CN112592880B - 一种产假尿苷工程菌及其应用 - Google Patents
一种产假尿苷工程菌及其应用 Download PDFInfo
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- CN112592880B CN112592880B CN202011637153.9A CN202011637153A CN112592880B CN 112592880 B CN112592880 B CN 112592880B CN 202011637153 A CN202011637153 A CN 202011637153A CN 112592880 B CN112592880 B CN 112592880B
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- pseudouridine
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Abstract
本发明提供了一种产假尿苷工程菌及其应用，所述工程菌是将来源于链霉菌Streptomyces sp.ID38640中的假尿苷合成基因pumH、pumJ和pumD基因克隆至大肠杆菌体内，转化得到可产假尿苷的工程菌，可产假尿苷达7g/L以上。本发明大肠杆菌基因构建方法简单，发酵过程中不需要添加诱导剂异丙基‑β‑D‑硫代吡喃半乳糖苷即可获得较高产量的假尿苷，应用前景广阔。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种产假尿苷工程菌及其应用。
背景技术
1957年，DAV I S等在核糖体RNA(ribosomal RNA，rRNA)和转运RNA(transferRNA，tRNA)中首次发现一种与尿嘧啶核苷天然结构类似的异构体，其核糖不与尿嘧啶(Uridine，U)N1相连，而与C5相连，形成假尿嘧啶核苷(Pseudouridine，PU)。(DAVIS F F，ALLEN FW.Ribonucleic acids from yeast which contain a fifth nucleotide[J].J BiolChem，1957，227(2)：907-915.)尿苷通过假尿苷合酶从核糖糖碱基裂解并重新连接生成C-C糖苷键，异构化成假尿苷。尿嘧啶核苷与假尿嘧啶核苷化学结构式如式1所示。
作为核苷尿苷的一种异构体，假尿苷存在于除信使RNA外的许多类RNA中，包括tRNA和rRNA。假尿苷是非编码RNA中含量最多的修饰核苷，通过稳定RNA结构来增强转移RNA和核糖体RNA的功能。假尿苷是由假尿苷合酶催化得来的，这种酶在转录后将RNA中特定的尿苷异构化，这个过程被称为假尿苷化。假尿苷作为核苷酸代谢物，对其的研究越来越受到学者们的重视。假尿苷可作为一个潜在的生物标志物，用于肾病与肿瘤的诊断、疗效监测，而且对于其他疾病的诊断也有一定的参考意义。研究表明，假尿苷可以减少辐射引起的人类淋巴细胞染色体畸变。近年来，科学家推测假尿苷在RNA稳定性和/或帮助氨基酰基转移酶与tRNAs相互作用方面发挥作用。另外，随着生物技术的飞速发展，利用核酸适配体、RNA干扰等手段发现的基因组药物引起了人们的兴趣，根据其特殊的生理功能，假尿苷被用作医药中间体，假尿苷的磷脒衍生物被用作合成此类低聚物的起始材料之一。
在假尿苷制备方面，现目前，只有传统的化学合成法可催化合成假尿苷，但是，针对假尿苷的化学合成，存在化学合成步骤长、收率低，且用到的试剂易燃易爆，较不安全等一系列的问题。在生物合成方面，尚未发现可专一大量用于假尿苷制备的微生物菌种和技术报道。因此，假尿苷的低成本便捷式获取存在一定的缺陷。
针对现有技术中假尿苷只能通过化学合成法制备，且化学合成过程中存在的合成步骤长、收率低，且用到的试剂易燃易爆，较不安全等一系列的问题，寻求一种通过基因工程构建可发酵产假尿苷的工程菌，并可通过简单的发酵和较低的成本实现假尿苷的生产。
发明内容
为了解决假尿苷现有制备方法不足的问题，本发明提供了一种产假尿苷工程菌及其应用，包括以下步骤：利用基因工程技术在大肠杆菌中表达假尿苷合成相关的基因，得到可产假尿苷的基因工程菌株；
所述假尿苷合成相关基因的蛋白序列为：腺苷酸激酶编码基因pumH蛋白序列、tRNA假尿苷合酶编码基因pumJ蛋白序列和HAD(卤代酸脱卤酶)蛋白家族水解酶编码基因pumD蛋白序列；
为了解决以上技术问题，本发明采用如下技术方案：
本发明的技术方案之一为：一种产假尿苷工程菌，利用基因工程技术在宿主菌中表达假尿苷合成相关的基因，得到可产假尿苷的基因工程菌。
本发明所述的宿主菌包括不限于细菌、藻和真菌，其中，细菌优选自大肠杆菌，进一步，优选为大肠杆菌DH5α。
进一步，作为优选的，所述假尿苷合成相关的基因为：腺苷酸激酶编码基因pumH、tRNA假尿苷合酶编码基因pumJ和HAD(卤代酸脱卤酶)蛋白家族水解酶编码基因pumD。
进一步，作为优选的，所述的编码基因pumH蛋白序列、编码基因pumJ蛋白序列和编码基因pumD蛋白序列均来源于链霉菌Streptomyces sp.ID38640，分别为SEQ ID No.1所示的基因pumH蛋白序列、SEQ ID No.2所示的基因pumJ蛋白序列和SEQ ID No.3所示的基因pumD蛋白序列。
进一步，作为优选的，所述的基因pumH、pumJ和pumD，其DNA序列均来源于链霉菌Streptomyces sp.ID38640，序列分别如SEQ ID No.4所示的核苷酸序列、SEQ ID No.5所示的核苷酸序列和SEQ ID No.6所示的核苷酸序列。
进一步，作为优选的，所述的基因pumH、pumJ和pumD，其DNA序列按照大肠杆菌密码子偏好性进行优化，优化后的序列分别如SEQ ID No.7所示的核苷酸序列、SEQ ID No.8所示的核苷酸序列和SEQ ID No.9所示的核苷酸序列。
进一步，作为优选的，包括对所述的密码子偏好性优化后的基因pumH、pumJ和pumD进行修饰，具体为pumH密码子优化后的核酸序列在起始密码子前连接启动子Ptrp序列，pumJ和pumD密码子优化后的核酸序列在起始密码子前连接RBS序列aaaggaggatatacat，修饰后序列分别如SEQ ID No.10所示的核苷酸序列、SEQ ID No.11所示的核苷酸序列和SEQID No.12所示的核苷酸序列。
本发明所述的产假尿苷工程菌的构建方法具体包括如下步骤：
(1)pumH+Ptrp序列和载体pUC18分别用HindIII+SphI酶切，然后用T4连接酶(Thermfisher)连接，连接产物转化大肠杆菌感受态细胞(优选为DH5α)，得到克隆pZH423；
(2)pumJ+RBS序列和载体pZH423分别用XbaI+SphI酶切，然后用T4连接酶(Thermfisher)连接，连接产物转化大肠杆菌感受态细胞(优选为DH5α)，得到克隆pZH424；
(3)pumD+RBS序列和载体pZH424分别用XbaI+KpnI酶切，然后用T4连接酶(Thermfisher)连接，连接产物转化大肠杆菌感受态细胞(优选为DH5α)，得到克隆pZH425，即产假尿苷工程菌。
本发明所述的产假尿苷工程菌在产假尿苷中的应用。
进一步，作为优选的，所述的产假尿苷的方法，包括如下步骤：将上述所述的基因工程菌进行发酵培养得到假尿苷。
