CN113621629B - 一种基于丙二酰辅酶a再生的柚皮素体外酶促合成方法 - Google Patents

一种基于丙二酰辅酶a再生的柚皮素体外酶促合成方法 Download PDF

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CN113621629B
CN113621629B CN202110870360.7A CN202110870360A CN113621629B CN 113621629 B CN113621629 B CN 113621629B CN 202110870360 A CN202110870360 A CN 202110870360A CN 113621629 B CN113621629 B CN 113621629B
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张新跃
聂也森
何妍之
张智萍
丁笠
陈磊
廖凯
赵晨宏
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Abstract

本发明设计一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，在构建乙酰辅酶A合成酶ACS基因与乙酰辅酶A羧化酶ACC1基因的重组表达质粒的基础上，将重组质粒分别转化大肠杆菌和酵母细胞，表达目的蛋白，并将纯化的ACS与ACC1重组蛋白加入柚皮素体外酶促合成体系中，实现了丙二酰辅酶A的再生，以及以4‑香豆酸为底物在体外低成本酶促合成柚皮素。本发明设计了一个新的反应体系，无需添加昂贵的丙二酰辅酶A便能对其进行再生利用，并最终生成柚皮素。

Description

一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法
技术领域
本发明涉及一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，分类号为C12N9，属于生物医药技术领域。
背景技术
丙二酰辅酶A（malonyl-coenzyme A）是一种辅酶A衍生物。作为一种关键的中间代谢物，丙二酰辅酶A在多种代谢途径中发挥重要作用，是细胞内合成脂肪酸和甘油三酯的重要前体分子（Kastaniotis A J, et al. Biochim Biophys Acta Mol Cell Biol Lipids,2017, 1862(1): 39-48.），也是合成聚酮类化合物（Shimizu Y, et al. Chembiochem,2017, 18(1): 50-65.）和许多平台化合物（Kildegaard K R, et al. Microb Cell Fact,2016, 15: 53.）的原材料。
一般而言，细胞内的辅酶A经乙酰辅酶A合成酶（acetyl-CoA synthase, ACS）和乙酰辅酶A羧化酶（acetyl-CoA carboxylase, ACC）的先后催化，形成丙二酰辅酶A（Qu Q, etal. Cell Death Dis, 2016, 7(5): e2226.）。丙二酰辅酶A在参与细胞反应并被消耗的同时，脱去羧基重新生成辅酶A（Waki T, et al. Nat Commun, 2020, 11(1): 870.）。因此，丙二酰辅酶A在细胞内处于一种动态平衡。由于丙二酰辅酶A的胞内含量低，分离和纯化难度较大，并且难以通过化学方法合成，导致其市场价格通常远高于其下游产品，因而制约了这一辅助因子在工业化生产中的广泛应用。
近年来，学者们尝试通过多种途径提高微生物细胞内丙二酰辅酶A的量，如通过提高细胞内乙酰辅酶A的含量（Krivoruchko A, et al. Metab Eng, 2015, 28: 28-42.）或乙酰辅酶A羧化酶的表达水平（Wang J C, et al. Biosci Biotechnol Biochem, 2016,80(6): 1214-1222.）等手段促进丙二酰辅酶A的形成，同时限制丙二酰辅酶A流向与目标分子无关的通路（Yang D，et al. Proc Natl Acad Sci U S A, 2018, 115(40): 9835-9844.），从而提高目标分子的产量。然而，目前在体外合成体系中尚无类似策略降低因丙二酰辅酶A带来的高生产成本。
发明内容
本发明所要解决的技术问题是，克服现有技术的不足而提供一种基于丙二酰辅酶A再生的柚皮素酶促合成方法，该方法首先建立一种丙二酰辅酶A的再生体系，以显著降低了体外酶促合成柚皮素时因昂贵的丙二酰辅酶A所带来的高成本，该体系同样适应于其他需要以丙二酰辅酶A为底物的生化反应；同时构建一种高活性三功能酶，并通过优化体外反应条件显著提高了目标分子的产量和底物的总转化率。
本发明提供了一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，具体包括以下步骤：
步骤1、克隆丙二酰辅酶A再生体系中关键酶基因
从大肠杆菌中克隆ACS基因，构建重组质粒pET-32a-ACS，从毕赤酵母中克隆ACC1基因，构建重组质粒pGAP-Neo-ACC1；
步骤2、重组蛋白ACS和ACC1的诱导表达与纯化
将重组质粒pET-32a-ACS转化感受态大肠杆菌，IPTG诱导表达、纯化，得到重组蛋白ACS；同时将重组质粒pGAP-Neo-ACC1转化毕赤酵母，经过表达和纯化，得到重组蛋白ACC1；
步骤3、从头合成柚皮素的重组三功能酶的构建、表达与纯化
利用重叠延伸PCR技术，将大豆的4-香豆酰辅酶A连接酶基因4CL1和查尔酮异构酶基因CHI以及高粱的查尔酮合酶基因CHS2用柔性连接肽(GGGGS)2相连接，构建原核表达质粒pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI，将原核表达质粒pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI转化大肠杆菌，IPTG诱导表达、纯化，得到重组三功能酶；
步骤4、建立基于丙二酰辅酶A再生的体外酶促合成体系
基于丙二酰辅酶A再生的体外酶促合成体系至少包含Tris-HCl、磷酸钾缓冲液、甘油、MgCl2、ATP、CoA、醋酸钠、NaHCO3、ACS、ACC1，并在该体系中加入用于合成柚皮素的重组三功能酶，然后在一定条件下反应得到反应产物。
本发明设计一种丙二酰辅酶A再生的反应体系，在构建乙酰辅酶A合成酶ACS基因与乙酰辅酶A羧化酶ACC1基因的重组表达质粒的基础上，将重组质粒分别转化大肠杆菌和酵母细胞，表达目的蛋白，并将纯化的ACS与ACC1重组蛋白加入柚皮素体外酶促合成体系中，实现了丙二酰辅酶A的再生，以及以4-香豆酸为底物在体外低成本酶促合成柚皮素。本发明设计了一个新的反应体系，无需添加昂贵的丙二酰辅酶A便能对其进行再生利用，并最终生成柚皮素。该体系仅需添加低成本的无机盐、ATP和能够维持丙二酰辅酶A循环生成的辅酶A，反应产物单一，易于分离纯化，成本可控，使柚皮素体外合成在生产层面成为可能，同时为其他利用丙二酰辅酶A进行生产的方案提供新思路。
