原位电离质谱技术在微生物菌株筛选中的应用进展

doi:10.12211/2096-8280.2023-018

合成生物学 ›› 2023, Vol. 4 ›› Issue (5): 980-999.DOI: 10.12211/2096-8280.2023-018

原位电离质谱技术在微生物菌株筛选中的应用进展

刘欢¹^,²^,³, 崔球¹^,²^,³

^1.中国科学院青岛生物能源与过程研究所，中国科学院生物燃料重点实验室，山东省合成生物学重点实验室，山东青岛 266101
^2.山东能源研究院，山东青岛 266101
^3.青岛新能源山东省实验室，山东青岛 266101

收稿日期:2023-03-01 修回日期:2023-07-27 出版日期:2023-10-31 发布日期:2023-11-15
通讯作者: 崔球
作者简介:刘欢（1988—），女，项目副研究员。研究方向为生物质谱、直接质谱的高通量筛选平台和方法体系。E-mail：liuhuan@qibebt.ac.cn
崔球（1975—），男，研究员，博士生导师。研究方向为蛋白质及代谢物层面的组学分析、低值生物质的生物降解利用及生物智造相关的自动化装备。E-mail：cuiqiu@qibebt.ac.cn
基金资助:
国家自然科学基金(32001053);山东能源研究院科研创新基金(SEI I202123);山东省重点研发计划(2021ZDSYS29)

Advances and applications of ambient ionization mass spectrometry in screening of microbial strains

Huan LIU¹^,²^,³, Qiu CUI¹^,²^,³

^1.Shandong Provincial Key Laboratory of Synthetic Biology，CAS Key Laboratory of Biofuels，Qingdao Institute of Bioenergy and Bioprocess Technology，Chinese Academy of Sciences，Qingdao 266101，Shandong，China
^2.Shandong Energy Institute，Qingdao 266101，Shandong，China
^3.Qingdao New Energy Shandong Laboratory，Qingdao 266101，Shandong，China

Received:2023-03-01 Revised:2023-07-27 Online:2023-10-31 Published:2023-11-15
Contact: Qiu CUI

摘要/Abstract

摘要：

质谱是一种强大的分析工具，可提供分子量和化学结构信息。它具有高特异性、高灵敏度、快速、普适性、微量和非标记等优点。在合成生物学的“设计-构建-测试-学习”工程化策略中，质谱具有重要的应用潜力。随着质谱仪器及其方法体系的不断发展，质谱，特别是原位电离质谱技术，已经成为检测和筛选微生物菌株的重要工具。它能够获取完整的细胞代谢表型，用于合成生物学中的“测试”环节高通量筛选和成像。本文重点介绍了经典的基质辅助激光解吸/电离质谱技术和基于电喷雾、激光和等离子体的原位电离质谱技术的工作机制。此外，还综述了这些质谱技术可以在无需样品预处理的情况下直接检测完整的微生物细胞，以及在微生物突变文库的高通量筛选和活微生物菌落的质谱成像方面的研究进展。最后，总结了原位电离质谱技术在合成生物学中的应用。原位电离质谱技术具有微量、无标记、高通量、普适性、高灵敏度等特点，将在合成生物学“测试”环节的高通量筛选装备中发挥重要作用。

关键词: 原位电离质谱技术, 微生物菌株, 细胞代谢表型, 高通量筛选, 合成生物学

Abstract:

Mass spectrometry (MS) is a powerful analytical tool that provides information on the molecular weight and chemical structure of analytes. With the advantages of high specificity, sensitivity, speed, universality, minimal sample requirements, and label-free detection, MS holds great potential in the "Design-Build-Test-Learn" engineering strategy employed in synthetic biology. MS, particularly ambient ionization MS (AI-MS) technology, with the continuous development of MS instruments and their methodologies, enables the detection of intact cellular metabolic phenotypes of microbial cells. This makes it an essential tool for high-throughput screening and imaging in the "test" link. Matrix-assisted desorption/ionization MS (MALDI-MS) is a well-established platform for rapid screening of microbial strains, with a throughput of about one second per sample, by directly analyzing intact cells. AI-MS, a novel set of analytical techniques, allows for direct desorption and ionization of cellular metabolic phenotypes from intact cells under open atmospheric pressure without the need for sample preparation. Its real-time, surface, and in situ capabilities make AI-MS suitable for high-throughput analysis and imaging of microbial strains with a throughput of about ten seconds per sample. In this review, we first introduce the desorption and ionization mechanisms of MALDI-MS and AI-MS based on electrospray, plasma, and laser, and illuminate these MS methods' analytical processes and their uniqueness for different intact microbial strains. We then summarize important research progresses of MALDI-MS and AI-MS in high-throughput screening of microbial mutant libraries and in situ MS imaging of living microbial colonies. Finally, we outline the advantages and limitations of different AI-MS methods for screening microbial strains, and discuss the application of AI-MS in synthetic biology. Compared to time-consuming and labor-intensive liquid or gas chromatography-based cell phenotype detection methods, AI-MS offers low sample requirements, rapid analysis, in situ capabilities, and environmental friendliness, providing an efficient and cost-effective analytic biotechnology platform for strain engineering. MS will play an important role in the development of high-throughput screening equipment in the "test" phase of synthetic biology.

