Application of FACS Technology in the Directed Evolution of Enzyme

doi:10.13560/j.cnki.biotech.bull.1985.2023-0486

Abstract

Abstract:

Flow cytometry（FCM）is a rapid and relatively quantitative multi-parameter analytical technique for analyzing a large number of cells at the single-cell level. The constructed fluorescence activated cell sorting（FACS）based on FCM can physically separate fluorescent-labeled single cells and has been widely used in the field of directed evolution of enzyme. Directed evolution has been proven to be an effective method for obtaining industrial enzymes with excellent properties, such as high enzyme activity, strong thermal stability and solvent resistance. To conduct directed evolution, mutant library firstly needs to be constructed by random mutagenesis or DNA recombination, and then be screened under manual selection pressure. Traditional screening methods such as plate and microplate methods have limitations of low throughput, high cost, large errors, and poor accuracy, and cannot achieve high-throughput screening of large mutant library. Compared with traditional screening methods, FACS has the advantages of high throughput, low cost, small error and high precision. In this article, we first summarized the development process of FCM and FACS, as well as the composition and classification of related instruments derived from them. Then, we discussed the application status and limitations of FACS in the directed evolution of enzyme. Finally, we summarized and prospected the development direction of FACS and its application in the field of directed evolution of enzyme.

Key words: flow cytometry, fluorescence-activated cell sorting, high-throughput screening

LIU Jin-sheng, CHEN Zhen-ya, HUO Yi-xin, GUO Shu-yuan. Application of FACS Technology in the Directed Evolution of Enzyme[J]. Biotechnology Bulletin, 2023, 39(10): 93-106.

Figures/Tables 8

References 94

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筛选方法 Screening method	通量 Throughput	突变文库 Mutant library	特点 Characteristic	参考文献 References
平板		10⁵-10⁶	操作简便；半定量分析；通量低；精度低	[44,45,46,47]
微孔板	1/s	10⁵	操作简便；定量分析；通量低；精度低	[13, 47]
FACS	10⁴/s	>10⁸	通量高；消耗少；精度高；单细胞定量分析；设备昂贵；不便携	[8, 48]
FADS	<10³/s	10⁷	通量高；消耗少；精度高；单细胞定量分析；工艺复杂度高；搭建难	[43, 49-51]

筛选方法 Screening method	通量 Throughput	突变文库 Mutant library	特点 Characteristic	参考文献 References
平板		10⁵-10⁶	操作简便；半定量分析；通量低；精度低	[44,45,46,47]
微孔板	1/s	10⁵	操作简便；定量分析；通量低；精度低	[13, 47]
FACS	10⁴/s	>10⁸	通量高；消耗少；精度高；单细胞定量分析；设备昂贵；不便携	[8, 48]
FADS	<10³/s	10⁷	通量高；消耗少；精度高；单细胞定量分析；工艺复杂度高；搭建难	[43, 49-51]

类别 Classification	目标酶 Target enzyme	结果 Result	宿主细胞 Host cell	参考文献 Reference
细胞表面酶	丝氨酸蛋白酶OmpT	k_cat/K_m值提高60倍	大肠杆菌E. coli	[54]
	分选酶A	k_cat/K_m值提高140倍	酿酒酵母S. cerevisiae	[55]
	半乳糖氧化酶	K_m值降低4.4倍	大肠杆菌E. coli	[56]
	D-氨基酸氧化酶	k_cat值提高4.2倍	大肠杆菌E. coli	[56]
胞内酶	β-1,3-半乳糖基转移酶	底物耐受性提高300倍	大肠杆菌E. coli	[57]
	α-1,3-岩藻糖基转移酶	合成Lewis X和3'岩藻糖基乳糖的k_cat/K_m值分别提高6倍和14倍	幽门螺旋杆菌Helicobacter pylori	[58]
	α-1,2-岩藻糖基转移酶	2'岩藻糖基乳糖的滴度和产量分别提高1.72倍和1.51倍	大肠杆菌E. coli	[59]
	多聚唾液酸转移酶	比酶活和热稳定性分别提高 1.5-2.1倍和9倍	奈瑟菌Neisseria	[60]
	葡萄糖氧化酶	V_max值分别提高1.3和2.3倍	酿酒酵母S. cerevisiae	[61]
	ATP磷酸核糖转移酶	L-组氨酸产量为0.1 mmol/L，野生型未生产L-组氨酸	谷氨酸棒状杆菌Corynebacterium glutamicum	[62]
	丝氨酸乙酰转移酶	L-半胱氨酸产量提高2.36倍	大肠杆菌E. coli	[63]
	3C蛋白酶	蛋白水解活性提高4倍	大肠杆菌E. coli	[64]
	咖啡因去甲基化酶	V_max/K_m值和产物选择性分别提高33和22倍	酿酒酵母S. cerevisiae	[65]
	丙酮酸羧化酶	赖氨酸滴度分别增加9%和19%	谷氨酸棒状杆菌C. glutamicum	[66]
	莽草酸生产菌株	莽草酸滴度达到（3.72±0.35）mmol/L，比野生型提高2.4倍	谷氨酸棒状杆菌C. glutamicum	[67]
	丝氨酸羟甲基转移酶	L-丝氨酸产量达到34.78 g/L，比野生型菌株提高35.9%	谷氨酸棒状杆菌C. glutamicum	[68]

