动态调控策略在代谢工程中的应用研究进展

doi:10.12211/2096-8280.2020-029

摘要/Abstract

摘要：

微生物细胞工厂作为一种可持续的生化反应器，被广泛应用于天然产物、药品、营养保健品等高附加值产物的生产中。为了使细胞工厂在生产过程中以最大的产量、产率和生产能力生产目标化合物，往往需要利用代谢工程方法对细胞工厂进行合理的改造和调控。以基因敲除和过表达为主要策略的静态调控不可避免地带来了细胞代谢流与能量流失衡、生长阻滞和毒性中间体积累等问题，限制了细胞工厂的生产能力、碳收率和产物产量。为了解决这一问题，构建调控元件并设计基因线路以精确调节物质流及能量流的动态调控策略被普遍应用于代谢工程领域，成为调控微生物细胞工厂的常用方法之一。本文依据不同动态调控策略的特点，将动态调控策略分为代谢物响应、群体感应响应、环境响应和蛋白质水平调控四种类型，重点介绍了各种调控元件的构建方法及其在代谢工程中的应用，分析了不同调控策略在工业化应用中面临的挑战。同时，指出了高通量筛选和蛋白质工程方法、计算机模拟和数学模型分析、耦合基因控制元件等方面的策略在解决动态调控工具响应阈值窄、调控范围有限等问题中的应用潜力。

关键词: 动态调控, 代谢物响应, 群体感应, 环境响应, 代谢工程, 蛋白水平调控

Abstract:

As a sustainable biochemical reactor, microbial cell factories are widely used in the production of value-added compounds such as natural products, pharmaceuticals and nutraceuticals. In order to make microbial cell factories produce target compounds with high titer, productivity and yield, metabolic engineering strategies are employed to rationally modify and regulate their metabolism. However, knockout and overexpression of genes inevitably bring stresses such as redox imbalance and toxic intermediate accumulation. While dynamic control strategy has been proven as a promising tool to address these challenges by balancing carbon flux and energy generation and dissipation for cell growth and generation of target compounds. As a result, many dynamic regulation elements and gene circuits have been developed and widely used in metabolic engineering so far. In this review, we summarize four types of attractive dynamic regulation systems based on metabolite-response, quorum sensing-response, environmental parameter-response and protein level regulation, with a focus on the construction method of various regulatory elements and their applications in metabolic engineering. In addition, challenges faced by different dynamic control strategies in industrial applications are analyzed. At the same time, we prospect the application potentials of some strategies such as high-throughput screening, protein engineering, computer simulation and mathematical model analysis coupled with gene control elements in solving the problems of narrow response threshold and limited control range of dynamic regulation tools. With the further development of synthetic biology and metabolic engineering, we believe that dynamic control strategy will be widely used for the construction of microbial cell factories in the near future.

Key words: dynamic regulation, metabolite-response, quorum sensing, environmental parameter-response, metabolic engineering, protein level regulation

中图分类号:

Q812

于政, 申晓林, 孙新晓, 王佳, 袁其朋. 动态调控策略在代谢工程中的应用研究进展[J]. 合成生物学, 2020, 1(4): 440-453.

Yu Zheng, SHEN Xiaolin, Sun Xinxiao, Wang Jia, Yuan Qipeng. Application of dynamic regulation strategies in metabolic engineering[J]. Synthetic Biology Journal, 2020, 1(4): 440-453.

