In our days, enzymes are highly important vehicles that are used to synthesize valuable chemical compounds. However, the optimization of enzymes, a key discipline in industrial biotechnology, struggles because of natural limitations. A new approach to overcome those restrictions is the high-level production of synthetic proteins containing non-canonical amino acids as performed in the Austrian Centre of Industrial Biotechnology.

Enzymatic processes are more specific and more eco-friendly than traditional chemical methods. Therefore, enzymes are valuable helpers in the chemical industry and biocatalysis. Enzymes are used in washing agents, in food and feed industries, to bleach jeans, to soften leather, or to clean contact lenses. However, natural enzymes lack important factors like a high stability or the ability to catalyze very specific reactions. Using genetically modifications, biotech researchers try to improve the enzyme functionalities but struggle with natural limitations. As enzymes are produced with microorganisms, protein composition is confined to 20 proteinogenic or natural amino acids. Researchers at the Austrian Centre of Industrial Biotechnology (acib) thrive to overcome this limitation by using non-canonical amino acids (ncAA) as protein building blocks.

“We used an auxotrophic variety of the bacterium Escherichia coli to produce a synthetic lipase and an oxidoreductase both containing different ncAAs”, explains Patrik Fladischer, a researcher at the acib. The auxotrophic bacterium (it has to be fed with one special amino acid) was grown to a high cell density. At this time, the auxotrophic amino acid was depleted. Starting the production of the target enzyme and simultaneously feeding the bacteria with a ncAA, the host incorporates the ncAA instead of the proteogenic amino acid into the target protein. The scientists achieved protein titers up to 4g/L which is quite good for proteins containing ncAAs, says Fladischer. However, the yield could be increased through process optimization. The researchers hope to improve the thermostability, durability in solvents, binding properties of enzymes and ligands and other physiochemical attributes when classical enzyme engineering is failing. Nevertheless, until industrial application some more some more development is needed.