进一步，作为优选的，向所述的发酵培养基中添加肌苷，以溶于氢氧化钠的水溶液的形式加入，所述的发酵培养基中肌苷的初始终浓度为1.0-10g/L。
进一步，作为优选的，所述的发酵过程pH维持在6.5-7.6之间，优选为采用柠檬酸水溶液或乙酸水溶液和氨水进行组合调节。
进一步，作为优选的，所述的柠檬酸或乙酸的浓度为50-100g/L；所述的氨水优选浓度为13mol/L。
进一步，作为具体的，所述发酵培养的培养基配方(g/L)含有如下组分：葡萄糖10-20，KH2PO4 2-6，酵母粉4-8，硫酸铵3-10，玉米浆干粉10-20，MgSO4·7H2O 2-6；培养基中其他物料组分(mg/L)：FeSO4·7H2O 100-200，MnSO4·7H2O 10-30，VB1 1.3-3.0，VH 0.6-2.0，氯化钴2-5，硫酸锌2-5，氯化钙5-10，壮观霉素50，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。
进一步，作为优选的，发酵过程中，阶段控温，发酵第10-20h，发酵温度保持在28-30℃，随后，发酵温度升高至35-37℃；发酵培养周期为72-96h。
与现有技术相比，本发明提供了一种产假尿苷工程菌及其应用，所述工程菌是将来源于链霉菌Streptomyces sp.ID38640中的假尿苷合成基因pumH、pumJ和pumD基因克隆至大肠杆菌体内，转化得到可产假尿苷的工程菌。本发明通过对大肠杆菌进行基因改造，实现了假尿苷在大肠杆菌体内的异源表达，进一步通过向培养基中添加肌苷，在发酵过程中采用阶段控温，并使用柠檬酸和氨水对pH进行调控，极大促进了假尿苷的积累，最终基因工程改造的大肠杆菌可产假尿苷达7g/L以上。本发明大肠杆菌基因构建方法简单，发酵过程中不需要添加诱导剂异丙基-β-D-硫代吡喃半乳糖苷即可获得较高产量的假尿苷，应用前景广阔。
附图说明
图1为假尿苷标准品的质谱图；
图2为假尿苷标准品的HPLC检测图谱；
图3为实施例4pZH425发酵液检测的HPLC图谱。
具体实施方式
以下实施例对本发明作进一步说明，但有必要指出以下实施例只用于对发明内容的描述，并不构成对本发明保护范围的限制，本发明保护范围以权利要求为准。
本发明所用菌种为大肠杆菌DH5α，菌种保藏编号为CCTCC AB2013329，购自中国典型培养物保藏中心(China Center for Type Culture Collection，简称CCTCC)。
除特别说明外，本发明实施例中所用的各种材料和试剂都是本领域种常用的材料和试剂，均可以通过常规的商业途径获得。
本发明假尿苷效价检测所用的液相检测方法条件如下：
色谱柱：Waters XBridge Amide(250mm×460mm,3.5μm)，流动相A：0.2％(质量体积比，单位为g/L)乙酸铵水溶液，流动相B：乙腈，保留时间：20min，流速：1mL/min，进样量：10μL，检测波长：255nm，柱温：(25±1)℃。
表1流动相A和B的洗脱程序体积比
| Min | A | B |
| 0 | 100 | 0 |
| 3 | 100 | 900 |
| 6 | 98 | 2 |
| 12 | 60 | 40 |
| 17 | 100 | 0 |
| 20 | 100 | 0 |
本发明实施例中所用的固体培养基(g/L)为：酵母提取物5，胰蛋白胨10，NaCl 10，壮观霉素0.05，其余为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。
本发明实施例中所用的种子培养基(g/L)为：酵母提取物5，胰蛋白胨10，NaCl 10，葡萄糖20，壮观霉素0.05，其余为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。
本发明实施例中所用发酵培养基配方(g/L)为：葡萄糖10-20，KH2PO4 2-6，酵母粉4-8，硫酸铵3-10，玉米浆干粉10-20，MgSO4·7H2O 2-6；培养基中其他物料组分(mg/L)：FeSO4·7H2O 100-200，MnSO4·7H2O 10-30，VB1 1.3-3.0，VH 0.6-2.0，氯化钴2-5，硫酸锌2-5，氯化钙5-10，另外培养基中加入壮观霉素50mg/L，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。
实施例1：pZH423的构建HindIII和SphI双酶切由上海生工合成的SEQ IDNo.10DNA片段，得到外源片段1；HindIII和SphI双酶切载体pUC18，得到载体片段1；将外源片段1与载体片段1连接转化大肠杆菌感受态细胞DH5α，得到重组菌落pZH423。
实施例2：pZH424的构建
XbaI和SphI双酶切上海生工合成的SEQ ID No.11DNA片段，得到外源片段2；XbaI和SphI双酶切载体pZH423，得到载体片段2；将外源片段2与载体片段2连接转化大肠杆菌感受态细胞DH5α，得到重组菌落pZH424。
实施例3：pZH425的构建
XbaI和KpnI双酶切上海生工合成的SEQ ID No.12DNA片段，得到外源片段3；XbaI和KpnI双酶切载体pZH424，得到载体片段3；将外源片段3与载体片段3连接转化大肠杆菌感受态细胞DH5α，得到重组菌落pZH425。
实施例4：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取4ml种子液至20ml的发酵培养基中，所述发酵培养的培养基配方(g/L)如下：葡萄糖10，KH2PO4 2，酵母粉4，硫酸铵3，玉米浆干粉10，MgSO4·7H2O 2；培养基中其他物料组分(mg/L)：FeSO4·7H2O 100，MnSO4·7H2O 10，VB1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，发酵温度保持在37℃，250rpm培养30h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
假尿苷标准品的质谱图(图1)，假尿苷标准品的HPLC检测图谱(图2，保留时间15min)。
pZH425发酵液检测的HPLC图谱(图3)，经与标准品对比测算，假尿苷发酵单位约为0.6g/L。
实施例5：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖15，KH2PO4 4，酵母粉6，硫酸铵8，玉米浆干粉15，MgSO4·7H2O 4；培养基中其他物料组分(mg/L)：FeSO4·7H2O 150，MnSO4·7H2O 20，VB1 2，VH 1，氯化钴3.5，硫酸锌3.5，氯化钙8，另外培养基中加入壮观霉素50mg/L，另，将10g肌苷溶解于100ml含有氢氧化钠1mol/L的水溶液中，再将含有10g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有50g/L的柠檬酸水溶液和含氨13mol/L的氨水调控在6.5，发酵温度在0-10h保持在28℃，发酵第11h至发酵结束，发酵温度保持在35℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期72h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位约为5.4g/L。