所述步骤1中，克隆ACS基因时，设计一对PCR引物：正向引物为5’-GCTGATATCGGATCC GAATTC atgagccaaattcacaaacac-3’，斜体碱基示酶切位点EcoRI；反向引物为5’-GTGGTGGTGGTGGTG CTCGAG ttacgatggcatcgcgatag-3’，斜体碱基示酶切位点XhoI；
克隆ACC1基因时，设计一对PCR引物：正向引物为5’-CTATTTCGAAACGAG GAATTC ATGAGCGAAGAAAGCTTATTC-3’，斜体碱基示酶切位点EcoRI；反向引物为5’-TCGGGCCCAAGCTG GCG GCCGC CTTTCAAAGTCTTCAACAATTTTTC-3’，斜体碱基示酶切位点NotI。
采用南京诺唯赞生物科技股份有限公司的非连接酶依赖型单片段快速克隆试剂盒（ClonExpress II One Step Cloning Kit）将PCR片段分别克隆至原核表达质粒pET-32a(+)和酵母表达质粒pGAP-Neo。
所述步骤2中，取重组质粒pET-32a-ACS，常规转化感受态大肠杆菌BL21(DE3)，37℃、220 rpm培养过夜；挑取3～5个菌落，接种至含有100 µg/mL氨苄青霉素的LB培养基中，37 ℃、250 rpm培养过夜；按照1:100转接至300 mL含有100 µg/mL氨苄青霉素的LB培养基，37 ℃ 250 rpm培养约2 h，至菌液OD600nm达0.4～0.6；向菌液中加入IPTG至终浓度0.1 mM，20 ℃诱导表达4 h，4 ℃离心收集菌体；参照碧云天BeyoGold™ His-tag PurificationResin试剂说明书纯化重组蛋白；用Tris-Cl缓冲液（pH 7.5）常规透析纯化的重组蛋白，再用PEG 20000进行浓缩，收集蛋白溶液，加入甘油至终浓度10%，分装，-80 ℃保存备用。
取重组质粒pGAP-Neo-ACC1，LiCl法转化毕赤酵母GS115，涂板含有500 µg/mL G-418的YPD平板，30 ℃恒温培养箱中倒置培养；3 d后，挑取单菌落，接种至2 mL含有500 µg/mL G-418的YPD液体培养基中，30 ℃、250 rpm培养24 h；按1：100转接300 mL含有500 µg/mL G-418的YPD液体培养基，30 ℃、250 rpm继续培养12 h；4 ℃、5000 g离心10 min，收集菌体；加入16.5 g 0.4 mm玻璃珠和21 mL非变性裂解液，在涡旋振荡仪中破碎酵母菌体；离心，收集裂解上清，进行硫酸铵沉淀，硫酸铵终浓度为40%饱和度；4 ℃、7000 g离心20 min，弃上清，沉淀用非变性裂解液溶解；参照碧云天BeyoGold™ His-tag Purification Resin试剂说明书纯化重组蛋白；配制pH 7.5透析缓冲液（含100 mM Tris-HCl和100 mM磷酸钾），对纯化后的蛋白透析3次，3 h/次；PEG 20000浓缩透析后的蛋白溶液，加入甘油至终浓度10%，分装，-80 ℃保存备用。
所述步骤3中，利用重叠延伸PCR技术拼接基因片段，将大豆的4-香豆酰辅酶A连接酶基因4CL1和查尔酮异构酶基因CHI，高梁的查尔酮合酶基因CHS2重组为单一序列，克隆至原核表达载体pET-32a-SUMO，构建重组质粒
pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI，各个关键酶之间通过连接肽(GGGGS)2相连接。将上述重组质粒按常规方法转化大肠杆菌BL21(DE3)或Rosetta(DE3),经0.05 mM IPTG于16 ℃诱导表达12 h，利用镍柱亲和纯化重组三功能酶蛋白，透析，PEG20000浓缩，加入甘油至终浓度10%，分装，-80 ℃保存备用。
构建重组质粒pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI过程中，克隆4CL1基因时，设计一对PCR引物：
正向引物为5’-GAACAGATTGGTGGT GGATCC ATGGCACCTTCTCCACAA-3’，斜体碱基示酶切位点BamHI；反向引物为5’-CGACCCACCTCCGCCCGACCCACCTCCGCCACTAGTATTGGCCACCACCAAACC-3’。
克隆CHS2基因时，设计一对PCR引物：
正向引物为5’-GGCGGAGGTGGGTCGGCTAGCATGGCCGGCGCGACTGTG-3’；反向引物为5’-ACTGCCCCCGCCACCCGATCCCCCGCCACCGGATCCGGCGGTGATGGCCGCTCCG-3’；
克隆CHI基因时，设计一对PCR引物：
正向引物为5’-GGTGGCGGGGGCAGTGAATTCATGGCAACGATCAGCGCG-3’；
反向引物为5’-GTGGTGGTGGTGGTG CTCGAG TCAGACTATAATGCCGTGGC-3’，斜体碱基示酶切位点XhoI。
利用重叠延伸PCR技术拼接各个基因片段，用南京诺唯赞生物科技股份有限公司的非连接酶依赖型单片段快速克隆试剂盒（ClonExpress II One Step Cloning Kit）将PCR片段克隆至原核表达质粒pET-32a-SUMO。
所述步骤4中，设计基于丙二酰辅酶A再生的柚皮素体外酶促合成体系，该体系包括Tris-HCl、磷酸钾缓冲液、甘油、MgCl2、BSA、DMSO、β-巯基乙醇、ATP、CoA、mM 醋酸钠、NaHCO3、4-香豆酸、ACS、ACC1、重组三功能酶。
所述步骤4中，设计基于丙二酰辅酶A再生的柚皮素体外酶促合成体系，该体系包括100 mM Tris-HCl（pH 6.8-8.1）、100 mM 磷酸钾缓冲液（pH 6.8-8.1）、10% 甘油、5 mMMgCl2、0.1 mg/mL BSA、1% DMSO、1 mM β-巯基乙醇、6 mM ATP、1 mM CoA、1～20 mM 醋酸钠、5～65 mM NaHCO3、0.3～1.5 mM 4-香豆酸、60 μg/mL ACS、60 μg/mL ACC1、60 μg/mL4CC，4CC为重组三功能酶；将配制的合成体系置于恒温摇床中，在25～35 ℃、600 rpm条件下反应6 h，加入NaOH溶液至终浓度为1 M，终止反应。
反应产物的检测：取上述已经终止反应的100 μL反应液，加入等体积乙酸乙酯，萃取2 h，吸取上层有机相。同上再次萃取，合并有机相，空气干燥，加入75 μL HPLC级甲醇溶解，再加入75 μL 50% HPLC级乙腈，混匀，经0.22 μm有机系微孔滤膜过滤，进行高效液相色谱分析（HPLC）。
