L-精氨酸的微生物合成研究进展

doi:10.12211/2096-8280.2024-068

合成生物学 ›› 2025, Vol. 6 ›› Issue (2): 290-305.DOI: 10.12211/2096-8280.2024-068

L-精氨酸的微生物合成研究进展

王倩¹^,², 果士婷², 辛波¹, 钟成¹, 王钰²

^1.天津科技大学生物工程学院，天津 300222
^2.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

收稿日期:2024-08-28 修回日期:2024-10-31 出版日期:2025-04-30 发布日期:2025-05-20
通讯作者: 钟成,王钰
作者简介:王倩（1998—），女，硕士研究生。研究方向为甲醇合成氨基酸菌种选育和合成调控机制。E-mail：wangqian23@tib.cas.cn
钟成（1979—），男，博士，教授，博士生导师。研究方向为微生物发酵合成生物纳米材料，纤维素的生物合成代谢与降解，固体废弃物资源综合利用等。E-mail：czhong@tust.edu.cn
王钰（1987—），男，博士，研究员，博士生导师。研究方向为工业微生物的基因编辑育种和一碳原料的生物转化利用研究。E-mail：wang_y@tib.cas.cn
基金资助:
国家重点研发计划(2023YFD1300700)

Advances in biosynthesis of L-arginine using engineered microorganisms

WANG Qian¹^,², GUO Shiting², XIN Bo¹, ZHONG Cheng¹, WANG Yu²

^1.College of Biotechnology，Tianjin University of Science and Technology，Tianjin 300222，China
^2.Key Laboratory of Engineering Biology for Low-Carbon Manufacturing，Tianjin Institute of Industrial Biotechnology，Chinese Academy of Sciences，Tianjin 300308，China

Received:2024-08-28 Revised:2024-10-31 Online:2025-04-30 Published:2025-05-20
Contact: ZHONG Cheng, WANG Yu

摘要/Abstract

摘要：

L-精氨酸是一种碱性氨基酸，是护肤产品中常用的中和剂、保湿剂和抗氧化剂，此外，L-精氨酸还广泛应用于饲料、医药、食品等领域。以工程化的谷氨酸棒杆菌和大肠杆菌等微生物为催化剂，以可再生的淀粉糖为原料，通过微生物发酵的方法生产L-精氨酸是目前该产品最主要的生产方法。为创制高效的工程微生物菌种，早期研究者通常采用诱变筛选的方法，但由于突变的不确定性和非定向性，育种效率较低。随着合成生物技术的发展，人工设计L-精氨酸的合成途径和调控机制，并通过基因编辑理性创制工程微生物菌种成为研究的主流。本文综述了不同微生物中发现的L-精氨酸合成途径及调控机制，以谷氨酸棒杆菌和大肠杆菌为主，介绍了设计创制L-精氨酸高产菌种的合成生物学代谢改造策略，以及基于生物传感器的高通量筛选在L-精氨酸高产菌种筛选中的应用。最后，展望了进一步提高L-精氨酸生物合成水平的潜在策略，以及一碳原料等新型非粮碳资源在未来L-精氨酸生产中的应用前景。

关键词: L-精氨酸, 代谢工程, 合成生物学, 一碳原料, 谷氨酸棒杆菌, 大肠杆菌

Abstract:

L-arginine is an alkaline amino acid that has been used as a neutralizer, moisturizer, and antioxidant in skin care products. In addition, L-arginine is also widely used in feed, medicine, and food industries. The wide range of applications for L-arginine has garnered significant attention for its robust production. L-arginine can be produced through protein hydrolysis and microbial fermentation. However, protein hydrolysis has drawbacks, including complicated operation, high purification cost, low recovery efficiency, and environmental pollution. In contrast, the microbial fermentation can use renewable and cheap feedstock. Besides, the process is performed under mild conditions, and thus is more environmentally friendly. At present, engineered microorganisms such as Corynebacteriumglutamicum and Escherichiacoli are major producers of L-arginine, and design and construction of microbial strains is the robust production of L-arginine through microbial fermentation. Random mutagenesis and screening strategies are used to develop L-arginine producing microbial strains, which are random with uncertainties, resulting in a low-efficiency for the breeding. With the development of synthetic biotechnology, development of L-arginine producing strains is empowered by the rational design of artificial synthetic pathways and regulatory machineries, taking advantages of advanced genome editing technologies. This paper reviews the progress in the studies of the synthetic pathways and regulatory mechanisms of L-arginine production that have been discovered in different microorganisms. Synthetic biology-guided metabolic engineering strategies for improving L-arginine production in C.glutamicum and E.coli are summarized. Besides, the application of the biosensor-based high-throughput screening strategy for selecting L-arginine producing strains is introduced. Finally, potential strategies to enhancing L-arginine production and the possibility of using new carbon resources such as non-food biomass and one-carbon feedstock for L-arginine production are discussed. It is envisioned that synthetic biology-guided strain engineering will further enhance the production of L-arginine, particularly using non-food feedstock in the near future.

