Systematic Engineering of Komagataella phaffii for Efficient Production of Sweet‐Tasting Protein Brazzein

ABSTRACT Excessive sugar consumption is associated with metabolic disorders such as obesity and diabetes, prompting the search for healthier alternatives. Sweet‐tasting proteins offer high sweetness, safety, and low caloric content, making them promising sugar substitutes. However, their application is hindered by limited natural abundance and challenging extraction processes from plant sources. In this study, the microorganism‐based sweet‐tasting protein was explored. Brazzein was first verified as an expressible protein for production using the Komagataella phaffii expression system. Through the evaluation of promoters and signal peptides, the P AOX1 →SP 0030 → PbBRZ →T das1 expression cassette was identified as optimal for brazzein production. Via the optimization of brazzein gene copy number and the modulation of protein translation, folding, and degradation processes, brazzein production was further improved to 113.24 and 166.54 mg/L, respectively. By performing fed‐batch fermentation in a 5 L bioreactor, the engineered strain produced brazzein at a titer of 639.11 mg/L. This is the highest level for microbial brazzein production. These findings highlight the effectiveness of our strain optimization strategies and provide a foundation for the industrial application of sweet‐tasting proteins.