多酶筛选使用高通量基因酶的筛选系统
Summary
This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate).
Abstract
The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.
Introduction
最近开发的高通量的单细胞分析技术允许新的酶从基于其功能活动1大规模的基因库进行筛选。在单细胞水平，调节转录的蛋白质采用通过检测所生产作为目标酶活性的结果，小分子引发报告基因的表达。一种早期方法涉及从富养产碱 E2使用底物诱导的基因表达筛选（SIGEX）方法，其中基材诱导报告蛋白2的表达的酚降解操纵子的分离。 恶臭假单胞菌 NhaR用于选择苯甲醛脱氢酶3，并从谷氨酸棒杆菌 LysG被用于从不同的突变体文库4新的L-赖氨酸生产菌株的高通量筛选。
先前，遗传酶screeniNG系统（GESS）提出了一个普遍适用的筛选平台5。该系统采用酚系识别二甲调节器，DMPR，P的恶臭 。 DMPR（E135K），和DMPR的突变体，也可以在GESS（PNP-GESS）检测p硝基苯酚（PNP）的使用。在生产酚醛化合物靶酶的存在下，GESS在大肠杆菌大肠杆菌细胞发出的荧光信号，能够使用荧光激活细胞分选仪（FACS）单细胞的快速分离。但宏基因组酶的表达似乎是比传统的重组酶的弱;因此，GESS旨在通过与最佳操作条件5沿调查核糖体结合位点（RBS）和终止子序列的组合以检测与最大灵敏度酚类化合物。
之一的GESS的基本优点是，这种单一的方法理论上允许OV的筛选器超过200种不同类型的BRENDA数据库酶（ 表1对于http：// www.brenda-enzymes.info，2013.7）通过简单地采用不同的基板。它表明，纤维素酶，脂肪酶，和甲基对硫磷水解酶（MPH）可使用的pNP-GESS与对硝基苯基丁酸酯，对硝基苯基- cellotrioside和甲基对硫磷，分别为5的适当底物进行检测。最近，证明了碱性磷酸酶（AP），其是使用的pNP-GESS确定的新酶之一，是在冷适应的海洋宏基因组6找到的第一个不耐热的AP。
这里，筛选过程的细节呈现的pNP-GESS检测三种不同类型的enzymes-脂肪酶，纤维素酶，和碱性磷酸酶的活动-and迅速从宏基因组文库5,6-识别新的候选酶。这些过程包括：宏基因组为PNP-GESS预处理和经营FL流式细胞仪分拣机。而获得的匹配将需要测序进一步鉴定，该协议覆盖的步骤执行到使用流式细胞仪的酶活性的确认的步骤。
Protocol
Representative Results
Discussion
提高生物催化剂的生产效率是生物化工为主业9和宏基因组成功的关键被认为是最好的天然酶源之一。在这个意义上说，它是从那里大多数遗传资源还没有被开发10的宏基因组筛选新酶是必不可少的。几种筛选方法已经开发了使用转录激活11,12直接检测酶的产品，但这些技术需要特定代谢物应答转录系统。另一方面，GESS根据使用的苯酚或PNP标记底物是广泛适用的。虽然大多?…
Declarações
The authors have nothing to disclose.
Acknowledgements
This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031944), the Next-Generation Biogreen 21 Program (PJ009524), NRF-2015M3D3A1A01064875 and the KRIBB Research Initiative Program.
Materials
| CopyControl | Epicentre | CCFOS110 | Fosmid library production kit |
| CopyControl Induction Solution | Epicentre | CCIS125 | Fosmid copy induction solution |
| EPI300 | Epicentre | EC300110 | Electrocompetent cell |
| pCC1FOS | Epicentre | CCFOS110 | Fosmid vector |
| Gene Pulser Mxcell | Bio-Rad | Electroporation cuvette and electroporate system | |
| FACSAria III | Becton Dickinson | Flow Cytometry (FACS machine) | |
| AZ100M | Nikon | Microscope | |
| UltraSlim | Maestrogen | LED illuminator | |
| 50-mL conical tube | BD Falcon | ||
| 14-mL round-bottom tube | BD Falcon | ||
| 5-mL round-bottom tube | BD Falcon | ||
| p-nitrophenyl phosphate | Sigma-Aldrich | N7653 | Substrate |
| p-nitrophenyl β-D-cellobioside | Sigma-Aldrich | N5759 | Substrate |
| p-nitrophenyl butylate | Sigma-Aldrich | N9876 | Substrate |
| Luria- Bertani (LB) | BD Difco | 244620 | Tryptone 10g/L, Yeast extract 5g/L, Sodium Chloride 10g/L |
| Super Optimal broth (SOB) | BD Difco | 244310 | Tryptone 20g/L, Yeast extract 5g/L, Sodium Chloride 0.5g/L, Magnesium Sulfate 2.4g/L, Potassium Chloride 186mg/L |
| Super Optimal broth with Catabolite repression (SOC) | SOB, 0.4 % glucose | ||
| 2x Yeast Extract Tryptone (2xYT) | BD Difco | 244020 | Pancreatic digest of Casein 16g/L, Yeast extract 10g/L, Yeast extract 5g/L |
| Cell storage media | 2xYT broth, 15 % Glycerol, 2 % Glucose | ||
| pGESS(E135K) | A DNA vector containing dmpR, egfp genes with their appropriate promoters, RBS, and terminator. See the reference 5 in the manuscript for more details. |
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| Chloramphenicol | Sigma | C0378 | |
| Ampicillin | Sigma | A9518 | |
| BD FACSDiva | Becton Dickinson | Flow Cytometry Software Version 7.0 | |
| PBS | Gibco | 70011-044 | 0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4 |
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