CN109234337B - 一种槲皮素糖苷的生物合成方法 - Google Patents

一种槲皮素糖苷的生物合成方法 Download PDF

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CN109234337B
CN109234337B CN201811073927.2A CN201811073927A CN109234337B CN 109234337 B CN109234337 B CN 109234337B CN 201811073927 A CN201811073927 A CN 201811073927A CN 109234337 B CN109234337 B CN 109234337B
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贾红华
李艳
蔡如鑫
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陈可泉
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Abstract

本发明公开了一种槲皮素糖苷的生物合成方法,将糖基转移酶UGT73G1突变体的基因与蔗糖合酶基因连接得到重组质粒，构建含双酶体系的重组菌；将重组菌接种于LB液体培养基中，培养，添加诱导剂乳糖诱导，发酵液离心收集菌体，菌体破碎，离心收集上清液得到粗酶液；将槲皮素或异槲皮素溶于DMSO中，加入粗酶液、蔗糖，反应，离心得上清槲皮素‑3，4’‑二葡萄糖糖苷。所述糖基转移酶UGT73G1突变体的氨基酸序列是SEQ ID NO:1所示序列发生V371A和/或F381Y突变的氨基酸序列。该突变体制备简单，产量大，实现了槲皮素‑3，4’二葡萄糖糖苷产量的提高。

Description

一种槲皮素糖苷的生物合成方法
技术领域
本发明属于基因工程技术领域，具体涉及一种槲皮素糖苷的生物合成方法。
背景技术
槲皮素（Quercetin），又称栎精、槲皮素黄，作为一种天然黄酮类化合物，广泛存在于植物的花、叶、果实中，多以甙的形式存在，具有很好的祛痰、止咳作用。此外还有降低血压、增强毛细血管抵抗力、减少毛细血管脆性、降血脂、扩张冠状动脉，增加冠脉血流量等作用。在美国，槲皮素治疗前列腺癌已是非处方药，但在中国，还没批准该药物作为前列腺治疗药物。槲皮素不溶于水，导入亲水性基团可增加其溶解性，便于吸收，从而增强其药理作用，将槲皮素糖基化是一种增加其溶解性的有效手段。专利201110343655.5报道了利用糖苷酶对槲皮素进行糖基化作用，产量0.148g/L,转化率14.81%.转化率及产量相对较低。目前国内外糖基化合物的研究热点主要是黄酮衍生物的合成，但是由于大多数黄酮苷元及其黄酮苷类在水相中溶解度低，限制了其制剂的开发。槲皮素-3，4’-二葡萄糖糖苷是槲皮素糖基化的产物，比槲皮素有更好的水溶性和药理作用。它存在于洋葱的表皮细胞中，但含量极少，表皮细胞干重含量只有0.2g-7.4g/kg，提取困难。由于槲皮素来源广泛，且芦丁水解成槲皮素和异槲皮素工艺成熟。因此利用微生物转化具有原料优势，便于槲皮素-3，4’-二葡萄糖糖生产的应用。微生物转化法通过一步转化，工艺简单，便于调控，目前国内关于制备槲皮素-3，4’-二葡萄糖糖苷报道较少。
发明内容
本发明的目的是提供一种槲皮素糖苷的生物合成方法；
本发明的另一目的是提供一种糖基转移酶突变体及其制备方法，利用该基因实现槲皮素的糖基化修饰，制备得到槲皮素-3，4’二葡萄糖糖苷，并为工业化生产提供一定的借鉴。
为了实现上述目的，本申请采用的技术方案为：
一种适用于槲皮素糖苷合成的糖基转移酶突变体，糖基转移酶突变体的氨基酸序列是SEQ ID NO:1所示发生突变的氨基酸序列，突变的氨基酸位点选自S151，T168，K274，T293，G361,S365,V371及F381中的一个或多个。
进一步地，突变的氨基酸位点发生的突变包括如下任意一种或多种：S151T，T168S，K274W/E，T293G/V，G361K,S365T,V371A/G,F381Y，其中，“/”表示“或”。
F381Y为SEQ ID NO:1序列中第381位氨基酸由苯丙氨酸（F）突变为酪氨酸（Y）；S151T为ID NO:1序列中第151位氨基酸由丝氨酸（S）突变为苏氨酸（T），T168S为ID NO:1序列中第168位氨基酸由苏氨酸（T）突变为丝氨酸（S），K274W/E为ID NO:1序列中第274位氨基酸由赖氨酸（K）突变为色氨酸（W）或谷氨酸（E），T293G/V为ID NO:1序列中第293位氨基酸由苏氨酸（T）突变为甘氨酸（G）缬氨酸（V），G361K为ID NO:1序列中第361位氨基酸由甘氨酸（G）突变为赖氨酸（K），S365T为ID NO:1序列中第365位氨基酸由丝氨酸（S）突变为苏氨酸（T），V371A为ID NO:1序列中第371位氨基酸由缬氨酸（V）突变为丙氨酸（A）或甘氨酸（G）。
一种表达基因，该基因编码上述任一种糖基转移酶突变体。
一种重组质粒，该重组质粒连接有上述表达基因和蔗糖合酶基因SUS。
一种重组细胞，该重组细胞包含有上述重组表达载体或上述糖基转移酶突变体的表达基因。
上述糖基转移酶突变体在制备槲皮素-3，4’-二葡萄糖糖苷的应用。
槲皮素-3，4’二葡萄糖糖苷的生物合成方法，包括如下步骤：
1）构建含双酶体系的重组菌：将糖基转移酶UGT73G1或者其突变体的基因与蔗糖合酶基因连接得到重组质粒，采用热激法将连接产物转化到大肠杆菌BL21（DE3）感受态细胞中，得到含双酶体系的重组菌；
2) 重组菌的发酵诱导产酶：将重组菌接种于LB液体培养基中，37℃，培养至OD600达到2-3时，添加诱导剂乳糖至终浓度1g/L，25℃，200r/min条件诱导20h，发酵液离心收集菌体，菌体破碎，离心收集上清液得到粗酶液；
3）将槲皮素或异槲皮素溶于DMSO中，加入粗酶液、蔗糖，20-40℃条件下反应8-30h，加入甲醇终止反应，离心得上清槲皮素-3，4’-二葡萄糖糖苷。
糖基转移酶UGT73G1的基因序列如SEQ ID 6所示，蔗糖合酶基因的序列如SEQ ID:7所示。
本发明以糖基转移酶UGT73G1为基础，对其活性中心进行关键氨基酸的定点突变，获得了糖基转移酶突变体。将该糖基转移酶突变体与蔗糖合成酶偶连，以槲皮素或异槲皮素为底物，添加适量蔗糖，催化生成槲皮素-3，4’-二葡萄糖糖苷。该突变体制备简单，产量大，实现了槲皮素-3，4’-二葡萄糖糖苷和异槲皮素的产量的提高，相同条件下，突变体活性较原始酶提高了约两倍，产槲皮素-3，4’-二葡萄糖糖苷和异槲皮素的转化率较原始酶有显著的提高。
本发明通过突变提高了糖基转移酶UGT73G1的有机溶剂耐受性，极大提高底物溶解性，进而极大提高转化率及产物量。
有益效果：
本发明通过突变提高了糖基转移酶UGT73G1的有机溶剂耐受性及酶活，利用该突变体实现了槲皮素-3，4’二葡萄糖糖苷的生物合成，合成条件温和，操作简单，转化率及产物量较现有方法有大幅提高，具有较好的应用前景。
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阐述，但本发明所保护范围不限于此。
以下实施例中所采用的检测方法如下：
HPLC测定方法：色谱柱Agilent TC-C18 (4 .6mm×250mm，5μm);流动性（A）：水+千分之一的三氟乙酸，（B）乙腈+千分之一的三氟乙酸;梯度洗脱；流速(1 .0mL/min)；
检测器：350nm，进样量20ul，柱温40℃。
以下实施例中所采用的确认突变体的方法：
本研究用UGT73G1序列BLAST PDB数据库，获得多条与目标序列UGT73G1同源性相近的已知结构序列，经过分析，同源建模。初步同源建模完成后，进行底物UDPG的对接，并采用pymol软件中的amber插件进行能量优化。将优化后的结构模型与已有晶体结构模板对齐，结合序列与结构进行比对分析，结果发现，模板序列中位于活性中心的第381位氨基酸为酪氨酸（Y），而原始UGT73G1序列相应位置为苯丙氨酸（F）。由于酪氨酸在结构上比苯丙氨酸多一个对位的羟基，因此在这个位置会比苯丙氨酸与底物UDPG有利于与底物的结合。其他的氨基酸位点突变与该方法类似。
实施例1 糖基转移酶突变体的制备
（1）单突变
来源于洋葱的糖基转移酶UGT73G1的两种突变体V371A，氨基酸序列式SEQ IDNO.2，F381Y，氨基酸序列式SEQ ID NO.3所示；
根据糖基转移酶UGT73G1的基因序列，进行定点突变，测定DNA编码序列，并导入大肠杆菌中进行表达，得到单突变糖基转移酶，单突变V371A,F381Y均由金斯瑞生物科技有限公司合成构建到pet28a载体上。
V371A的定点突变引物为：
正向引物：
TAAGAAGGAGATATACATATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTG
反向引物：
GGTTTCTTTACCAGACTCGAGTCATTAGTGGTGGTGGTGGTGGTGTTTGTTGCGACGGTC
F381Y的定点突变引物为：
正向引物：
TAAGAAGGAGATATACATATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTG
反向引物：
GGTTTCTTTACCAGACTCGAGTCATTAGTGGTGGTGGTGGTGGTGTTTGTTGCGACGGTC
将构建好的质粒（质粒由金斯瑞公司构建，质粒模板来源Novagen公司）转化到E.coli BL21（DE3）感受态细胞中，转化液涂布于含有50ug/mL卡那霉素的LB固体培养基，37℃过夜培养，随机挑取单克隆至含5mL LB培养基(含50μg/mL卡那霉素)的摇管中，过夜培养，测序验证序列正确。
（2）双突变
洋葱来源的糖基转移酶的双突变体酶V371A/F381Y，氨基酸序列是SEQ ID NO.4：双突变体酶的制备方法，根据糖基转移酶UGT73G1的基因序列，进行定点突变，测定DNA编码序列，并导入大肠杆菌中进行表达。得到双突变糖基转移酶。双突变V371A/F381Y均由公司合成，构建到pet28a载体上。