Key words: L-arginine, metabolic engineering, synthetic biology, one-carbon feedstocks, Corynebacteriumglutamicum, Escherichiacoli

中图分类号:

Q816

王倩, 果士婷, 辛波, 钟成, 王钰. L-精氨酸的微生物合成研究进展[J]. 合成生物学, 2025, 6(2): 290-305.

WANG Qian, GUO Shiting, XIN Bo, ZHONG Cheng, WANG Yu. Advances in biosynthesis of L-arginine using engineered microorganisms[J]. Synthetic Biology Journal, 2025, 6(2): 290-305.

图/表 3

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菌种	代谢改造策略	原料	产量 /(g/L)	转化率 /(g/g)	生产强度 /[g/(L·h)]	发酵方式	参考文献
谷氨酸棒杆菌	增强辅因子NADPH的供应：过表达pntAB和ppnk；解除阻遏蛋白对L-精氨酸操纵子的转录抑制：敲除argR和farR；阻断副产物合成途径：敲除ldh（编码乳酸脱氢酶）	葡萄糖	67.01	0.35	0.89	补料分批发酵	[38]
谷氨酸棒杆菌	诱变育种；增强辅因子NADPH的供应：下调pgi的表达，过表达tkt、tal、zwf、opcA、pgl；强化L-精氨酸合成途径：过表达argGH、carAB；解除阻遏蛋白对L-精氨酸操纵子的转录抑制：敲除argR和farR；增强前体物质L-谷氨酸的供应：敲除ncgl1221	葡萄糖；蔗糖	92.50	0.40	1.28	补料分批发酵	[39]
钝齿棒杆菌	解除终产物对L-精氨酸合成关键酶的反馈抑制：定点突变ArgB^{E19Y/I74V/F91H/K234T}	葡萄糖	61.20	0.43	0.64	补料分批发酵	[40]
大肠杆菌	解除阻遏蛋白对L-精氨酸操纵子的转录抑制：敲除argR；解除终产物对L-精氨酸合成关键酶的反馈抑制：外源表达argJ；强化L-精氨酸合成途径：外源表达argCJBDF	葡萄糖	70.10	0.33	1.17	补料分批发酵	[41]
钝齿棒杆菌	增强前体物质L-谷氨酸的供应：过表达iolT1、ptsG、ppgk、pyc、gltA、gdh，下调odhA的表达；强化L-精氨酸合成途径：过表达argCJBDF、argGH；阻断副产物合成途径：敲除proB，下调lysC的表达；增强辅因子NADPH的供应：下调pgi的表达	葡萄糖	87.30	0.43	1.21	补料分批发酵	[42]
钝齿棒杆菌	增强辅因子ATP的供应：过表达pyk、pgk，敲除frd12、nox、amn	葡萄糖	57.30	0.33	0.58	补料分批发酵	[43]
钝齿棒杆菌	阻断副产物合成途径：敲除proB；解除终产物对L-精氨酸合成关键酶的反馈抑制：定点突变ArgB^{E19R,H26E,D311,D312R}	葡萄糖	16.50	0.39	0.15	摇瓶发酵	[44]
钝齿棒杆菌	增强前体物质L-谷氨酸的供应：敲除putP、pta、ncg12310、ncgl1221；增强L-精氨酸转运：过表达lysE	葡萄糖	24.85	0.57	0.23	摇瓶发酵	[45]
钝齿棒杆菌	增强氮源供应：过表达glnA、aspA、gdh	葡萄糖	53.20	0.32	0.55	补料分批发酵	[46]
钝齿棒杆菌	增强氮源供应：敲除amtR，过表达amtB2	葡萄糖	60.90	0.36	0.63	补料分批发酵	[47]
大肠杆菌	阻断L-精氨酸降解途径：敲除adiA、speC、speF；解除阻遏蛋白对L-精氨酸操纵子的转录抑制：敲除argR；解除终产物对L-精氨酸合成关键酶的反馈抑制：过表达ArgA^H15Y；增强L-精氨酸转运：过表达argO	葡萄糖	11.64	1.18	0.24	补料分批发酵	[48]
钝齿棒杆菌	增强L-精氨酸转运：过表达lysE	葡萄糖	35.91	—	0.37	摇瓶发酵	[49]
钝齿棒杆菌	解除终产物对L-精氨酸合成关键酶的反馈抑制：过表达glnK		49.98	0.34	0.52	补料分批发酵	[50]
大肠杆菌	解除终产物对L-精氨酸合成关键酶的反馈抑制：敲除argA；阻断副产物合成途径：敲除pflB（编码丙酮酸-甲酸裂解酶）、ldhA（编码乳酸脱氢酶）、poxB（编码丙酮酸氧化酶）、adhE（编码乙醇脱氢酶）、aceE（编码丙酮酸脱氢酶）、speF；阻断L-精氨酸降解途径：敲除speB、astA；解除阻遏蛋白对L-精氨酸操纵子的转录抑制：敲除argR；强化L-精氨酸合成途径：过表达argCBI、argD、argG、argH、carAB	葡萄糖；乙酰谷氨酸	4.00	—	—	摇瓶发酵	[51]