类别 Classification	目标酶 Target enzyme	结果 Result	宿主细胞 Host cell	参考文献 Reference
细胞表面酶	丝氨酸蛋白酶OmpT	k_cat/K_m值提高60倍	大肠杆菌E. coli	[54]
	分选酶A	k_cat/K_m值提高140倍	酿酒酵母S. cerevisiae	[55]
	半乳糖氧化酶	K_m值降低4.4倍	大肠杆菌E. coli	[56]
	D-氨基酸氧化酶	k_cat值提高4.2倍	大肠杆菌E. coli	[56]
胞内酶	β-1,3-半乳糖基转移酶	底物耐受性提高300倍	大肠杆菌E. coli	[57]
	α-1,3-岩藻糖基转移酶	合成Lewis X和3'岩藻糖基乳糖的k_cat/K_m值分别提高6倍和14倍	幽门螺旋杆菌Helicobacter pylori	[58]
	α-1,2-岩藻糖基转移酶	2'岩藻糖基乳糖的滴度和产量分别提高1.72倍和1.51倍	大肠杆菌E. coli	[59]
	多聚唾液酸转移酶	比酶活和热稳定性分别提高 1.5-2.1倍和9倍	奈瑟菌Neisseria	[60]
	葡萄糖氧化酶	V_max值分别提高1.3和2.3倍	酿酒酵母S. cerevisiae	[61]
	ATP磷酸核糖转移酶	L-组氨酸产量为0.1 mmol/L，野生型未生产L-组氨酸	谷氨酸棒状杆菌Corynebacterium glutamicum	[62]
	丝氨酸乙酰转移酶	L-半胱氨酸产量提高2.36倍	大肠杆菌E. coli	[63]
	3C蛋白酶	蛋白水解活性提高4倍	大肠杆菌E. coli	[64]
	咖啡因去甲基化酶	V_max/K_m值和产物选择性分别提高33和22倍	酿酒酵母S. cerevisiae	[65]
	丙酮酸羧化酶	赖氨酸滴度分别增加9%和19%	谷氨酸棒状杆菌C. glutamicum	[66]
	莽草酸生产菌株	莽草酸滴度达到（3.72±0.35）mmol/L，比野生型提高2.4倍	谷氨酸棒状杆菌C. glutamicum	[67]
	丝氨酸羟甲基转移酶	L-丝氨酸产量达到34.78 g/L，比野生型菌株提高35.9%	谷氨酸棒状杆菌C. glutamicum	[68]

类别 Classification	目标酶 Target enzyme	结果 Result	宿主细胞 Host cell	参考文献 Reference
双乳化液滴法	血清对氧磷酶	k_cat/K_m值提高100倍	大肠杆菌E. coli	[74]
	碱性磷酸酶	实现活性碱性磷酸酶的100万倍富集	大肠杆菌E. coli	[75]
	α-L-苏糖核酸聚合酶	获得一个不依赖于锰的α-L-苏氨酸核糖核酸（TNA）聚合酶突变体	大肠杆菌E. coli	[76]
	几丁质酶A	比酶活提高2倍	大肠杆菌E. coli	[77]
	人工金属酶	建立FACS筛选双乳化液滴中人工金属酶的方法	大肠杆菌E. coli	[78]
水凝胶微珠法	RNA聚合酶	获得一个对利福霉素有耐药性的突变体	大肠杆菌E. coli	[79]
	植酸酶	k_cat值提高31%	大肠杆菌E. coli	[80]
	木聚糖酶	比酶活提高1.3倍	毕赤酵母Pichia pastoris	[81]
	转肽酶	k_cat/K_m值提高114倍	大肠杆菌E. coli	[82]