Expression, structural studies and protein engineering of lipases from Basidiomycota for better understanding of their sequence- and structure-guided specificity and applications in cheese

• Applicant: Dr. Martin Gand, Ph.D.
• Subject Area: Food Chemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 514527182

In cheese production, the formation of certain flavors and texture plays a crucial role in obtaining an appealing product. The production of flavor compounds inside the cheese is an extremely complex process, wherein free fatty acids that are generated through lipolysis, performed by lipases, act as the character impact compounds for the flavor of various cheeses. In previous works, I have shown that fungi of the phylum Basidiomycota can produce lipases, with similar chain-length specificity in comparison to animal lipases that are isolated from the tongues of kids, lamb and calves or animal rennet ("paste rennet") from the aforementioned animals. Lipases from animal sources are the current state-of-the-art in the cheese industry for production of certain flavors from milk fats. However, in the recent years, changes in the dairy industry are required, due to increased demands for microbial and fermentation-derived enzymes and additives as an alternative to animal products. One of our identified basidiomycetous lipases also tend to have a higher activity towards milk fats with long-chain fatty acids, so within this project the generation of basidiomycetous lipase variants by in silico molecular modeling followed by protein engineering in the laboratory will be covered. The variants will be heterologously expressed and characterized. Moreover, their performance will be investigated, particularly with respect to the modulation of the chain-length specificity and their stability for applications in cheese production using cow’s milk will be studied. Since no crystal structures of lipases from higher edible fungi are currently available, the structure of these and their variants will be predicted by the software AlphaFold2 and afterwards the structural information will be solved and subsequently used for the next rounds of rational design. Furthermore, by matching the specificities of the free fatty acids produced of the variants with structural information, a better sequence and structure-guided specificity understanding of lipases from Basidiomycota in general will be gained. Noteworthy, one of our lipase, according to analysis of the "lipases engineered database" v.4 (https://led.biocatnet.de/), falls into the family of these enzymes similar to cholesterol esterase, but none of the basidiomycetal enzymes have been biochemically characterized. Furthermore, no cholesterol esterase activity was found for this enzyme in our laboratory. Therefore, the probability of a new subgroup for lipases is high. From this point of view, the lipases of Basidiomycota are an interesting research target.

• DFG Programme: Research Grants
• International Connection: China
• Cooperation Partners: Professor Dr. Binglin Li, Ph.D.; Professorin Dr. Fengjiao Xin, Ph.D.