V371A/F381Y的定点突变引物为：
正向引物：
TAAGAAGGAGATATACATATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTG
反向引物：
GGTTTCTTTACCAGACTCGAGTCATTAGTGGTGGTGGTGGTGGTGTTTGTTGCGACGGTC
反应条件及突变基因的测序方法同单突变体的方法。
（3）将突变体与蔗糖合酶偶连构建双酶体系
将突变后UGT73G1直接克隆到pET-28a（+）的Nde I和Xho I酶切位点间，得到重组质粒pET28a-UGT（pET-28a质粒来源于Novagen公司），此过程交由金斯瑞公司完成，将重组质粒pET28a-UGT用Nco I和EcoR I酶切，得到线性化载体，对带有蔗糖合酶（蔗糖合酶氨基酸序列如SEQ ID NO.5所示）的质粒pETDuet-SUS (来自Novagen公司)做同样的酶切处理，得到SUS基因片段。用同样的方法将SUS基因片段和线性化载体进行胶回收，将UGT和SUS，16℃过夜连接。次日采用热激法将连接产物转化到大肠杆菌BL21（DE3）感受态细胞中，筛选阳性克隆，得到新的重组质粒pRSF-UGT-SUS，然后导入到感受态细胞BL21（DE3）中，得到含双酶体系的重组菌E. coli BL21(DE3) /pRSF-UGT-SUS。实验步骤如下：
1.将感受态细胞BL21（DE3）从-80℃拿出，在冰上溶解完全后轻轻将细胞悬浮均匀；
2.取出重组质粒pRSF-UGT-SUS（20ul），取10ul加入感受态中，冰上放置30min；42℃水浴锅热激90s；冰上放置2min；向其中加入400ulLB培养液，37℃，220rpm振荡培养30min；取100ul菌液，涂布在固体LB培养基中，剩余的400ul 4℃保存；平板在37℃下正放30min，待接种的液体吸收进琼脂后，将平板倒置，培养过夜；次日观察平板，若菌落太少，将存放的菌液离心去300ul上清，用余下的100ul轻柔悬浮细胞，涂板；晚上取出平板观察，平板上的多数菌落白色略大于针尖，即可用封口膜封好，4℃保存。
实施例2 突变体酶的发酵诱导
将有重组质粒pRSF-UGT-SUS的BL21（DE）大肠杆菌接种于100mL LB液体培养基(含50μg/mL卡那霉素)，37℃，200r/min培养至OD600达到2-3时，添加诱导剂乳糖至终浓度1g/L，25℃，200r/min条件诱导20h。发酵液于4℃，6000 /min条件下离心3min，8mL 100mM 磷酸钾缓冲(pH8 .0)重悬菌体，利用超声破碎仪提取突变酶，破碎条件：300W，工作时间1s，间歇2s，全程15min。破碎液4℃，8000r/min离25min，收集上清液。心10min，收集上清液。
实施例3 糖基转移酶突变体与蔗糖合酶偶连双酶的转化率测定方法
糖基转移酶酶活测定方法为：在220 μl的反应体系中（0.5 mM槲皮素、5 mM UDPG、5 mM MgCl2、ph-7.2磷酸钾缓冲），加入蛋白终浓度为3 mg/ml的粗酶液反应，37℃反应30min后，取220 μl反应液，加入180 μl甲醇终止反应，12000 rpm离心1 min后上清液用高效液相色谱（HPLC）进行分析。酶活力单位定义为：在上述反应条件下，1 min催化形成1 μmol异槲皮苷所需要的酶量为1个活力单位（U）。
蔗糖合酶的酶活测定方法：在3ml的反应体系中（500mM蔗糖，10mM UDP, ph=7.2磷酸钾缓冲），加入6mg蛋白。30℃反应一小时，取1ml反应液，95℃下煮沸10分钟，12000 rpm离心1 min后上清液用DNS法测定。酶活力单位定义为：在给定的测定条件下每分钟释放1μmol还原糖的酶量为一个活力单位(U）。
由于槲皮素几乎不溶于水，使用DMSO可以极大的提高它的溶解性，溶解度可提高至约1g/L。且该酶在DMSO中的耐受性很好，不会出现酶活下降的情况。测定结果如表1所示。
表1：
样品名 酶活(U/g)（水） 酶活(U/g)（DMSO）
突变前 5.24 4.03
V371A 9.98 9.43
F381Y 8,34 8.23
V371AF381Y 8.15 8.13
实施例4
取6ml的酶液（酶活分别是UGT (15.3 mU/mg) ，SUS (61.0 mU/mg)）转化底物浓度1g/L的槲皮素或异槲皮素（15%DMSO），蔗糖10g/L（pH=8.0），30℃条件下反应16h，取100ul反应液，加入900ul甲醇终止反应。室温12000 r / m i n离心1 m i n ，取上清，利用高效液相色谱(H ig hPerformance Liquid Chromatography，HPLC)检测转化体系中槲皮素，异槲皮素，槲皮素-3，4’-二葡萄糖糖苷含量。取平均值，测定结果如表2所示（定义原始酶的产量为100%）。
表2：
产量（槲皮素为底物）（g/L） 产量（异槲皮素为底物）(g/L)
原始酶 100%（0.23） 100%(0.27)
V371A 255%（0.59） 193%(0.52)
F381Y 165%(0.38) 148%(0.40)
V371AF381Y 130%(0.30) 124%(0.33)
将上述突变体表达获得的突变酶与原始酶相比，可以发现，突变体实现了槲皮素和异槲皮素产量的提高。突变酶槲皮素产量相对于原始酶提高至255%，165%，130%。突变酶异槲皮素产量相对于原始酶提高至193%，148%，124%。
实施例3
按照上述方法，将同样位于活性中心附近的S151，T168，K274，T293，G361,S365，V371，F381突变为如下任意一种或多种：S151T，T168S，K274W/E，T293G/V，G361K,S365T,V371G。按相同方法检测。测定结果如表3所示,这些突变酶产量相对于原始酶均提高至120%-300%。
表3：
产量1（槲皮素为底物） 产量2（异槲皮素为底物）
原始酶 100% 100%
S151T 173% 165%
T168S 150% 143%
K274W 194% 176%
K274E 130% 126%
T293G 188% 170%
T293V 186% 170%
G361K 176% 154%
S365T 130% 123%
V371G 234% 207%
序列表
<110> 南京工业大学
<120> 一种槲皮素糖苷的生物合成方法
<141> 2018-09-14
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 479
<212> PRT
<213> 糖基转移酶(glycosyltransferase)
<400> 1
Met Asn Ser Asn His His Pro Leu His Val Ile Ile Phe Pro Phe Leu
1 5 10 15
Ala Gln Gly His Ile Ile Pro Thr Ile Asp Leu Cys Lys Leu Phe Ala
20 25 30
Ser His Gly Val Lys Val Thr Val Leu Thr Thr Lys Gly Asn Leu Ser
35 40 45
Arg Phe His Ser Pro Leu Thr Arg Ala Asn Glu Leu Ser Thr Phe Leu
50 55 60
His Pro Ile Gln Ile Ser Leu Ile Pro Phe Pro Ser Val Ser Gly Leu
65 70 75 80
Pro Glu Asn Cys Glu Asn Met Ala Thr Val Pro Pro His Leu Lys Ser
85 90 95
Leu Phe Phe Asp Ala Val Ala Met Leu Gln Gln Pro Phe Arg Ala Phe
100 105 110
Leu Lys Glu Thr Asn Pro Asp Cys Val Val Ala Gly Leu Phe Leu Ala
115 120 125
Trp Ile His Asn Val Ala Ser Glu Leu Asn Ile Pro Ser Leu Asp Phe
130 135 140
His Gly Ser Asn Phe Ser Ser Lys Cys Met Ser His Thr Val Glu His
145 150 155 160
His Asn Leu Leu Asp Asn Ser Thr Ala Glu Thr Val Leu Leu Pro Asn
165 170 175
Leu Pro His Lys Ile Glu Met Arg Arg Ala Leu Ile Pro Asp Phe Arg
180 185 190
Lys Val Ala Pro Ser Val Phe Gln Leu Leu Ile Lys Gln Lys Glu Ala
195 200 205
Glu Lys Leu Ser Tyr Gly Leu Ile Ile Asn Ser Phe Tyr Glu Leu Glu
210 215 220
Pro Gly Tyr Val Asp Tyr Phe Arg Asn Val Val Gly Arg Lys Ala Trp
225 230 235 240
His Val Gly Pro Leu Leu Leu Asn Asp Lys Asn Val Asn Thr Phe Asp
245 250 255
Arg Gly Ser Lys Ser Ala Ile Asp Glu Ala Ser Cys Leu Ser Trp Leu
260 265 270
Gly Lys Lys Ser Ala Gly Ser Val Leu Tyr Val Cys Phe Gly Ser Ala
275 280 285
Ser Phe Phe Thr Thr Arg Gln Leu Arg Glu Ile Ala Val Gly Leu Glu
290 295 300