实施例6：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖15，KH2PO4 4，酵母粉6，硫酸铵8，玉米浆干粉15，MgSO4·7H2O 4；培养基中其他物料组分(mg/L)：FeSO4·7H2O 200，MnSO4·7H2O 30，VB1 3，VH 2，氯化钴5，硫酸锌5，氯化钙10，另外培养基中加入壮观霉素50mg/L，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有60g/L的乙酸水溶液和含氨13mol/L的氨水调控在6.8，发酵温度在0-10h保持在28℃，发酵第11h至发酵结束，发酵温度保持在35℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期72h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位约为2.9g/L。
实施例7：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖20，KH2PO4 6，酵母粉8，硫酸铵10，玉米浆干粉20，MgSO4·7H2O 6；培养基中其他物料组分(mg/L)：FeSO4·7H2O 100，MnSO4·7H2O 10，VB1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50，另，将60g肌苷溶解于200ml含有氢氧化钠1mol/L的水溶液中，再将含有60g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有80g/L的柠檬酸水溶液和含氨13mol/L的氨水调控在7.0，发酵温度在前15h保持在30℃，发酵第16h至发酵结束，发酵温度保持在37℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期84h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位约为6.1g/L。
实施例8：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖20，KH2PO4 6，酵母粉8，硫酸铵10，玉米浆干粉20，MgSO4·7H2O 6；培养基中其他物料组分(mg/L)：FeSO4·7H2O 100，MnSO4·7H2O 10，VB1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50mg/L，另，将60g肌苷溶解于200ml含有氢氧化钠1mol/L的水溶液中，再将含有60g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有80g/L的乙酸水溶液和含氨13mol/L的氨水调控在7.0，发酵温度在前15h保持在30℃，发酵第16h至发酵结束，发酵温度保持在37℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期84h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位约为3.3g/L。
实施例9：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖20，KH2PO4 6，酵母粉7，硫酸铵10，玉米浆干粉18，MgSO4·7H2O 6；培养基中其他物料组分(mg/L)：FeSO4·7H2O 200，MnSO4·7H2O 20，VB1 2，VH 1，氯化钴4，硫酸锌4，氯化钙8，另外培养基中加入壮观霉素50mg/L，另，将100g肌苷溶解于300ml含有氢氧化钠1mol/L的水溶液中，再将含有100g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有100g/L的柠檬酸水溶液和含氨13mol/L的氨水调控在7.6，发酵温度在前20h保持在30℃，发酵第20h至发酵结束，发酵温度保持在37℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期96h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位约为7.2g/L。
对比实施例1：改造前大肠杆菌发酵产假尿苷检测
(1)改造前大肠杆菌的发酵培养
取改造前大肠杆菌菌落接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取4ml种子液至20ml的发酵培养基中，所述发酵培养的培养基配方(g/L)如下：葡萄糖10，KH2PO4 2，酵母粉4，硫酸铵3，玉米浆干粉10，MgSO4·7H2O 2；培养基中其他物料组分(mg/L)：FeSO4·7H2O 100，MnSO4·7H2O 10，VB1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50μg/ml，余量为水，pH 7.0。发酵过程中，发酵温度保持在37℃，250rpm培养30h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
经过对HPLC检测图谱的分析，改造前大肠杆菌发酵液中未检测有假尿苷，说明改造前，大肠杆菌不产假尿苷。
对比实施例2：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖20，KH2PO4 6，酵母粉8，硫酸铵10，玉米浆干粉20，MgSO4·7H2O 6；培养基中其他物料组分(mg/L)：FeSO4·7H2O 100，MnSO4·7H2O 10，VB1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50mg/L，另，将80g肌苷溶解于200ml含有氢氧化钠1mol/L的水溶液中，再将含有80g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用含有100g/L的乙酸水溶液和含氨13mol/L的氨水调控在8.0，发酵温度在前20h保持在30℃，发酵第20h至发酵结束，发酵温度保持在37℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期96h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位基本检测不到。
对比实施例3：pZH425发酵产假尿苷
(1)pZH425的发酵培养
