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.
Astaxanthin is a keto-carotenoid with high added value. In this study, we aimed to biosynthesize 3S,3′S-astaxanthin efficiently and sustainably from the renewable single-carbon (C1) feedstock methanol through multiplex metabolic engineering strategies in Komagataella phaffii. First, the K. phaffii cell-free terpene synthesis system was established successfully and applied to evaluate the astaxanthin synthase combinations rapidly. We then systematically engineered K. phaffii for the overproduction of 3S,3′S-astaxanthin from methanol by tuning the carotenoid synthesis module rationally, optimizing the precursor supply and carotenoid storage globally, thereby achieving a significant elevation in astaxanthin content from 3.910 mg/g to 7.513 mg/g. Thereafter, key node enzyme assembly, branch route reconstruction, and cofactor engineering were employed to further improve astaxanthin accumulation, achieving a significant increase of astaxanthin content to 11.397 mg/g. Finally, the astaxanthin production reached 4.75g/L under fed-batch fermentation, which is the highest astaxanthin level reported in an engineered microbe to date. In addition, the synthesized astaxanthin was successfully and effectively applied in shrimp farming for color enhancement and antioxidant effects. These results demonstrate the potential of K. phaffii as a promising platform for sustainable green production of value-added terpenoid compounds from organic one-carbon feedstocks and will pave the way for astaxanthin industrial production.
