Metabolic Engineering of Komagataella Phaffii for de Novo Synthesis of Retinol From Methanol

Abstract

Retinol, a derivative of vitamin A with potent antioxidant and therapeutic properties, is in high market demand. In response to the low productivity of conventional methods, metabolic engineering has been explored for microbial retinol production. However, systematic engineering strategies for high‐level retinol synthesis in Komagataella phaffii remain limited. In this study, the methylotrophic yeast K. phaffii was developed as an engineered chassis for efficient de novo retinol biosynthesis. Based on a previously constructed β‐carotene‐producing strain, β‐carotene‐15,15′‐dioxygenase (Blh) and retinol dehydrogenase (RDH12) were screened and introduced to establish the synthetic pathway of retinol. To increase precursor supply, key genes in the β‐carotene biosynthetic pathway were overexpressed. The mevalonate (MVA) pathway was further optimized, and central carbon metabolism was reprogrammed to enhance metabolic flux toward retinol. Transport engineering was also performed to improve retinol secretion. Several candidate transporters were overexpressed, and the protein encoded by chr1‐4_0619 in K. phaffii was identified as an endogenous retinol transporter. The final engineered strain produced 3.38 g/L retinol with BHT supplementation in fed‐batch fermentation using a 1.5 L bioreactor. This work represents de novo microbial synthesis of retinol from a one‐carbon feedstock, demonstrating the formidable potential of K. phaffii as a sustainable chassis for retinol production.