通过建立单罐双酶级联,从弗拉瓦内生物合成一种黄酮
1College of Bioscience and Biotechnology,Yangzhou University, 2Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of China,Yangzhou University, 3Joint International Research Laboratory of Agriculture & Agri-Product Safety,Yangzhou University, 4Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, 5The Testing Center,Yangzhou University
Summary
黄酮醇的衍生对于其在医疗保健和食品工业中的应用至关重要。在这里,我们为黄酮中黄酮的生物合成提供详细的方案,并讨论与其他方法不同的关键步骤及其优势。
Abstract
黄酮类是黄酮类的主要亚类,具有多种生物和药理活性。在这里,我们提供了一种体外酶合成黄酮醇的方法。在这种方法中,Atf3h和Atfls1是黄酮醇生物合成途径中的两个关键基因,在大肠杆菌中被克隆和过度表达。重组酶通过亲和柱进行纯化,然后在特定的合成缓冲液中建立双酶级联。本系统中合成了两种黄酮醇,并通过TLC和HPLC/LC/MS分析确定。该方法在黄酮醇的衍生中表现出明显的优势。既省时又省力,而且极具成本效益。反应易于精确控制,从而达到批量生产的规模。由于系统中的组件简单,目标产品易于纯化。然而,这个系统通常仅限于生产黄酮黄酮黄酮。
Introduction
黄酮类是植物类黄酮的主要亚类,涉及植物发育和色素沉着1,2,3。更重要的是,这些化合物具有广泛的有益健康活性,如抗癌4、5、抗氧化6、抗炎7、抗肥胖8、抗高血压9,和记忆回忆属性10,导致大量研究这些植物衍生的二次代谢物。传统上,这些化合物主要来自使用有机溶剂的植物萃取物。然而,由于在植物11、12、13中含量极低,大多数黄酮醇的生产成本仍然很高,这限制了其在医疗保健和食品中的应用。行业。
在过去的几十年里,科学家们已经开发出许多方法来衍生黄酮类化合物14,15。然而,这些复杂分子的化学合成具有各种内在缺点16。它不仅需要毒试剂和极端反应条件,还需要许多步骤来产生靶向黄酮类化合物14、17。此外,这一策略的另一个重要挑战是活性类黄酮分子的手性合成。因此,通过化学合成16、17,在商业规模上生产类黄酮不是理想的策略。
最近,科学家已经开发出一种有前途的替代策略,通过工程微生物生产这些复杂的天然化合物,为黄酮类生物合成途径18,19,20,21,22,这已被成功破译在植物23。例如,Duan等人将一种生物合成途径引入萌芽的酵母糖精,以产生kaempferol(KMF)24。马拉等人通过引入黄烷3-羟基酶(f3h)、黄酮合成酶(fls1)和UDP葡萄糖:黄酮3-O-葡萄糖转移酶UGTT78K1基因,生产出一种甘基化黄酮黄酮。大肠杆菌BL21(DE3)17 .尽管有相当多的模式,但并非所有基因工程微生物都生产感兴趣的产品,因为细胞平台的复杂性,人工合成的基因元素和宿主之间的不兼容,抑制目标产品对宿主细胞的影响,以及工程细胞系统本身的不稳定性16。
黄酮类化合物生产的另一个有前途的替代策略是在体外建立多酶级联。程等人曾报告说,肠多基肽可以通过在一锅25中组装一个完整的酶通路而成功合成。这种无细胞合成策略规避了微生物生产工厂的限制,因此可以大量生产一些类黄酮。
最近,我们成功地开发了一种双酶合成系统,将纳林金(NRN)在一罐16中转化为KMF。在这里,我们将详细介绍此系统以及分析产品所涉及的方法。我们还提供了两个示例,使用该系统从 NRN 和曲霉素 (QRC) 从二恶极醇 (ERD) 生成 KMF。此外,我们还讨论了该方法的关键步骤以及黄酮类化合物生物合成的未来研究方向。
Protocol
1. 从植物组织中分离总RNA26,27 使植物组织同质化。 收集100毫克的新鲜植物组织(例如,4周大的幼苗从阿拉伯拟南芥)。用液氮冷冻组织和虫子和砂浆,然后将组织研磨成粉末。 将1mL的RNA分离试剂(见材料表)加入砂浆中。试剂将立即冷冻。当冷冻试剂熔化时,将组织样品与虫子同质化。 将均质转移到1.5-mL管中,?…
Representative Results
F3H和FLS1是植物中黄酮转化为黄酮醇的两种重要关键酶,如图1所示。开发一种体外生物合成系统,用于从黄酮Atf3h(GenBank加入号)中生产黄酮醇。NM_114983.3)和Atfls1(根银行加入号NM_120951.3)基因从4周大的A.thaliana幼苗中克隆成原核表达载体pET-32a(+)。重组质粒转化为大肠杆菌BL21(DE3),在IPTG诱导后表达融合蛋白,然后用Ni-IDA抗氧化树脂进行纯化。…
Discussion
相当多的研究侧重于黄酮醇的衍生,因为它们在保健和食品工业中具有潜在的应用。然而,传统的植物提取使用有机溶剂和化学合成具有内在的缺点,限制了其在黄酮醇的生产中使用。在这里,我们报告一种通过建立体外双酶级联从一个锅中从黄酮中产生黄酮的详细方法。该协议的关键步骤是:1)获得具有高活性的纯重组酶,2)建立一锅双酶反应级联。一般来说,植物源基因在细菌中的表达倾向于形成内含?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由扬州大学特聘教授创业基金、江苏省特聘教授创业基金、江苏省六大人才高峰项目(授权2014-SWYY-016)和江苏省资助项目资助。江苏省高等学校(兽医)重点学术项目发展。感谢扬州大学黄酮类化合物的HPLC和MS分析测试中心。
Materials
| 2× Pfu MasterMix | Beijing CoWin Biotech Co., Ltd | CW0717A | PCR amplification of genes with high fidelity |
| Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole LC/MS system | Agilent Technologies, Inc | N/A | an equipment for analysis of flavonoids by HPLC/MS |
| Agilent MassHunter Workstation (version B.03.01) | Agilent Technologies, Inc | N/A | a software for collection of the data from the Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole LC/MS system |