本发明建立了一种丙二酰辅酶A的体外再生体系，并在此基础上以4-香豆酸为原料在重组三功能酶的催化下从头合成柚皮素，其产量高达3.99 mg/L；并且本发明的柚皮素体外酶促合成体系成分简单，不存在工程菌细胞内复杂的调控作用，反应过程易于精确控制、副产物少、产品分离相对简单、生产周期明显缩短、生产成本显著降低。
附图说明
图1为本发明三功能酶纯化蛋白的SDS-PAGE电泳图。图中：1表示ACS，2表示ACC1，3表示4CC。
图2为本发明中HPLC法分析三功能酶活性的结果图。图中：A表示4-香豆酸标准品，出峰时间为6.11 min；B表示柚皮素标准品，出峰时间为11.22 min；C表示实验组。
具体实施方式
下面结合实施例对本发明的技术方案做进一步的详细说明：本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护权限不限于下述的实施例。
实施例中所涉及材料的来源：
G-418购自Gibco公司；0.4 mm玻璃珠购自柏奥易杰EASYBIO公司。毕赤酵母GS115购自Invitrogen公司；非变性裂解液由本实验室配制，每升含6.9 g NaH2PO4•H2O，17.54 gNaCl，蒸馏水溶解后用10 M氢氧化钠调节pH值至8.0；pET-32a-SUMO载体由本实验室构建：将Novagen公司的pET-32a(+)载体中的硫氧还蛋白TrxA替换为小分子泛素样修饰蛋白SUMO后得到；pGAP-Neo载体由本实验室构建：将Invitrogen公司的pGAPZ A载体的博来霉素抗性基因替换为新霉素抗性基因后得到。
本实施例所涉及的其余试剂及材料均为市购，此处不再一一列举。本实施例中所涉及的“%”若没有特殊说明，一般为质量百分比。
实施例1
1.重组质粒的构建
根据GenBank数据库提供的大肠杆菌乙酰辅酶A合成酶基因ACS和酵母乙酰辅酶A羧化酶基因ACC1的核苷酸序列信息，设计两对引物序列，用于将ACS基因的编码区克隆至大肠杆菌表达载体pET-32a(+),将ACC1基因的编码区克隆至毕赤酵母表达载体pGAP-Neo：
PCR扩增ACS基因的正向引物为5’-GCTGATATCGGATCCGAATTCatgagccaaattcacaaacac-3’（如SEQ ID No.3所示），斜体碱基表示酶切位点EcoRI；反向引物为5’-GTGGTGGTGGTGGTGCTCGAGttacgatggcatcgcgatag-3’（如SEQ ID No.4所示），斜体碱基表示酶切位点XhoI。
PCR扩增ACC1基因的正向引物为
5’-CTATTTCGAAACGAGGAATTCATGAGCGAAGAAAGCTTATTC-3’（如SEQ ID No.5所示），斜体碱基表示酶切位点EcoRI；反向引物为5’-TCGGGCCCAAGCTGGCGGCCGCCTTTCAAAGTCTTCAACAATTTTTC-3’（如SEQ ID No.6所示），斜体碱基表示酶切位点NotI。
参照康为世纪Super Pfx DNA Polymerase操作说明建立如下PCR扩增体系：
试剂 100 μL反应体系
5×Super Pfx HF Buffer 20 μL
dNTP Mix，10 mM each 2 μL
Forward Primer，10 μM 5 μL
Reverse Primer，10 μM 5 μL
Template DNA 1 μL
Super Pfx DNA Polymerase 1 μL
ddH2O 66 μL
扩增ACS基因的模板DNA为培养4 h的大肠杆菌DH5α菌液1 μL，扩增ACC1基因的模板DNA为毕赤酵母GS115的cDNA，PCR反应条件如下：
步骤 温度 时间
预变性 98 ℃ 3 min
变性 98 ℃ 5 s
退火 45 ℃ 15 s
延伸 72 ℃ 2 min 30 s
终延伸 72 ℃ 10 min
PCR反应产物进行DNA凝胶电泳并对目的条带进行胶回收。载体pET-32a(+)和pGAP-Neo分别经EcoRI/XhoI和EcoRI/NotI双酶切，进行DNA凝胶电泳并对目的条带进行胶回收。使用南京诺唯赞生物科技股份有限公司的非连接酶依赖型单片段快速克隆试剂盒（ClonExpress II One Step Cloning Kit）连接目的片段和载体，常规转化感受态大肠杆菌DH5α，均匀涂布于氨苄抗性的LB平板，37 ℃过夜培养。第二天挑单菌落，接种至3 mL氨苄抗性的LB液体培养基中，37 ℃ 250 rpm培养15 h，提取质粒后进行测序鉴定，获得重组质粒pET-32a-ACS 和pGAP-Neo-ACC1。
用重叠延伸PCR技术拼接关键酶基因4CL1、CHS2和CHI，用南京诺唯赞生物科技股份有限公司的非连接酶依赖型单片段快速克隆试剂盒（ClonExpress II One StepCloning Kit）将PCR片段克隆至原核表达质粒pET-32a-SUMO，构建重组质粒pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI。构建重组质粒pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI过程中，根据黄豆的4-香豆酰辅酶A连接酶基因4CL1和查尔酮异构酶基因CHI以及高粱的查尔酮合酶基因CHS2设计引物，克隆4CL1基因时，设计一对PCR引物：
正向引物为5’-GAACAGATTGGTGGT GGATCC ATGGCACCTTCTCCACAA-3’，斜体碱基示酶切位点BamHI；反向引物为5’-CGACCCACCTCCGCCCGACCCACCTCCGCCACTAGTATTGGCCACCACCAAACC-3’。
克隆CHS2基因时，设计一对PCR引物：
正向引物为5’-GGCGGAGGTGGGTCGGCTAGCATGGCCGGCGCGACTGTG-3’；反向引物为5’-ACTGCCCCCGCCACCCGATCCCCCGCCACCGGATCCGGCGGTGATGGCCGCTCCG-3’；
克隆CHI基因时，设计一对PCR引物：
正向引物为5’-GGTGGCGGGGGCAGTGAATTCATGGCAACGATCAGCGCG-3’；
反向引物为5’-GTGGTGGTGGTGGTG CTCGAG TCAGACTATAATGCCGTGGC-3’，斜体碱基示酶切位点XhoI。
2.重组蛋白的诱导表达与纯化
2.1ACS的诱导表达与纯化
取重组质粒pET-32a-ACS，常规转化感受态大肠杆菌BL21(DE3)，37 ℃、220 rpm培养过夜；挑取3～5个菌落，接种至含有100 µg/mL氨苄青霉素的LB培养基中，37 ℃、250 rpm培养过夜；按照1:100转接至300 mL含有100 µg/mL氨苄青霉素的LB培养基，37 ℃ 250 rpm培养约2 h，至菌液OD600nm达0.4～0.6；向菌液中加入IPTG至终浓度0.1 mM，20 ℃诱导表达4h，4 ℃离心收集菌体；参照碧云天BeyoGold™ His-tag Purification Resin试剂说明书纯化重组蛋白；用Tris-Cl缓冲液（pH 7.5）常规透析纯化的重组蛋白，再用PEG 20000进行浓缩，收集蛋白溶液，加入甘油至终浓度10%，分装，-80 ℃保存备用。重组蛋白ACS的氨基酸序列如SEQ ID No.1所示。
