Evolutionary and Metabolic Engineering of Lactococcus lactis for Improved Food and Flavor Compound Production

Soon after the beginning of agriculture and animal husbandry, lactic acid bacteria (LAB) were used for fermenting milk or other food materials to improve storability and ensure safe consumption. One of these products, which is still of immense importance, is cheese. Nowadays, the fermentation with LAB not only provides food with long lifetimes but also results in delicious products, which are valued throughout the world.

Many cheese varieties owe their unique flavors to mesophilic LAB, especially Lactococcus lactis (L. lactis), which thrive at temperatures below 40°C. For producing hard cheeses, often temperatures higher than 40°C are required, suitable only for thermophilic LAB, which have different flavor forming properties.

In this project, the possibility of using the mesophilic L. lactis for making hard cheeses was explored. For this, thermotolerant mutants of an industrial L. lactis strain (SD96) were created by using a natural selection method, namely adaptive laboratory evolution (ALE). Three mutants were characterized in detail using genomics and transcriptomics analysis.

By comparison with the parent strain SD96, mechanisms for overcoming the temperature limitation were identified. Unexpectedly, the mutants were found to be more autolytic, which is beneficial for cheese ripening, and this observation was further investigated. Overall, the thermotolerant mutants are promising candidates for producing hard cheese variants with the unique flavor profile provided by mesophilic LAB.

Generally, this project shows that it is possible to obtain thermotolerant mutants of mesophilic LAB with superior industrially relevant properties. Besides applications for food fermentations, LAB have become increasingly important for the industrial biotechnology sector. Due to their long-term and safe application in food products, many LAB are have Generally Recognized As Safe (GRAS) status and are therefore promising candidates for overproducing ingredients for food or cosmetic products.

Moreover, due to their simple metabolism and comparably high robustness, LAB are rising candidates as biocatalysts for producing valuable chemicals using waste streams from the food industry as starting material. Metabolic engineering, by genome editing, facilitates the streamlining of metabolic pathways for converting carbohydrates into desired chemicals.

For several decades, such applications had been restricted to a handful of laboratory strains due to the limited availability of genome editing tools. More recent progress in the field, which allowed metabolic engineering of wild LAB, was reviewed in this thesis and provides a comprehensive overview of recent developments in the field.

Building on these advances, in the current project, metabolically engineered L. lactis were constructed and used for producing large amounts of α-acetolactate from a dairy waste stream. This compound quickly degrades to diacetyl, which provides a rich butter flavor. A two-step bioprocess was developed, combining fermentation and whole-cell biocatalysis, which turned out to be superior to one-step processes solely relying on fermentation.

Furthermore, the optimization of this process using permeabilized cells and initial experiments towards using another low-value waste stream from the dairy industry for α-acetolactate production are reported.