| dihydrokaempferol | Sigma-Aldrich Co. LLC | 91216 | intermediate product for producing kaempferol from naringenin |
| dihydroquercetin | Sichuan Provincial Standard Substance Center for Chinese Herbal Medicine | PCS0371 | intermediate product for producing quercetin from eriodictyol |
| DNA Clean-up Kit | Beijing CoWin Biotech Co., Ltd | CW2301 | purification of PCR-amplified or gel-purified DNA |
| eriodictyol | Shanghai Yuan Ye Biotechnology Co., Ltd. | B21160 | substrate for producing quercetin |
| Escherichia coli BL21(DE3) | Beijing CoWin Biotech Co., Ltd | CW0809 | bacteria strain for expressing target genes |
| Escherichia coli DH5α | Beijing CoWin Biotech Co., Ltd | CW0808 | bacteria strain for plasmid proliferation |
| FreeZone 1 Liter Benchtop Freeze-Dry System | Labconco Corporation | 7740020 | an equipment for freeze-drying of flavonoids dissolved in organic solvent |
| Gel Extraction Kit | Beijing CoWin Biotech Co., Ltd | CW2302 | purification of a DNA band from an agarose gel |
| Gel Imaging System | Shanghai Tanon Science & Technology Co. Ltd. | Tanon- 2500 |
an equipment for visualization of DNA band on an agarose gel or flavonoid spot on a polyamide TLC plate |
| GenElute Plasmid Miniprep Kit | Sigma-Aldrich Co. LLC | PLN350-1KT | minipreparation of plasmids |
| kaempferol | Sigma-Aldrich Co. LLC | 60010 | final reaction product and standard substance |
| MassHunter Quanlitative Analysis (version B.01.04) | Agilent Technologies, Inc | N/A | a software for analysis of HPLC/LC/MS data |
| NanoDrop Microvolume UV-Vis Spectrophotometer | Thermo Fisher Scientific | ND-8000-GL | an equipment for determination of DNA/RNA concentration |
| naringenin | Sigma-Aldrich Co. LLC | N5893 | substrate for producing kaempferol |
| Ni-IDA Agarose Resin | Beijing CoWin Biotech Co., Ltd | CW0010 | purification of His-tagged fusion proteins |
| pET-32a(+) | Novagen | 69015-3 | plasmid for cloning and expressing target genes |
| plasmid sequencing | GENEWIZ Suzhou | N/A | sequencing of recombinant plasmids |
| primer synthesis | GENEWIZ Suzhou | N/A | synthesis of PCR primers |
| quercetin | Shanghai Aladdin Biochemical Technology Co.,Ltd. | Q111273 | final reaction product and standard substance |
| SuperRT cDNA Synthesis Kit | Beijing CoWin Biotech Co., Ltd | CW0741 | synthesis of the first strand of cDNA from total RNA |
| T4 DNA Ligase | Thermo Fisher Scientific | EL0016 | ligation of an insert into a linearized vector DNA |
| Trizol | Thermo Fisher Scientific | 15596018 | isolation of total RNA |
| Vector NTI Advance | Thermo Fisher Scientific | 12605099 | a software for PCR primer design and DNA sequence analysis |
| Xcalibur v2.0.7 | Thermo Fisher Scientific | N/A | a software for analysis of HPLC data |
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Cite This Article
Zhang, Z., Fan, S., Chen, Z., He, Y., Huang, M., Ding, L., Zhang, Y., Chen, L., Zhang, X. Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade. J. Vis. Exp. (150), e59336, doi:10.3791/59336 (2019).