酵母合成单萜类化合物的研究进展

doi:10.12211/2096-8280.2024-049

摘要/Abstract

摘要：

单萜类化合物是一类由两个异戊二烯单元缩合而成的萜类化合物，被广泛应用于医药、食品、香料、化妆品、农业和能源等行业中。相较于植物提取和化学合成，利用微生物异源合成单萜类化合物提供了一种高效、可持续及生态友好的可替代途径。酵母细胞由于具有短暂的生长周期、内源甲羟戊酸路径和完整的蛋白后修饰体系等优势，成为生物合成单萜类化合物的潜在宿主。随着合成生物学关键技术的发展，研究者们已经成功构建了合成单萜的微生物细胞工厂，但与大规模工业化生产之间还有很大距离。本文介绍了单萜的生物合成途径，除酵母内源甲羟戊酸途径外，人工构建的异源异戊烯醇利用途径与醇依赖型半萜途径也可用于单萜前体香叶基二磷酸的合成，随后围绕提高单萜前体供应、关键酶的改造和调控、区室化工程、缓解单萜的细胞毒性等几个方面阐述了利用酵母细胞合成单萜类化合物的策略和研究进展。最后基于目前单萜类化合物合成仍面临的前体供给不足与单萜及中间代谢物的细胞毒性等挑战，对未来酵母合成单萜类化合物的发展方向进行了展望，包括对单萜产生细胞毒性的具体机制进一步解析、更高效单萜合酶的挖掘与改造、动态调控单萜合成的代谢途径以及更稳定高效合成单萜宿主细胞的探索等，旨在为以后利用酵母合成单萜提供一定的指导。

关键词: 单萜类化合物, 酵母, 代谢工程, 酶, 区室化工程, 细胞毒性

Abstract:

Monoterpenoids constitute a significant subclass of terpenoids, known for their volatility and strong aromatic properties. These compounds are extensively employed across multiple sectors, including pharmaceuticals, foods, flavors, cosmetics, agriculture, and energy, due to their diverse pharmacological and biological activities. Currently, monoterpenoids are primarily sourced from plant extracts or chemical synthesis. However, low yield and high cost associated with plant extracts as well as low purity and high energy consumption with chemical synthesis cannot address the growing demand. As a result, the heterologous synthesis of monoterpenoids using microorganisms presents an alternative pathway that is efficient, sustainable, and eco-friendly. Yeasts show promise as hosts for monoterpenoid biosynthesis due to their fast growth, inherent mevalonate (MVA) pathway, and robust post-translational modification systems. Currently, the industrial production of the artemisinin precursor artemisinic acid and the sesquiterpene farnesene has been achieved using Saccharomyces cerevisiae. Advances in synthetic biology have enabled the construction of microbial cell factories for monoterpenoid synthesis. However, challenges remain in scaling up production due to limited precursor availability and monoterpene cytotoxicity. This review first introduces the foundational pathways of monoterpenoid biosynthesis in yeast, followed by discussion on engineering strategies and advancements in yeast-mediated monoterpenoid synthesis, which include enhancing the supply and utilization of acetyl coenzyme A and geranyl pyrophosphate (GPP), regulating and modifying key enzymes such as GPP synthase and monoterpene synthase, optimizing subcellular organelle localization and compartmentalization of MVA pathway genes and monoterpenoid synthases, and implementing exocytosis and tolerance engineering to mitigate monoterpene cytotoxicity. Future directions and strategies to overcome bottlenecks in microbial synthesis are explored to guide research in yeast synthesis of monoterpenoids.

Key words: monoterpenoids, yeast, metabolic engineering, enzymes, compartmentalization engineering, cell toxicity

中图分类号:

Q816

高琪, 肖文海. 酵母合成单萜类化合物的研究进展[J]. 合成生物学, 2025, 6(2): 357-372.

GAO Qi, XIAO Wenhai. Advances in the biosynthesis of monoterpenes by yeast[J]. Synthetic Biology Journal, 2025, 6(2): 357-372.

