紫杉醇生物合成机制研究进展

doi:10.12211/2096-8280.2023-085

合成生物学

• 特约评述 •

紫杉醇生物合成机制研究进展

刘晓楠¹^,², 李静¹^,²^,³, 祝晓熙¹^,², 徐子硕¹^,²^,⁴^,⁵, 齐健¹^,², 江会锋¹^,²

^1.中国科学院天津工业生物技术研究所，低碳合成工程生物学重点实验室，天津 300308
^2.国家合成生物技术创新中心，天津 300308
^3.南开大学化学学院，天津 300071
^4.东北林业大学，东北盐碱植被恢复与重建教育部重点实验室，黑龙江哈尔滨 150040
^5.东北林业大学，黑龙江省植物天然活性物质的合成与利用重点实验室，黑龙江哈尔滨 150040

收稿日期:2023-11-17 修回日期:2024-04-12 出版日期:2024-04-17
通讯作者: 刘晓楠,江会锋
作者简介:刘晓楠（1990—），女，博士，副研究员，硕士生导师。研究方向为植物天然产物合成、酵母基因组工程。E-mail：liu_xn@tib.cas.cn
江会锋（1981—），男，博士，研究员，博士生导师。研究方向为新酶设计与酵母基因组工程。中科院百人计划入选者，曾获国家自然科学基金优秀青年基金、天津市杰出青年基金、2020年云南省科学技术进步奖特等奖、2021年中国科学院年度团队，2022年度天津市自然科学特等奖等。已在Science、Molecular Plant、Nature Communications、ACS Catalysis、等刊物上发表SCI论文50余篇，申请发明专利40余项。E-mail：jiang_hf@tib.cas.cn
基金资助:
国家自然科学基金(32371499);中国博士后科学基金(2019M661032);北京生命科技研究院有限公司重点项目(2023200CB0090)

Recent advances on paclitaxel biosynthesis

Xiaonan LIU¹^,², Jing LI¹^,²^,³, Xiaoxi ZHU¹^,², Zishuo XU¹^,²^,⁴^,⁵, Jian QI¹^,², Huifeng JIANG¹^,²

^1.Engineering Biology for Low-Carbon Manufacturing，Tianjin Institute of Industrial Biotechnology，Chinese Academy of Sciences，Tianjin 300308，China
^2.National Center of Technology Innovation for Synthetic Biology，Tianjin 300308，China
^3.College of Chemistry，Nankai University，Tianjin 300071，China
^4.Key Laboratory of Saline-Alkali Vegetation Ecology Restoration，Northeast Forestry University，Harbin 150040，Heilongjiang，China
^5.Heilongjiang Key Laboratory of Plant Bioactive Substance Biosynthesis and Utilization，Northeast Forestry University，Harbin 150040，Heilongjiang，China

Received:2023-11-17 Revised:2024-04-12 Online:2024-04-17
Contact: Xiaonan LIU, Huifeng JIANG

摘要/Abstract

摘要：

紫杉醇是目前已发现的最具抗癌活性的天然广谱抗癌药物之一，其生产方式主要依赖于从珍稀植物红豆杉中进行分离提取以及化学半合成，因其含量稀少，生产能力受到严重的限制。随着红豆杉基因组的全解析和合成生物学的迅速发展，通过合成生物学技术，构建重组工程细胞合成紫杉醇及其关键前体成为解决当前供需不平衡和资源有限性的有效方法。本文针对紫杉醇生物合成途径解析、红豆杉组学分析、底盘细胞构建、关键前体合成、紫杉醇合成途径关键酶的改造及催化机理解析等相关研究进展开展系统性的综述，尤其对近期发表的关于氧杂环丁烷环形成的相关突破性研究进行了详细介绍，并基于相关进展探讨当前紫杉醇合成生物学研究面临的关键酶催化效率低下、产物杂泛性严重、具体反应顺序未知等技术挑战及生物合成紫杉醇关键中间体的未来前景。助力加强对紫杉醇合成通路和催化过程的理解，进一步实现紫杉醇的绿色、高效生物合成。