2.2ACC1的表达与纯化
取重组质粒pGAP-Neo-ACC1，采用LiCl法转化毕赤酵母GS115（购自Invitrogen公司），涂板为含有500 µg/mL遗传霉素G-418（购自Gibco公司）的YPD平板，30 ℃恒温培养箱中倒置培养；3 d后，挑取单菌落，接种至2 mL含有500 µg/mL G-418的YPD液体培养基中，30℃、250 rpm培养24 h；按质量比1：100转接到300 mL含有500 µg/mL G-418的YPD液体培养基，30 ℃、250 rpm继续培养12 h；4 ℃、5000 g离心10 min，收集菌体；加入16.5 g 0.4 mm玻璃珠（购自柏奥易杰EASYBIO公司）和21 mL非变性裂解液（由本实验室配制，称取6.9 gNaH2PO4•H2O和17.54 g NaCl，溶于800 mL蒸馏水，用10 M氢氧化钠调节pH值至8.0，再用蒸馏水定容至1 L），在涡旋振荡仪中破碎酵母菌体；离心，收集裂解上清，进行硫酸铵沉淀，硫酸铵终浓度为40%饱和度；4 ℃、7000 g离心20 min，弃上清，沉淀用非变性裂解液溶解；参照碧云天BeyoGold™ His-tag Purification Resin试剂说明书纯化重组蛋白；配制pH7.5透析缓冲液（含100 mM Tris-HCl和100 mM磷酸钾），对纯化后的蛋白透析3次，3 h/次；PEG 20000浓缩透析后的蛋白溶液，加入甘油至终浓度10%，分装，-80 ℃保存备用。重组蛋白ACC1的氨基酸序列如SEQ ID No.2所示。
2.3重组三功能酶的油道表达与纯化
取原核表达质粒pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI，按常规方法转化大肠杆菌BL21(DE3)或Rosetta(DE3),经0.05 mM IPTG于16 ℃诱导表达12 h，利用镍柱亲和纯化重组三功能酶蛋白4CC，透析，PEG 20000浓缩，加入甘油至终浓度10%，分装，-80℃保存备用。
对上述三种纯化蛋白进行SDS-PAGE电泳，结果见图1，图中1表示ACS，2表示ACC1,3表示4CC。
3.基于丙二酰辅酶A再生的柚皮素体外酶促合成体系的建立
设计丙二酰辅酶A的体外合成体系，该体系含有100 mM Tris-HCl（pH 7.5）、100mM 磷酸钾缓冲液（pH 7.5）、10% 甘油、5 mM MgCl2、0.1 mg/mL BSA、1% DMSO、1 mM β-巯基乙醇、6 mM ATP、1 mM CoA、10 mM 醋酸钠、50 mM NaHCO3、0.6 mM 4-香豆酸、60 μg/mLACS、60 μg/mL ACC1、60 μg/mL 4CC。将配制的合成体系置恒温摇床中， 30 ℃、600 rpm反应6 h，加入NaOH溶液至终浓度为1 M，终止反应。
4.产品柚皮素的检测条件
取上述已经终止反应的反应液100 μL，加入等体积乙酸乙酯，萃取2 h，吸取上层有机相。同上再次萃取，合并有机相，空气干燥，加入75 μL HPLC级甲醇溶解，再加入75 μL50% HPLC级乙腈，混匀，经0.22 μm有机系微孔滤膜过滤，进行高效液相色谱分析（HPLC），HPLC检测结果如图2所示，图中A表示4-香豆酸标准品，出峰时间为6.11 min；B表示柚皮素标准品，出峰时间为11.22 min；C表示实验组（即上述丙二酰辅酶A的体外合成体系反应后的产物）。HPLC检测条件如下：
仪器设备：美国安捷伦公司1260液质联用仪(Agilent 1260)。
色谱柱：Agilent C18column(150×4.6 mm，5 μm，ThermoFisherScientific.Inc.，San Jose，CA，USA)。柱温：35 ℃。
流动相：A(乙腈)，B(Milli Q水)，梯度洗脱：0-4 min，A：20％-22％，B：80％-78％；4-7 min，A：22％-42％，B：78％-58％；7-10 min，A：42％-38％，B：58％-62％；10-15 min，A：38％-95％，B：62％-5％；15-24 min，A：95％，B：5％；24-25 min，A：95％-20％，B：5％-80％。
进样量：20 μL；流速：1 mL/min；
检测器：紫外检测器(VWD)；
检测波长：λ＝280 nm；330 nm。
经测定，柚皮素的产量为3.99 mg/L。
柚皮素具有多种药理生理活性，在生物医药领域具有广泛的应用前景。与微生物细胞工厂发酵法相比较，本发明提供了一种操作简便、产品分离相对简单、生产周期明显缩短、生产成本显著降低的制备方法。以酿酒酵母生产柚皮素为例，发酵培养一般超过48小时，而使用本发明的制备方法，每次耗时仅3小时，受原材料和人力的限制较小，易于放大。如果算上人工成本，则相对生产成本还会进一步降低，因此潜在的经济效益将是相当可观的。
以上所述，仅为本发明中的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉该技术的人在本发明所揭露的技术范围内，可理解想到的变换或替换，都应涵盖在本发明的包含范围之内，因此，本发明的保护范围应该以权利要求书的保护范围为准。
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Ala Lys Ala Glu Thr Phe His Glu Met Glu Lys Ala Ala Val Arg Leu
340 345 350
Gly Lys Leu Val Gly Tyr Val Ser Ala Gly Thr Val Glu Tyr Leu Tyr
355 360 365
Ser His Asp Asp Gly Lys Phe Tyr Phe Leu Glu Leu Asn Pro Arg Leu
370 375 380
Gln Val Glu His Pro Thr Thr Glu Met Val Ser Gly Val Asn Leu Pro
385 390 395 400
Ala Ala Gln Leu Gln Ile Ala Met Gly Ile Pro Met His Arg Ile Ser
405 410 415
Asp Ile Arg Thr Leu Tyr Gly Met Asn Pro His Ser Ala Ser Glu Ile
420 425 430
Asp Phe Glu Phe Lys Thr Gln Asp Ala Thr Lys Lys Gln Arg Arg Pro
435 440 445
Ile Pro Lys Gly His Cys Thr Ala Cys Arg Ile Thr Ser Glu Asp Pro
450 455 460
