Reprogramming Komagataella phaffii Cell Factory for High-Yield Production of 3S,3′S-Astaxanthin via Multiplex Metabolic Engineering and Its Application

Abstract 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.