A new chemoenzymatic route from carboxylic acids to nitriles

Nitriles are important compounds in chemistry. Citronellyl nitrile, as an example, has a pleasant lemon-like smell, and is widely used in home-care products. The classical way to prepare nitriles is by the reaction of a precursor molecule with cyanide. However, cyanide is highly toxic. In Agatha Christies murder stories, the murderer takes the victims breath with "sparkling cyanide": a bit of the inconspicuous white powder potassium cyanide mixed into sparkling wine. To avoid the use of cyanide in chemical syntheses, we propose to apply enzymes. They are non-toxic and typically operate at ecologically benign conditions. The aim of this project is, to combine two enzymes (carboxylate reductase and aldoxime dehydratase) and a simple chemical reaction to sequentially convert carboxylic acids to aldehydes, oximes and finally to produce nitriles. Carboxylate reductase enzymes can selectively make an aldehyde from a carboxylic acid using cofactors as the energy source. These cofactors, however, are too expensive to be added to the reaction and therefore, we will use microorganisms, which easily produce these cofactors from sugar and air. The product of this first step the aldehyde is very reactive and will be trapped immediately in a chemical reaction as an oxime. This compound is then recognized by aldoxime dehydratase as a substrate. Aldoxime dehydratases are remarkable enzymes, because of their unique reaction mechanism, the removal of water from the oxime in aqueous solution. This is a mild alternative to the harsh chemical equivalent to this reaction which proceeds at high temperature and requires high energy input. We will engineer the bacterium Escherichia coli such that it will catalyze the reaction cascade described above to give nitriles. Our method will not only be useful to prepare fragrance nitriles, but may also be valuable for the synthesis of pharmaceutical products. One of the key outcomes of this project will be a better understanding of the interplay between enzymes and chemicals within the complex system of a living microorganism. This knowledge allows us to spot bottlenecks in the reaction cascade and to find solutions how to overcome them. Our results will provide valuable input for other cascade reactions with aldehyde intermediates. From genome sequencing projects we know that hundreds of genes encoding aldoxime dehydratases are present in bacteria and fungi. However only a handful of these enzymes have been studied. Therefore, one of our goals is to revive slumbering aldoxime dehydratase genes. An important additional outcome will be a deeper knowledge of the so far rarely studied enzyme class of aldoxime dehydratases and new variants of these highly interesting biocatalysts.

Nitriles are important compounds in chemistry. Citronellyl nitrile, as an example, has a pleasant lemon-like smell, and is widely used in home-care products. The classical way to prepare nitriles is by the reaction of a precursor molecule with cyanide. However, cyanide is highly toxic. In Agatha Christies murder stories, the murderer takes the victim's breath with "sparkling cyanide": a bit of the inconspicuous white powder potassium cyanide mixed into sparkling wine. To avoid the use of cyanide in chemical syntheses, we propose to apply enzymes. They are non-toxic and typically operate at ecologically benign conditions. The aim of this project is, to combine two enzymes (carboxylate reductase and aldoxime dehydratase) and a simple chemical reaction to sequentially convert carboxylic acids to aldehydes, oximes and finally to produce nitriles. Carboxylate reductase enzymes can selectively make an aldehyde from a carboxylic acid using cofactors as the energy source. These cofactors, however, are too expensive to be added to the reaction and therefore, we used microorganisms, which easily produce these cofactors from sugar and air. The product of this first step- the aldehyde - is very reactive and will be trapped immediately in a chemical reaction as an oxime. This compound is then recognized by aldoxime dehydratase as a substrate. Aldoxime dehydratases are remarkable enzymes, because of their unique reaction mechanism, the removal of water from the oxime in aqueous solution. This is a mild alternative to the harsh chemical equivalent to this reaction which proceeds at high temperature and requires high energy input. We engineered the bacterium Escherichia coli such that it can catalyze the reaction cascade described above to give nitriles and produced two nitriles on a preparative scale. We gained a lot of knowledge regarding compatibilites of these reactions, various possible reaction scenarios and the two involved protein classes.