Metabolic engineering of yeasts for the production of natural food colorants

The color of foods and beverages is often associated with the products’ flavor, freshness, and quality. Food manufacturers therefore often add colorants to processed foods and beverages to enhance or correct their appearance, ensuring that they align with consumer expectations. Synthetic dyes currently dominate the food colorant market, but consumer concerns regarding their safety and sustainability is driving a demand for natural food-color alternatives. However, natural food colorants are significantly more costly than synthetics, since the pigment content in the raw organic material is typically very low and extraction can be challenging.

The production of natural food colorants from renewable substrates by fermentation of engineered microorganisms has been proposed as a sustainable alternative to conventional extraction from organic materials - yielding natural pigments that are less costly and have improved purity. However, most biotechnological processes for the production of natural food colorants are too inefficient to compete with extraction-based processes. This thesis seeks to advance the field of natural food colorant production by metabolic engineering and fermentation. Specifically, this thesis is framed around the production of red beet betalains by genetic engineering and fermentation of the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica.

Initially we engineer the oleaginous yeast Y. lipolytica for high-level production of betanin, the red pigment typically found in beetroots. We then apply life cycle and techno-economic assessment to evaluate the sustainability performance and economic viability of our proposed fermentation-based production process. We find that at industrial scale, our proposed process for red beet colorant production would require significantly less land, energy, and resources compared with conventional pigment extraction from beetroot crops. We also find that our fermentation-based process could be economically feasible in the existing market conditions.

With indications that red beet colorant production by fermentation is a sustainable and economically viable alternative to extraction from red beets, we next sought to improve our understanding of betalain metabolism to aid further engineering. While Y. lipolytica is an excellent and safe production host for food ingredients, such as omega-3-fatty acids and steviol glycosides, its synthetic biology tools are less mature compared to S. cerevisiae. We therefore used S. cerevisiae as a discovery host, interrogating the betalain biosynthesis pathway, as well as the native S. cerevisiae metabolism involved in the synthesis of the betalain precursors. Here we develop combinatorial libraries targeting various parts of metabolism and screen thousands of colonies to gain novel insights. Additionally, we demonstrate the use of absorbance-activated droplet sorting for screening pigment-producing microorganisms, which will be useful for accelerating future high-throughput engineering campaigns for improved pigment production.

In summary, this thesis advances the broader field of metabolic engineering by contributing to the development of a fermentation-based process for producing red beet colorant from renewable substrates using engineered yeasts.