Key words: ambient ionization mass spectrometry, microbial strains, cellular metabolic phenotypes, high-throughput screening, synthetic biology

中图分类号:

Q816

刘欢, 崔球. 原位电离质谱技术在微生物菌株筛选中的应用进展[J]. 合成生物学, 2023, 4(5): 980-999.

Huan LIU, Qiu CUI. Advances and applications of ambient ionization mass spectrometry in screening of microbial strains[J]. Synthetic Biology Journal, 2023, 4(5): 980-999.

图/表 5

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电离技术	电离机理	应用场景	应用案例
DESI	电喷雾电离气动辅助	细胞液干燥	亚种分化：大肠杆菌，鼠伤寒沙门氏菌菌株^[24]
		菌落	菌株表征：枯草芽孢杆菌^[25]
			微生物相互作用：大肠杆菌和黄色黏球菌^[26]
			表型分析：大肠杆菌^[27]
nano-DESI	电喷雾电离	菌落	代谢分析：希瓦氏菌、枯草芽孢杆菌、链霉菌^[28]；聚球藻属PCC^[29]
nano-DESI	电喷雾电离	菌落	分子网络：枯草芽孢杆菌，链霉菌，分枝杆菌和铜绿假单胞菌^[30]
PSI	电喷雾电离纸基	菌落置于纸基	细菌区分：革兰氏阳性菌和革兰氏阴性菌的8个属16个物种^[31]
PSI	电喷雾电离纸基	藻液置于纸基	微藻表征：小球藻和微拟球藻^[32]
MISI	电喷雾电离多级纸基	菌置于纸基	细菌鉴别：枯草芽孢杆菌、大肠杆菌和恶臭假单胞菌^[33]
TD-ESI	热解吸-电喷雾电离	菌落	细菌区分：5种革兰氏阳性菌和5种革兰氏阴性菌^[34]
EASI	声波电离	细胞液	膜脂质分析：蓝藻^[35-36]
ITSME	电喷雾电离	细胞脂滴	油脂检测：HepG2细胞^[37]
Sciex Echo-MS	电喷雾电离	细胞上清液或粗细胞提取液	酶筛选：二酰基甘油酰基转移酶2抑制剂^[38]
微流体-ESI 微流体-MALDI	电喷雾电离激光电离	纳升液滴纳升液滴	酶促反应：转氨酶ATA-117^[39] 酶分泌分析：酵母细胞中植酸酶^[40]
LA-ESI	激光烧蚀电喷雾电离	菌落	生长、代谢和抗生素抑制研究的分子成像：枯草芽孢杆菌和大肠杆菌^[41]
			表征：蓝藻^[42]
			微生物周转率：莱茵衣藻^[43]
LA-REI	激光蒸发电离	菌落	菌株分类：细菌和酵母^[44]
MALDESI	激光解吸电喷雾电离	细胞分散于载玻片上	脂质组学分析：HeLa细胞^[45]
AP-LSI	激光解吸电离	细胞液与基质共结晶	细菌区分：12个属的21种食源性细菌^[46]
AP-MALDI	激光解吸电离	菌落	成像：球状芽孢杆菌^[47]
DART	等离子体	全细胞悬浮液	细菌鉴定：化脓链球菌、大肠杆菌、γ-射线贝氏考克斯菌^[48]
DBDI	等离子体	细胞	化合物检测：洋葱细胞和PANC-1细胞^[49]
LTP	等离子体	细胞液沉积载玻片上	细菌区分：16种细菌的脂肪酸乙酯检测^[50]