Gly Ser Gly His Ala Phe Ile Trp Val Val Arg Asp Asp Gly Asp Glu
305 310 315 320
Gln Trp Met Pro Glu Gly Cys Glu Glu Arg Ile Glu Gly Arg Gly Leu
325 330 335
Ile Ile Lys Gly Trp Ala Pro Gln Met Met Ile Leu Asn His Glu Ala
340 345 350
Val Gly Gly Tyr Leu Thr His Cys Gly Trp Asn Ser Ser Leu Glu Gly
355 360 365
Ile Cys Val Gly Leu Pro Phe Val Thr Trp Pro Leu Phe Ala Glu Gln
370 375 380
Pro Tyr Asn Glu Arg Leu Ile Val Asp Val Leu Lys Val Gly Val Ala
385 390 395 400
Val Gly Val Lys Glu Tyr Ser Phe Asp Pro Glu Glu Arg Thr Val Ile
405 410 415
Glu Ala Gly Ser Ile Glu Thr Ala Val Lys Lys Leu Met Gly Asp Asp
420 425 430
Glu Glu Ala Glu Glu Arg Arg Arg Arg Ala Lys Glu Leu Ala Ala Met
435 440 445
Ala Arg Lys Ala Val Glu Glu Gly Gly Ser Ser Tyr Glu Leu Met Ser
450 455 460
Asp Leu Ile Arg Glu Leu Glu Gly Leu Arg Asp Arg Arg Asn Lys
465 470 475
<210> 2
<211> 479
<212> PRT
<213> 糖基转移酶(glycosyltransferase)
<400> 2
Met Asn Ser Asn His His Pro Leu His Val Ile Ile Phe Pro Phe Leu
1 5 10 15
Ala Gln Gly His Ile Ile Pro Thr Ile Asp Leu Cys Lys Leu Phe Ala
20 25 30
Ser His Gly Val Lys Val Thr Val Leu Thr Thr Lys Gly Asn Leu Ser
35 40 45
Arg Phe His Ser Pro Leu Thr Arg Ala Asn Glu Leu Ser Thr Phe Leu
50 55 60
His Pro Ile Gln Ile Ser Leu Ile Pro Phe Pro Ser Val Ser Gly Leu
65 70 75 80
Pro Glu Asn Cys Glu Asn Met Ala Thr Val Pro Pro His Leu Lys Ser
85 90 95
Leu Phe Phe Asp Ala Val Ala Met Leu Gln Gln Pro Phe Arg Ala Phe
100 105 110
Leu Lys Glu Thr Asn Pro Asp Cys Val Val Ala Gly Leu Phe Leu Ala
115 120 125
Trp Ile His Asn Val Ala Ser Glu Leu Asn Ile Pro Ser Leu Asp Phe
130 135 140
His Gly Ser Asn Phe Ser Ser Lys Cys Met Ser His Thr Val Glu His
145 150 155 160
His Asn Leu Leu Asp Asn Ser Thr Ala Glu Thr Val Leu Leu Pro Asn
165 170 175
Leu Pro His Lys Ile Glu Met Arg Arg Ala Leu Ile Pro Asp Phe Arg
180 185 190
Lys Val Ala Pro Ser Val Phe Gln Leu Leu Ile Lys Gln Lys Glu Ala
195 200 205
Glu Lys Leu Ser Tyr Gly Leu Ile Ile Asn Ser Phe Tyr Glu Leu Glu
210 215 220
Pro Gly Tyr Val Asp Tyr Phe Arg Asn Val Val Gly Arg Lys Ala Trp
225 230 235 240
His Val Gly Pro Leu Leu Leu Asn Asp Lys Asn Val Asn Thr Phe Asp
245 250 255
Arg Gly Ser Lys Ser Ala Ile Asp Glu Ala Ser Cys Leu Ser Trp Leu
260 265 270
Gly Lys Lys Ser Ala Gly Ser Val Leu Tyr Val Cys Phe Gly Ser Ala
275 280 285
Ser Phe Phe Thr Thr Arg Gln Leu Arg Glu Ile Ala Val Gly Leu Glu
290 295 300
Gly Ser Gly His Ala Phe Ile Trp Val Val Arg Asp Asp Gly Asp Glu
305 310 315 320
Gln Trp Met Pro Glu Gly Cys Glu Glu Arg Ile Glu Gly Arg Gly Leu
325 330 335
Ile Ile Lys Gly Trp Ala Pro Gln Met Met Ile Leu Asn His Glu Ala
340 345 350
Val Gly Gly Tyr Leu Thr His Cys Gly Trp Asn Ser Ser Leu Glu Gly
355 360 365
Ile Cys Ala Gly Leu Pro Phe Val Thr Trp Pro Leu Phe Ala Glu Gln
370 375 380
Pro Tyr Asn Glu Arg Leu Ile Val Asp Val Leu Lys Val Gly Val Ala
385 390 395 400
Val Gly Val Lys Glu Tyr Ser Phe Asp Pro Glu Glu Arg Thr Val Ile
405 410 415
Glu Ala Gly Ser Ile Glu Thr Ala Val Lys Lys Leu Met Gly Asp Asp
420 425 430
Glu Glu Ala Glu Glu Arg Arg Arg Arg Ala Lys Glu Leu Ala Ala Met
435 440 445
Ala Arg Lys Ala Val Glu Glu Gly Gly Ser Ser Tyr Glu Leu Met Ser
450 455 460
Asp Leu Ile Arg Glu Leu Glu Gly Leu Arg Asp Arg Arg Asn Lys
465 470 475
<210> 3
<211> 479
<212> PRT
<213> 糖基转移酶(glycosyltransferase)
<400> 3
Met Asn Ser Asn His His Pro Leu His Val Ile Ile Phe Pro Phe Leu
1 5 10 15
Ala Gln Gly His Ile Ile Pro Thr Ile Asp Leu Cys Lys Leu Phe Ala
20 25 30
Ser His Gly Val Lys Val Thr Val Leu Thr Thr Lys Gly Asn Leu Ser
35 40 45
Arg Phe His Ser Pro Leu Thr Arg Ala Asn Glu Leu Ser Thr Phe Leu
50 55 60
His Pro Ile Gln Ile Ser Leu Ile Pro Phe Pro Ser Val Ser Gly Leu
65 70 75 80
Pro Glu Asn Cys Glu Asn Met Ala Thr Val Pro Pro His Leu Lys Ser
85 90 95
Leu Phe Phe Asp Ala Val Ala Met Leu Gln Gln Pro Phe Arg Ala Phe
100 105 110
Leu Lys Glu Thr Asn Pro Asp Cys Val Val Ala Gly Leu Phe Leu Ala
115 120 125
Trp Ile His Asn Val Ala Ser Glu Leu Asn Ile Pro Ser Leu Asp Phe
130 135 140
His Gly Ser Asn Phe Ser Ser Lys Cys Met Ser His Thr Val Glu His
145 150 155 160
His Asn Leu Leu Asp Asn Ser Thr Ala Glu Thr Val Leu Leu Pro Asn
165 170 175
Leu Pro His Lys Ile Glu Met Arg Arg Ala Leu Ile Pro Asp Phe Arg
180 185 190
Lys Val Ala Pro Ser Val Phe Gln Leu Leu Ile Lys Gln Lys Glu Ala
195 200 205
Glu Lys Leu Ser Tyr Gly Leu Ile Ile Asn Ser Phe Tyr Glu Leu Glu
210 215 220
Pro Gly Tyr Val Asp Tyr Phe Arg Asn Val Val Gly Arg Lys Ala Trp
225 230 235 240
