代谢工程调控策略在生物合成氨基酸及其衍生物中的应用

doi:10.11949/0438-1157.20200460

摘要/Abstract

摘要：

氨基酸的工业化生产已有上百年历史，多用于动物饲料和食品添加剂；很多种类的氨基酸如L-半胱氨酸、β-丙氨酸、S-腺苷甲硫氨酸、4-羟基异亮氨酸和高丝氨酸等也具有很高的应用价值。相较于化工合成或分离提取的方式，利用微生物细胞作为平台生产氨基酸及其衍生物具有绿色安全、可持续等独特的优势。本文综述了近年来微生物合成氨基酸及其衍生物的研究进展，分别介绍了碳源的高效利用、限速步骤的调节、碳通量的调节、转录和反馈抑制调节以及转运调节等代谢调控策略在提高微生物生产氨基酸及其衍生物效率的研究及应用，分析了不同调控策略的优势和缺点，总结了不同氨基酸及其衍生物的应用价值，最后展望了微生物作为细胞工厂生产各类氨基酸及其衍生物的广阔前景。

关键词: 合成生物学, 氨基酸及其衍生物, 代谢调控, 转运调控

Abstract:

The industrial production of amino acids has a history of more than 100 years, and is mostly used in animal feed and food additives. Many types of amino acids such as L-cysteine, β-alanine, S-adenosylmethionine, 4-hydroxyisocyanine acid and homoserine also have broad applications. Plant extraction and chemical synthesis are the commonly used methods to produce these amino acids and their derivatives. Compared with the way of chemical synthesis or separation and extraction, the use of microbial cells as a platform for the production of amino acids and derivatives has unique advantages such as green safety and sustainability. This review highlights the recent advances in developing metabolic engineering strategies including increase of the carbon sources uptake rate, elimination of the rate-limiting steps, enhancement of the carbon flux through the target pathway, remove of the feed-back inhibition effect and regulation of the intermediates and products transportation for biological production of amino acids and their derivatives. Our goals are to provide a landscape of current works and present guidelines to address future challenges in biosynthesis of amino acids and their derivatives using engineered microorganisms.

Key words: synthetic biology, amino acids and their derivatives, metabolic regulation, transport regulation

中图分类号:

Q 812

高虎涛, 申晓林, 孙新晓, 王佳, 袁其朋. 代谢工程调控策略在生物合成氨基酸及其衍生物中的应用[J]. 化工学报, 2020, 71(9): 4058-4070.

Hutao GAO, Xiaolin SHEN, Xinxiao SUN, Jia WANG, Qipeng YUAN. Metabolic engineering strategies in biosynthesis of amino acids and their derivatives[J]. CIESC Journal, 2020, 71(9): 4058-4070.

图/表 5

图1 碳源高效利用策略的应用（a）L-赖氨酸在谷氨酸棒状杆菌中的部分生物合成途径；（b）L-3,4-二羟基苯丙氨酸在大肠杆菌中的生物合成途径；（c）S-腺苷甲硫氨酸在解淀粉芽孢杆菌中的生物合成途径；（d）4-羟基异亮氨酸在大肠杆菌中的生物合成途径IolT1, IolT2—肌醇通透酶；EIIbglF—β-葡萄糖苷-PTS渗透酶；GlK,PpgK,Cgl2647—葡萄糖激酶； IICBGlc—整合膜葡萄糖通透酶；galP—半乳糖渗透酶基因； glk—葡萄糖激酶基因； sucC—琥珀酰-CoA合成酶基因；metA—高丝氨酸O-琥珀酰转移酶基因；metB—胱硫醚-γ-半胱氨酸酶基因； IDH—异柠檬酸脱氢酶；IDO—L-异亮氨酸双加氧酶；AceA—异柠檬酸连接酶；AceK—异柠檬酸脱氢酶激酶；SucAB—α-酮戊二酸脱氢酶

Fig.1 Application of carbon source efficient utilization strategy(a) partial biosynthetic pathway of L-lysine in Corynebacterium glutamicum; (b) biosynthetic pathway of L-3,4-dihydroxyphenylalanine in E. coli;(c) biosynthetic pathway of S-adenosylmethionine in B. amyloliquefaciens; (d) biosynthetic pathway of 4-hydroxyisoleucine in E. coli