电离技术	电离机理	应用场景	应用案例
DESI	电喷雾电离气动辅助	细胞液干燥	亚种分化：大肠杆菌，鼠伤寒沙门氏菌菌株^[24]
		菌落	菌株表征：枯草芽孢杆菌^[25]
			微生物相互作用：大肠杆菌和黄色黏球菌^[26]
			表型分析：大肠杆菌^[27]
nano-DESI	电喷雾电离	菌落	代谢分析：希瓦氏菌、枯草芽孢杆菌、链霉菌^[28]；聚球藻属PCC^[29]
nano-DESI	电喷雾电离	菌落	分子网络：枯草芽孢杆菌，链霉菌，分枝杆菌和铜绿假单胞菌^[30]
PSI	电喷雾电离纸基	菌落置于纸基	细菌区分：革兰氏阳性菌和革兰氏阴性菌的8个属16个物种^[31]
PSI	电喷雾电离纸基	藻液置于纸基	微藻表征：小球藻和微拟球藻^[32]
MISI	电喷雾电离多级纸基	菌置于纸基	细菌鉴别：枯草芽孢杆菌、大肠杆菌和恶臭假单胞菌^[33]
TD-ESI	热解吸-电喷雾电离	菌落	细菌区分：5种革兰氏阳性菌和5种革兰氏阴性菌^[34]
EASI	声波电离	细胞液	膜脂质分析：蓝藻^[35-36]
ITSME	电喷雾电离	细胞脂滴	油脂检测：HepG2细胞^[37]
Sciex Echo-MS	电喷雾电离	细胞上清液或粗细胞提取液	酶筛选：二酰基甘油酰基转移酶2抑制剂^[38]
微流体-ESI 微流体-MALDI	电喷雾电离激光电离	纳升液滴纳升液滴	酶促反应：转氨酶ATA-117^[39] 酶分泌分析：酵母细胞中植酸酶^[40]
LA-ESI	激光烧蚀电喷雾电离	菌落	生长、代谢和抗生素抑制研究的分子成像：枯草芽孢杆菌和大肠杆菌^[41]
			表征：蓝藻^[42]
			微生物周转率：莱茵衣藻^[43]
LA-REI	激光蒸发电离	菌落	菌株分类：细菌和酵母^[44]
MALDESI	激光解吸电喷雾电离	细胞分散于载玻片上	脂质组学分析：HeLa细胞^[45]
AP-LSI	激光解吸电离	细胞液与基质共结晶	细菌区分：12个属的21种食源性细菌^[46]
AP-MALDI	激光解吸电离	菌落	成像：球状芽孢杆菌^[47]
DART	等离子体	全细胞悬浮液	细菌鉴定：化脓链球菌、大肠杆菌、γ-射线贝氏考克斯菌^[48]
DBDI	等离子体	细胞	化合物检测：洋葱细胞和PANC-1细胞^[49]
LTP	等离子体	细胞液沉积载玻片上	细菌区分：16种细菌的脂肪酸乙酯检测^[50]

电离技术	优劣势及适用场景	单个菌样分析时间	空间分辨率	应用案例
MALDI	优势：方法成熟、样品制备简单、高通量劣势：真空取样，限制不稳定或挥发性小分子的检测适用场景：菌落	约2 s		中链脂肪酸合成酶突变体库筛选：酿酒酵母（m/z 730/758峰比值＞1）^[21] 脂肪酸合成酶突变文库筛选：酿酒酵母（脂肪酸酰基片段C_16:1/C_18:1比值为18.1±1.1）、大肠杆菌（脂肪酸酰基片段C_16:1/C₁₆比值为1.11±0.04）和荧光假单胞菌（C_16:1/C₁₆比值为0.45±0.06）^[104]
		1～2.5 s		多步酶促反应：产肽类抗生素类似物的大肠杆菌和产鼠李糖脂的大肠杆菌^[22]
		5 s		环二肽合酶定向进化：产二酮哌嗪的大肠杆菌^[103]
		约1 s	120 μm× 120 μm	菌落成像：铜绿假单胞菌、枯草芽孢杆菌和金黄色葡萄球菌^[105]
		约1 s	250 μm	菌落生物膜成像：枯草芽孢杆菌^[106]
DESI	优势：常压敞开条件下取样，原位、实时、高通量，适于细胞外泌代谢物和细胞膜相关分子劣势：难以直接提供细胞内代谢物适用场景：菌落、完整微生物细胞	10 s	100 μm	生物催化剂筛选：表达氨裂解酶和P450单加氧酶的大肠杆菌（L-苯丙氨酸检测限为>50 nmol/mm²）^[107]
DESI		约7 s	50 μm× 250 μm	菌落成像：生产过量游离脂肪酸的大肠杆菌^[27]
Nano-DESI	同DESI		1 mm × 1 mm	质谱成像：希瓦氏菌、枯草芽孢杆菌和链霉菌^[28]
Nano-DESI	同DESI	20 s	约200 μm	菌落质谱分子网络：枯草芽孢杆菌、链霉菌、分枝杆菌和铜绿假单胞菌^[30]
LA-REI	优势：常压敞开条件下取样，原位、实时、高通量、高分辨劣势：限制细胞内代谢物直接分析适用场景：菌落	8 s	约500 μm	微生物菌种分类：大肠杆菌等25种微生物^[44]
LA-REI	优势：常压敞开条件下取样，原位、实时、高通量、高分辨劣势：限制细胞内代谢物直接分析适用场景：菌落	10 s	0.8 cm²	突变文库筛选：产紫色素和白桦酸的酿酒酵母（白桦酸线性范围2～2×10^-5 g/L）^[108]
LAESI	优势：常压敞开条件下取样，原位、实时、高通量、高分辨劣势：限制低极性化合物的分析适用场景：菌落		150 μm	菌落成像：枯草芽孢杆菌和大肠杆菌^[41]

