Synthetic Biology Journal ›› 2023, Vol. 4 ›› Issue (5): 947-965.DOI: 10.12211/2096-8280.2023-017
• Invited Review • Previous Articles Next Articles
Mengchu SUN, Liangyu LU, Xiaolin SHEN, Xinxiao SUN, Jia WANG, Qipeng YUAN
Received:
2023-02-28
Revised:
2023-04-17
Online:
2023-11-15
Published:
2023-10-31
Contact:
Jia WANG, Qipeng YUAN
孙梦楚, 陆亮宇, 申晓林, 孙新晓, 王佳, 袁其朋
通讯作者:
王佳,袁其朋
作者简介:
基金资助:
CLC Number:
Mengchu SUN, Liangyu LU, Xiaolin SHEN, Xinxiao SUN, Jia WANG, Qipeng YUAN. Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction[J]. Synthetic Biology Journal, 2023, 4(5): 947-965.
孙梦楚, 陆亮宇, 申晓林, 孙新晓, 王佳, 袁其朋. 基于荧光检测的高通量筛选技术和装备助力细胞工厂构建[J]. 合成生物学, 2023, 4(5): 947-965.
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URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2023-017
菌种 | 目标产物 | 成果 | 技术 | 信号检测 | 文献 |
---|---|---|---|---|---|
酿酒酵母 | 左旋多巴 | 左旋多巴(L-DOPA)产量增加 | FACS | 左旋多巴的酶偶联生物传感器 | [ |
芽孢杆菌 | 抗生素Ami | 通过基因挖掘鉴定抗生素Ami | FACS (液滴) | 生长偶联 | [ |
大肠杆菌 | L-半胱氨酸 | 诱导半胱氨酸产生增加约2.67倍 | FACS | L-半胱氨酸响应性转录因子生物传感器 | [ |
解脂耶氏酵母 | β-胡萝卜素 | 产生9.4 g/L β-胡萝卜素的菌株 | FACS | β-胡萝卜素的菌株自身荧光 | [ |
里氏木霉 | 十六烷醇 | 构建首个丝状真菌脂肪醇生产细胞工厂 | FACS | GFP-融合耦合荧光 | [ |
凝结芽孢杆菌 | 乳酸 | 乳酸滴度增加52% | FACS (液滴) | 荧光染料(探针1) | [ |
大肠杆菌 | 丙二酰-CoA | 显著提高了柚皮素的产量,达到(523.7±51.8)mg/L | FACS | 基于柚皮素和对香豆酸生物传感器 | [ |
毕赤酵母 | 木聚糖酶 | 木聚糖酶活性提高1.3倍 | FACS | 荧光标记底物 | [ |
光滑念珠菌 | 丙酮酸 | 丙酮酸产量增加73.6% | FACS (液滴) | pH敏感荧光蛋白 | [ |
谷氨酸棒状杆菌 | L-赖氨酸 | 获得高产L-赖氨酸的丙酮酸羧化酶突变体 | FACS | 基于赖氨酸转录因子生物传感器 | [ |
大肠杆菌 | 引入非天然氨基酸 | 成功筛选到可将非天然氨基酸插入到蛋白质特定位点的功能性PylRS突变体 | FACS | 荧光蛋白融合表达 | [ |
大肠杆菌 | α-1,3-岩藻糖基转移酶 | 成功筛选到活性提高14倍的α-1,3-岩藻糖基转移酶突变 | FACS | 荧光标记底物及利用细胞膜的选择性转运蛋白实现细胞捕获荧光产物 | [ |