关键词: 紫杉醇, 途径解析, P450酶, 酶改造, 合成生物学

Abstract:

Paclitaxel (Taxol) is a natural broad-spectrum anticancer drug, which is well-known for its potent anticancer activity. Its production mainly relies on the isolation and extraction from the rare Taxus plant, coupled with chemical semi-synthesis. The limited natural abundance of paclitaxel imposes significant constraints on its production capacity. In recent years, with the complete decoding of the Taxus genome and the rapid development of synthetic biology, constructing recombinant cells through synthetic biology techniques has emerged as an effective method to address the current imbalance in supply and demand as well as the limitation of resources. Since the process of paclitaxel biosynthesis involves more than 20 steps of complex enzyme-catalyzed reactions and about half of them are P450 enzyme-mediated hydroxylation reactions, the complete elucidation of its biosynthetic pathway remains elusive. Meanwhile, the production of paclitaxel by engineered microbes is still in the initial stage, and there are numerous by-products, which seriously hindered the efficient synthesis of paclitaxel. Therefore, this paper systematically reviewed the research progress related to paclitaxel synthesis pathway analysis, Taxus omics analysis, diverse chassis cells construction, key precursors synthesis, crucial enzyme modifications and catalytic mechanisms analysis in the paclitaxel biosynthetic pathway. Special attention is given to the recent breakthrough that elucidates the formation of oxetane ring and the discovery process of Taxane 1β-hydroxylase and Taxane 9α-hydroxylase will be also introduced. Recent advances in the study of the catalytic mechanism of Taxadiene-5α-hydroxylase and significant progress in tobacco and yeast chassis engineering will also be included. Based on the relevant research, the current challenges and future prospects involved in the paclitaxel synthetic biology research are discussed, such as the issues of low enzyme catalytic efficiency, significant product promiscuity, unknown specific reaction sequences, and the biosynthesis of critical paclitaxel intermediates, aiming to enhance the understandings of paclitaxel biosynthetic pathway and catalytic processes, and contributing to the pursuit of a greener and more efficient production mode of paclitaxel.

Key words: Paclitaxel, Pathway analysis, P450 enzyme, Enzyme modification, Synthetic biology

中图分类号:

R932

刘晓楠, 李静, 祝晓熙, 徐子硕, 齐健, 江会锋. 紫杉醇生物合成机制研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2023-085.

Xiaonan LIU, Jing LI, Xiaoxi ZHU, Zishuo XU, Jian QI, Huifeng JIANG. Recent advances on paclitaxel biosynthesis[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2023-085.

图/表 3

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89	BIGGS B W, ROUCK J E, KAMBALYAL A, et al. Orthogonal assays clarify the oxidative biochemistry of taxol P450 CYP725A4[J]. ACS Chemical Biology, 2016, 11(5): 1445-1451.
90	BARTON N A, MARSH B J, LEWIS W, et al. Accessing low-oxidation state taxanes: is taxadiene-4(5)-epoxide on the taxol biosynthetic pathway?[J]. Chemical Science, 2016, 7(5): 3102-3107.
91	SONG X T, WANG Q, ZHU X X, et al. Unraveling the catalytic mechanism of taxadiene-5α-hydroxylase from crystallography and computational analyses[J]. ACS Catalysis, 2024, 14(6): 3912-3925.
92	LI B J, WANG H, GONG T, et al. Improving 10-deacetylbaccatin III-10-β-O-acetyltransferase catalytic fitness for Taxol production[J]. Nature Communications, 2017, 8: 15544.
93	LIN S L, WEI T, LIN J F, et al. Bio-production of baccatin III, an important precursor of paclitaxel by a cost-effective approach[J]. Molecular Biotechnology, 2018, 60(7): 492-505.
94	CHEN J J, LIANG X, WANG F, et al. Combinatorial mutation on the β-glycosidase specific to 7- β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast[J]. Acta Pharmaceutica Sinica B, 2019, 9(3): 626-638.
95	戴黄益, 何德, 刘明志. 产紫杉醇内生真菌研究的回顾、进展及趋势[J]. 西部林业科学, 2017, 46(3): 169-176, 186.
	DAI H Y, HE D, LIU M Z. Progress and trends on researches of taxol-producing endophytic fungi[J]. Journal of West China Forestry Science, 2017, 46(3): 169-176, 186.
96	徐志荣, 王婷, 娄佳兰, 等. 南方红豆杉细胞悬浮培养体系优化及动力学研究[J]. 林业科学研究, 2019, 32(1): 8-14.
	XU Z R, WANG T, LOU J L, et al. Study on optimization of cell suspension culture system and kinetics of Taxus chinensis var. mairer [J]. Forest Research, 2019, 32(1): 8-14.
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	WU Y M, LIAO Q G, SHANG Y, et al. Recent progress of paclitaxel biosynthesis aided by multi-omics[J]. Plant Science Journal, 2022, 40(6): 853-866.

