紫杉醇（Taxol®）是紫杉属植物中最常见的抗癌二萜类化合物。紫杉醇的核心骨架经过了高度改变，但对于作用于其后修饰过程的细胞色素P450酶的研究非常有限。本研究利用计算辅助蛋白工程和代谢工程，对来自锐齿红豆杉的紫杉烯-10β-羟化酶（T10βH）进行了调查，该酶是第三个催化将紫杉烯-5α-乙酸酯（T5OAc）转化为紫杉烯-5α-乙酰氧基-10β-醇（T10OH）的后修饰酶。T10βH的变体M3（I75F/L226K/S345V）在蛋白表达方面表现出了显著的9.5倍增加，伴随着翻倍的催化频率（TOF）和总催化次数（TTN）的1.3倍和2.1倍的提高。将变体M3集成到工程菌株中后，T10OH的产量从0.97增加到2.23 mg/L。最终，在5升生物反应器的积分培养中，T10OH的滴定达到了3.89 mg/L，这是迄今报告的微生物全新合成这一关键紫杉醇中间体的最高水平。本研究对于进一步探索与紫杉醇和其他萜类化合物的人工生物合成有关的其他细胞色素P450酶具有重要参考价值.关键点： • 首次在E. coli中表达和改造了来自T. cuspidata的T10βH。 • 通过蛋白工程提高了T10βH的表达和活性。 • 在E. coli中实现了T10OH的全新生物合成，产量达到了3.89 mg/L.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

Paclitaxel (Taxol®) is the most popular anticancer diterpenoid predominantly present in Taxus. The core skeleton of paclitaxel is highly modified, but researches on the cytochrome P450s involved in post-modification process remain exceedingly limited. Herein, the taxane-10β-hydroxylase (T10βH) from Taxus cuspidata, which is the third post-modification enzyme that catalyzes the conversion of taxadiene-5α-yl-acetate (T5OAc) to taxadiene-5α-yl-acetoxy-10β-ol (T10OH), was investigated in Escherichia coli by combining computation-assisted protein engineering and metabolic engineering. The variant of T10βH, M3 (I75F/L226K/S345V), exhibited a remarkable 9.5-fold increase in protein expression, accompanied by respective 1.3-fold and 2.1-fold improvements in turnover frequency (TOF) and total turnover number (TTN). Upon integration into the engineered strain, the variant M3 resulted in a substantial enhancement in T10OH production from 0.97 to 2.23 mg/L. Ultimately, the titer of T10OH reached 3.89 mg/L by fed-batch culture in a 5-L bioreactor, representing the highest level reported so far for the microbial de novo synthesis of this key paclitaxel intermediate. This study can serve as a valuable reference for further investigation of other P450s associated with the artificial biosynthesis of paclitaxel and other terpenoids. KEY POINTS: • The T10βH from T. cuspidata was expressed and engineered in E. coli unprecedentedly. • The expression and activity of T10βH were improved through protein engineering. • De novo biosynthesis of T10OH was achieved in E. coli with a titer of 3.89 mg/L.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.