Asn Asp Gly Phe Lys Pro Ser Gly Gly Thr Leu His Glu Leu Asn Phe
465 470 475 480
Arg Ser Ser Ser Asn Val Trp Gly Tyr Phe Ser Val Gly Asn Asn Gly
485 490 495
Asn Ile His Ser Phe Ser Asp Ser Gln Phe Gly His Ile Phe Ala Phe
500 505 510
Gly Glu Asn Arg Gln Ala Ser Arg Lys His Met Val Val Ala Leu Lys
515 520 525
Glu Leu Ser Ile Arg Gly Asp Phe Arg Thr Thr Val Glu Tyr Leu Ile
530 535 540
Lys Leu Leu Glu Thr Glu Asp Phe Glu Asp Asn Thr Ile Thr Thr Gly
545 550 555 560
Trp Leu Asp Asp Leu Ile Thr His Lys Met Thr Ala Glu Lys Pro Asp
565 570 575
Pro Thr Leu Ala Val Ile Cys Gly Ala Ala Thr Lys Ala Phe Leu Ala
580 585 590
Ser Glu Glu Ala Arg His Lys Tyr Ile Glu Ser Leu Gln Lys Gly Gln
595 600 605
Val Leu Ser Lys Asp Leu Leu Gln Thr Met Phe Pro Val Asp Phe Ile
610 615 620
His Glu Gly Lys Arg Tyr Lys Phe Thr Val Ala Lys Ser Gly Asn Asp
625 630 635 640
Arg Tyr Thr Leu Phe Ile Asn Gly Ser Lys Cys Asp Ile Ile Leu Arg
645 650 655
Gln Leu Ser Asp Gly Gly Leu Leu Ile Ala Ile Gly Gly Lys Ser His
660 665 670
Thr Ile Tyr Trp Lys Glu Glu Val Ala Ala Thr Arg Leu Ser Val Asp
675 680 685
Ser Met Thr Thr Leu Leu Glu Val Glu Asn Asp Pro Thr Gln Leu Arg
690 695 700
Thr Pro Ser Pro Gly Lys Leu Val Lys Phe Leu Val Glu Asn Gly Glu
705 710 715 720
His Ile Ile Lys Gly Gln Pro Tyr Ala Glu Ile Glu Val Met Lys Met
725 730 735
Gln Met Pro Leu Val Ser Gln Glu Asn Gly Ile Val Gln Leu Leu Lys
740 745 750
Gln Pro Gly Ser Thr Ile Val Ala Gly Asp Ile Met Ala Ile Met Thr
755 760 765
Leu Asp Asp Pro Ser Lys Val Lys His Ala Leu Pro Phe Glu Gly Met
770 775 780
Leu Pro Asp Phe Gly Ser Pro Val Ile Glu Gly Thr Lys Pro Ala Tyr
785 790 795 800
Lys Phe Lys Ser Leu Val Ser Thr Leu Glu Asn Ile Leu Lys Gly Tyr
805 810 815
Asp Asn Gln Val Ile Met Asn Ala Ser Leu Gln Gln Leu Ile Glu Val
820 825 830
Leu Arg Asn Pro Lys Leu Pro Tyr Ser Glu Trp Lys Leu His Ile Ser
835 840 845
Ala Leu His Ser Arg Leu Pro Ala Lys Leu Asp Glu Gln Met Glu Glu
850 855 860
Leu Val Ala Arg Ser Leu Arg Arg Gly Ala Val Phe Pro Ala Arg Gln
865 870 875 880
Leu Ser Lys Leu Ile Asp Met Ala Val Lys Asn Pro Glu Tyr Asn Pro
885 890 895
Asp Lys Leu Leu Gly Ala Val Val Glu Pro Leu Ala Asp Ile Ala His
900 905 910
Lys Tyr Ser Asn Gly Leu Glu Ala His Glu His Ser Ile Phe Val His
915 920 925
Phe Leu Glu Glu Tyr Tyr Glu Val Glu Lys Leu Phe Asn Gly Pro Asn
930 935 940
Val Arg Glu Glu Asn Ile Ile Leu Lys Leu Arg Asp Glu Asn Pro Lys
945 950 955 960
Asp Leu Asp Lys Val Ala Leu Thr Val Leu Ser His Ser Lys Val Ser
965 970 975
Ala Lys Asn Asn Leu Ile Leu Ala Ile Leu Lys His Tyr Gln Pro Leu
980 985 990
Cys Lys Leu Ser Ser Lys Val Ser Ala Ile Phe Ser Thr Pro Leu Gln
995 1000 1005
His Ile Val Glu Leu Glu Ser Lys Ala Thr Ala Lys Val Ala Leu Gln
1010 1015 1020
Ala Arg Glu Ile Leu Ile Gln Gly Ala Leu Pro Ser Val Lys Glu Arg
1025 1030 1035 1040
Thr Glu Gln Ile Glu His Ile Leu Lys Ser Ser Val Val Lys Val Ala
1045 1050 1055
Tyr Gly Ser Ser Asn Pro Lys Arg Ser Glu Pro Asp Leu Asn Ile Leu
1060 1065 1070
Lys Asp Leu Ile Asp Ser Asn Tyr Val Val Phe Asp Val Leu Leu Gln
1075 1080 1085