	酶名称	简写	登录号	参考文献
1	Geranylgeranyl diphosphate synthase	GGPPS	AF081514	[14]
2	Taxadiene synthase	TS	AY364469	[15]
3	Taxadiene-5α-hydroxylase	T5αOH	AY289209	[16]
4	Taxadiene-13α-hydroxylase	T13αOH	AY056019	[17]
5	Taxadiene-5α-ol-O-acetyl transferase	TAT	AF190130	[18]
6	Taxadiene-10β-hydroxylase	T10βOH	AF318211/AY563635	[19-20]
7	Taxadiene-7β-hydroxylase	T7βOH	AY307951	[21]
8	Taxadiene-2α-hydroxylase	T2αOH	AY518383	[22]
9	Taxane-2α-O-benzoyl transferase	TBT	AF297618	[23]
10	10-deacetylbaccatin III-10-O-acetyl transferase	DBAT	AF193765	[23]
11	Phenylalanine aminomutase	PAM	AY582743	[24]
12	β-phenylalanoyl-CoA ligase	PCL	KM593667	[25]
13	Baccatin III: 3-amino, 13-phenylpropanoyltransferase	BAPT	AY082804	[25]
14	Taxane-2’α-hydroxylase	T2’αOH	KP178208	[26]
15	N-benzoyl transferase	DBTNBT	AF466397	[27]
16	Taxadiene-14β-hydroxylase	T14βOH	AY188177	[28]
17	Cytochrome P450 reductase	TcCPR	AY571340	[29]
18	C4β-C20 epoxidase			[12]
19	Taxane 1β-hydroxylase	T1βOH		[12]
20	Taxane 9α-hydroxylase	T9αOH		[12]
21	Taxane 9α-dioxygenase			[12]
22	Phenylalanine-CoA ligase	PCL		[12]
23	Taxane oxetanase	TOT		[13]
24	Taxane 9α-hydroxylase	T9αH1		[13]
25	Taxane 9α-hydroxylase	CYP725A37	PP197199/PP197200	[30]
26	Taxane oxetanase	CYP725A55	PP197201	[30]
27	Acyltransferase	AT5	PP197202	[30]

