Catalytic cascades for selective aldehyde syntheses in vivo

Many fragrances that we know from daily life share the same chemical functional group the aldehyde. The most prominent example is probably vanillin, which is used in a huge number of food products, cosmetics and perfumes but there are also others like cinnamaldehyde (which gives the smell to cinnamon) and derivatives thereof. As many of these fragrances are difficult to obtain in sufficient amounts from natural sources, there is a lot of interest to find ways how to prepare them. Carboxylic acids could be promising precursors, but it is very difficult to reduce them selectively to aldehydes. Chemistry has some possibilities to do that, but they all suffer from drawbacks that enzymatic methods can circumvent. Enzymes are non-toxic and typically operate at ecologically benign conditions. So called carboxylate reductase enzymes can selectively make an aldehyde from an acid at the expense of cofactors. These cofactors, however, are too expensive to be added to a reaction and therefore, we will use microorganisms, which produce these cofactors from sugar and air. The microorganism E. coli will be engineered such that they are able to catalyze a cascade reaction to fragrances like for example TropionalTM, which has an ocean like smell, from precursors that were synthesized with catalytic chemical methods. Ultimately, the aim is to integrate the chemical step into the biotransformation step and run the reaction in a chemoenzymatic one pot fashion. One of the main outcomes from this project will be a better understanding of the complex interplay between the foreign enzymes and chemicals with the microorganisms metabolism. Only with this knowledge it will be possible to find bottlenecks and to develop ideas how to alleviate them. This will not only be for the benefit of fragrance synthesis but also generally for other cascade reactions with aldehyde intermediates.

Many compounds we know from their pleasant smell contain an aldehyde group. Examples are vanillin, the typical smell of vanilla, or cinnamaldehyde, a constituent of cinnamon smell. A totally different aldehyde compound has the smell of grass and there are many, many more. To get hold of these compounds is a challenging task, especially when large amounts are needed. This holds true for chemical production and even more so for natural routes. Carboxylic acids could be promising precursors of aldehydes, because they are abundant in renewable feedstock. Our aim in this project was to find new concepts for the preparation of fragrance compounds by combining bioreduction of carboxylic acids with a biocompatible synthetic step that stiches simple precursor molecules together to derivatives of cinnamic acid. For the bioreduction, we used enzymes called carboxylate reductases, also known by their nick-name CARs. These enzymes were embedded in the well known microorganism Escherichia coli, who is providing energy and reduction power to fuel the reduction reaction. One of the main challenges in this project was to gain better understanding of the complex interplay between foreign enzymes and chemicals with the microorganism's metabolism. Another challenge was that no efficient methods for the detection and quantification of aldehydes in the presence of water and living cells existed. This prompted us to develop a new method we named the 'ABAO-assay', that allowed us to look at thousands of reactions in a very short time. The ABAO assay will not only be for the benefit of fragrance synthesis but also generally for other cascade reactions with aldehydes as end products or intermediates.