyproline is accumulated in the cell and the insertion of hydroxyproline is increased. Thus, human-like collagen can be produced [4].

We favor the second approach as it is much easier to realize. However, as the cloning and characterization of our parts took more time than expected, we focused on cloning and the following tests.


1. Kuehn, K., Struktur und Biochemie des Kollagens, Chemie in unserer Zeit, 1974

2. Pinkas, D., Tunable, Post-translational Hydroxylation of Collagen Domains in Escherichia coli, ACS Chemical Biology, 2011

3. Buechter, D., Co-translational Incorporation of Trans-4-hydroxyproline into Recombinant Proteins in Bacteria The journal of biological chemistry, 2003

4. Báez, J., Mini-Review: Recombinant microbial systems for the production of human collagen and gelatin, Appl. Microbiol. Biotechnolgy.l 2005