菌种	代谢改造策略	原料	产量 /(g/L)	转化率 /(g/g)	生产强度 /[g/(L·h)]	发酵方式	参考文献
谷氨酸棒杆菌	增强辅因子NADPH的供应：过表达pntAB和ppnk；解除阻遏蛋白对L-精氨酸操纵子的转录抑制：敲除argR和farR；阻断副产物合成途径：敲除ldh（编码乳酸脱氢酶）	葡萄糖	67.01	0.35	0.89	补料分批发酵	[38]
谷氨酸棒杆菌	诱变育种；增强辅因子NADPH的供应：下调pgi的表达，过表达tkt、tal、zwf、opcA、pgl；强化L-精氨酸合成途径：过表达argGH、carAB；解除阻遏蛋白对L-精氨酸操纵子的转录抑制：敲除argR和farR；增强前体物质L-谷氨酸的供应：敲除ncgl1221	葡萄糖；蔗糖	92.50	0.40	1.28	补料分批发酵	[39]
钝齿棒杆菌	解除终产物对L-精氨酸合成关键酶的反馈抑制：定点突变ArgB^{E19Y/I74V/F91H/K234T}	葡萄糖	61.20	0.43	0.64	补料分批发酵	[40]
大肠杆菌	解除阻遏蛋白对L-精氨酸操纵子的转录抑制：敲除argR；解除终产物对L-精氨酸合成关键酶的反馈抑制：外源表达argJ；强化L-精氨酸合成途径：外源表达argCJBDF	葡萄糖	70.10	0.33	1.17	补料分批发酵	[41]
钝齿棒杆菌	增强前体物质L-谷氨酸的供应：过表达iolT1、ptsG、ppgk、pyc、gltA、gdh，下调odhA的表达；强化L-精氨酸合成途径：过表达argCJBDF、argGH；阻断副产物合成途径：敲除proB，下调lysC的表达；增强辅因子NADPH的供应：下调pgi的表达	葡萄糖	87.30	0.43	1.21	补料分批发酵	[42]
钝齿棒杆菌	增强辅因子ATP的供应：过表达pyk、pgk，敲除frd12、nox、amn	葡萄糖	57.30	0.33	0.58	补料分批发酵	[43]
钝齿棒杆菌	阻断副产物合成途径：敲除proB；解除终产物对L-精氨酸合成关键酶的反馈抑制：定点突变ArgB^{E19R,H26E,D311,D312R}	葡萄糖	16.50	0.39	0.15	摇瓶发酵	[44]
钝齿棒杆菌	增强前体物质L-谷氨酸的供应：敲除putP、pta、ncg12310、ncgl1221；增强L-精氨酸转运：过表达lysE	葡萄糖	24.85	0.57	0.23	摇瓶发酵	[45]
钝齿棒杆菌	增强氮源供应：过表达glnA、aspA、gdh	葡萄糖	53.20	0.32	0.55	补料分批发酵	[46]
钝齿棒杆菌	增强氮源供应：敲除amtR，过表达amtB2	葡萄糖	60.90	0.36	0.63	补料分批发酵	[47]
大肠杆菌	阻断L-精氨酸降解途径：敲除adiA、speC、speF；解除阻遏蛋白对L-精氨酸操纵子的转录抑制：敲除argR；解除终产物对L-精氨酸合成关键酶的反馈抑制：过表达ArgA^H15Y；增强L-精氨酸转运：过表达argO	葡萄糖	11.64	1.18	0.24	补料分批发酵	[48]
钝齿棒杆菌	增强L-精氨酸转运：过表达lysE	葡萄糖	35.91	—	0.37	摇瓶发酵	[49]
钝齿棒杆菌	解除终产物对L-精氨酸合成关键酶的反馈抑制：过表达glnK		49.98	0.34	0.52	补料分批发酵	[50]
大肠杆菌	解除终产物对L-精氨酸合成关键酶的反馈抑制：敲除argA；阻断副产物合成途径：敲除pflB（编码丙酮酸-甲酸裂解酶）、ldhA（编码乳酸脱氢酶）、poxB（编码丙酮酸氧化酶）、adhE（编码乙醇脱氢酶）、aceE（编码丙酮酸脱氢酶）、speF；阻断L-精氨酸降解途径：敲除speB、astA；解除阻遏蛋白对L-精氨酸操纵子的转录抑制：敲除argR；强化L-精氨酸合成途径：过表达argCBI、argD、argG、argH、carAB	葡萄糖；乙酰谷氨酸	4.00	—	—	摇瓶发酵	[51]

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L-精氨酸的微生物合成研究进展

Advances in biosynthesis of L-arginine using engineered microorganisms

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