	酶名称	简写	登录号	参考文献
1	Geranylgeranyl diphosphate synthase	GGPPS	AF081514	[14]
2	Taxadiene synthase	TS	AY364469	[15]
3	Taxadiene-5α-hydroxylase	T5αOH	AY289209	[16]
4	Taxadiene-13α-hydroxylase	T13αOH	AY056019	[17]
5	Taxadiene-5α-ol-O-acetyl transferase	TAT	AF190130	[18]
6	Taxadiene-10β-hydroxylase	T10βOH	AF318211/AY563635	[19-20]
7	Taxadiene-7β-hydroxylase	T7βOH	AY307951	[21]
8	Taxadiene-2α-hydroxylase	T2αOH	AY518383	[22]
9	Taxane-2α-O-benzoyl transferase	TBT	AF297618	[23]
10	10-deacetylbaccatin III-10-O-acetyl transferase	DBAT	AF193765	[23]
11	Phenylalanine aminomutase	PAM	AY582743	[24]
12	β-phenylalanoyl-CoA ligase	PCL	KM593667	[25]
13	Baccatin III: 3-amino, 13-phenylpropanoyltransferase	BAPT	AY082804	[25]
14	Taxane-2’α-hydroxylase	T2’αOH	KP178208	[26]
15	N-benzoyl transferase	DBTNBT	AF466397	[27]
16	Taxadiene-14β-hydroxylase	T14βOH	AY188177	[28]
17	Cytochrome P450 reductase	TcCPR	AY571340	[29]
18	C4β-C20 epoxidase			[12]
19	Taxane 1β-hydroxylase	T1βOH		[12]
20	Taxane 9α-hydroxylase	T9αOH		[12]
21	Taxane 9α-dioxygenase			[12]
22	Phenylalanine-CoA ligase	PCL		[12]
23	Taxane oxetanase	TOT		[13]
24	Taxane 9α-hydroxylase	T9αH1		[13]
25	Taxane 9α-hydroxylase	CYP725A37	PP197199/PP197200	[30]
26	Taxane oxetanase	CYP725A55	PP197201	[30]
27	Acyltransferase	AT5	PP197202	[30]

