Production of biopolymers, as well as the 3D-printing technology, have gained increasing relevance in the last years, but so far there are few efforts to combine the biotechnological production of specialized polymers for 3D-printing purposes. In our project we planned to provide a toolbox of monomers with different influences on the characteristics of a later produced resin. While current biotechnological production of applicable monomers is often based on sugarcrane or other food products which may limit its capacity, all our pathways are based on xylose. Xylose is largely available in the form of xylane, one of the most common hemicelluloses. In this manner we were able to produce the three monomers itaconic acid, ethylene glycol and xylitol. This toolbox is already able to be combined to a functional polymer and can easily be enlarged in the future.
3D-printing demands specified characteristics for the used resin. Beside the structural properties of the printing product viscosity of the resin the punctual hardening needs to be controlled tightly for providing a high resolution. The combination of monomers with different reacting groups makes it possible to design a polymer with the exactly needed properties. In the case of the commonly used polyesterfication one way to control the characteristics of the product is the use of alcohols and carboxylic acids with different numbers of reactive groups and different length of aliphatic intermediary sections. The inclusion of monomers with more than two groups allows controlled crosslinking while monoalkohols or acids would stop the polymerisation and limit the length. Adding photoactive sides like double bonds is necessary for lightcontrolled 3D-printing technologies because this way the crosslinking and also the hardening can be regiocontrolled by simply lightening specific spots.
While there already excist a huge variety of molecules with all the descripted characteristics most of these are produced in petroleum based chemical processes. In order to reduce dependence on fossil resources a lot of research is done in the developement of biotechnological processes substituting these classical synthesis.
However fermantative production has problems of its own. An often discussed problematic are “food versus fuel” concerns because many processes run on food products like sugarcane. Moreover only few model organisms used in the production are understood in detail which limits concerted host modification for yield increasement.
In order to avoid these issues we used only pathways running on xylose which can be produced by degradation of the non-eatable hemicellulose xylan. Xylan moreover is one of the most common compounds in all plants and therefore widely available.
As a host we used E.Coli which is the best understood model organism. Howether all pathways are designed in a way that they can easily be used in other hosts.
Of course the choices of host and educt limit the number of producable monomers. Nethertheless in our research we found the enzymes for three pathways which would form itaconic acid ,ethylene glycol and xylitol from xylose without using any co-factors not present in E.Coli. Those monomers include all basic parts needed for a prepolymer and can already form an usable resin for a stereolithography 3D-printer
Our main goal was the formation of biobrick compatible operons. With those we wanted to proof that all the enzymes would be overexpressed in E.Coli and in combination perform the predicted reactions. Therefore all enzymes which do not originally occure in E.Coli were synthesized in codon optimized form and combined in one operon per monomer which was controlled by a T7-promotor. Finally we wanted to show the presence of proteins by SDS-Page and production of monomers by instrumental analytic with HPLC and GC-MS.