基于非天然途径以7-木糖-10-去乙酰紫杉醇为前体生物合成紫杉醇

Paclitaxel (Taxol®) is one of the most effective antitumor drugs ever developed. The compound was first isolated from the bark of yew tree with extremely low concentration. The early destructive collection from the wild tree limited its supply, which prompted intensive efforts to develop alternative means of production. Up to now, one of the practical means is to isolate paclitaxel from the renewable twigs of yew seedlings through nursery plantation. However, the amount of paclitaxel in the yew twigs is still low. On the other hand, the amount of its by-product—7-β-xylosyl-10-deacetyltaxol (XDT) is dozens of times higher than that of paclitaxel.The C7-xylosyl group of XDT can be removed to form 10-deacetyltaxol (DT), while the C10-hydroxy group of DT can be acetylated to manufacture paclitaxel. We have cloned the glycoside hydrolase LXYL-P1-2 from Lentinula edodes and the enzyme can efficiently bioconvert XDT to DT. The engineered yeast GS115-3.5K-P1-2 harboring the Lxyl-p1-2 gene has also been constructed. In this project, we will optimize 10-deacetylbaccatin III 10β-O-acetyltransferase (DBAT) from yew tree to creat DBATm by protein engineering, which greatly improves the enzyme’s acetylation on the C10-hydroxy residue of the unnatural substrate DT. We will also construct the chassis cells of Saccharomyces cerevisiae via metabolic pathway engineering, which dramatically increases the concentration of the innate acetyl-CoA of the chassis cells. Then we will firstly construct the engineered yeast Q1 and Q2 by introducing dbatm and dbatm plus Lxyl-p1-2 respectively into the yeast chassis cells with the yeast episomal plasmids, and secondly construct the chromosomal integration strain Q1 or Q2, creating the unnatural biosynthetic pathway from the exogenous XDT through DT to paclitaxel (the DT origin of Q1 is from the hydrolized product of XDT by GS115-3.5K-P1-2) by using the yeast innate acetyl-CoA as the dornor of acetyl group to produce paclitaxel. This project will lay the foundation for the production of paclitaxel from the precursor XDT, relying entirely on the bio-enzymatic strategy.

紫杉醇天然含量低微但其副产物7-木糖-10-去乙酰紫杉醇XDT含量丰富，已克隆到能高效脱除XDT木糖基生成10-去乙酰紫杉醇DT的水解酶LXYL-P1-2，并建成携带Lxyl-p1-2的工程酵母GS115-3.5K-P1-2。本项目通过蛋白质工程对红豆杉来源10-去乙酰巴卡亭Ⅲ 10β-O-乙酰转移酶DBAT进行改构，提高DBATm对非天然底物DT的C10位羟基乙酰化能力，以高效合成紫杉醇；通过代谢途径工程构建内源性乙酰辅酶A胞内浓度显著提高的酿酒酵母底盘细胞；将dbatm、dbatm+Lxyl-p1-2分别导入底盘细胞，构建工程菌Q1和Q2，进而构建染色体整合型Q1或Q2，创建由前体XDT到DT再到紫杉醇的非天然生物合成途径（Q1的DT来自GS115-3.5K-P1-2对XDT的水解），以酵母内源性乙酰辅酶A为乙酰基供体生物合成紫杉醇，为完全依赖生物酶法以XDT为前体制备紫杉醇奠定基础。

紫杉醇（Taxol）主要由红豆杉产生，其生物合成从GGPP开始，经由10-去乙酰巴卡亭III（10-DAB）和巴卡亭III（BCT-III）等最后形成Taxol。从10-DAB到BCT-III由10-去乙酰巴卡亭III-10-O-乙酰转移酶（DBAT）催化。树皮等部位的Taxol含量~0.02%；但10-DAB在欧洲红豆杉枝叶中含量可达0.1%以上，是化学半合成Taxol的重要前体；7-木糖-10-去乙酰紫杉醇（XDT）在一些种类红豆杉树皮中含量可高达0.5%。XDT并非Taxol生物合成中间体，但可通过专一性β-木糖苷酶（LXYL-P1-2）脱除木糖基产生10-去乙酰紫杉醇（DT）进而通过DBAT在DT的C-10位乙酰化而被转化成Taxol。我们克隆表达了6种红豆杉的DBAT，证明它们均可将DT乙酰化为Taxol，但催化活性相比其催化10-DAB低近3个数量级。以东北红豆杉DBAT为模板，通过同源模建生成DBAT三维结构，利用丙氨酸扫描确定活性位点及发现高活性突变体，通过饱和突变与重组设计出DBATG38R/F301V，其催化DT的效率比DBAT提高近6倍。利用DBATG38R/F301V和LXYL-P1-2建立以XDT为底物的一锅法酶促反应体系，Taxol产量达0.64mg/mL（50mL体积）。.在化学半合成Taxol时多个基团需要保护与解保护，且从10-DAB到BCT-III要用到吡啶、乙酰氯等化学试剂和苛刻的反应条件。DBAT可一步完成乙酰化反应并可减少有毒有害试剂使用，但酰基供体来源问题是重要限制因素，且规模化生产条件尚未建立。通过模块工程优化大肠杆菌乙酰辅酶A合成通路并导入DBAT，促使胞内乙酰辅酶A流向以10-DAB为底物的乙酰化途径生成BCT-III。在工程菌高密度发酵试验中，当10-DAB投料量分别为3g/L和6g/L时BCT-III产量分别为3g/L和4.6g/L。.通过一锅法酶促反应体系把废弃物XDT直接转变成高附加值的Taxol，具原始创新性，代表着用绿色环保方法以其类似物生产Taxol的一种全新替代模式。将大肠杆菌乙酰辅酶A合成通路优化与DBAT酶促反应结合起来构建10-DAB乙酰化途径并建立BCT-III高产的高密度发酵工艺亦未见报道，该技术绿色环保，可望被应用到Taxol的化学半合成中以减少有毒有害试剂的使用和减少反应步骤。