Phe Leu Thr His Gln Asp Pro Val Val Thr Ala Ala Ala Ala Gln Val
1090 1095 1100
Tyr Ile Arg Arg Ala Tyr Arg Ala Tyr Thr Ile Gly Asp Ile Arg Val
1105 1110 1115 1120
His Glu Gly Val Thr Val Pro Ile Val Glu Trp Lys Phe Gln Leu Pro
1125 1130 1135
Ser Ala Ala Phe Ser Thr Phe Pro Thr Val Lys Ser Lys Met Gly Met
1140 1145 1150
Asn Arg Ala Val Ser Val Ser Asp Leu Ser Tyr Val Ala Asn Ser Gln
1155 1160 1165
Ser Ser Pro Leu Arg Glu Gly Ile Leu Met Ala Val Asp His Leu Asp
1170 1175 1180
Asp Val Asp Glu Ile Leu Ser Gln Ser Leu Glu Val Ile Pro Arg His
1185 1190 1195 1200
Gln Ser Ser Ser Asn Gly Pro Ala Pro Asp Arg Ser Gly Ser Ser Ala
1205 1210 1215
Ser Leu Ser Asn Val Ala Asn Val Cys Val Ala Ser Thr Glu Gly Phe
1220 1225 1230
Glu Ser Glu Glu Glu Ile Leu Val Arg Leu Arg Glu Ile Leu Asp Leu
1235 1240 1245
Asn Lys Gln Glu Leu Ile Asn Ala Ser Ile Arg Arg Ile Thr Phe Met
1250 1255 1260
Phe Gly Phe Lys Asp Gly Ser Tyr Pro Lys Tyr Tyr Thr Phe Asn Gly
1265 1270 1275 1280
Pro Asn Tyr Asn Glu Asn Glu Thr Ile Arg His Ile Glu Pro Ala Leu
1285 1290 1295
Ala Phe Gln Leu Glu Leu Gly Arg Leu Ser Asn Phe Asn Ile Lys Pro
1300 1305 1310
Ile Phe Thr Asp Asn Arg Asn Ile His Val Tyr Glu Ala Val Ser Lys
1315 1320 1325
Thr Ser Pro Leu Asp Lys Arg Phe Phe Thr Arg Gly Ile Ile Arg Thr
1330 1335 1340
Gly His Ile Arg Asp Asp Ile Ser Ile Gln Glu Tyr Leu Thr Ser Glu
1345 1350 1355 1360
Ala Asn Arg Leu Met Ser Asp Ile Leu Asp Asn Leu Glu Val Thr Asp
1365 1370 1375
Thr Ser Asn Ser Asp Leu Asn His Ile Phe Ile Asn Phe Ile Ala Val
1380 1385 1390
Phe Asp Ile Ser Pro Glu Asp Val Glu Ala Ala Phe Gly Gly Phe Leu
1395 1400 1405
Glu Arg Phe Gly Lys Arg Leu Leu Arg Leu Arg Val Ser Ser Ala Glu
1410 1415 1420
Ile Arg Ile Ile Ile Lys Asp Pro Gln Thr Gly Ala Pro Val Pro Leu
1425 1430 1435 1440
Arg Ala Leu Ile Asn Asn Val Ser Gly Tyr Val Ile Lys Thr Glu Met
1445 1450 1455
Tyr Thr Glu Val Lys Asn Ala Lys Gly Glu Trp Val Phe Lys Ser Leu
1460 1465 1470
Gly Lys Pro Gly Ser Met His Leu Arg Pro Ile Ala Thr Pro Tyr Pro
1475 1480 1485
Val Lys Glu Trp Leu Gln Pro Lys Arg Tyr Lys Ala His Leu Met Gly
1490 1495 1500
Thr Thr Tyr Val Tyr Asp Phe Pro Glu Leu Phe Arg Gln Ala Ser Ser
1505 1510 1515 1520
Ser Gln Trp Lys Asn Phe Ser Ala Asp Val Lys Leu Thr Asp Asp Phe
1525 1530 1535
Phe Ile Ser Asn Glu Leu Ile Glu Asp Glu Asn Gly Glu Leu Thr Glu
1540 1545 1550
Val Glu Arg Glu Pro Gly Ala Asn Ala Ile Gly Met Val Ala Phe Lys
1555 1560 1565
Ile Thr Val Lys Thr Pro Glu Tyr Pro Arg Gly Arg Gln Phe Val Val
1570 1575 1580
Val Ala Asn Asp Ile Thr Phe Lys Ile Gly Ser Phe Gly Pro Gln Glu
1585 1590 1595 1600
Asp Glu Phe Phe Asn Lys Val Thr Glu Tyr Ala Arg Lys Arg Gly Ile
1605 1610 1615
Pro Arg Ile Tyr Leu Ala Ala Asn Ser Gly Ala Arg Ile Gly Met Ala
1620 1625 1630
Glu Glu Ile Val Pro Leu Phe Gln Val Ala Trp Asn Asp Ala Ala Asn
1635 1640 1645
Pro Asp Lys Gly Phe Gln Tyr Leu Tyr Leu Thr Ser Glu Gly Met Glu
1650 1655 1660
Thr Leu Lys Lys Phe Asp Lys Glu Asn Ser Val Leu Thr Glu Arg Thr
1665 1670 1675 1680
