在寻求创新的癌症治疗方法的过程中，紫杉醇仍然是临床肿瘤学的基石。然而，在最后一种紫杉烷羟化酶表征后的几十年里，其复杂的生物合成途径，特别是复杂的氧化步骤，仍然是一个谜。所涉及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进行的分子对接研究发现 TB574 是紫杉醇生物合成途径中潜在缺失的酶，证明了良好的相互作用。利用工程酿酒酵母菌株作为新型微生物细胞工厂联盟的补充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T5αAc-1β,10β-二醇，浓度约为 40 mg/L。这一成就非常有前景，为进一步探索利用微生物群落的新型代谢工程方法提供了新方向。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，而且为全面了解紫杉醇生物合成及其异源生产开辟了一条开拓性的道路。 T1βOH 的表征强调了使用异源系统改善癌症治疗和药品生产紫杉醇生产的未来进步的重大飞跃，从而为提高癌症治疗的功效和药品生产的效率带来了巨大的希望。版权所有 © 2024。出版者爱思唯尔公司

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.Copyright © 2024. Published by Elsevier Inc.