合成体系	产物	产量	研究方法	发表时间	参考文献
大肠杆菌	紫杉二烯	1.3 mg/L	过表达IDI、GGPPS、TS、DXP	2001	[49]
大肠杆菌	紫杉二烯	1 g/L	多变量模块化代谢工程，过表达TS和T5αOH	2010	[50]
大肠杆菌	紫杉二烯-5α-醇	58±3 mg/L	多变量模块化代谢工程，过表达TS和T5αOH	2010	[50]
大肠杆菌	紫杉二烯	4.5 mg/g DW	JM109(DE3)菌株，22°C	2012	[51]
链格孢菌TPF6	紫杉二烯	61.9 ± 6.3 μg/L	alcA启动子，过表达IDI、tHMGR	2017	[8]
大肠杆菌	氧化紫杉烷	27 mg/L	TbrTS，Taxus CPR，T5αOH-GSTGS-CPR，引入异源MVA途径	2022	[52]
大肠杆菌	紫杉二烯-5α-醇	7 mg/L	TbrTS，Taxus CPR，T5αOH-GSTGS-CPR，引入异源MVA途径	2022	[52]
大肠杆菌	紫杉二烯	93.5 mg/L	融合表达GGPP和TS	2022	[53]
大肠杆菌	氧化紫杉烷	570 ± 45 mg/L	P450酶N端修饰	2016	[54]
枯草芽孢杆菌	紫杉二烯	17.8 mg/L	过表达MEP途径，GGPPS和TS	2019	[55]
酿酒酵母	紫杉二烯	8.7 mg/L	共表达tHMGR、突变调节蛋白 UPC2-1、GGPPS和TS	2008	[56]
酿酒酵母	紫杉二烯	72.8 mg/L	YSG50菌株，GGPPSbc	2014	[57]
酿酒酵母	氧化紫杉烷	33 mg/L	大肠杆菌和酿酒酵母共培养	2015	[58]
酿酒酵母	紫杉二烯	129 mg/L	增加MBP标签的多拷贝TS，20 ℃	2020	[59]
酿酒酵母	紫杉二烯-5α-醇	20 mg/L	对发酵工艺的改进, pH优化	2020	[37]
酿酒酵母	紫杉二烯-5α-yl-乙酸酯	3.7 mg/L
酿酒酵母	氧化紫杉烷	78 mg/L
酿酒酵母	紫杉二烯-5α-醇	42 mg/L	2×YP, 统计学确定性筛选设计	2022	[60]
酿酒酵母	紫杉二烯-5α-yl-乙酸酯	22 mg/L	2×YP, 统计学确定性筛选设计	2022	[60]
酿酒酵母	紫杉二烯-5α-醇	38.1±8.4 mg/L	启动子pHXT7,融合表达T5OH和CPR，中性pH条件下静息细胞测定	2022	[61]
酿酒酵母	氧化紫杉烷	361.4±52.4 mg/L	启动子pHXT7,融合表达T5OH和CPR，中性pH条件下静息细胞测定	2022	[61]
解脂耶式酵母	紫杉二烯	101.4 mg/L	融合表达SUMO与TS，过表达tHMG1、GGS1和TS	2023	[62]
酿酒酵母	紫杉二烯	215 mg/L	计算代谢工程	2023	[63]
酿酒酵母	紫杉二烯-5α-醇	43.65 mg/L
酿酒酵母	紫杉二烯-5α-乙酸酯	26.2 mg/L
酿酒酵母	1β-dehydroxybaccatin VI		细胞共共表达已鉴定的12个基因并饲喂紫杉二烯-5α-醇	2024	[30]
拟南芥	紫杉二烯	600 ng/g DW	糖皮质激素诱导表达TS	2004	[64]
本氏烟草	紫杉二烯	50 μg/g DW	表达TS，MeJA诱导，沉默PSY和PDS	2014	[65]
本氏烟草	紫杉二烯	56.6 ± 3.2 µg/g fresh weight	区室化策略, DXS和GGPPS共表达	2019	[66]
本氏烟草	紫杉二烯-5α-醇	1.3 ± 0.5 µg/g fresh weight	区室化策略, DXS和GGPPS共表达	2019	[66]
红花烟草	紫杉二烯	87.8 μg/g DW	TS的N端融合叶绿体转运肽	2021	[67]
本氏烟草	巴卡亭III	154.87 ng/g fresh weight	瞬时表达C4β-C20环氧化酶、T9αOH、T1βOH和T9OX与其它9个已知基因	2023	[12]
	紫杉醇	64.29 ng/g fresh weight	瞬时表达PCL与BAPT、PAM、DBTNBT、T2’OH
本氏烟草	紫杉二烯-5α-醇		弱组成型启动子NOS弱化T5αOH的表达
	5α,10β-二乙酰氧基-紫杉二烯-13α-醇	42 µg/g DW	（NOS）T10βOH、DBAT和（NOS）T13αOH	2024	[39]
本氏烟草	巴卡亭Ⅲ		瞬时表达TOT和T9αOH-1与其他7个已知合成基因	2024	[13]

