Yeast-Based Expression System Design for the Characterization of Fatty Acid Photodecarboxylase (YesFAP)

• Applicant: Professorin Dr.-Ing. Selin Kara
• Subject Area: Biological Process Engineering
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 567047711

Fatty Acid Photodecarboxylase (FAP), discovered in 2017 from the green microalgae Chlorella variabilis, is a unique photoenzyme that requires continuous light absorption to sustain its catalytic activity, becoming inactive in darkness. FAP catalyzes the direct decarboxylation of fatty acids and other carboxylic acids into hydrocarbons using blue light, operating without coenzymes, selectively and irreversibly. FAP is highly valued for synthesizing biobased hydrocarbons, such as renewable diesel and therapeutic products, and also for use in enzymatic kinetic resolution. Despite its promise, several challenges limit its widespread application: low expression levels, limited photostability, limited enzyme selectivity, and the requirement for tailored reaction conditions to ensure high productivity. To date, research has primarily focused on broadening the range of substrates, improving substrate selectivity, enhancing stability through immobilization, and employing immobilized FAP formulations. However, the main challenge remains FAP's inherently low expression levels and its inefficient production in both native and heterologous E. coli systems. An understanding of designing effective overexpression strategies to achieve high enzyme yield is needed to address this limitation, highlighting a significant fundamental knowledge gap. The YesFAP project addresses this main challenge by employing Komagataella phaffii as an alternative host. K. phaffii offers several benefits over the classical E. coli system: (i) improved protein folding and stability, supporting FAP's complex structure and enhancing yields; (ii) direct secretion of proteins into the culture medium, simplifying purification; and (iii) high cell density capability and increasing protein yields. The YesFAP project aims to create an innovative platform using K. phaffii for high-yield FAP expression, structured into six work packages (WPs): WP1 involves constructing FAP expression vectors with the AOX1 (alcohol oxidase 1) promoter to boost efficiency. WP2 focuses on the transformation of K. phaffii. WP3 covers extraction, purification, and structural analysis of FAP. WP4 tests FAP's decarboxylation capabilities. WP5, in collaboration with the Enzyme Engineering Lab at Aarhus University, Denmark, conducts enzyme engineering and comparative expression analysis in yeast and bacterial systems. WP6 involves data processing, drafting two manuscripts on novel contributions, and archiving research materials. Expected outcomes include a deep understanding of the transition to K. phaffii for FAP expression should open new opportunities for FAP’s application in synthetic biology and industrial biotechnology, thereby fostering sustainable enzyme production methods.

• DFG Programme: Research Grants
• International Connection: Denmark
• Cooperation Partner: Professor Dr. Bekir Engin Eser