De novo biosynthesis of [beta]-Arbutin in Komagataella phaffii based on metabolic engineering strategies

[beta]-Arbutin, found in the leaves of bearberry, stands out as one of the globally acknowledged eco-friendly whitening additives in recent years. However, the natural abundance of [beta]-Arbutin is low, and the cost-effectiveness of using chemical synthesis or plant extraction methods is low, which cannot meet the requirements. While modifying the [beta]-Arbutin synthesis pathway of existing strains is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application. In this study, we established a biosynthetic pathway in Komagataella phaffii for [beta]-Arbutin production with a titer of 1.58 g/L. Through diverse metabolic strategies, including fusion protein construction, enhancing shikimate pathway flux, and augmenting precursor supplies (PEP, E4P, and UDPG), we significantly increased [beta]-Arbutin titer to 4.32 g/L. Further optimization of methanol concentration in shake flasks led to a titer of 6.32 g/L titer after 120 h of fermentation, representing a fourfold increase over the initial titer. In fed-batch fermentation, strain UA3-10 set a record with the highest production to date, reaching 128.6 g/L in a 5 L fermenter. This is the highest yield in the fermentation tank level of using microbial cell factories for de novo synthesis of [beta]-Arbutin. Applying combinatorial engineering strategies has significantly improved the [beta]-Arbutin yield in K. phaffii and is a promising approach for synthesizing functional products using a microbial cell factory. This study not only advances low-cost fermentation-based production of [beta]-Arbutin but also establishes K. phaffii as a promising chassis cell for synthesizing other aromatic amino acid metabolites.