图2 限速步骤调节策略的应用（a）高丝氨酸在大肠杆菌中的生产代谢途径；（b）L-精氨酸在谷氨酸棒状杆菌中的生产代谢途径；（c）5-羟基色氨酸在大肠杆菌中的生产代谢途径aspC—天冬氨酸转氨酶基因；asd—天门冬氨酸半醛脱氢酶基因；lysA—二氨基庚二酸酯脱羧酶基因；thrA—高丝氨酸脱氢酶I基因；metL—高丝氨酸脱氢酶II基因；thrBC—高丝氨酸激酶基因；metA—高丝氨酸琥珀酰转移酶基因；gdh—谷氨酸脱氢酶基因；argB—N-乙酰谷氨酸激酶基因；argC—N-乙酰-γ-谷氨酰磷酸还原酶基因；argD—乙酰鸟氨酸氨基转移酶基因；argJ—双功能鸟氨酸乙酰转移酶基因；argF—鸟氨酸氨基甲酰基转移酶基因；argG—精氨酸琥珀酸合酶基因；argH—精氨酸琥珀酸合酶基因；pntAB—膜结合转氢酶基因；ppnK—NAD+激酶基因；DNMR—二氢莫那还原酶；P4H—苯丙氨酸4-羟化酶；PCD—蝶呤4a-甲醇胺脱水酶

Fig.2 Application of speed-limiting step adjustment strategy(a) the metabolic pathway of homoserine production in E. coli; (b) the metabolic pathway of L-arginine in C. glutamicum; (c) the metabolic pathway of 5-hydroxytryptophan in E. coli

图3 碳通量调节策略的应用（a）L-鸟氨酸在谷氨酸棒状杆菌中的生产代谢途径；（b）β-鸟氨酸在大肠杆菌中的生产代谢途径odhA—酮戊二酸脱氢酶基因； argCJBD—精氨酸合成的操纵子基因；argF—鸟氨酸氨基甲酰基转移酶基因；标记紫色为下调基因，绿色为过表达基因，红色为被敲除基因；ldhA—D-乳酸脱氢酶基因；pflB—丙酮酸甲酸酯裂解酶基因；adhE—酒精/醛脱氢酶基因；pta—磷酸乙酰基转移酶基因；gluDH—谷氨酸脱氢酶；akI，akII和akIII—天冬氨酸激酶基因；BtADC—L-天冬氨酸α-脱羧酶；AspA—天冬氨酸酶；标记蓝色箭头表示基因过表达，红色箭头表示基因敲除

Fig.3 Application of carbon flux regulation strategy(a) the metabolic pathway of L-ornithine in C. glutamicum; (b) production and metabolism pathway of β-ornithine in E. coli

图4 转录调节与反馈抑制调节策略的应用（a）2-氨基丁酸在大肠杆菌中的生产代谢途径；（b）L-亮氨酸在谷氨酸棒状杆菌中的生产代谢途径ilvA—苏氨酸脱水酶基因；ilvIH—乙酰羟酸合酶Ⅲ基因；leuDH—2-酮丁酸脱氢酶基因；减号表示受到抑制，虚线表示反馈抑制，标记蓝色箭头表示过表达；leuA—2-异丙基苹果酸合酶（IPMS）基因；leuCD—3-异丙基苹果酸脱水酶（IPMD）基因；leuB—3-异丙基苹果酸脱氢酶（IPMDH）基因；ilvE—支链氨基酸转氨酶（BCAAT）基因

Fig.4 Application of transcription regulation and feedback inhibition regulation strategies(a) the metabolic pathway of 2-aminobutyric acid in E. coli; (b) L-leucine production and metabolic pathways in C. glutamicum