大肠杆菌 | 单胺氧化酶 | 获得对仲胺底物具有催化活性的突变体 | FACS | 多级酶偶联反应,过氧化氢与荧光探针反应释放荧光基团 | [ |
自来水样本 | 酯酶 | 挖掘具有高催化效率新型酯酶EstWY | FACS (液滴) | 荧光标记底物 | [ |
Table 1 Summary of recent FACS applications for microbial cell factories and directed evolution
菌种 | 目标产物 | 成果 | 技术 | 信号检测 | 文献 |
---|---|---|---|---|---|
酿酒酵母 | 左旋多巴 | 左旋多巴(L-DOPA)产量增加 | FACS | 左旋多巴的酶偶联生物传感器 | [ |
芽孢杆菌 | 抗生素Ami | 通过基因挖掘鉴定抗生素Ami | FACS (液滴) | 生长偶联 | [ |
大肠杆菌 | L-半胱氨酸 | 诱导半胱氨酸产生增加约2.67倍 | FACS | L-半胱氨酸响应性转录因子生物传感器 | [ |
解脂耶氏酵母 | β-胡萝卜素 | 产生9.4 g/L β-胡萝卜素的菌株 | FACS | β-胡萝卜素的菌株自身荧光 | [ |
里氏木霉 | 十六烷醇 | 构建首个丝状真菌脂肪醇生产细胞工厂 | FACS | GFP-融合耦合荧光 | [ |
凝结芽孢杆菌 | 乳酸 | 乳酸滴度增加52% | FACS (液滴) | 荧光染料(探针1) | [ |
大肠杆菌 | 丙二酰-CoA | 显著提高了柚皮素的产量,达到(523.7±51.8)mg/L | FACS | 基于柚皮素和对香豆酸生物传感器 | [ |
毕赤酵母 | 木聚糖酶 | 木聚糖酶活性提高1.3倍 | FACS | 荧光标记底物 | [ |
光滑念珠菌 | 丙酮酸 | 丙酮酸产量增加73.6% | FACS (液滴) | pH敏感荧光蛋白 | [ |
谷氨酸棒状杆菌 | L-赖氨酸 | 获得高产L-赖氨酸的丙酮酸羧化酶突变体 | FACS | 基于赖氨酸转录因子生物传感器 | [ |
大肠杆菌 | 引入非天然氨基酸 | 成功筛选到可将非天然氨基酸插入到蛋白质特定位点的功能性PylRS突变体 | FACS | 荧光蛋白融合表达 | [ |
大肠杆菌 | α-1,3-岩藻糖基转移酶 | 成功筛选到活性提高14倍的α-1,3-岩藻糖基转移酶突变 | FACS | 荧光标记底物及利用细胞膜的选择性转运蛋白实现细胞捕获荧光产物 | [ |
大肠杆菌 | 单胺氧化酶 | 获得对仲胺底物具有催化活性的突变体 | FACS | 多级酶偶联反应,过氧化氢与荧光探针反应释放荧光基团 | [ |
自来水样本 | 酯酶 | 挖掘具有高催化效率新型酯酶EstWY | FACS (液滴) | 荧光标记底物 | [ |
化合物/酶分子 | 表达菌株 | 成果 | 信号检测 | 文献 |
---|---|---|---|---|
微生物菌群 | 筛选PET降解菌株,挖掘并验证两种PET降解酶 | 荧光探针 | [ | |
L-色氨酸 | 大肠杆菌 | 筛选色氨酸增加165.9%突变菌株 | 核糖体开关生物传感器 | [ |
α-L-苏糖核酸 | 大肠杆菌 | α-L-苏糖核酸合成提高10倍 | 荧光标记底物 | [ |
纤维素 | 里氏木霉 | 纤维素产量增加46% | 荧光标记底物 | [ |
3-脱氢莽草酸 | 大肠杆菌 | 3-脱氢莽草酸产量增加30% | 荧光生物传感器 | [ |
纳米抗体VHH | 谷氨酸棒状杆菌 | 挖掘蛋白分泌关联基因位点并构建了VHH产量提高2.78倍的底盘菌株 | 氧化还原反应转录因子生物传感器 | [ |
硫酸酯酶 | 大肠杆菌 | 获得的酶变体对4-硝基苯基硫酸盐催化活性提高6.2倍,对二硫酸荧光素催化活性提高30倍 | 荧光标记底物 | [ |