取实施例3中构建成功的pZH425接入平板固体培养基中，36℃培养20h后，从固体培养基中挑取单菌落接入种子培养基中，36℃，250rpm培养15h得到种子液。再按照20％的接种量，吸取2L种子液至发酵培养基中，接种后培养基总体积为10L。所述发酵培养的培养基配方(g/L)如下：葡萄糖20，KH2PO4 6，酵母粉7，硫酸铵10，玉米浆干粉18，MgSO4·7H2O 6；培养基中其他物料组分(mg/L)：FeSO4·7H2O 100，MnSO4·7H2O 10，VB1 1.3，VH 0.6，氯化钴2，硫酸锌2，氯化钙5，另外培养基中加入壮观霉素50mg/L，另，将50g肌苷溶解于200ml含有氢氧化钠1mol/L的水溶液中，再将含有50g肌苷的氢氧化钠水溶液加入发酵培养基中，余量为水，灭菌前，发酵培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调至7.0。发酵过程中，培养基pH用2mol/L的盐酸和2mol/L的氢氧化钠调控在7.2，发酵温度保持在37℃，发酵过程中通气设定为1.0vvm，通过搅拌转速和溶氧联动，控制溶氧在30％-40％，发酵周期96h，得到发酵液。
(2)假尿苷的HPLC检测
发酵结束后，将发酵液进行14000×g离心10min，取离心后的上清过膜处理(0.22um水系滤膜)，过膜后的上清液进行HPLC检测。以假尿苷的标准品为对照，进行发酵液中假尿苷发酵单位的计算。
pZH425发酵液检测的HPLC图谱，经与标准品对比测算，假尿苷发酵单位基本检测不到。
SEQ ID No.1：
腺苷酸激酶编码基因pumH蛋白序列
SEQ ID No.2：
tRNA假尿苷合酶编码基因pumJ蛋白序列
SEQ ID No.3：
HAD(卤代酸脱卤酶)蛋白家族水解酶编码基因pumD蛋白序列
SEQ ID No.4：
基因pumH核苷酸序列
SEQ ID No.5：
基因pumJ核苷酸序列
SEQ ID No.6：
基因pumD核苷酸序列
SEQ ID No.7：pumH密码子优化后的核酸序列
SEQ ID No.8：pumJ密码子优化后的核酸序列
SEQ ID No.9：
pumD密码子优化后的核酸序列
SEQ ID No.10：
人工合成的pumH和启动子Ptrp序列
SEQ ID No.11：
人工合成的pumJ和RBS序列
SEQ ID No.12：
人工合成的pumD和RBS序列
序列表
<110> 浙江珲达生物科技有限公司
<120> 一种产假尿苷工程菌及其应用
<130> 1
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 233
<212> PRT
<213> Streptomyces sp. ID 38640
<400> 1
Met Ile Ile Glu Gly Ala Gly Gly Arg Gln Arg Pro Ala Asn Gly Pro
1 5 10 15
Arg Gly Lys Pro Thr Val Thr Leu His Val Leu Asn Pro Ala Ala Ala
20 25 30
Leu Pro Arg Arg Pro Asp Arg Val Leu Ile Ala Gly Gly Ser Gly Ala
35 40 45
Gly Lys Thr Ala Val Ala Asp Lys Leu Ala Thr Leu Leu Ala Leu Pro
50 55 60
Arg Ile Glu Leu Asp Glu Leu Tyr Tyr Gln Pro Gly Trp Glu Pro Arg
65 70 75 80
Pro Glu Phe Ala Glu Asp Val Ala Arg Val Ser Gly Ala Pro Arg Trp
85 90 95
Ile Thr Glu Trp His Tyr Pro Glu Val Ser Gly Leu Leu Ala Arg Arg
100 105 110
Ala Asp Leu Leu Val Trp Leu Asp Tyr Pro Thr Arg Thr Thr Met Thr
115 120 125
Ser Leu Val Leu Arg Thr Leu Leu Arg Ser Leu His Lys Glu Val Leu
130 135 140
Trp Ser Gly Asn Gln Glu Pro Arg Leu Arg Ala Val Leu Thr Asp Pro
145 150 155 160
Glu His Ile Leu Arg Tyr Gly Trp Ser Thr Arg His Ser Ala Arg Asp
165 170 175
Glu Val Arg Thr Leu Ala Arg Arg Ala Pro Asp Ile Thr Leu Asp Leu
180 185 190
Leu Arg Phe Ala Arg Pro Ala Glu Leu Ser Asp Trp Leu Leu Arg Leu
195 200 205
Ala Gly Thr Ser Tyr Ala Pro Ala Pro Ser Gly Gln Pro Ala Thr Glu
210 215 220
Arg Ser Thr Asp Ala Ser Gly Leu Arg
225 230
<210> 2
<211> 342
<212> PRT
<213> Streptomyces sp. ID38640
<400> 2
Met Thr Gly Thr Leu Val Leu Lys His Arg Gln Glu Asp Phe Arg Val
1 5 10 15
Arg Glu Asn Leu Val Val Ala Leu Thr Asp Ala Ala Ala Ala Thr His
20 25 30
Arg Tyr Leu Leu Leu His Lys Arg Gly His Thr Thr Met Glu Ala Val
35 40 45
Arg Leu Val Ala Asp Arg Leu Gly Val Ala Arg Glu Asp Val Gly Tyr
50 55 60
Ala Gly Leu Lys Asp Glu Asp Gly Ile Thr Glu Gln Leu Leu Ser Val
65 70 75 80
Pro Leu Ala Ser Leu Ala Ala Asp Ala Leu Pro Ala Ala Gly Leu Val
85 90 95
Glu Gly Ala Gly Pro Glu Arg Met Leu Ser Leu Ser His Tyr Gly Phe
100 105 110
Gly Arg Glu Pro Leu Thr Val Gly Gln Leu Asn Gly Asn Gly Phe Arg
115 120 125
Val Val Leu Arg Asp Leu Asp Glu Ala Ala Ala Ala Arg Leu Val Asp
130 135 140
Arg Gln Arg Ile Asn Leu Leu Phe Val Asn Tyr Tyr Asp Thr Gln Arg
145 150 155 160
Phe Gly Val Pro Gly Gly Pro Lys Arg Thr His Leu Val Gly Glu Ala
165 170 175
Leu Leu Lys Glu Asp Trp Ala Leu Ala Arg Gln Glu Leu Ala Gly Leu
180 185 190
Gly Ala Pro Glu Ser Gly Glu Ala Ala Arg Trp Thr Gly Gln Asp Arg