His Val Gly Pro Leu Leu Leu Asn Asp Lys Asn Val Asn Thr Phe Asp
245 250 255
Arg Gly Ser Lys Ser Ala Ile Asp Glu Ala Ser Cys Leu Ser Trp Leu
260 265 270
Gly Lys Lys Ser Ala Gly Ser Val Leu Tyr Val Cys Phe Gly Ser Ala
275 280 285
Ser Phe Phe Thr Thr Arg Gln Leu Arg Glu Ile Ala Val Gly Leu Glu
290 295 300
Gly Ser Gly His Ala Phe Ile Trp Val Val Arg Asp Asp Gly Asp Glu
305 310 315 320
Gln Trp Met Pro Glu Gly Cys Glu Glu Arg Ile Glu Gly Arg Gly Leu
325 330 335
Ile Ile Lys Gly Trp Ala Pro Gln Met Met Ile Leu Asn His Glu Ala
340 345 350
Val Gly Gly Tyr Leu Thr His Cys Gly Trp Asn Ser Ser Leu Glu Gly
355 360 365
Ile Cys Val Gly Leu Pro Phe Val Thr Trp Pro Leu Tyr Ala Glu Gln
370 375 380
Pro Tyr Asn Glu Arg Leu Ile Val Asp Val Leu Lys Val Gly Val Ala
385 390 395 400
Val Gly Val Lys Glu Tyr Ser Phe Asp Pro Glu Glu Arg Thr Val Ile
405 410 415
Glu Ala Gly Ser Ile Glu Thr Ala Val Lys Lys Leu Met Gly Asp Asp
420 425 430
Glu Glu Ala Glu Glu Arg Arg Arg Arg Ala Lys Glu Leu Ala Ala Met
435 440 445
Ala Arg Lys Ala Val Glu Glu Gly Gly Ser Ser Tyr Glu Leu Met Ser
450 455 460
Asp Leu Ile Arg Glu Leu Glu Gly Leu Arg Asp Arg Arg Asn Lys
465 470 475
<210> 4
<211> 479
<212> PRT
<213> 糖基转移酶(glycosyltransferase)
<400> 4
Met Asn Ser Asn His His Pro Leu His Val Ile Ile Phe Pro Phe Leu
1 5 10 15
Ala Gln Gly His Ile Ile Pro Thr Ile Asp Leu Cys Lys Leu Phe Ala
20 25 30
Ser His Gly Val Lys Val Thr Val Leu Thr Thr Lys Gly Asn Leu Ser
35 40 45
Arg Phe His Ser Pro Leu Thr Arg Ala Asn Glu Leu Ser Thr Phe Leu
50 55 60
His Pro Ile Gln Ile Ser Leu Ile Pro Phe Pro Ser Val Ser Gly Leu
65 70 75 80
Pro Glu Asn Cys Glu Asn Met Ala Thr Val Pro Pro His Leu Lys Ser
85 90 95
Leu Phe Phe Asp Ala Val Ala Met Leu Gln Gln Pro Phe Arg Ala Phe
100 105 110
Leu Lys Glu Thr Asn Pro Asp Cys Val Val Ala Gly Leu Phe Leu Ala
115 120 125
Trp Ile His Asn Val Ala Ser Glu Leu Asn Ile Pro Ser Leu Asp Phe
130 135 140
His Gly Ser Asn Phe Ser Ser Lys Cys Met Ser His Thr Val Glu His
145 150 155 160
His Asn Leu Leu Asp Asn Ser Thr Ala Glu Thr Val Leu Leu Pro Asn
165 170 175
Leu Pro His Lys Ile Glu Met Arg Arg Ala Leu Ile Pro Asp Phe Arg
180 185 190
Lys Val Ala Pro Ser Val Phe Gln Leu Leu Ile Lys Gln Lys Glu Ala
195 200 205
Glu Lys Leu Ser Tyr Gly Leu Ile Ile Asn Ser Phe Tyr Glu Leu Glu
210 215 220
Pro Gly Tyr Val Asp Tyr Phe Arg Asn Val Val Gly Arg Lys Ala Trp
225 230 235 240
His Val Gly Pro Leu Leu Leu Asn Asp Lys Asn Val Asn Thr Phe Asp
245 250 255
Arg Gly Ser Lys Ser Ala Ile Asp Glu Ala Ser Cys Leu Ser Trp Leu
260 265 270
Gly Lys Lys Ser Ala Gly Ser Val Leu Tyr Val Cys Phe Gly Ser Ala
275 280 285
Ser Phe Phe Thr Thr Arg Gln Leu Arg Glu Ile Ala Val Gly Leu Glu
290 295 300
Gly Ser Gly His Ala Phe Ile Trp Val Val Arg Asp Asp Gly Asp Glu
305 310 315 320
Gln Trp Met Pro Glu Gly Cys Glu Glu Arg Ile Glu Gly Arg Gly Leu
325 330 335
Ile Ile Lys Gly Trp Ala Pro Gln Met Met Ile Leu Asn His Glu Ala
340 345 350
Val Gly Gly Tyr Leu Thr His Cys Gly Trp Asn Ser Ser Leu Glu Gly
355 360 365
Ile Cys Ala Gly Leu Pro Phe Val Thr Trp Pro Leu Tyr Ala Glu Gln
370 375 380
Pro Tyr Asn Glu Arg Leu Ile Val Asp Val Leu Lys Val Gly Val Ala
385 390 395 400
Val Gly Val Lys Glu Tyr Ser Phe Asp Pro Glu Glu Arg Thr Val Ile
405 410 415
Glu Ala Gly Ser Ile Glu Thr Ala Val Lys Lys Leu Met Gly Asp Asp
420 425 430
Glu Glu Ala Glu Glu Arg Arg Arg Arg Ala Lys Glu Leu Ala Ala Met
435 440 445
Ala Arg Lys Ala Val Glu Glu Gly Gly Ser Ser Tyr Glu Leu Met Ser
450 455 460
Asp Leu Ile Arg Glu Leu Glu Gly Leu Arg Asp Arg Arg Asn Lys
465 470 475
<210> 5
<211> 2418
<212> PRT
<213> 蔗糖合酶(sucrose synthase)
<400> 5
Ala Thr Gly Gly Cys Cys Gly Ala Ala Cys Gly Thr Gly Thr Cys Cys
1 5 10 15
Thr Gly Ala Cys Cys Cys Gly Thr Gly Thr Cys Cys Ala Thr Ala Gly
20 25 30
Thr Cys Thr Gly Cys Gly Thr Gly Ala Ala Cys Gly Thr Gly Thr Thr
35 40 45
Gly Ala Thr Gly Cys Thr Ala Cys Cys Cys Thr Gly Gly Cys Thr Gly
50 55 60
Cys Cys Cys Ala Cys Cys Gly Thr Ala Ala Thr Gly Ala Ala Ala Thr
65 70 75 80
Cys Cys Thr Gly Cys Thr Gly Thr Thr Thr Cys Thr Gly Ala Gly Thr
85 90 95
Cys Gly Thr Ala Thr Thr Gly Ala Ala Ala Gly Cys Cys Ala Cys Gly
100 105 110
Gly Cys Ala Ala Ala Gly Gly Thr Ala Thr Cys Cys Thr Gly Ala Ala
115 120 125
Ala Cys Cys Gly Cys Ala Cys Gly Ala Ala Cys Thr Gly Cys Thr Gly
130 135 140
Gly Cys Ala Gly Ala Ala Thr Thr Thr Gly Ala Thr Gly Cys Thr Ala
145 150 155 160
Thr Thr Cys Gly Cys Cys Ala Gly Gly Ala Thr Gly Ala Cys Ala Ala