表1 本文涉及到的氨基酸及其衍生物摘要

Table 1 Summary of amino acids and their derivatives involved in this article

代谢工程策略	氨基酸及其衍生物	功能	工程菌	产量	文献
碳源的高效利用	左旋鸟氨酸	一种非蛋白质氨基酸，能够加速伤口愈合并能有效维持心脏健康	谷氨酸棒状杆菌S9114	43.6 g/L	[20]
	L-亮氨酸	在刺激肌肉蛋白质合成和葡萄糖稳态中具有重要作用	谷氨酸棒状杆菌	23.7 g/L	[22]
	L-赖氨酸	促进人体的发育、增强免疫力，并有提高中枢神经功能的作用	谷氨酸棒状杆菌 ZL-19	201.6 g/L	[17]
	L-3，4-二羟基苯丙氨酸	用于治疗帕金森氏疾病类药物的主要前体	大肠杆菌	1.51 g/L	[26]
	苯丙氨酸	用于生产人造甜味剂阿斯巴甜	大肠杆菌	1.4 g/L	[29]
	S-腺苷甲硫氨酸	被用作预防和治疗骨关节炎的功能性营养品或药物	解淀粉芽孢杆菌	107.47mg/L	[31]
	4-羟基异亮氨酸	在治疗和预防2-型糖尿病中有广阔应用前景	大肠杆菌	24.1 g/L	[32]
限速步骤的调节	高丝氨酸	诱导免疫系统，增强植物对疾病的抵抗力	大肠杆菌W3110	1.20 g/L	[41]
	L-高丙氨酸	抗癫痫药物左乙拉西坦和溴拉西坦的直接前体	大肠杆菌	5.4 g/L	[42]
	L-精氨酸	具有舒张因子的作用，临床上用于舒张和扩张血管	谷氨酸棒状杆菌	61.13 g/L	[45]
	L-赖氨酸	促进人体的发育、增强免疫力，并有提高中枢神经功能的作用	谷氨酸棒状杆菌	8.76 g/L	[46]
	5-羟基色氨酸	用于治疗和缓解抑郁症，有效治疗失眠和慢性头痛	大肠杆菌	1.11 g/L	[47]
碳通量的调节	L-鸟氨酸	用于治疗肝脏疾病和创伤，在肝脏保护中起着有效的作用	谷氨酸棒状杆菌	16 g/L	[60]
	左旋肉碱	用于治疗功能障碍疾病	大肠杆菌	61.3 g/L	[61]
	β-丙氨酸	生物体中泛酸生产的主要前体	大肠杆菌	43.12 g/L	[27]
	5-氨基戊酸酯	生产尼龙的结构单元	谷氨酸棒状杆菌	28 g/L	[62]
	5-氨基乙酰丙酸	用于多种癌症的光动力学诊断和治疗中	谷氨酸棒状杆菌	2.06 g/L	[63]
转录调节与反馈抑制调节	L-酪氨酸	食品和饲料添加剂	酿酒酵母	36.2 mg/L	[73]
	2-氨基丁酸	抗结核性乙胺丁醇的重要前体	大肠杆菌	5.39 g/L	[74]
	L-精氨酸	在食品和补充保健食品，制药和化妆品行业中有重要应用	谷氨酸棒状杆菌	61.9 g/L	[75]
	L-亮氨酸	在刺激肌肉蛋白质合成和葡萄糖稳态中具有重要作用	谷氨酸棒状杆菌	5.7 g/L	[22]
	L-苏氨酸	广泛用于食品、制药和化妆品行业	大肠杆菌	26.0 g/L	[77]
转运系统的调节	L-异亮氨酸	形成血红蛋白必需氨基酸，在调节血糖与能量方面起到关键作用	谷氨酸棒状杆菌	26.8 g/L	[84]
	高丝氨酸	诱导免疫系统，增强植物对疾病的抵抗力	大肠杆菌W3110	39.54 g/L	[41]
	L-半胱氨酸	在提高动物的免疫调节和促进动物生长发育方面起着重要作用	大肠杆菌	634.4 mg/L	[87]
	L-蛋氨酸	用于脂肪肝，以及酒精等引起的肝损害	大肠杆菌	593.5 mg/L	[78]
	L-瓜氨酸	对于保持肌肉和肝脏健康有重要作用	谷氨酸棒状杆菌	21 g/L	[88]
	L-苏氨酸	促进人体发育和抗脂肪肝的药用效能	谷氨酸棒状杆菌	8.1 g/L	[89]