图/表 3

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单萜	底盘	策略	产量 /（mg/L）	参考文献
香叶醇	酿酒酵母	①过表达截短的tHMGR和IDI1 ②利用计算机结构分析和建模来截短CrGES酶的N端转运肽 ③反向融合ERG20^ww /t3CrGES与另一拷贝ERG20^ww 共表达 ④补料分批发酵	1680	[37]
	解脂耶氏酵母	①过表达截短的HMG1、IDI和tCrGES ②过表达3拷贝的tCrGES和单拷贝的ERG10、HMGS、tHMG1、IDI1	1000	[26]
	甘油假丝酵母	①MVA与IUP双途径 ②设计癸烷响应杂交启动子调控基因表达：将P_CgALK1的ARR1元件串联至P_GAP的核心启动子（P_{GAP (core-477)}）	1194.6	[32]
香茅醇	酿酒酵母	①表达CrIS还原酶并敲除ATF1 ②内源ERG20突变为ERG20^F96W ③对融合蛋白、CrIS酶、IDI1使用蛋白支架SF1(SH3₁PDZ₁GBD₁)	8300	[38]
芳樟醇	酿酒酵母	①对芳樟醇合成酶(t67OMcLIS_M)底物结合口袋的入口处氨基酸位点F447E突变 ②利用细胞质和过氧化物酶体促进芳樟醇合成 ③5 L补料分批发酵	2600	[39]
月桂烯	酿酒酵母	①使用弱启动子P_HXT1替换ERG20的启动子 ②将ERG20^F96W 与MS/OS进行融合表达 ③优化两相发酵中有机相的添加量	8.12	[40]
罗勒烯	酿酒酵母		34.56	[40]
柠檬烯	酿酒酵母	①动态抑制竞争性旁路 ②优化tLimS拷贝数 ③增加乙酰辅酶A和NADPH供应	2630	[41]
柠檬烯	解脂耶氏酵母	①引入额外拷贝的柠檬烯合成基因 ②甘油和柠檬酸作为碳源	165.3	[42]
薄荷醇	酿酒酵母	①薄荷醇从头合成路径的构建 ②过表达MVA路径基因 ③使用弱启动子P_HXT1替换ERG20的启动子 ④增加限速酶IPDH与KSI拷贝数	6.28	[43]
蒎烯	酿酒酵母	①表达ERG20^WW +tPtPS ②过表达IDI1和MAF1	11.7	[44]
	解脂耶氏酵母	①构建非正交生物合成途径 ②利用餐厨废油和木质纤维素水解液作为碳源	36.1	[27]
	甘油假丝酵母	①强化MVA路径并引入NPP合酶 ②过表达Hog1基因与外源磷酸酶 ③对Pt30进行理性设计——点突变（T376R） ④添加NaCl升高渗透压促使角鲨烯应答 ⑤优化培养基及5 L发酵罐扩大	16.4	[31]
桧烯	酿酒酵母	①在细胞质和线粒体中同时表达t34SabS1 ②过表达线粒体相关基因AIM25	154.9	[45]

单萜	底盘	策略	产量 /（mg/L）	参考文献
香叶醇	酿酒酵母	①过表达截短的tHMGR和IDI1 ②利用计算机结构分析和建模来截短CrGES酶的N端转运肽 ③反向融合ERG20^ww /t3CrGES与另一拷贝ERG20^ww 共表达 ④补料分批发酵	1680	[37]
	解脂耶氏酵母	①过表达截短的HMG1、IDI和tCrGES ②过表达3拷贝的tCrGES和单拷贝的ERG10、HMGS、tHMG1、IDI1	1000	[26]
	甘油假丝酵母	①MVA与IUP双途径 ②设计癸烷响应杂交启动子调控基因表达：将P_CgALK1的ARR1元件串联至P_GAP的核心启动子（P_{GAP (core-477)}）	1194.6	[32]
香茅醇	酿酒酵母	①表达CrIS还原酶并敲除ATF1 ②内源ERG20突变为ERG20^F96W ③对融合蛋白、CrIS酶、IDI1使用蛋白支架SF1(SH3₁PDZ₁GBD₁)	8300	[38]
芳樟醇	酿酒酵母	①对芳樟醇合成酶(t67OMcLIS_M)底物结合口袋的入口处氨基酸位点F447E突变 ②利用细胞质和过氧化物酶体促进芳樟醇合成 ③5 L补料分批发酵	2600	[39]
月桂烯	酿酒酵母	①使用弱启动子P_HXT1替换ERG20的启动子 ②将ERG20^F96W 与MS/OS进行融合表达 ③优化两相发酵中有机相的添加量	8.12	[40]
罗勒烯	酿酒酵母		34.56	[40]
柠檬烯	酿酒酵母	①动态抑制竞争性旁路 ②优化tLimS拷贝数 ③增加乙酰辅酶A和NADPH供应	2630	[41]
柠檬烯	解脂耶氏酵母	①引入额外拷贝的柠檬烯合成基因 ②甘油和柠檬酸作为碳源	165.3	[42]
薄荷醇	酿酒酵母	①薄荷醇从头合成路径的构建 ②过表达MVA路径基因 ③使用弱启动子P_HXT1替换ERG20的启动子 ④增加限速酶IPDH与KSI拷贝数	6.28	[43]
蒎烯	酿酒酵母	①表达ERG20^WW +tPtPS ②过表达IDI1和MAF1	11.7	[44]
	解脂耶氏酵母	①构建非正交生物合成途径 ②利用餐厨废油和木质纤维素水解液作为碳源	36.1	[27]
	甘油假丝酵母	①强化MVA路径并引入NPP合酶 ②过表达Hog1基因与外源磷酸酶 ③对Pt30进行理性设计——点突变（T376R） ④添加NaCl升高渗透压促使角鲨烯应答 ⑤优化培养基及5 L发酵罐扩大	16.4	[31]
桧烯	酿酒酵母	①在细胞质和线粒体中同时表达t34SabS1 ②过表达线粒体相关基因AIM25	154.9	[45]

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