合成体系	产物	产量	研究方法	发表时间	参考文献
大肠杆菌	紫杉二烯	1.3 mg/L	过表达IDI、GGPPS、TS、DXP	2001	[49]
大肠杆菌	紫杉二烯	1 g/L	多变量模块化代谢工程，过表达TS和T5αOH	2010	[50]
大肠杆菌	紫杉二烯-5α-醇	58±3 mg/L	多变量模块化代谢工程，过表达TS和T5αOH	2010	[50]
大肠杆菌	紫杉二烯	4.5 mg/g DW	JM109(DE3)菌株，22°C	2012	[51]
链格孢菌TPF6	紫杉二烯	61.9 ± 6.3 μg/L	alcA启动子，过表达IDI、tHMGR	2017	[8]
大肠杆菌	氧化紫杉烷	27 mg/L	TbrTS，Taxus CPR，T5αOH-GSTGS-CPR，引入异源MVA途径	2022	[52]
大肠杆菌	紫杉二烯-5α-醇	7 mg/L	TbrTS，Taxus CPR，T5αOH-GSTGS-CPR，引入异源MVA途径	2022	[52]
大肠杆菌	紫杉二烯	93.5 mg/L	融合表达GGPP和TS	2022	[53]
大肠杆菌	氧化紫杉烷	570 ± 45 mg/L	P450酶N端修饰	2016	[54]
枯草芽孢杆菌	紫杉二烯	17.8 mg/L	过表达MEP途径，GGPPS和TS	2019	[55]
酿酒酵母	紫杉二烯	8.7 mg/L	共表达tHMGR、突变调节蛋白 UPC2-1、GGPPS和TS	2008	[56]
酿酒酵母	紫杉二烯	72.8 mg/L	YSG50菌株，GGPPSbc	2014	[57]
酿酒酵母	氧化紫杉烷	33 mg/L	大肠杆菌和酿酒酵母共培养	2015	[58]
酿酒酵母	紫杉二烯	129 mg/L	增加MBP标签的多拷贝TS，20 ℃	2020	[59]
酿酒酵母	紫杉二烯-5α-醇	20 mg/L	对发酵工艺的改进, pH优化	2020	[37]
酿酒酵母	紫杉二烯-5α-yl-乙酸酯	3.7 mg/L
酿酒酵母	氧化紫杉烷	78 mg/L
酿酒酵母	紫杉二烯-5α-醇	42 mg/L	2×YP, 统计学确定性筛选设计	2022	[60]
酿酒酵母	紫杉二烯-5α-yl-乙酸酯	22 mg/L	2×YP, 统计学确定性筛选设计	2022	[60]
酿酒酵母	紫杉二烯-5α-醇	38.1±8.4 mg/L	启动子pHXT7,融合表达T5OH和CPR，中性pH条件下静息细胞测定	2022	[61]
酿酒酵母	氧化紫杉烷	361.4±52.4 mg/L	启动子pHXT7,融合表达T5OH和CPR，中性pH条件下静息细胞测定	2022	[61]
解脂耶式酵母	紫杉二烯	101.4 mg/L	融合表达SUMO与TS，过表达tHMG1、GGS1和TS	2023	[62]
酿酒酵母	紫杉二烯	215 mg/L	计算代谢工程	2023	[63]
酿酒酵母	紫杉二烯-5α-醇	43.65 mg/L
酿酒酵母	紫杉二烯-5α-乙酸酯	26.2 mg/L
酿酒酵母	1β-dehydroxybaccatin VI		细胞共共表达已鉴定的12个基因并饲喂紫杉二烯-5α-醇	2024	[30]
拟南芥	紫杉二烯	600 ng/g DW	糖皮质激素诱导表达TS	2004	[64]
本氏烟草	紫杉二烯	50 μg/g DW	表达TS，MeJA诱导，沉默PSY和PDS	2014	[65]
本氏烟草	紫杉二烯	56.6 ± 3.2 µg/g fresh weight	区室化策略, DXS和GGPPS共表达	2019	[66]
本氏烟草	紫杉二烯-5α-醇	1.3 ± 0.5 µg/g fresh weight	区室化策略, DXS和GGPPS共表达	2019	[66]
红花烟草	紫杉二烯	87.8 μg/g DW	TS的N端融合叶绿体转运肽	2021	[67]
本氏烟草	巴卡亭III	154.87 ng/g fresh weight	瞬时表达C4β-C20环氧化酶、T9αOH、T1βOH和T9OX与其它9个已知基因	2023	[12]
	紫杉醇	64.29 ng/g fresh weight	瞬时表达PCL与BAPT、PAM、DBTNBT、T2’OH
本氏烟草	紫杉二烯-5α-醇		弱组成型启动子NOS弱化T5αOH的表达
	5α,10β-二乙酰氧基-紫杉二烯-13α-醇	42 µg/g DW	（NOS）T10βOH、DBAT和（NOS）T13αOH	2024	[39]
本氏烟草	巴卡亭Ⅲ		瞬时表达TOT和T9αOH-1与其他7个已知合成基因	2024	[13]

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紫杉醇生物合成机制研究进展

Recent advances on paclitaxel biosynthesis

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