Val Ile Asn Gly Glu Glu Arg Phe Val Ile Lys Thr Ile Ile Gly Ser
1685 1690 1695
Glu Asp Gly Leu Gly Val Glu Cys Leu Arg Gly Ser Gly Leu Ile Ala
1700 1705 1710
Gly Ala Thr Ser Arg Ala Tyr His Asp Ile Phe Thr Ile Thr Leu Val
1715 1720 1725
Thr Cys Arg Ser Val Gly Ile Gly Ala Tyr Leu Val Arg Leu Gly Gln
1730 1735 1740
Arg Ala Ile Gln Val Glu Gly Gln Pro Ile Ile Leu Thr Gly Ala Pro
1745 1750 1755 1760
Ala Ile Asn Lys Met Leu Gly Arg Glu Val Tyr Thr Ser Asn Leu Gln
1765 1770 1775
Leu Gly Gly Thr Gln Ile Met Tyr Asn Asn Gly Val Ser His Leu Thr
1780 1785 1790
Ala Val Asp Asp Leu Ala Gly Val Glu Lys Ile Val Glu Trp Met Ser
1795 1800 1805
Tyr Val Pro Ala Lys Arg Asn Met Pro Val Pro Ile Leu Glu Thr Lys
1810 1815 1820
Asp Thr Trp Asp Arg Pro Val Asp Phe Thr Pro Thr Asn Asp Glu Thr
1825 1830 1835 1840
Tyr Asp Val Arg Trp Met Ile Glu Gly Arg Glu Thr Glu Ser Gly Phe
1845 1850 1855
Glu Tyr Gly Leu Phe Asp Lys Gly Ser Phe Phe Glu Thr Leu Ser Gly
1860 1865 1870
Trp Ala Lys Gly Val Val Val Gly Arg Ala Arg Leu Gly Gly Ile Pro
1875 1880 1885
Leu Gly Val Ile Gly Val Glu Thr Arg Thr Val Glu Asn Leu Ile Pro
1890 1895 1900
Ala Asp Pro Ala Asn Pro Asn Ser Ala Glu Thr Leu Ile Gln Glu Pro
1905 1910 1915 1920
Gly Gln Val Trp His Pro Asn Ser Ala Phe Lys Thr Ala Gln Ala Ile
1925 1930 1935
Asn Asp Phe Asn Asn Gly Glu Gln Leu Pro Met Met Ile Leu Ala Asn
1940 1945 1950
Trp Arg Gly Phe Ser Gly Gly Gln Arg Asp Met Phe Asn Glu Val Leu
1955 1960 1965
Lys Tyr Gly Ser Phe Ile Val Asp Ala Leu Val Asp Tyr Lys Gln Pro
1970 1975 1980
Ile Ile Ile Tyr Ile Pro Pro Thr Gly Glu Leu Arg Gly Gly Ser Trp
1985 1990 1995 2000
Val Val Val Asp Pro Thr Ile Asn Ala Asp Gln Met Glu Met Tyr Ala
2005 2010 2015
Asp Val Asn Ala Arg Ala Gly Val Leu Glu Pro Gln Gly Met Val Gly
2020 2025 2030
Ile Lys Phe Arg Arg Glu Lys Leu Leu Asp Thr Met Asn Arg Leu Asp
2035 2040 2045
Asp Lys Tyr Arg Glu Leu Arg Ser Gln Leu Ser Asn Lys Ser Leu Ala
2050 2055 2060
Pro Glu Val His Gln Gln Ile Ser Lys Gln Leu Ala Asp Arg Glu Arg
2065 2070 2075 2080
Glu Leu Leu Pro Ile Tyr Gly Gln Ile Ser Leu Gln Phe Ala Asp Leu
2085 2090 2095
His Asp Arg Ser Ser Arg Met Val Ala Lys Gly Val Ile Ser Lys Glu
2100 2105 2110
Leu Glu Trp Thr Glu Ala Arg Arg Phe Phe Phe Trp Arg Leu Arg Arg
2115 2120 2125
Arg Leu Asn Glu Glu Tyr Leu Ile Lys Arg Leu Ser His Gln Val Gly
2130 2135 2140
Glu Ala Ser Arg Leu Glu Lys Ile Ala Arg Ile Arg Ser Trp Tyr Pro
2145 2150 2155 2160
Ala Ser Val Asp His Glu Asp Asp Arg Gln Val Ala Thr Trp Ile Glu
2165 2170 2175
Glu Asn Tyr Lys Thr Leu Asp Asp Lys Leu Lys Gly Leu Lys Leu Glu
2180 2185 2190
Ser Phe Ala Gln Asp Leu Ala Lys Lys Ile Arg Ser Asp His Asp Asn
2195 2200 2205
Ala Ile Asp Gly Leu Ser Glu Val Ile Lys Met Leu Ser Thr Asp Asp
2210 2215 2220
Lys Glu Lys Leu Leu Lys Thr Leu Lys Ala Ala Ala Ser Phe Leu Glu
2225 2230 2235 2240
Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His
2245 2250 2255
His His His His
2260
<210> 3
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gctgatatcg gatccgaatt catgagccaa attcacaaac ac 42
<210> 4