L-色氨酸 | 大肠杆菌 | 获得产量提高145%的变体菌株 | 核糖开关生物传感器 | [ |
醛缩酶 | 大肠杆菌 | 单轮筛选中醛缩酶RA95.0催化活性大幅跃升高达80倍 | 荧光标记底物 | [ |
谷氨酸 | 谷氨酸棒杆菌 | 获得谷氨酸产量分别提升了25.8%和19.1%的变体菌 | 谷氨酸感应荧光报告器 | [ |
谷氨酰胺酶 | 解淀粉芽孢杆菌 | 筛选产生了谷氨酰胺酶产量增加47%的菌株 | 响应谷氨酸iGluSnFR生物传感器 | [ |
环己胺氧化酶 | 大肠杆菌 | 环己胺氧化酶在一轮定向进化后催化效率提高了960倍 | 多酶级联反应辅助荧光底物释放荧光基团 | [ |
丙酮酸 | 光滑球拟酵母 | 最终获得了1株高产突变菌株4H2,摇瓶产量达48.6 g/L,相比出发菌株提高了73.6% | pH敏感传感器 | [ |
D-阿洛酮糖 | 大肠杆菌将 | 获得催化效率提高了17倍的突变体 | D-阿洛酮糖响应转录因子生物传感器 | [ |
抗生素红霉素 | 放线菌 | 筛选出高产红霉素放线菌 | 全细胞转录因子生物传感器 | [ |
α-淀粉酶 | 地衣芽孢杆菌 | 成功鉴定出α-淀粉酶生产能力较高的突变体 | 荧光标记底物 | [ |
Table 2 Summary of recent FADS applications for microbial cell factories and directed evolution
化合物/酶分子 | 表达菌株 | 成果 | 信号检测 | 文献 |
---|---|---|---|---|
微生物菌群 | 筛选PET降解菌株,挖掘并验证两种PET降解酶 | 荧光探针 | [ | |
L-色氨酸 | 大肠杆菌 | 筛选色氨酸增加165.9%突变菌株 | 核糖体开关生物传感器 | [ |
α-L-苏糖核酸 | 大肠杆菌 | α-L-苏糖核酸合成提高10倍 | 荧光标记底物 | [ |
纤维素 | 里氏木霉 | 纤维素产量增加46% | 荧光标记底物 | [ |
3-脱氢莽草酸 | 大肠杆菌 | 3-脱氢莽草酸产量增加30% | 荧光生物传感器 | [ |
纳米抗体VHH | 谷氨酸棒状杆菌 | 挖掘蛋白分泌关联基因位点并构建了VHH产量提高2.78倍的底盘菌株 | 氧化还原反应转录因子生物传感器 | [ |
硫酸酯酶 | 大肠杆菌 | 获得的酶变体对4-硝基苯基硫酸盐催化活性提高6.2倍,对二硫酸荧光素催化活性提高30倍 | 荧光标记底物 | [ |
L-色氨酸 | 大肠杆菌 | 获得产量提高145%的变体菌株 | 核糖开关生物传感器 | [ |
醛缩酶 | 大肠杆菌 | 单轮筛选中醛缩酶RA95.0催化活性大幅跃升高达80倍 | 荧光标记底物 | [ |
谷氨酸 | 谷氨酸棒杆菌 | 获得谷氨酸产量分别提升了25.8%和19.1%的变体菌 | 谷氨酸感应荧光报告器 | [ |
谷氨酰胺酶 | 解淀粉芽孢杆菌 | 筛选产生了谷氨酰胺酶产量增加47%的菌株 | 响应谷氨酸iGluSnFR生物传感器 | [ |
环己胺氧化酶 | 大肠杆菌 | 环己胺氧化酶在一轮定向进化后催化效率提高了960倍 | 多酶级联反应辅助荧光底物释放荧光基团 | [ |
丙酮酸 | 光滑球拟酵母 | 最终获得了1株高产突变菌株4H2,摇瓶产量达48.6 g/L,相比出发菌株提高了73.6% | pH敏感传感器 | [ |
D-阿洛酮糖 | 大肠杆菌将 | 获得催化效率提高了17倍的突变体 | D-阿洛酮糖响应转录因子生物传感器 | [ |
抗生素红霉素 | 放线菌 | 筛选出高产红霉素放线菌 | 全细胞转录因子生物传感器 | [ |
α-淀粉酶 | 地衣芽孢杆菌 | 成功鉴定出α-淀粉酶生产能力较高的突变体 | 荧光标记底物 | [ |
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