195 200 205
Ala Leu Phe Arg Ala Leu Asp Pro Arg Thr Val Ala Phe Tyr Leu Ala
210 215 220
Ala Tyr Ser Ser Tyr Asp Trp Asn Ala Arg Val Arg Asp Leu Ile Gly
225 230 235 240
Ser Leu Cys Pro Asp Asp Ala Pro Glu Ser Ala Val Asp Gly Leu Pro
245 250 255
Tyr Arg Phe Pro Thr Thr Ala Glu Gly Val Ala Ala Leu Leu Ala Ala
260 265 270
Cys His Glu Leu Pro Tyr Thr Arg Tyr Ala Tyr Arg Asp Gly Pro Val
275 280 285
Glu Arg Pro Thr Thr Arg Pro Thr Val Ile Gln Thr Ala Ile Thr Val
290 295 300
Gly Ala Asp Gly Pro Asp Asp Ala Phe Pro Gly Arg Arg Ala Val Glu
305 310 315 320
Val Ser Phe Leu Leu Pro Ser Gly Cys Tyr Ala Thr Ala Ala Leu Arg
325 330 335
Gln Leu Val Leu Arg Arg
340
<210> 3
<211> 200
<212> PRT
<213> Streptomyces sp. ID38640
<400> 3
Met Thr Gly Thr Val Leu Phe Asp Leu Phe Gly Val Ile Ala Arg His
1 5 10 15
Gln Ser Thr Glu Gly Lys Asn Arg Leu Thr Arg Thr Ala Gly Val Ala
20 25 30
Gly Pro Ala Phe Trp Asp Ala Tyr Trp Glu Leu Arg Pro Pro Tyr Asp
35 40 45
Arg Gly Glu Val Asn Gly Pro Gly Tyr Trp Arg Gln Val Ala Asp Ala
50 55 60
Ile Gly Val Arg Phe Asp Asp His Arg Ile Ala Asp Leu Val Glu Ala
65 70 75 80
Asp Ile Ala Ser Trp Ser Ala Val Asp Asp Thr Met Val Ala Leu Ile
85 90 95
Glu Glu Leu Thr Ala Thr Gly Arg His Met Gly Leu Leu Ser Asn Ile
100 105 110
Pro Glu Glu Leu Ala Ser His Tyr Glu Ala His His Ala Trp Leu Lys
115 120 125
His Phe Pro Val Arg Ala Phe Ser Cys Arg Met Gly His Ala Lys Pro
130 135 140
Glu Arg Ala Ala Tyr Glu Trp Cys Gln His Ala Leu Arg Thr Glu Pro
145 150 155 160
Asp Arg Ile Leu Phe Val Asp Asp Arg Ala Asp Asn Val Arg Ala Ala
165 170 175
Glu Glu Leu Gly Met Gln Gly His Leu Phe Thr Thr Pro Asp Arg Leu
180 185 190
Arg Gln Ala Leu Ser Gln Trp Thr
195 200
<210> 4
<211> 702
<212> DNA
<213> Streptomyces sp. ID38640
<400> 4
gtgatcattg agggcgccgg cgggcgacag cggccggcga acggaccgag aggaaagccg 60
acggtgactc tgcacgtgct caacccggcg gcggccctgc cgcgccgccc ggacagggtg 120
ctgatcgcgg gcggcagcgg cgcgggcaag acggctgtcg ccgacaaact cgccacgctg 180
ctggcgctgc cccgcatcga actggacgag ctgtactacc agcccggctg ggagccgcgc 240
cccgagttcg ccgaggacgt cgcgcgggtc tccggggcgc cccggtggat caccgagtgg 300
cactacccgg aggtctccgg cctcctcgcg cgccgcgccg atctgctcgt ctggctcgac 360
tacccgaccc gcacgacgat gaccagcctg gtgctgcgga ccctgctgcg gtcgctgcac 420
aaggaggtgc tgtggagcgg caaccaggaa ccgcggttgc gggcggtgct caccgacccg 480
gagcacatcc tgcgctacgg ctggtcgacc cggcactcgg cccgtgacga ggtgcggaca 540
ctggcccgac gggctcccga catcacgctg gacctgctgc ggttcgcccg gcccgccgag 600
ttgtcggact ggctgctgcg gctggccgga acctcgtacg ccccggcgcc gtccgggcaa 660
cccgccacgg agaggagtac cgatgcgtca gggcttcgat ga 702
<210> 5
<211> 1029
<212> DNA
<213> Streptomyces sp. ID38640
<400> 5
gtgaccggca ccctggtgct caagcaccgc caggaggact tccgggtacg ggagaacctc 60
gtcgtcgcgc tgaccgacgc cgcagcggcc acccaccgct acctcctgct gcacaaacgc 120
ggccacacga cgatggaggc cgtgcggctc gtggcggacc ggctcggtgt ggcccgcgag 180
gacgtcggct acgcgggcct caaggacgag gacggcatca cggagcagct gctgtccgtg 240
ccgcttgctt cccttgccgc cgacgcgctg cccgccgccg gtctcgtcga aggggcgggg 300
ccggagcgga tgctgagcct gagccactac ggtttcggtc gtgagccgct gaccgtcggg 360
cagctcaacg gcaacggctt ccgggtggtg ctgcgggacc tcgacgaggc ggccgccgcc 420
cggctcgtgg accggcagcg gatcaacctg ctgttcgtca actactacga cacccagcgc 480