图/表 7

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调控类型	输入信号	调控元件	菌株	产物	调控效果
转录因子	乙酰磷酸	NRI	大肠杆菌	番茄红素	50%^[9]
	丙二酰辅酶A	FapR	大肠杆菌	脂肪酸	2.1倍^[18]
	丙二酰辅酶A	FapR	大肠杆菌	脂肪酸	34%^[19]
	衣康酸	ItcR	大肠杆菌	衣康酸	0.78mmol/L^[20]
	阿魏酸、香草醛	HucR	大肠杆菌	香草醛	11mmol/L^[22]
	葡糖胺-6-磷酸	GamR	枯草芽孢杆菌	N-乙酰葡糖胺	131.6 g/L^[28]
	黏糠酸	CatR	大肠杆菌	黏糠酸	16.3倍^[29]
核糖开关	茶碱	茶碱核糖开关	大肠杆菌	—	3倍^[35]
	硫胺素焦磷酸	硫胺素焦磷酸核糖开关	米曲霉	—	4.7倍^[36]
	赖氨酸	赖氨酸核糖开关	谷氨酸棒状杆菌	赖氨酸	63%^[37]
	甘氨酸	甘氨酸核糖开关	大肠杆菌	5-氨基酮戊酸	11%^[38]
群体感应	AHLs	luxI/luxR	大肠杆菌	红没药烯	44%^[45]
	AHLs	luxI/luxR	大肠杆菌	—	6%～76%^[46]
	AHLs	esaI/esaR	大肠杆菌	4-羟基苯乙酸	46.4%^[49]
	AHLs	esaI/esaR	大肠杆菌	聚-β-羟丁酸	6倍^[50]
	AHLs	esaI/esaR	大肠杆菌	葡萄糖二酸	2g/L^[52]
	AHLs	lux、esa	大肠杆菌	水杨酸、柚皮素	1.8、6倍^[53]
发酵条件	温度	CI857	大肠杆菌	衣康酸	47g/L^[58]
	温度	Gal4M9	酵母	番茄红素	1.12g/L^[59]
	溶氧	P_nar	大肠杆菌	D-乳酸	113.12g/L^[60]
	pH	P_gas	黑曲霉	衣康酸	4.92g/L^[61]
	pH	CadCΔ	大肠杆菌	乙二醇	170%^[62]
	光	UirS/UirR	大肠杆菌	—	6.24倍^[65]
	光	Magnets	大肠杆菌	—	300倍^[66]
	光	OptoEXP	酵母	异丁醇	4倍^[67]
	葡萄糖	P_HXT1	酵母	3-羟基丙酸	10倍^[70]
蛋白水平	赖氨酸	高丝氨酸脱氢酶	大肠杆菌	赖氨酸	—^[73]
	—	振荡器	大肠杆菌	D-木糖酸	199.44g/L^[74]
	—	SsrA	大肠杆菌	肌醇	2倍^[75]

调控类型	输入信号	调控元件	菌株	产物	调控效果
转录因子	乙酰磷酸	NRI	大肠杆菌	番茄红素	50%^[9]
	丙二酰辅酶A	FapR	大肠杆菌	脂肪酸	2.1倍^[18]
	丙二酰辅酶A	FapR	大肠杆菌	脂肪酸	34%^[19]
	衣康酸	ItcR	大肠杆菌	衣康酸	0.78mmol/L^[20]
	阿魏酸、香草醛	HucR	大肠杆菌	香草醛	11mmol/L^[22]
	葡糖胺-6-磷酸	GamR	枯草芽孢杆菌	N-乙酰葡糖胺	131.6 g/L^[28]
	黏糠酸	CatR	大肠杆菌	黏糠酸	16.3倍^[29]
核糖开关	茶碱	茶碱核糖开关	大肠杆菌	—	3倍^[35]
	硫胺素焦磷酸	硫胺素焦磷酸核糖开关	米曲霉	—	4.7倍^[36]
	赖氨酸	赖氨酸核糖开关	谷氨酸棒状杆菌	赖氨酸	63%^[37]
	甘氨酸	甘氨酸核糖开关	大肠杆菌	5-氨基酮戊酸	11%^[38]
群体感应	AHLs	luxI/luxR	大肠杆菌	红没药烯	44%^[45]
	AHLs	luxI/luxR	大肠杆菌	—	6%～76%^[46]
	AHLs	esaI/esaR	大肠杆菌	4-羟基苯乙酸	46.4%^[49]
	AHLs	esaI/esaR	大肠杆菌	聚-β-羟丁酸	6倍^[50]
	AHLs	esaI/esaR	大肠杆菌	葡萄糖二酸	2g/L^[52]
	AHLs	lux、esa	大肠杆菌	水杨酸、柚皮素	1.8、6倍^[53]
发酵条件	温度	CI857	大肠杆菌	衣康酸	47g/L^[58]
	温度	Gal4M9	酵母	番茄红素	1.12g/L^[59]
	溶氧	P_nar	大肠杆菌	D-乳酸	113.12g/L^[60]
	pH	P_gas	黑曲霉	衣康酸	4.92g/L^[61]
	pH	CadCΔ	大肠杆菌	乙二醇	170%^[62]
	光	UirS/UirR	大肠杆菌	—	6.24倍^[65]
	光	Magnets	大肠杆菌	—	300倍^[66]
	光	OptoEXP	酵母	异丁醇	4倍^[67]
	葡萄糖	P_HXT1	酵母	3-羟基丙酸	10倍^[70]
蛋白水平	赖氨酸	高丝氨酸脱氢酶	大肠杆菌	赖氨酸	—^[73]
	—	振荡器	大肠杆菌	D-木糖酸	199.44g/L^[74]
	—	SsrA	大肠杆菌	肌醇	2倍^[75]

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