Abstract:Efficient β-carotene biosynthesis was achieved by reconstructing the metabolic network of Pichia pastoris. The synthesis pathway was established through heterologous expression of geranylgeranyl diphosphate synthase (CrtE), lycopene cyclase/phytoene synthase (CrtYB), and phytoene desaturase (CrtI) in the GS115 strain. Systematic gene screening and dosage optimization led to the development of the high-yield recombinant strain G04B, which produced 309.49 mg/L (21.71 mg/g dry cell weight [DCW]) of β-carotene. Further, based on this result, β-carotene synthesis efficiency was significantly improved by enhancing the mevalonate pathway, optimizing nicotinamide adenine dinucleotide phosphate hydrogen supply, and overexpressing lipid synthesis-related genes. The optimized strain achieved a yield of 366.55 mg/L (24.34 mg/g DCW) under shake-flask conditions and 6.15 g/L (49.16 mg/g DCW) in a 5 L fermenter. The Together, the results demonstrated that optimizing the enzyme pathway, tuning gene dosage, and regulating lipid metabolism are effective strategies for increasing β-carotene production in P. pastoris, providing a foundation for natural terpenoid synthesis engineering.
