1. Provide an abstract/summary for your project.
Chocolate is one of the most popular flavors world wide. Currently the production of cacao beans is being harmed by climate change: the price of chocolate is rising while the quality of the beans is decreasing. Substitutes for this flavoring can be chemically synthesized but they provide no nutritional value for the consumer. The aim of this project is to produce theobromine and linalool, the main components of chocolate flavor, in Saccharomyces cerevisiae, in order to use this modified yeast as a nutritional supplement with this popular flavor. Once achieved, the next aim is to improve the pathways to maximize the production of these components without compromising the livelyhood of the cells and determine the conditions for an optimal batch growth. The final goal of this project is to establish the protocol for freeze drying the transformed yeast in order to comercialize it as a nutritional supplement. This will provide an alternative to chemically synthesized flavoring, with the added benefit that it provides a nutritional boost. In the future, this system can be used to produce different flavorings, mainly of products that are harder to cultivate or more rare.
2. Outline three aims of your final project
The first aim of my final project is to express the biosynthetic pathways for the produciton of theobromine and linalool in S. cerevisiae through genomic integration of the expression cassettes. The cassettes will be flanked by homology arms that will facilitate the recombination into genome safe harbor sites within the chromosome. The design of the constructs will be done in silico and they will be chemically synthezised. After the introduction of the constructs, there will be an evaluation of the cellular density and of the production of linalool and theobromine.
The second aim of this project is to identify ways in which the biosynthetic pathways can be improved to increase the amount of linalool and theobromine produced, while keeping a healthy cell metabolism. The areas of improvement will be identified with the results of the measurements recorded form the cellular density and the production of components. The changes will be made to the promoters and ribosome binding sites used, as well as the amount copies of the expression cassettes. The improvements have to ensure that the yeast can be cultivated in larger batch reactors. Finding the optimal conditions for their growth in a batch reactor is part of the second aim.
The third aim of this project is to establish a protocol for freeze drying the cells to be able to comercialize them as a nutritional supplement with an endogenous chocolate flavor. This aim is focused on finding the best way to preserve the cells to sell them on the market. Future studies would include evaluating the organoleptic qualities of the yeast and, making the changes necessary for there to be a strong chocolate flavor. Furthermore, if the project is successful, it will open up the posibilities of the flavors that can be expressed in microbial platforms and, even complex metabolic routes for the production rare/difficult to obtain compounds.
3. Provide background research for your final project.
Agriculture has been significantly impacted by climate change and the consequences have become more apparent in the last few decades. As the temperature rises and the weather patterns become more irregular, the crops yield less product with a lower quality. These negative consequences are more evident in the tropical regions and not only affect staple crops, but they also affect plants that produce luxury goods, like vanilla and chocolate.
Chocolate is one of the most popular flavors world wide and the price of cacao beans are on the rise. The increase in the price is not atributed to an improvement in the quality of the bean but on the increasing demand and decreasing crop yield. Climate change causes significant droughts and changes in the sunshine hours, which leads to a lower quality and yield of crops (Yuan et al., 2024).
As an alternative to these products and others that have similar characteristics (expensive or hard to come-by), chemically synthezised versions have been developed. Artificial flavors are often used because they are similar enough to the original flavors and are cheaper and easier to produce. However, artificial flavors lack the nutritional benefits that come from consuming the source product and the methods of production are not very sustainable. Due to these shortcomings, researchers have been looking into the production of these compounds in microbial platforms.
In 2009, Hansen et al. reported the first de novo production of vanillin in Saccharomyces cerevisiae. This was done through the cloning of the biosynthetic pathway into the yeast. Many researchers, like Brochado et al. (2010) and Qiu et al. (2022), have improved on the original design, with the end goal of creating a more environmentally friendly and economic option for the synthesis of this flavor. The production of this compound was done through the integration to the yeast genome of the enzymes involved in the metabolic pathway for the biosynthesis of vanillin. There was also an improvement in the type and amount of susbtrate added to the yeast. By optimizing the conditions in which yeast expressing vanillin synthesizing enzymes grows, it offers an alternative for artificial vanilla flavor and sets the precedent that flavor compounds can be produced in microbial platforms
The goal of this project is to synthesize the compounds that produce chocolate flavor in Saccharomyces cerevisiae to obtain chocolate flavored yeast.
Chocolate flavor is not attributed to a single molecule (like vanillin), but is a mixture of a variety of compounds present in a cacao bean and those that are formed during the process of producing chocolate. Fermentation and toasting create new compounds that contribute to the characteristic flavor. Various studies about the compounds that contribute to chocolate’s flavor profile of have shown that the most abundant are theobromine and linalool. Theobromine is an alkaloid that has a bitter taste (Barišić et al., 2019). Linalool is a monoterpene alcohol that provides a floral aroma to chocolate (Seyfried & Granvogl, 2019).
The study done by Jin et al. (2014) was able to produce theobromine in Saccharomyces cerevisiae through metabolic engineering. The study showed that the synthesis of theobromine was a 3 step process (a fourth step was considered for the synthesis of caffeine). In this pathway 3 enzymes intervene: xanthosine methyltransferase transforms xanthosine to 7-methylxanthosine, N-methyl-nucleosidase transforms 7-methylxanthosine into 7-methylxanthine, and caffeine synthase methylates 7-methylxanthine to obtain theobromine (this enzyme also facilitates the last step where the methylation of theobromine results in caffeine). The enzyme xanthosine methyltransferase (CaXMT1) was found to have dual activity, being able to carry out steps 1 and 2 of the pathway.