165 170 175
Ala Ala Ala Cys Ala Ala Ala Cys Thr Gly Ala Ala Cys Gly Ala Ala
180 185 190
Cys Ala Thr Gly Cys Ala Thr Thr Cys Gly Ala Ala Gly Ala Ala Cys
195 200 205
Thr Gly Cys Thr Gly Ala Ala Ala Ala Gly Cys Ala Cys Cys Cys Ala
210 215 220
Ala Gly Ala Ala Gly Cys Thr Ala Thr Cys Gly Thr Cys Cys Thr Gly
225 230 235 240
Cys Cys Gly Cys Cys Gly Thr Gly Gly Gly Thr Gly Gly Cys Ala Cys
245 250 255
Thr Gly Gly Cys Ala Ala Thr Thr Cys Gly Thr Cys Thr Gly Cys Gly
260 265 270
Cys Cys Cys Gly Gly Gly Cys Gly Thr Thr Thr Gly Gly Gly Ala Ala
275 280 285
Thr Ala Cys Ala Thr Cys Cys Gly Thr Gly Thr Thr Ala Ala Cys Gly
290 295 300
Thr Cys Ala Ala Thr Gly Cys Gly Cys Thr Gly Gly Thr Thr Gly Thr
305 310 315 320
Gly Gly Ala Ala Gly Ala Ala Cys Thr Gly Ala Gly Thr Gly Thr Gly
325 330 335
Cys Cys Gly Gly Ala Ala Thr Ala Thr Cys Thr Gly Cys Ala Gly Thr
340 345 350
Thr Thr Ala Ala Ala Gly Ala Ala Gly Ala Ala Cys Thr Gly Gly Thr
355 360 365
Cys Gly Ala Thr Gly Gly Cys Gly Cys Gly Thr Cys Cys Ala Ala Cys
370 375 380
Gly Gly Thr Ala Ala Thr Thr Thr Cys Gly Thr Gly Cys Thr Gly Gly
385 390 395 400
Ala Ala Cys Thr Gly Gly Ala Cys Thr Thr Thr Gly Ala Ala Cys Cys
405 410 415
Gly Thr Thr Cys Ala Cys Cys Gly Cys Cys Thr Cys Ala Thr Thr Thr
420 425 430
Cys Cys Gly Ala Ala Ala Cys Cys Gly Ala Cys Cys Cys Thr Gly Ala
435 440 445
Cys Gly Ala Ala Ala Thr Cys Gly Ala Thr Thr Gly Gly Cys Ala Ala
450 455 460
Cys Gly Gly Thr Gly Thr Thr Gly Ala Ala Thr Thr Thr Cys Thr Gly
465 470 475 480
Ala Ala Thr Cys Gly Thr Cys Ala Thr Cys Thr Gly Ala Gly Cys Gly
485 490 495
Cys Cys Ala Ala Ala Ala Thr Gly Thr Thr Cys Cys Ala Cys Gly Ala
500 505 510
Thr Ala Ala Ala Gly Ala Ala Thr Cys Thr Ala Thr Gly Ala Cys Cys
515 520 525
Cys Cys Gly Cys Thr Gly Cys Thr Gly Gly Ala Ala Thr Thr Thr Cys
530 535 540
Thr Gly Cys Gly Cys Gly Cys Ala Cys Ala Thr Cys Ala Cys Thr Ala
545 550 555 560
Thr Ala Ala Ala Gly Gly Thr Ala Ala Ala Ala Cys Cys Ala Thr Gly
565 570 575
Ala Thr Gly Cys Thr Gly Ala Ala Cys Gly Ala Thr Cys Gly Thr Ala
580 585 590
Thr Thr Cys Ala Gly Ala Ala Cys Ala Gly Cys Ala Ala Thr Ala Cys
595 600 605
Gly Cys Thr Gly Cys Ala Ala Ala Ala Thr Gly Thr Gly Cys Thr Gly
610 615 620
Cys Gly Cys Ala Ala Ala Gly Cys Gly Gly Ala Ala Gly Ala Ala Thr
625 630 635 640
Ala Cys Cys Thr Gly Ala Thr Cys Ala Thr Gly Cys Thr Gly Cys Cys
645 650 655
Gly Cys Cys Gly Gly Ala Ala Ala Cys Cys Cys Cys Gly Thr Ala Cys
660 665 670
Thr Thr Cys Gly Ala Ala Thr Thr Thr Gly Ala Ala Cys Ala Thr Ala
675 680 685
Ala Ala Thr Thr Cys Cys Ala Gly Gly Ala Ala Ala Thr Thr Gly Gly
690 695 700
Cys Cys Thr Gly Gly Ala Ala Ala Ala Ala Gly Gly Cys Thr Gly Gly
705 710 715 720
Gly Gly Thr Gly Ala Thr Ala Cys Gly Gly Cys Ala Gly Ala Ala Cys
725 730 735
Gly Thr Gly Thr Gly Cys Thr Gly Gly Ala Ala Ala Thr Gly Gly Thr
740 745 750
Thr Thr Gly Cys Ala Thr Gly Cys Thr Gly Cys Thr Gly Gly Ala Thr
755 760 765
Cys Thr Gly Cys Thr Gly Gly Ala Ala Gly Cys Thr Cys Cys Gly Gly
770 775 780
Ala Cys Ala Gly Cys Thr Gly Thr Ala Cys Cys Cys Thr Gly Gly Ala
785 790 795 800
Ala Ala Ala Ala Thr Thr Thr Cys Thr Gly Gly Gly Thr Cys Gly Cys
805 810 815
Ala Thr Thr Cys Cys Gly Ala Thr Gly Gly Thr Thr Thr Thr Cys Ala
820 825 830
Ala Cys Gly Thr Cys Gly Thr Gly Ala Thr Cys Cys Thr Gly Thr Cys
835 840 845
Thr Cys Cys Gly Cys Ala Cys Gly Gly Cys Thr Ala Thr Thr Thr Thr
850 855 860
Gly Cys Gly Cys Ala Gly Gly Ala Ala Ala Ala Thr Gly Thr Cys Cys
865 870 875 880
Thr Gly Gly Gly Thr Thr Ala Cys Cys Cys Gly Gly Ala Thr Ala Cys
885 890 895
Cys Gly Gly Cys Gly Gly Thr Cys Ala Gly Gly Thr Thr Gly Thr Cys
900 905 910
Thr Ala Thr Ala Thr Thr Cys Thr Gly Gly Ala Cys Cys Ala Ala Gly
915 920 925
Thr Gly Cys Cys Gly Gly Cys Cys Cys Thr Gly Gly Ala Ala Cys Gly
930 935 940
Thr Gly Ala Ala Ala Thr Gly Cys Thr Gly Ala Ala Ala Cys Gly Cys
945 950 955 960
Ala Thr Cys Ala Ala Ala Gly Ala Ala Cys Ala Gly Gly Gly Cys Cys
965 970 975
Thr Gly Gly Ala Thr Ala Thr Thr Ala Thr Cys Cys Cys Gly Cys Gly
980 985 990
Thr Ala Thr Thr Cys Thr Gly Ala Thr Cys Gly Thr Cys Ala Cys Cys
995 1000 1005
Cys Gly Thr Cys Thr Gly Cys Thr Gly Cys Cys Gly Gly Ala Cys Gly
1010 1015 1020
Cys Ala Gly Thr Gly Gly Gly Cys Ala Cys Cys Ala Cys Gly Thr Gly
1025 1030 1035 1040
Cys Gly Gly Thr Cys Ala Ala Cys Gly Thr Ala Thr Thr Gly Ala Ala
1045 1050 1055
Ala Ala Ala Gly Thr Gly Thr Ala Thr Gly Gly Cys Gly Cys Thr Gly
1060 1065 1070
