In the quest for innovative cancer therapeutics, paclitaxel remains a cornerstone in clinical oncology. However, its complex biosynthetic pathway, particularly the intricate oxygenation steps, has remained a puzzle in the decades following the characterization of the last taxane hydroxylase. The high divergence and promiscuity of enzymes involved have posed significant challenges. In this study, we adopted an innovative approach, combining in silico methods and functional gene analysis, to shed light on this elusive pathway. Our molecular docking investigations using a library of potential ligands uncovered TB574 as a potential missing enzyme in the paclitaxel biosynthetic pathway, demonstrating auspicious interactions. Complementary in vivo assays utilizing engineered S. cerevisiae strains as novel microbial cell factory consortia not only validated TB574's critical role in forging the elusive paclitaxel intermediate, T5αAc-1β,10β-diol, but also achieved the biosynthesis of paclitaxel precursors at an unprecedented yield including T5αAc-1β,10β-diol with approximately 40 mg/L. This achievement is highly promising, offering a new direction for further exploration of a novel metabolic engineering approaches using microbial consortia. In conclusion, our study not only furthers study the roles of previously uncharacterized enzymes in paclitaxel biosynthesis but also forges a path for pioneering advancements in the complete understanding of paclitaxel biosynthesis and its heterologous production. The characterization of T1βOH underscores a significant leap forward for future advancements in paclitaxel production using heterologous systems to improve cancer treatment and pharmaceutical production, thereby holding immense promise for enhancing the efficacy of cancer therapies and the efficiency of pharmaceutical manufacturing.

中文翻译：

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紫杉醇异源生物合成的新步骤：T1βOH 紫杉烷羟化酶的表征


在寻求创新癌症疗法的过程中，紫杉醇仍然是临床肿瘤学的基石。然而，其复杂的生物合成途径，尤其是复杂的氧合步骤，在最后一个紫杉烷羟化酶表征后的几十年里仍然是一个谜。所涉及的酶的高度分歧和混杂性带来了重大挑战。在这项研究中，我们采用了一种创新方法，将计算机模拟方法和功能基因分析相结合，以阐明这一难以捉摸的途径。我们使用潜在配体库的分子对接研究发现 TB574 是紫杉醇生物合成途径中潜在的缺失酶，证明了吉祥的相互作用。利用工程酿酒酵母菌株作为新型微生物细胞工厂联盟的互补体内测定不仅验证了 TB574 在锻造难以捉摸的紫杉醇中间体 T5αAc-1β，10β-二醇中的关键作用，而且还以前所未有的产量实现了紫杉醇前体的生物合成，包括约 40 mg/L 的 T5αAc-1β，10β-二醇。这一成就非常有前景，为进一步探索利用微生物联盟的新型代谢工程方法提供了新的方向。总之，我们的研究不仅进一步研究了以前未表征的酶在紫杉醇生物合成中的作用，而且还为完全理解紫杉醇生物合成及其异源生产开辟了一条开创性进展的道路。 T1βOH 的表征强调了紫杉醇生产未来进步的重大飞跃，使用异源系统来改善癌症治疗和药物生产，从而为提高癌症治疗的疗效和药物生产的效率带来巨大的希望。