表1 本文涉及到的氨基酸及其衍生物摘要

Table 1 Summary of amino acids and their derivatives involved in this article

代谢工程策略	氨基酸及其衍生物	功能	工程菌	产量	文献
碳源的高效利用	左旋鸟氨酸	一种非蛋白质氨基酸，能够加速伤口愈合并能有效维持心脏健康	谷氨酸棒状杆菌S9114	43.6 g/L	[20]
	L-亮氨酸	在刺激肌肉蛋白质合成和葡萄糖稳态中具有重要作用	谷氨酸棒状杆菌	23.7 g/L	[22]
	L-赖氨酸	促进人体的发育、增强免疫力，并有提高中枢神经功能的作用	谷氨酸棒状杆菌 ZL-19	201.6 g/L	[17]
	L-3，4-二羟基苯丙氨酸	用于治疗帕金森氏疾病类药物的主要前体	大肠杆菌	1.51 g/L	[26]
	苯丙氨酸	用于生产人造甜味剂阿斯巴甜	大肠杆菌	1.4 g/L	[29]
	S-腺苷甲硫氨酸	被用作预防和治疗骨关节炎的功能性营养品或药物	解淀粉芽孢杆菌	107.47mg/L	[31]
	4-羟基异亮氨酸	在治疗和预防2-型糖尿病中有广阔应用前景	大肠杆菌	24.1 g/L	[32]
限速步骤的调节	高丝氨酸	诱导免疫系统，增强植物对疾病的抵抗力	大肠杆菌W3110	1.20 g/L	[41]
	L-高丙氨酸	抗癫痫药物左乙拉西坦和溴拉西坦的直接前体	大肠杆菌	5.4 g/L	[42]
	L-精氨酸	具有舒张因子的作用，临床上用于舒张和扩张血管	谷氨酸棒状杆菌	61.13 g/L	[45]
	L-赖氨酸	促进人体的发育、增强免疫力，并有提高中枢神经功能的作用	谷氨酸棒状杆菌	8.76 g/L	[46]
	5-羟基色氨酸	用于治疗和缓解抑郁症，有效治疗失眠和慢性头痛	大肠杆菌	1.11 g/L	[47]
碳通量的调节	L-鸟氨酸	用于治疗肝脏疾病和创伤，在肝脏保护中起着有效的作用	谷氨酸棒状杆菌	16 g/L	[60]
	左旋肉碱	用于治疗功能障碍疾病	大肠杆菌	61.3 g/L	[61]
	β-丙氨酸	生物体中泛酸生产的主要前体	大肠杆菌	43.12 g/L	[27]
	5-氨基戊酸酯	生产尼龙的结构单元	谷氨酸棒状杆菌	28 g/L	[62]
	5-氨基乙酰丙酸	用于多种癌症的光动力学诊断和治疗中	谷氨酸棒状杆菌	2.06 g/L	[63]
转录调节与反馈抑制调节	L-酪氨酸	食品和饲料添加剂	酿酒酵母	36.2 mg/L	[73]
	2-氨基丁酸	抗结核性乙胺丁醇的重要前体	大肠杆菌	5.39 g/L	[74]
	L-精氨酸	在食品和补充保健食品，制药和化妆品行业中有重要应用	谷氨酸棒状杆菌	61.9 g/L	[75]
	L-亮氨酸	在刺激肌肉蛋白质合成和葡萄糖稳态中具有重要作用	谷氨酸棒状杆菌	5.7 g/L	[22]
	L-苏氨酸	广泛用于食品、制药和化妆品行业	大肠杆菌	26.0 g/L	[77]
转运系统的调节	L-异亮氨酸	形成血红蛋白必需氨基酸，在调节血糖与能量方面起到关键作用	谷氨酸棒状杆菌	26.8 g/L	[84]
	高丝氨酸	诱导免疫系统，增强植物对疾病的抵抗力	大肠杆菌W3110	39.54 g/L	[41]
	L-半胱氨酸	在提高动物的免疫调节和促进动物生长发育方面起着重要作用	大肠杆菌	634.4 mg/L	[87]
	L-蛋氨酸	用于脂肪肝，以及酒精等引起的肝损害	大肠杆菌	593.5 mg/L	[78]
	L-瓜氨酸	对于保持肌肉和肝脏健康有重要作用	谷氨酸棒状杆菌	21 g/L	[88]
	L-苏氨酸	促进人体发育和抗脂肪肝的药用效能	谷氨酸棒状杆菌	8.1 g/L	[89]

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