Ala Ala Cys Ala Thr Thr Cys Ala Cys Ala Cys Ala Thr Cys Cys Thr
1075 1080 1085
Gly Cys Gly Thr Gly Thr Thr Cys Cys Gly Thr Thr Thr Cys Gly Cys
1090 1095 1100
Ala Cys Cys Gly Ala Ala Ala Ala Ala Gly Gly Thr Ala Thr Thr Gly
1105 1110 1115 1120
Thr Cys Cys Gly Thr Ala Ala Ala Thr Gly Gly Ala Thr Cys Thr Cys
1125 1130 1135
Gly Cys Gly Cys Thr Thr Thr Gly Ala Ala Gly Thr Gly Thr Gly Gly
1140 1145 1150
Cys Cys Gly Thr Ala Cys Ala Thr Gly Gly Ala Ala Ala Cys Gly Thr
1155 1160 1165
Thr Cys Ala Thr Thr Gly Ala Ala Gly Ala Thr Gly Thr Thr Gly Cys
1170 1175 1180
Ala Ala Ala Ala Gly Ala Ala Ala Thr Cys Thr Cys Ala Gly Cys Gly
1185 1190 1195 1200
Gly Ala Ala Cys Thr Gly Cys Ala Gly Gly Cys Cys Ala Ala Ala Cys
1205 1210 1215
Cys Gly Gly Ala Cys Cys Thr Gly Ala Thr Thr Ala Thr Cys Gly Gly
1220 1225 1230
Cys Ala Ala Cys Thr Ala Thr Ala Gly Cys Gly Ala Ala Gly Gly Thr
1235 1240 1245
Ala Ala Thr Cys Thr Gly Gly Cys Gly Gly Cys Cys Thr Cys Thr Cys
1250 1255 1260
Thr Gly Cys Thr Gly Gly Cys Cys Cys Ala Thr Ala Ala Ala Cys Thr
1265 1270 1275 1280
Gly Gly Gly Cys Gly Thr Gly Ala Cys Cys Cys Ala Ala Thr Gly Thr
1285 1290 1295
Ala Cys Gly Ala Thr Thr Gly Cys Ala Cys Ala Cys Gly Cys Thr Cys
1300 1305 1310
Thr Gly Gly Ala Ala Ala Ala Ala Ala Cys Cys Ala Ala Ala Thr Ala
1315 1320 1325
Thr Cys Cys Gly Gly Ala Thr Thr Cys Gly Gly Ala Cys Ala Thr Cys
1330 1335 1340
Thr Ala Cys Thr Gly Gly Ala Ala Ala Ala Ala Ala Thr Thr Cys Gly
1345 1350 1355 1360
Ala Thr Gly Ala Ala Ala Ala Ala Thr Ala Cys Cys Ala Thr Thr Thr
1365 1370 1375
Cys Ala Gly Cys Thr Cys Thr Cys Ala Gly Thr Thr Cys Ala Cys Cys
1380 1385 1390
Gly Cys Ala Gly Ala Thr Cys Thr Gly Ala Thr Thr Gly Cys Thr Ala
1395 1400 1405
Thr Gly Ala Ala Cys Cys Ala Cys Ala Cys Gly Gly Ala Cys Thr Thr
1410 1415 1420
Thr Ala Thr Thr Ala Thr Cys Ala Cys Cys Ala Gly Thr Ala Cys Gly
1425 1430 1435 1440
Thr Thr Cys Cys Ala Gly Gly Ala Ala Ala Thr Cys Gly Cys Gly Gly
1445 1450 1455
Gly Cys Thr Cys Cys Ala Ala Ala Gly Ala Thr Ala Cys Cys Gly Thr
1460 1465 1470
Gly Gly Gly Thr Cys Ala Ala Thr Ala Cys Gly Ala Ala Ala Gly Thr
1475 1480 1485
Cys Ala Thr Ala Thr Gly Gly Cys Cys Thr Thr Thr Ala Cys Gly Ala
1490 1495 1500
Thr Gly Cys Cys Gly Gly Gly Cys Cys Thr Gly Thr Ala Thr Cys Gly
1505 1510 1515 1520
Cys Gly Thr Gly Gly Thr Thr Cys Ala Cys Gly Gly Thr Ala Thr Cys
1525 1530 1535
Ala Ala Cys Gly Thr Thr Thr Thr Cys Gly Ala Thr Cys Cys Gly Ala
1540 1545 1550
Ala Ala Thr Thr Cys Ala Ala Cys Ala Thr Thr Gly Thr Cys Thr Cys
1555 1560 1565
Cys Cys Cys Gly Gly Gly Thr Gly Cys Ala Gly Ala Cys Ala Thr Cys
1570 1575 1580
Ala Ala Thr Cys Thr Gly Thr Ala Thr Thr Thr Thr Thr Cys Ala Thr
1585 1590 1595 1600
Ala Cys Thr Cys Gly Gly Ala Ala Ala Cys Cys Gly Ala Ala Ala Ala
1605 1610 1615
Ala Cys Gly Thr Cys Thr Gly Ala Cys Gly Gly Cys Thr Thr Thr Cys
1620 1625 1630
Cys Ala Thr Cys Cys Gly Gly Ala Ala Ala Thr Cys Gly Ala Thr Gly
1635 1640 1645
Ala Ala Cys Thr Gly Cys Thr Gly Thr Ala Thr Ala Gly Cys Gly Ala
1650 1655 1660
Thr Gly Thr Gly Gly Ala Ala Ala Ala Cys Gly Ala Cys Gly Ala Ala
1665 1670 1675 1680
Cys Ala Cys Cys Thr Gly Thr Gly Cys Gly Thr Thr Cys Thr Gly Ala
1685 1690 1695
Ala Ala Gly Ala Thr Cys Gly Cys Ala Cys Cys Ala Ala Ala Cys Cys
1700 1705 1710
Gly Ala Thr Thr Cys Thr Gly Thr Thr Thr Ala Cys Gly Ala Thr Gly
1715 1720 1725
Gly Cys Gly Cys Gly Thr Cys Thr Gly Gly Ala Cys Cys Gly Cys Gly
1730 1735 1740
Thr Thr Ala Ala Ala Ala Ala Thr Cys Thr Gly Ala Cys Cys Gly Gly
1745 1750 1755 1760
Cys Cys Thr Gly Gly Thr Cys Gly Ala Ala Thr Gly Gly Thr Ala Cys
1765 1770 1775
Gly Cys Cys Ala Ala Ala Ala Ala Cys Cys Cys Gly Cys Gly Thr Cys
1780 1785 1790
Thr Gly Cys Gly Cys Gly Gly Thr Cys Thr Gly Gly Thr Gly Ala Ala
1795 1800 1805
Thr Cys Thr Gly Gly Thr Cys Gly Thr Gly Gly Thr Thr Gly Gly Cys
1810 1815 1820
Gly Gly Thr Gly Ala Thr Cys Gly Thr Cys Gly Cys Ala Ala Ala Gly
1825 1830 1835 1840
Ala Ala Thr Cys Thr Ala Ala Ala Gly Ala Cys Cys Thr Gly Gly Ala
1845 1850 1855
Ala Gly Ala Ala Cys Ala Gly Gly Cys Gly Gly Ala Ala Ala Thr Gly
1860 1865 1870
Ala Ala Gly Ala Ala Ala Ala Thr Gly Thr Ala Cys Gly Ala Ala Cys
1875 1880 1885
Thr Gly Ala Thr Cys Gly Ala Ala Ala Cys Cys Cys Ala Thr Ala Ala
1890 1895 1900
Cys Cys Thr Gly Ala Ala Thr Gly Gly Cys Cys Ala Gly Thr Thr Cys
1905 1910 1915 1920
Cys Gly Thr Thr Gly Gly Ala Thr Cys Ala Gly Thr Thr Cys Cys Cys
1925 1930 1935
Ala Ala Ala Thr Gly Ala Ala Cys Cys Gly Thr Gly Thr Thr Cys Gly