ttcggcgtac ccggcggccc caaacgcacc cacctggtcg gcgaggcgct gctgaaggag 540
gactgggccc tcgcacggca ggagttggcg gggctcggcg ctccggagag cggcgaggcc 600
gcccggtgga ccgggcagga ccgggccctc ttccgggcgc tcgacccccg gaccgtggcc 660
ttctacctgg ccgcgtactc ctcctacgac tggaacgcgc gggtccggga cctgatcggc 720
tctctctgcc cggacgacgc ccccgagagc gccgtggacg gactgcccta ccgctttccc 780
accacggcgg aaggcgtggc cgcgctcctc gcggcctgtc acgaactgcc gtacacccgc 840
tacgcgtacc gggacggccc cgtcgagcgg ccgacgaccc ggcccacggt catccagacg 900
gcgatcaccg tcggagcgga cggaccggac gacgccttcc ccgggcgccg ggcggtggag 960
gtctccttcc tgttgccctc ggggtgttac gccacggcgg cgctgcggca gttggtcctc 1020
cgccgctga 1029
<210> 6
<211> 603
<212> DNA
<213> Streptomyces sp. ID38640
<400> 6
gtgacgggca ccgtgctctt cgacctcttc ggcgtcatcg cacgccatca gtccaccgaa 60
ggcaagaacc ggctcacccg gacggcagga gtggcggggc cggccttctg ggacgcctac 120
tgggagctgc gcccgcccta cgaccgtggt gaggtgaacg gccccggcta ctggcgtcag 180
gtggccgacg ccatcggcgt ccgcttcgac gaccaccgga tcgccgacct cgtcgaggcc 240
gacatcgcga gctggagtgc ggtcgacgac acgatggtcg ccctgataga ggaactcacc 300
gccacaggcc gccacatggg gctgctgtcc aacatccccg aagagctggc ctcgcactat 360
gaagcgcatc atgcctggct caagcacttc ccggtacggg ccttctcatg tcgcatgggc 420
cacgccaagc ccgagcgcgc cgcctacgag tggtgtcagc acgccctgcg tacggagccg 480
gaccgcatcc tctttgtcga cgaccgcgcg gacaacgtac gtgccgccga agaactcggc 540
atgcagggac acctcttcac caccccggac cggctgaggc aggctctcag tcaatggacc 600
tga 603
<210> 7
<211> 702
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgataattg aaggtgctgg agggaggcaa agaccggcaa atggtccgcg tggtaagccg 60
accgttaccc ttcacgtcct gaacccggcg gcggcgctgc cgcgtcgtcc tgatcgtgtg 120
ctgattgcag gcggtagcgg tgcgggcaag accgcagttg ctgacaaact ggccaccttg 180
ctggctctgc cgcgcatcga attggacgag ctgtactacc agccgggttg ggaaccgcga 240
ccggagttcg ccgaagatgt tgcgcgtgtt tcgggcgcac cgcgttggat taccgagtgg 300
cattatccgg aggtaagcgg cctgttggcg cgtcgtgcgg atttgctggt gtggctggac 360
tacccgactc gcacgaccat gacctctctg gttctgcgca cgctgctgcg ctccctgcac 420
aaagaagtgc tgtggagcgg aaaccaggag ccgcgtttac gtgctgtgtt gaccgatccg 480
gagcacatcc tgcgttatgg ttggtcaacc cgtcatagcg caagagatga ggtgcgcact 540
ttggcgcgtc gcgcgccaga catcaccctc gacctgttgc gttttgcccg cccagctgaa 600
ctctctgatt ggctgcttcg cctggctggt acaagctatg caccggcgcc gtccggccaa 660
ccggcgaccg aacgttccac ggacgcgagc ggcctgcgtt aa 702
<210> 8
<211> 1029
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgactggaa cactagtttt aaaacacagg caagaagatt ttcgtgttcg tgagaacctg 60
gtggtggcgc ttaccgatgc cgcggcggcg acccatcgtt acctgttact gcacaaacga 120
ggtcacacta ccatggaggc agtgcgcctg gttgcggacc gtctgggcgt ggcgcgtgaa 180
gatgttggtt atgcaggcct gaaggacgaa gatggcatca ccgagcagct gctttccgtg 240
ccgttggcta gcctggccgc ggacgccctg ccggcagcgg gtctcgtaga gggcgctggt 300
ccggagcgta tgctgtcttt gtcgcattat ggtttcggcc gtgagccgtt gaccgtgggc 360
cagctgaacg gtaatggctt ccgcgttgtt ctgcgcgacc tggatgaagc cgccgctgcg 420
cgtctggtcg atcgtcaacg cattaacctg ctgttcgtga attattacga cacccagcgt 480
tttggtgttc cgggtggtcc gaaacgtact cacctggttg gcgaagcgtt gttgaaggag 540
gactgggcac tcgctcgcca agagctggcg ggtctgggtg ctccggagtc cggcgaagcg 600
gcgcgttgga ccggtcagga ccgtgcattg tttcgcgcat tggatccgcg taccgtcgcc 660
