毕赤酵母合成β-胡萝卜素代谢重构

Metabolic Reconstruction of Pichia pastoris for β-Carotene Biosynthesis

摘要:

该研究通过毕赤酵母代谢网络重构，实现β-胡萝卜素的高效生物合成。将GGPP合酶（CrtE）、双功能番茄红素环化酶/八氢番茄红素合成酶（CrtYB）和八氢番茄红素去饱和酶（CrtI）在毕赤酵母GS115异源表达，成功构建了β-胡萝卜素合成途径。接着，系统筛选不同来源的功能基因并进行基因剂量优化，获得了一株高产β-胡萝卜素的重组菌株G04B，其产量达到309.49 mg/L（21.71 mg/g DCW）。在此基础上，通过强化甲羟戊酸（MVA）途径、优化辅因子NADPH供给及过表达脂质合成相关基因，显著提高了β-胡萝卜素的合成效率。优化后的菌株在摇瓶培养条件下产量提升至366.55 mg/L（24.34 mg/g DCW），在5 L发酵罐中产量达到6.15 g/L（49.16 mg/g DCW）。研究结果表明，途径酶的优化、关键基因剂量优化以及脂质代谢调控是提高毕赤酵母中β-胡萝卜素产量的有效策略，进而为毕赤酵母合成天然萜类化合物的代谢工程改造提供了理论依据和技术参考。

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.