1940 1945 1950
Cys Ala Ala Thr Gly Gly Cys Gly Ala Ala Cys Thr Gly Thr Ala Thr
1955 1960 1965
Cys Gly Cys Thr Ala Cys Ala Thr Cys Gly Cys Ala Gly Ala Thr Ala
1970 1975 1980
Cys Gly Ala Ala Ala Gly Gly Thr Gly Cys Thr Thr Thr Thr Gly Thr
1985 1990 1995 2000
Cys Cys Ala Gly Cys Cys Gly Gly Cys Gly Thr Thr Thr Thr Ala Cys
2005 2010 2015
Gly Ala Ala Gly Cys Cys Thr Thr Cys Gly Gly Cys Cys Thr Gly Ala
2020 2025 2030
Cys Cys Gly Thr Cys Gly Thr Gly Gly Ala Ala Gly Cys Gly Ala Thr
2035 2040 2045
Gly Ala Cys Gly Thr Gly Cys Gly Gly Thr Cys Thr Gly Cys Cys Gly
2050 2055 2060
Ala Cys Cys Thr Thr Cys Gly Cys Ala Ala Cys Gly Ala Ala Thr Cys
2065 2070 2075 2080
Ala Thr Gly Gly Cys Gly Gly Cys Cys Cys Gly Gly Cys Ala Gly Ala
2085 2090 2095
Ala Ala Thr Thr Ala Thr Cys Gly Thr Thr Cys Ala Cys Gly Gly Cys
2100 2105 2110
Ala Ala Ala Ala Gly Thr Gly Gly Thr Thr Thr Thr Cys Ala Thr Ala
2115 2120 2125
Thr Thr Gly Ala Thr Cys Cys Gly Thr Ala Thr Cys Ala Cys Gly Gly
2130 2135 2140
Cys Gly Ala Ala Cys Ala Gly Gly Cys Ala Gly Cys Thr Gly Ala Thr
2145 2150 2155 2160
Cys Thr Gly Cys Thr Gly Gly Cys Cys Gly Ala Cys Thr Thr Thr Thr
2165 2170 2175
Thr Cys Gly Ala Ala Ala Ala Ala Thr Gly Thr Ala Ala Ala Ala Ala
2180 2185 2190
Ala Gly Ala Cys Cys Cys Gly Thr Cys Ala Cys Ala Thr Thr Gly Gly
2195 2200 2205
Gly Ala Ala Ala Cys Cys Ala Thr Thr Thr Cys Gly Ala Thr Gly Gly
2210 2215 2220
Gly Cys Gly Gly Thr Cys Thr Gly Ala Ala Ala Cys Gly Cys Ala Thr
2225 2230 2235 2240
Cys Gly Ala Ala Gly Ala Ala Ala Ala Ala Thr Ala Thr Ala Cys Cys
2245 2250 2255
Thr Gly Gly Cys Ala Ala Ala Thr Thr Thr Ala Cys Ala Gly Cys Gly
2260 2265 2270
Ala Ala Thr Cys Thr Cys Thr Gly Cys Thr Gly Ala Cys Gly Cys Thr
2275 2280 2285
Gly Gly Cys Gly Gly Cys Cys Gly Thr Gly Thr Ala Cys Gly Gly Thr
2290 2295 2300
Thr Thr Cys Thr Gly Gly Ala Ala Ala Cys Ala Cys Gly Thr Thr Thr
2305 2310 2315 2320
Cys Thr Ala Ala Ala Cys Thr Gly Gly Ala Thr Cys Gly Thr Cys Thr
2325 2330 2335
Gly Gly Ala Ala Ala Thr Thr Cys Gly Thr Cys Gly Cys Thr Ala Thr
2340 2345 2350
Cys Thr Gly Gly Ala Ala Ala Thr Gly Thr Thr Thr Thr Ala Thr Gly
2355 2360 2365
Cys Gly Cys Thr Gly Ala Ala Ala Thr Ala Cys Cys Gly Cys Ala Ala
2370 2375 2380
Ala Ala Thr Gly Gly Cys Gly Gly Ala Ala Gly Cys Cys Gly Thr Gly
2385 2390 2395 2400
Cys Cys Gly Cys Thr Gly Gly Cys Ala Gly Cys Thr Gly Ala Ala Thr
2405 2410 2415
Ala Ala
<210> 6
<211> 1440
<212> DNA/RNA
<213> 糖基转移酶(glycosyltransferase)
<400> 6
atgaattcta atcatcatcc tctccatgtc atcatcttcc cattcctcgc tcaaggccac 60
atcatcccca ccatcgactt atgcaagctc ttcgcttctc acggagtaaa agtcactgtc 120
ctcaccacca aaggcaacct ctccagattc cactccccat taactcgagc taacgagctt 180
tctaccttcc tccaccccat ccagatctcc ctcatccctt tcccttcagt ctccggctta 240
cctgaaaact gtgaaaacat ggctaccgtt cctccacacc tcaaatccct cttcttcgac 300
gccgtggcca tgcttcaaca accctttcgt gccttcctta aagaaacgaa ccctgactgc 360
gtagttgccg gtcttttcct tgcatggatc cacaacgtcg cctctgagct caacatcccc 420
agcttagact ttcatggatc caacttctct tccaagtgta tgtcccacac cgtagaacac 480
cacaacctcc tcgacaattc cactgcagaa actgtactcc taccaaacct ccctcacaaa 540
atagagatgc gtcgagctct tatcccagac ttcagaaagg tcgctccttc tgttttccag 600
ctactcataa agcagaagga agccgagaaa ctcagctatg ggttaataat caatagcttc 660
tacgagctcg aacccggtta cgttgactac ttcagaaacg ttgtaggaag aaaagcttgg 720
cacgttggtc ccttgttgct taacgataag aatgtgaata cttttgatag agggagtaag 780
tctgctattg atgaagcttc atgtttgagc tggctgggga agaagagtgc tggctctgtt 840
ttgtacgttt gttttgggag tgcgagtttc ttcactacaa ggcagctgcg tgagatagct 900
gtagggctcg aagggtcggg gcatgcattc atttgggtgg tcagagacga tggagatgag 960
cagtggatgc cggagggatg tgaagagaga attgaaggga gaggactaat tattaaaggg 1020
tgggcgcccc agatgatgat attgaatcat gaggcggttg gagggtactt gacccactgt 1080
ggatggaact caagtctgga agggatatgt gtagggctgc ccttcgtgac atggccattg 1140
tttgcagagc agccttacaa tgagaggttg attgtggatg tattgaaggt tggagtggcg 1200
gttggagtga aggagtattc tttcgaccca gaagagagga ctgtgatcga agcaggaagt 1260
atagagactg cagtgaagaa gttgatgggg gatgatgaag aagcggaaga gaggaggagg 1320
agagcaaagg agctggcagc catggctagg aaggcggtgg aggaaggtgg gtcttcttat 1380
gagttgatga gtgatttgat tcgagagttg gagggtttgc gagatagaag aaataaatga 1440
<210> 7
<211> 2414
<212> DNA/RNA
<213> 蔗糖合酶(sucrose synthase)
<400> 7
ccgaacgtgt cctgacccgt gtccatagtc tgcgtgaacg tgttgatgct accctggctg 60
cccaccgtaa tgaaatcctg ctgtttctga gtcgtattga aagccacggc aaaggtatcc 120