ttctacctgg cggcttactc cagctatgac tggaacgctc gtgttcgtga tctgattggt 720
tctttatgcc cggacgatgc gccggaaagc gcggtagacg gcctgccgta ccgcttcccg 780
actacggctg aaggtgtggc cgctttgctc gcggcgtgtc atgaactgcc gtatacccgt 840
tacgcctatc gcgacggccc ggttgagcgc cctaccacgc gcccgacggt gatccagacc 900
gcgatcacgg tgggtgcgga tggcccagac gacgccttcc cgggtcgtcg tgcagtggag 960
gtcagctttc tgttgccaag cgggtgctac gcaaccgctg cgttaagaca actggttctg 1020
cgtagataa 1029
<210> 9
<211> 603
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgactggaa cagttttatt tgatctattc ggcgttatcg cccgtcacca gagcaccgag 60
ggtaaaaacc gtttgacgcg tactgccggc gtcgccggtc cggcattctg ggatgcgtac 120
tgggaattgc gccctccgta tgatcgcggt gaagttaatg gcccaggtta ttggcgtcaa 180
gttgccgacg caattggcgt ccgttttgat gatcatcgta tcgcagacct ggtggaagcg 240
gacatcgctt cttggagcgc ggttgacgac accatggttg cgctgattga agagttgacg 300
gcgaccggtc gtcacatggg cctgctgagc aacattccgg aggagcttgc ttcccattac 360
gaggcgcatc acgcgtggct gaagcacttt ccggtgcgtg cgttcagctg ccgtatgggc 420
cacgctaaac cggaacgtgc tgcgtacgag tggtgtcagc atgcgctgcg taccgaaccg 480
gatcgcatcc tgttcgtgga tgaccgggct gacaacgtga gagcggcgga agagttaggt 540
atgcagggtc acctgtttac caccccggat cgcctccgcc aagcactgtc gcaatggacc 600
taa 603
<210> 10
<211> 766
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tgttgacaat taatcatcga actagttaac tagtacgcaa gttcacgtaa aaagggtatc 60
gacaatgata attgaaggtg ctggagggag gcaaagaccg gcaaatggtc cgcgtggtaa 120
gccgaccgtt acccttcacg tcctgaaccc ggcggcggcg ctgccgcgtc gtcctgatcg 180
tgtgctgatt gcaggcggta gcggtgcggg caagaccgca gttgctgaca aactggccac 240
cttgctggct ctgccgcgca tcgaattgga cgagctgtac taccagccgg gttgggaacc 300
gcgaccggag ttcgccgaag atgttgcgcg tgtttcgggc gcaccgcgtt ggattaccga 360
gtggcattat ccggaggtaa gcggcctgtt ggcgcgtcgt gcggatttgc tggtgtggct 420
ggactacccg actcgcacga ccatgacctc tctggttctg cgcacgctgc tgcgctccct 480
gcacaaagaa gtgctgtgga gcggaaacca ggagccgcgt ttacgtgctg tgttgaccga 540
tccggagcac atcctgcgtt atggttggtc aacccgtcat agcgcaagag atgaggtgcg 600
cactttggcg cgtcgcgcgc cagacatcac cctcgacctg ttgcgttttg cccgcccagc 660
tgaactctct gattggctgc ttcgcctggc tggtacaagc tatgcaccgg cgccgtccgg 720
ccaaccggcg accgaacgtt ccacggacgc gagcggcctg cgttaa 766
<210> 11
<211> 1045
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aaaggaggat atacatatga ctggaacact agttttaaaa cacaggcaag aagattttcg 60
tgttcgtgag aacctggtgg tggcgcttac cgatgccgcg gcggcgaccc atcgttacct 120
gttactgcac aaacgaggtc acactaccat ggaggcagtg cgcctggttg cggaccgtct 180
gggcgtggcg cgtgaagatg ttggttatgc aggcctgaag gacgaagatg gcatcaccga 240
gcagctgctt tccgtgccgt tggctagcct ggccgcggac gccctgccgg cagcgggtct 300
cgtagagggc gctggtccgg agcgtatgct gtctttgtcg cattatggtt tcggccgtga 360
gccgttgacc gtgggccagc tgaacggtaa tggcttccgc gttgttctgc gcgacctgga 420
tgaagccgcc gctgcgcgtc tggtcgatcg tcaacgcatt aacctgctgt tcgtgaatta 480
ttacgacacc cagcgttttg gtgttccggg tggtccgaaa cgtactcacc tggttggcga 540
agcgttgttg aaggaggact gggcactcgc tcgccaagag ctggcgggtc tgggtgctcc 600
ggagtccggc gaagcggcgc gttggaccgg tcaggaccgt gcattgtttc gcgcattgga 660
tccgcgtacc gtcgccttct acctggcggc ttactccagc tatgactgga acgctcgtgt 720
