Multidimensional Engineering of Komagataella phaffii for Efficient Synthesis of Retinal

Abstract

Retinal is a fundamental molecule in the visual system and a critical regulator of skin health and cellular physiology. The sustainable production of retinal via engineered microbial cell factories offers a promising alternative to chemical synthesis. In this study, we established the biosynthetic pathway in Komagataella phaffii, providing the first experimental demonstration of the membrane-associated nature of β-carotene 15,15′-mono(di)oxygenase (BCMO). To maximize productivity, we implemented a multi-layered engineering strategy, including the amplification of key gene dosages, the balancing of NADPH cofactor supply, and the augmentation of precursor availability. Leveraging the unique sub-cellular architecture of K. phaffii, an anchoring motif from the peroxin PEX15 was used to achieve peroxisome surface display of BCMO. This spatial scaffolding effectively achieved in vivo enzyme immobilization, significantly enhancing catalytic activity. To harmonize cellular fitness with the oxygen-dependent requirements of BCMO, heterologous expression of Vitreoscilla hemoglobin (VHb) led to a 58.13% increase in biomass and a 41.73% improvement in retinal titer. In addition, we identified the Pdr5 transporter as a key mediator for retinal efflux, thereby alleviating intracellular cytotoxicity. The integration of surfactants facilitated the recovery of over 90% of intracellular retinal into the extracellular space, substantially reducing downstream processing complexity. Finally, utilizing glucose and methanol as carbon sources, the engineered strain achieved retinal titers of 764.22 mg/L and 1406.69 mg/L in a 5 L bioreactor, respectively. The methanol-derived titer represents the highest reported to date, 3 underscoring the potential of K. phaffii as a versatile one-carbon biomanufacturing platform for industrial terpenoid biosynthesis.