tgaaaccgca cgaactgctg gcagaatttg atgctattcg ccaggatgac aaaaacaaac 180
tgaacgaaca tgcattcgaa gaactgctga aaagcaccca agaagctatc gtcctgccgc 240
cgtgggtggc actggcaatt cgtctgcgcc cgggcgtttg ggaatacatc cgtgttaacg 300
tcaatgcgct ggttgtggaa gaactgagtg tgccggaata tctgcagttt aaagaagaac 360
tggtcgatgg cgcgtccaac ggtaatttcg tgctggaact ggactttgaa ccgttcaccg 420
cctcatttcc gaaaccgacc ctgacgaaat cgattggcaa cggtgttgaa tttctgaatc 480
gtcatctgag cgccaaaatg ttccacgata aagaatctat gaccccgctg ctggaatttc 540
tgcgcgcaca tcactataaa ggtaaaacca tgatgctgaa cgatcgtatt cagaacagca 600
atacgctgca aaatgtgctg cgcaaagcgg aagaatacct gatcatgctg ccgccggaaa 660
ccccgtactt cgaatttgaa cataaattcc aggaaattgg cctggaaaaa ggctggggtg 720
atacggcaga acgtgtgctg gaaatggttt gcatgctgct ggatctgctg gaagctccgg 780
acagctgtac cctggaaaaa tttctgggtc gcattccgat ggttttcaac gtcgtgatcc 840
tgtctccgca cggctatttt gcgcaggaaa atgtcctggg ttacccggat accggcggtc 900
aggttgtcta tattctggac caagtgccgg ccctggaacg tgaaatgctg aaacgcatca 960
aagaacaggg cctggatatt atcccgcgta ttctgatcgt cacccgtctg ctgccggacg 1020
cagtgggcac cacgtgcggt caacgtattg aaaaagtgta tggcgctgaa cattcacaca 1080
tcctgcgtgt tccgtttcgc accgaaaaag gtattgtccg taaatggatc tcgcgctttg 1140
aagtgtggcc gtacatggaa acgttcattg aagatgttgc aaaagaaatc tcagcggaac 1200
tgcaggccaa accggacctg attatcggca actatagcga aggtaatctg gcggcctctc 1260
tgctggccca taaactgggc gtgacccaat gtacgattgc acacgctctg gaaaaaacca 1320
aatatccgga ttcggacatc tactggaaaa aattcgatga aaaataccat ttcagctctc 1380
agttcaccgc agatctgatt gctatgaacc acacggactt tattatcacc agtacgttcc 1440
aggaaatcgc gggctccaaa gataccgtgg gtcaatacga aagtcatatg gcctttacga 1500
tgccgggcct gtatcgcgtg gttcacggta tcaacgtttt cgatccgaaa ttcaacattg 1560
tctccccggg tgcagacatc aatctgtatt tttcatactc ggaaaccgaa aaacgtctga 1620
cggctttcca tccggaaatc gatgaactgc tgtatagcga tgtggaaaac gacgaacacc 1680
tgtgcgttct gaaagatcgc accaaaccga ttctgtttac gatggcgcgt ctggaccgcg 1740
ttaaaaatct gaccggcctg gtcgaatggt acgccaaaaa cccgcgtctg cgcggtctgg 1800
tgaatctggt cgtggttggc ggtgatcgtc gcaaagaatc taaagacctg gaagaacagg 1860
cggaaatgaa gaaaatgtac gaactgatcg aaacccataa cctgaatggc cagttccgtt 1920
ggatcagttc ccaaatgaac cgtgttcgca atggcgaact gtatcgctac atcgcagata 1980
cgaaaggtgc ttttgtccag ccggcgtttt acgaagcctt cggcctgacc gtcgtggaag 2040
cgatgacgtg cggtctgccg accttcgcaa cgaatcatgg cggcccggca gaaattatcg 2100
ttcacggcaa aagtggtttt catattgatc cgtatcacgg cgaacaggca gctgatctgc 2160
tggccgactt tttcgaaaaa tgtaaaaaag acccgtcaca ttgggaaacc atttcgatgg 2220
gcggtctgaa acgcatcgaa gaaaaatata cctggcaaat ttacagcgaa tctctgctga 2280
cgctggcggc cgtgtacggt ttctggaaac acgtttctaa actggatcgt ctggaaattc 2340
gtcgctatct ggaaatgttt tatgcgctga aataccgcaa aatggcggaa gccgtgccgc 2400
tggcagctga ataa 2414
<210> 8
<211> 60
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 8
taagaaggag atatacatat gggcagcagc catcatcatc atcatcacag cagcggcctg 60
<210> 9
<211> 60
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggtttcttta ccagactcga gtcattagtg gtggtggtgg tggtgtttgt tgcgacggtc 60
<210> 10
<211> 60
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 10
taagaaggag atatacatat gggcagcagc catcatcatc atcatcacag cagcggcctg 60
<210> 11
<211> 60
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggtttcttta ccagactcga gtcattagtg gtggtggtgg tggtgtttgt tgcgacggtc 60
<210> 12
<211> 60
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 12
taagaaggag atatacatat gggcagcagc catcatcatc atcatcacag cagcggcctg 60
<210> 13
<211> 60
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggtttcttta ccagactcga gtcattagtg gtggtggtgg tggtgtttgt tgcgacggtc 60

Claims (7)

1.一种槲皮素糖苷的生物合成方法，其特征在于，包括如下步骤：
1）构建含双酶体系的重组菌：将糖基转移酶UGT73G1突变体的基因与蔗糖合酶基因连接得到重组质粒，采用热激法将连接产物转化到大肠杆菌BL21（DE3）感受态细胞中，得到含双酶体系的重组菌；
2) 重组菌的发酵诱导产酶：将重组菌接种于LB液体培养基中，25~40℃，培养至OD600达到2-3时，添加诱导剂乳糖至终浓度0.25~1g/L，20~37℃，150~300r/min条件诱导12~20h，发酵液离心收集菌体，菌体破碎，离心收集上清液得到粗酶液；
3）将槲皮素或异槲皮素溶于DMSO中，加入粗酶液、蔗糖，20-40℃条件下反应8-30h，加入甲醇终止反应，离心得上清槲皮素-3，4’-二葡萄糖糖苷；
其中，所述糖基转移酶UGT73G1突变体的氨基酸序列是SEQ ID NO:1所示序列发生V371A和/或F381Y突变的氨基酸序列。
2.根据权利要求1所述的槲皮素糖苷的生物合成方法，其特征在于，诱导剂乳糖的终浓度为1g/L，诱导时间为20h。
3.根据权利要求1所述的槲皮素糖苷的生物合成方法，其特征在于，槲皮素或异槲皮素溶于DMSO中浓度为1g/L；蔗糖浓度为10g/L；粗酶液加入量为8mg/ml。
4.一种适用于槲皮素糖苷合成的糖基转移酶突变体，糖基转移酶突变体的氨基酸序列是SEQ ID NO:1所示发生突变的氨基酸序列，突变的氨基酸位点选自V371及F381中的一个或两个。
5.一种表达基因，该基因编码权利要求4所述的糖基转移酶突变体。
6.一种重组质粒，该重组质粒连接有权利要求5所述的表达基因和蔗糖合酶基因，所述蔗糖合酶基因序列如SEQ ID:7所示。
7.一种重组细胞，该重组细胞包含权利要求6的重组质粒或权利要求5的表达基因。
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