Improved Astaxanthin Synthesis in Komagataella phaffii through β‑Carotene Ketolase Screening and β‑Carotene Hydroxylase Mutagenesis

Astaxanthin is highly valued for its superior antioxidant properties and a variety of applications, prompting extensive research into its synthesis via microbial metabolic engineering. Commonly, organisms such as Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica have been studied for astaxanthin production. Komagataella phaffii, known for its efficacy in heterologous protein expression, has been less explored for astaxanthin synthesis. This study investigates the potential of K. phaffii, leveraging its membrane protein expression capability and suitability for high-density fermentation in industrial settings. We evaluated the compatibility of β-carotene hydroxylases (CrtW) and β-carotene ketolases (CrtZ) from different species within K. phaffii, finding that CrtW from Paracoccus sp. N81106 and CrtZ from Pantoea ananatis (PaCrtZ) pair most effectively. A semirational design was utilized for site-specific mutagenesis of the rate-limiting enzyme PaCrtZ, resulting in a 39% increase in astaxanthin production. Using a low-cost mineral salt medium in fed-batch fermentation, the astaxanthin yield reached 0.56 mg/g DCW. This study not only underscores the robust tolerance of K. phaffii to hydrophobic carotenoids and its proficiency in expressing membrane proteins within the astaxanthin biosynthetic pathway but also lays the groundwork for employing K. phaffii as a versatile chassis for the efficient synthesis of astaxanthin and other hydrophobic compounds.