tcgtgatctg attggttctt tatgcccgga cgatgcgccg gaaagcgcgg tagacggcct 780
gccgtaccgc ttcccgacta cggctgaagg tgtggccgct ttgctcgcgg cgtgtcatga 840
actgccgtat acccgttacg cctatcgcga cggcccggtt gagcgcccta ccacgcgccc 900
gacggtgatc cagaccgcga tcacggtggg tgcggatggc ccagacgacg ccttcccggg 960
tcgtcgtgca gtggaggtca gctttctgtt gccaagcggg tgctacgcaa ccgctgcgtt 1020
aagacaactg gttctgcgta gataa 1045
<210> 12
<211> 619
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aaaggaggat atacatatga ctggaacagt tttatttgat ctattcggcg ttatcgcccg 60
tcaccagagc accgagggta aaaaccgttt gacgcgtact gccggcgtcg ccggtccggc 120
attctgggat gcgtactggg aattgcgccc tccgtatgat cgcggtgaag ttaatggccc 180
aggttattgg cgtcaagttg ccgacgcaat tggcgtccgt tttgatgatc atcgtatcgc 240
agacctggtg gaagcggaca tcgcttcttg gagcgcggtt gacgacacca tggttgcgct 300
gattgaagag ttgacggcga ccggtcgtca catgggcctg ctgagcaaca ttccggagga 360
gcttgcttcc cattacgagg cgcatcacgc gtggctgaag cactttccgg tgcgtgcgtt 420
cagctgccgt atgggccacg ctaaaccgga acgtgctgcg tacgagtggt gtcagcatgc 480
gctgcgtacc gaaccggatc gcatcctgtt cgtggatgac cgggctgaca acgtgagagc 540
ggcggaagag ttaggtatgc agggtcacct gtttaccacc ccggatcgcc tccgccaagc 600
actgtcgcaa tggacctaa 619
Claims (13)
1.一种产假尿苷工程菌，其特征在于，利用基因工程技术在宿主菌中表达假尿苷合成相关的基因，得到可产假尿苷的基因工程菌；
所述宿主菌为大肠杆菌；
所述假尿苷合成相关的基因为：腺苷酸激酶编码基因pumH、tRNA假尿苷合酶编码基因pumJ和HAD（卤代酸脱卤酶）蛋白家族水解酶编码基因pumD；
所述基因pumH、pumJ和pumD，其DNA序列均来源于链霉菌Streptomycessp. ID38640，序列分别如SEQ ID No. 4所示的核苷酸序列、 SEQ ID No. 5所示的核苷酸序列和SEQ IDNo. 6所示的核苷酸序列。
2.根据权利要求1所述的产假尿苷工程菌，其特征在于，所述的宿主菌为大肠杆菌DH5α。
3.根据权利要求1所述的产假尿苷工程菌，其特征在于，所述的编码基因pumH蛋白序列、编码基因pumJ蛋白序列和编码基因pumD蛋白序列均来源于链霉菌Streptomyces sp.ID38640，分别为SEQ ID No. 1 所示的基因pumH蛋白序列、SEQ ID No. 2所示的基因pumJ蛋白序列和SEQ ID No. 3所示的基因pumD蛋白序列。
4.一种产假尿苷工程菌，其特征在于，利用基因工程技术在宿主菌中表达假尿苷合成相关的基因，得到可产假尿苷的基因工程菌；
所述宿主菌为大肠杆菌；
所述假尿苷合成相关的基因为：腺苷酸激酶编码基因pumH、tRNA假尿苷合酶编码基因pumJ和HAD（卤代酸脱卤酶）蛋白家族水解酶编码基因pumD；
所述的基因pumH、pumJ和pumD，其DNA序列按照大肠杆菌密码子偏好性进行优化，优化后的序列分别如SEQ ID No. 7所示的核苷酸序列、 SEQ ID No. 8所示的核苷酸序列和SEQID No. 9所示的核苷酸序列。
5.根据权利要求4所述的产假尿苷工程菌，其特征在于，包括对所述的密码子偏好性优化后的基因pumH、pumJ和pumD进行修饰，具体为pumH密码子优化后的核酸序列在起始密码子前连接启动子Ptrp序列，pumJ和pumD密码子优化后的核酸序列在起始密码子前连接RBS序列aaaggaggatatacat，修饰后序列分别如SEQ ID No. 10所示的核苷酸序列、 SEQ IDNo. 11所示的核苷酸序列和SEQ ID No. 12所示的核苷酸序列。
6.根据权利要求5所述的产假尿苷工程菌的构建方法，其特征在于，所述方法包括如下步骤：
（1）pumH+Ptrp序列和载体pUC18分别用HindIII+SphI酶切，然后用T4连接酶连接，连接产物转化大肠杆菌感受态细胞，得到重组菌落pZH423；
（2）pumJ+RBS序列和载体pZH423分别用XbaI+SphI酶切，然后用T4连接酶连接，连接产物转化大肠杆菌感受态细胞，得到重组菌落pZH424；
（3）pumD+RBS序列和载体pZH424分别用XbaI+KpnI酶切，然后用T4连接酶连接，连接产物转化大肠杆菌感受态细胞，得到重组菌落pZH425，即产假尿苷工程菌。
7.一种如权利要求1所述的产假尿苷工程菌在产假尿苷中的应用。
8.一种产假尿苷的方法，其特征在于：将权利要求1~5之一所述的产假尿苷工程菌进行发酵培养得到假尿苷；
所述的发酵过程pH维持在6.5-7.6之间，采用柠檬酸水溶液或乙酸水溶液与氨水组合进行调节。
9.根据权利要求8所述的方法，其特征在于：所述的发酵培养基中添加肌苷。
10.根据权利要求9所述的方法，其特征在于：所述的肌苷以溶于氢氧化钠的水溶液的形式加入，所述的发酵培养基中肌苷的初始终浓度为1.0-10 g/L。
11.根据权利要求8所述的方法，其特征在于：所述的柠檬酸或乙酸的浓度为50-100 g/L。
12.根据权利要求8所述的方法，其特征在于：所述发酵培养的培养基配方含有如下组分：葡萄糖 10-20g/L，KH2PO4 2-6g/L，酵母粉 4-8g/L，硫酸铵 3-10g/L，玉米浆干粉 10-20g/L，MgSO4·7H2O 2-6g/L；所述发酵培养基中还包括其他物料组分：FeSO4·7H2O 100-200mg/L，MnSO4·7H2O 10-30mg/L，VB1 1.3-3.0mg/L，VH 0.6-2.0mg/L，氯化钴 2-5mg/L，硫酸锌 2-5mg/L，氯化钙 5-10mg/L，壮观霉素50mg/L，余量为水，pH7.0。
13.根据权利要求8所述的方法，其特征在于：发酵过程中，阶段控温，发酵第10-20 h，发酵温度保持在28-30℃，随后，发酵温度升高至35-37℃；发酵培养周期为72-96 h。
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