Introducing a NeuNAc synthesis pathway into Trichoderma

N-Acetylneuraminic acid (NeuNAc), a C9 mono-saccharide, is the most prevalent exponent of sialic acids. Currently, more than 50 derivatives of sialic acids are known to exist in nature. NeuNAc is believed to serve as a precursor of all these derivatives as all biochemical pathways proceed via this substance. In biological systems salic acids are mostly terminal components of glycoproteins presented presented at the respective cell surface. In such cases salic acids serve as recognition sites for respective endgenous and exogenouse proteins all of them belonging to the class of lectins. Because of this exposed position of salic acides in cellular systems they play an important role in infection cycles of important viral diseases such as influenza viruses A and B. In such a case de novo synthesized virus particles attach to respective salic acids presented at the cell surface. For a propagation of the virus in the host a neuraminidase (sailidase) activity is needed, which cleaves the linkage between the salic acid and the glycol protein. Therefore salic acid derivatives are nowadays successfully applied in the therapy of such virus born diseases. In particular they are used as neuraminidase inhibitors to prevent a further propagation of the virus. The to date best known preparations on the market are "Tamiflu" (active pharmaceutical ingredient: "Osemtalvir") by Hoffmann la Roche and "Relenza" (active pharmaceutical ingredient: "Zanamivir") by GlaxoSmithKline both functioning as neuraminidase inhibitors and the latter produced from the precursor NeuNAc. It is noteworthy that NeuNAc is currently believed as a promising aspirant in antagonizing bird flu. Synthesis of NeuNAc is costly. It is currently carried out by a two step enzyme catalysed process. Production costs are especially detrimentally influenced by the second step of biosynthesis in which N-acetylmannosamine is metabolized to NeuNAc via a balance reaction. In this reaction step an excess of pyruvate has to be added to move the equilibrium to NeuNAc. However the removal of pyruvate from the reaction is a cost-intensiv downstream processing step. To optimise the synthesis of NeuNAc, the proposed work package focuses on the introduction of a whole cell catalysed process. In particular, it is planned to genetically engineer the filamentous ascomycete Trichoderma in such a way that it can synthesize NeuNAc under standard fermentation conditions using chitin (a cheep renewable biopolymer) as a carbon source. In comparison to the currently applied enzyme catalysed process, one major advantage has to be mentioned. In contrary to the application of an aldolase for the second synthesis step, which requires the use of an excess of pyruvate, the newly designed in vivo synthesis would allow the application of N- acetylneuraminate synthase in the process. This enzymatic step implies the use of phosphoenol-pyruvate instead of pyruvate (which in the intended in vivo process would be supplied by the organism) thereby leading to an irreversible process. Consequently, the insertion of an excess of pyruvate becomes obsolete and the resulting downstream process is significantly simplified. In addition, the application of Trichoderma as a whole cell catalyst offers the advantage of using cheap renewable raw materials, such as chitin. As a summary, the proposed work package follows the concept of transforming renewable raw materials into fine chemicals.

During the study supported by the Austrian Science Fund we succeeded in synthesizing a pharmaceutically relevant precursor substance for antiviral drugs by a biotechnological process using the natural resource chitin as starting material. In therapy of viral infections (as influenza) so-called neuraminidase inhibitors are commonly applied. They prevent the cleavage of a neuraminicacid from a glycoprotein antenna, and this hinders a virus from propagation in its host organism. Derivatives of N-acetylneuraminicacid are frequently used as neuraminidase inhibitors. One example is Zanamivir, which is the active compound of the drug Relenza, an antiviral product from GlaxoSmithKline. So far, N-acetylneuramincacid is produced by extraction from natural resources or is chemically synthesised and therefore, has a relative high market price (approx. 1000 Euro per gram). Chitin is the second most abundant biopolymer on earth and occurs in the exoskeletons of crustaceans and insects, in the radula of molluscs, in the beaks of the cephalopods, and in the cell walls of fungi. It is estimated that 1011 tons are annually produced only from maritime resources. With respect to principles of sustainability chitin is as a renewable, non-food resource obviously an optimal starting material for synthesis processes. The fungus Trichoderma is widely abundant in soils, wood and meadows and thrives on biomass. It has the capability to degrade chitin to its monomeric aminosugar (N-acetylglucosamine). For this reason this organism was chosen and a synthetic pathway for production of N-acetylneuraminicacid was introduced. This newly developed strain produces in a whole-cell catalysis approach, i.e. by cultivation in a bioreactor, the substance from chitin. Vienna University of Technology aims to valorise the promising invention and has filled a corresponding national and international patent.