Advances in the biosynthesis of monoterpenes by yeast

doi:10.12211/2096-8280.2024-049

Abstract

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, food, flavor, cosmetics, agriculture, and energy, due to their diverse pharmacological and biological activities. Currently, monoterpenoids are primarily sourced from plant extracts or chemical synthesis. However, the limited yield and elevated cost associated with plant extracts, along with the low purity and high energy consumption of chemical synthesis, are insufficient to satisfy the growing demand. The heterologous synthesis of monoterpenoids using microorganisms presents a highly efficient, sustainable, and eco-friendly alternative pathway. Yeasts show promise as hosts for monoterpenoid biosynthesis due to their short growth cycles, 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 a discussion of engineering strategies and research advancements in yeast-mediated monoterpenoid synthesis. These strategies 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 advancements in yeast synthesis of monoterpenoids.

Key words: Monoterpenoids, Yeast, Metabolic engineering, Enzymes, Compartmentalization engineering, Cell toxicity

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

CLC Number:

Q816

Qi GAO, Wenhai XIAO. Advances in the biosynthesis of monoterpenes by yeast[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-049.

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, DOI: 10.12211/2096-8280.2024-049.

Figures/Tables 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ERG20ww / t3CrGES与另一拷贝ERG20ww共表达 4.补料分批发酵	1680	[58]
	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	1.MVA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	[64]
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 3.5L补料分批发酵	2600	[100]
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	[101]
罗勒烯	酿酒酵母		34.56	[101]
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	[71]
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	[102]
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PHXT1替换Erg20的启动子 4.增加限速酶IPDH与KSI拷贝数	6.28	[103]
蒎烯	酿酒酵母	1.表达ERG20^WW+ tPtPS 2.过表达IDI1 和 MAF1	11.7	[104]
	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5L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	[72]

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ERG20ww / t3CrGES与另一拷贝ERG20ww共表达 4.补料分批发酵	1680	[58]
	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	1.MVA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	[64]
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 3.5L补料分批发酵	2600	[100]
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	[101]
罗勒烯	酿酒酵母		34.56	[101]
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	[71]
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	[102]
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PHXT1替换Erg20的启动子 4.增加限速酶IPDH与KSI拷贝数	6.28	[103]
蒎烯	酿酒酵母	1.表达ERG20^WW+ tPtPS 2.过表达IDI1 和 MAF1	11.7	[104]
	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5L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	[72]

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