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gtggtggtgg tggtgctcga gttacgatgg catcgcgata g 41
<210> 5
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctatttcgaa acgaggaatt catgagcgaa gaaagcttat tc 42
<210> 6
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tcgggcccaa gctggcggcc gcctttcaaa gtcttcaaca atttttc 47

Claims (9)

1.一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，其特征在于，包括以下步骤：
步骤1、从大肠杆菌中克隆ACS基因，构建重组质粒pET-32a-ACS，从毕赤酵母中克隆ACC1基因，构建重组质粒pGAP-Neo-ACC1；
步骤2、将重组质粒pET-32a-ACS转化感受态大肠杆菌，IPTG诱导表达、纯化，得到重组蛋白ACS；同时将重组质粒pGAP-Neo-ACC1转化毕赤酵母，经过表达和纯化，得到重组蛋白ACC1；
步骤3、将大豆的4-香豆酰辅酶A连接酶基因4CL1和查尔酮异构酶基因CHI以及高粱的查尔酮合酶基因CHS2用柔性连接肽(GGGGS)2相连接，构建原核表达质粒pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI，将原核表达质粒pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI转化大肠杆菌，IPTG诱导表达、纯化，得到重组三功能酶；
步骤4、设计基于丙二酰辅酶A再生的柚皮素体外酶促合成体系，该体系包括Tris-HCl、磷酸钾缓冲液、甘油、MgCl2、BSA、DMSO、β-巯基乙醇、ATP、CoA、醋酸钠、NaHCO3、4-香豆酸、ACS、ACC1、重组三功能酶，然后在一定条件下反应得到反应产物。
2.根据权利要求1所述一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，其特征在于：所述步骤3中，利用重叠延伸PCR技术将大豆的4-香豆酰辅酶A连接酶基因4CL1和查尔酮异构酶基因CHI，高梁的查尔酮合酶基因CHS2克隆至原核表达载体pET-32a-SUMO，构建重组质粒
pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI，将该重组质粒按常规方法转化大肠杆菌BL21(DE3)或Rosetta(DE3),经IPTG诱导表达，利用镍柱亲和纯化获得重组三功能酶蛋白。
3.根据权利要求2所述一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，其特征在于，构建重组质粒pET-32a-SUMO-4CL1-(GGGGS)2-CHS2-(GGGGS)2-CHI过程中，克隆4CL1基因时，设计一对PCR引物：
正向引物为5’-GAACAGATTGGTGGT GGATCC ATGGCACCTTCTCCACAA-3’；反向引物为5’-CGACCCACCTCCGCCCGACCCACCTCCGCCACTAGTATTGGCCACCACCAAACC-3’。
4.根据权利要求2所述一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，其特征在于，
克隆CHS2基因时，设计一对PCR引物：
正向引物为5’-GGCGGAGGTGGGTCGGCTAGCATGGCCGGCGCGACTGTG-3’；反向引物为5’-ACTGCCCCCGCCACCCGATCCCCCGCCACCGGATCCGGCGGTGATGGCCGCTCCG-3’。
5.根据权利要求2所述一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，其特征在于，克隆CHI基因时，设计一对PCR引物：
正向引物为5’-GGTGGCGGGGGCAGTGAATTCATGGCAACGATCAGCGCG-3’；
反向引物为5’-GTGGTGGTGGTGGTG CTCGAG TCAGACTATAATGCCGTGGC-3’。
6. 根据权利要求5所述一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，其特征在于，所述步骤4中，设计基于丙二酰辅酶A再生的柚皮素体外酶促合成体系，该体系包括100 mM Tris-HCl、100 mM 磷酸钾缓冲液、10% 甘油、5 mM MgCl2、0.1 mg/mL BSA、1%DMSO、1 mM β-巯基乙醇、6 mM ATP、1 mM CoA、1～20 mM 醋酸钠、5～65 mM NaHCO3、0.3～1.5 mM 4-香豆酸、60 μg/mL ACS、60 μg/mL ACC1、60 μg/mL重组三功能酶。
7. 根据权利要求6所述一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，其特征在于，将配制的合成体系置于恒温摇床中，在25～35 ℃、600 rpm条件下反应6 h，加入NaOH至终浓度为1 M，终止反应。
8. 根据权利要求1所述一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，其特征在于，所述步骤1中，克隆ACS基因时，设计一对PCR引物：正向引物为5’-GCTGATATCGGATCC GAATTC atgagccaaattcacaaacac-3’；反向引物为5’-GTGGTGGTGGTGGTG CTCGAG ttacgatggcatcgcgatag-3’。
9. 根据权利要求1所述一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法，其特征在于，所述步骤1中，克隆
ACC1
基因时，设计一对PCR引物：正向引物为5’-CTATTTCGAAACGAG
GAATTC
ATGAGCGAAGAAAGCTTATTC-3’；反向引物为5’-TCGGGCCCAAGCTG
GCGGCCGC
CTTTCAAAGTCTTCAACAATTTTTC-3’。
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WO2003000898A1 (en) * 2001-06-22 2003-01-03 Syngenta Participations Ag Plant genes involved in defense against pathogens
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