本研究以毕赤酵母为底盘，β-胡萝卜素为目标产物，探索了在毕赤酵母中合成β-胡萝卜素的能力。首先是β-胡萝卜素合成毕赤酵母菌株的构建：以毕赤酵母 GS115 为底盘细胞过表达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）合成β-胡萝卜素，并通过基因来源筛选和基因剂量优化，得到一株β-胡萝卜素产量相对较高的重组酵母工程菌株G04B，产量为309.49 mg/L（21.71 mg/g DCW）。其次通过甲羟戊酸途径优化、辅因子NADPH供给优化以及过表达脂质合成优化基因改善脂质合成三方面策略对毕赤酵母β-胡萝卜素合成途径进行优化重构，使β-胡萝卜素产量在摇瓶水平提高到366.55 mg/L（24.34 mg/g DCW），5 L 发酵罐发酵时，β-胡萝卜素产量为6.15 g/L（49.16 mg/g DCW）。本研究表明异源基因筛选、增加关键基因拷贝数和改善脂类合成是提高β-胡萝卜素表达量的有效手段，为毕赤酵母天然萜类合成的代谢优化提供了参考。

This study utilized?Pichia pastoris?as a host system to construct an efficient β-carotene biosynthesis pathway through metabolic engineering strategies. Initially, the β-carotene synthesis pathway was established in?Pichia pastoris?GS115 by heterologously expressing GGPP synthase (CrtE), bifunctional lycopene cyclase/phytoene synthase (CrtYB), and phytoene desaturase (CrtI). Through systematic screening of functional genes from different sources and optimization of gene dosage, a high-yielding recombinant strain, G04B, was obtained, achieving a β-carotene production of 309.49 mg/L (21.71 mg/g DCW). Subsequently, a multi-dimensional metabolic engineering approach was employed to optimize the pathway: enhancing the mevalonate (MVA) pathway, optimizing NADPH cofactor supply, and overexpressing lipid biosynthesis-related genes, which significantly improved β-carotene production. The optimized strain achieved a yield of 366.55 mg/L (24.34 mg/g DCW) in shake-flask cultures and 6.15 g/L (49.16 mg/g DCW) in a 5 L fermenter. The results demonstrate that heterologous gene screening, key gene copy number optimization, and lipid metabolism regulation are effective strategies for enhancing β-carotene production in?Pichia pastoris. This study provides theoretical insights and technical references for the metabolic engineering of?Pichia pastoris?in the synthesis of natural terpenoids.