紫杉醇是迄今为止开发的最有效的抗癌药物之一。尽管植物细胞培养提供了最可持续的生产方法，但产量受到紫杉烷生物合成途径中瓶颈酶的限制：浆果赤霉素-氨基苯丙酰-13-O-转移酶 (BAPT) 和 3'-N-去苯甲酰紫杉醇 N-苯甲酰转移酶（DBTNBT）。为了通过克服这一瓶颈来提高紫杉醇产量，我们获得了不同的红豆杉体外根系，通过发根根瘤菌 A4 介导的转化，每个系独立地过表达两个通量限制基因 BAPT 或 DBTNBT 中的一个。由于转基因红豆杉根的生长速度缓慢，对其进行去分化以获得愈伤组织系并建立细胞悬浮液。转基因细胞在两阶段系统中培养，并通过用 1μm 冠菌素加 50mm 随机甲基化-β-环糊精的双重诱导处理来刺激紫杉烷的产生。在转基因细胞培养物中观察到 BAPT（48 小时高出 59.72 倍）和 DBTNBT（72 小时高出 61.93 倍）基因的高过表达，以及紫杉烷产量的提高。与野生型品系（71.01mg/L）相比，DBTNBT品系产生的紫杉醇含量（310mg/L）高出四倍多，而BAPT品系中紫杉烷的含量几乎翻倍（135mg/L）。 ）。紫杉烷生物合成基因的转录谱显示，GGPPS、TXS 和 DBAT 基因对 DBTNBT 过表达和双重诱导反应最敏感，其表达逐渐持续增加。相同的基因在引发的 BAPT 过表达系中表现出孤立的表达峰模式。© 2023 作者。植物生物技术杂志由实验生物学学会和应用生物学家协会和约翰·威利出版

Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 μm coronatine plus 50 mm of randomly methylated-β-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line.© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.