电离技术	优劣势及适用场景	单个菌样分析时间	空间分辨率	应用案例
MALDI	优势：方法成熟、样品制备简单、高通量劣势：真空取样，限制不稳定或挥发性小分子的检测适用场景：菌落	约2 s		中链脂肪酸合成酶突变体库筛选：酿酒酵母（m/z 730/758峰比值＞1）^[21] 脂肪酸合成酶突变文库筛选：酿酒酵母（脂肪酸酰基片段C_16:1/C_18:1比值为18.1±1.1）、大肠杆菌（脂肪酸酰基片段C_16:1/C₁₆比值为1.11±0.04）和荧光假单胞菌（C_16:1/C₁₆比值为0.45±0.06）^[104]
		1～2.5 s		多步酶促反应：产肽类抗生素类似物的大肠杆菌和产鼠李糖脂的大肠杆菌^[22]
		5 s		环二肽合酶定向进化：产二酮哌嗪的大肠杆菌^[103]
		约1 s	120 μm× 120 μm	菌落成像：铜绿假单胞菌、枯草芽孢杆菌和金黄色葡萄球菌^[105]
		约1 s	250 μm	菌落生物膜成像：枯草芽孢杆菌^[106]
DESI	优势：常压敞开条件下取样，原位、实时、高通量，适于细胞外泌代谢物和细胞膜相关分子劣势：难以直接提供细胞内代谢物适用场景：菌落、完整微生物细胞	10 s	100 μm	生物催化剂筛选：表达氨裂解酶和P450单加氧酶的大肠杆菌（L-苯丙氨酸检测限为>50 nmol/mm²）^[107]
DESI		约7 s	50 μm× 250 μm	菌落成像：生产过量游离脂肪酸的大肠杆菌^[27]
Nano-DESI	同DESI		1 mm × 1 mm	质谱成像：希瓦氏菌、枯草芽孢杆菌和链霉菌^[28]
Nano-DESI	同DESI	20 s	约200 μm	菌落质谱分子网络：枯草芽孢杆菌、链霉菌、分枝杆菌和铜绿假单胞菌^[30]
LA-REI	优势：常压敞开条件下取样，原位、实时、高通量、高分辨劣势：限制细胞内代谢物直接分析适用场景：菌落	8 s	约500 μm	微生物菌种分类：大肠杆菌等25种微生物^[44]
LA-REI	优势：常压敞开条件下取样，原位、实时、高通量、高分辨劣势：限制细胞内代谢物直接分析适用场景：菌落	10 s	0.8 cm²	突变文库筛选：产紫色素和白桦酸的酿酒酵母（白桦酸线性范围2～2×10^-5 g/L）^[108]
LAESI	优势：常压敞开条件下取样，原位、实时、高通量、高分辨劣势：限制低极性化合物的分析适用场景：菌落		150 μm	菌落成像：枯草芽孢杆菌和大肠杆菌^[41]

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原位电离质谱技术在微生物菌株筛选中的应用进展

Advances and applications of ambient ionization mass spectrometry in screening of microbial strains

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