Taxadien‐5α‐hydroxylase and taxadien‐5α‐ol O‐acetyltransferase catalyze the oxidation of taxadiene to taxadien‐5α‐ol and subsequent acetylation to taxadien‐5α‐yl‐acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High‐throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien‐5α‐ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography‐mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien‐5α‐yl‐acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7‐fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.

中文翻译：

---

使用先进的生物加工策略优化酿酒酵母中氧化和乙酰化紫杉醇前体的生物合成。

在重磅抗癌药物紫杉醇 (Taxol®) 的生物合成中，紫杉二烯-5α-羟化酶和紫杉二烯-5α-ol O-乙酰转移酶催化紫杉二烯氧化为紫杉二烯-5α-醇，随后乙酰化为紫杉二烯-5α-基乙酸。尽管经过数十年的研究，混杂且多特异性的 CYP725A4 酶仍然是微生物生物合成途径开发的主要瓶颈。在这项研究中，采用跨学科方法在一系列生物反应器规模上构建和优化酿酒酵母的早期途径。高通量微尺度优化将总含氧紫杉烷滴度提高至 39.0 ± 5.7 mg/L，并且在微型和微型生物反应器规模下总紫杉烷产物滴度分别为 95.4 ± 18.0 和 98.9 mg/L。pH 控制的引入成功地缓解了含氧紫杉烷产量的减少，将潜在的紫杉烷-5α-醇异构体滴度提高至 19.2 mg/L，与微量达到的 23.8 ± 3.7 mg/L 相当。生物工艺优化和 1 L 生物反应器规模的气相色谱-质谱分辨率的提高相结合，促进了紫杉二烯-5α-乙酸酯的检测，最终滴度为 3.7 mg/L。在此规模下，总含氧紫杉烷滴度提高了 2.7 倍，达到 78 mg/L，这是酵母中报道的最高滴度。在一系列范围内确定了影响工程菌株生产力的关键参数，为开发稳健的集成生物过程控制系统奠定了基础。