CN101914482A - 一株异戊酰螺旋霉素i组分高含量、高产量基因工程菌 - Google Patents

一株异戊酰螺旋霉素i组分高含量、高产量基因工程菌 Download PDF

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CN101914482A
CN101914482A CN2010102375954A CN201010237595A CN101914482A CN 101914482 A CN101914482 A CN 101914482A CN 2010102375954 A CN2010102375954 A CN 2010102375954A CN 201010237595 A CN201010237595 A CN 201010237595A CN 101914482 A CN101914482 A CN 101914482A
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spiramycin
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isovaleryl
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王以光
武临专
姜洋
郝玉有
杨生武
林灵
周红霞
戴剑漉
赫卫清
马春燕
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Shenyang Tonglian Group Co Ltd
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Institute of Medicinal Biotechnology of CAMS
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Abstract

本发明涉及利用调节基因技术构建获得抗生素单一组分高含量、高产量菌株，具体来讲，是将含有ist-acyB2双基因重组质粒pSET52-ia转入异戊酰螺旋霉素I产生菌，获得异戊酰螺旋霉素I组分高含量、高产量的基因工程菌株，为其工业化生产和制备注射剂型创造了良好条件。

Description

一株异戊酰螺旋霉素I组分高含量、高产量基因工程菌
技术领域：
本发明涉及利用调节基因技术构建获得抗生素单一组分高含量、高产量菌株。
背景技术：
必特螺旋霉素(原名生技霉素)[专利号：ZL97104440.6]是利用基因工程技术研制的螺旋霉素衍生物。必特螺旋霉素对革兰氏阳性菌有较强的活性，尤其对肺炎链球菌、肺炎支原体、衣原体活性强，对红霉素和β-内酰胺类抗生素耐药菌、流感杆菌、军团菌、产气荚膜梭菌有抗菌活性；与同类药没有完全交叉耐药性。它有较高的亲脂性，口服吸收快，组织渗透性强，分布广，体内维持时间长，有较好的抗生素后效应。目前已完成基因工程必特螺旋霉素的三期临床试验研究，结果显示良好的疗效和安全性，目前正在申报国家一类新药证书。
临床研究已经证明，必特螺旋霉素是一个服用安全有效的抗生素，其疗效不亚于目前公认最好的大环内酯类抗生素阿奇霉素。然而，其本身是多组分药物，尽管按照目前建立的片剂生产工艺，可以得到产品组分比例可控、质量稳定的必特螺旋霉素，然而其对提取、纯化工艺的控制要求比较高，质量检测过程也比较繁锁。同时为了提高其在临床的治疗效率，有必要研制其注射剂，众所周注射剂药效迅速，适用于对危重病人或不宜口服的病人。注射剂在质量控制方面要求更高。通常对于经发酵产生的多组分抗生素，按照化学药品的质控方式很难控制其质量，而且工艺条件的细小变化均有可能导致物质基础的变化，而这些质量上的变化较难通过终产品的质量标准加以控制，从而可能会引发不可预测的不良反应。这就是至今尚无发酵产生的多组分注射剂问世的原因。
必特螺旋霉素产生菌产生多组分是源于螺旋霉素的多组分。螺旋霉素是一个大环内酯类抗生素，其主核是一个由16元环组成的内酯环，与普拉特内酯同质，含有三个糖分子福洛糖胺、碳霉糖胺和碳霉糖，而必特螺旋霉素主组分是螺旋霉素碳霉糖4’位羟基被异戊酰基酯化的衍生物。由于螺旋霉素产生菌中存在3-O 酰基转移酶基因，因而使螺旋霉素内酯环3位羟基可以进行乙酰化，形成螺旋霉素II组分和丙酰化，形成螺旋霉素III组分，内酯环3位为羟基时为螺旋霉素I组分，所以必特螺旋霉素至少也含有异戊酰螺旋霉素I、II、III三个组分。必特螺旋霉素主组分的化学结构如下：
Figure BSA00000206192800021
福洛糖胺                  普拉特内酯          碳霉糖胺       碳霉糖
                             必特螺旋霉素结构
其中：
异戊酰螺旋霉素III   R＝COCH2CH3   R’＝COCH2CH(CH3)2
异戊酰螺旋霉素II    R＝COCH3      R’＝COCH2CH(CH3)2
异戊酰螺旋霉素I     R＝H          R’＝COCH2CH(CH3)2
已有研究表明，螺旋霉素的三种组分之间及异戊酰螺旋霉素的三种组分之间，其抗菌活性无显著差异。为了减少菌种产生多种组分，本实验室利用基因重组技术，在必特螺旋霉素产生菌WSJ-1中，通过阻断3-O酰基转移酶基因，构建了只产生异戊酰螺旋霉素I组分的菌种WSJ-2[Chunyan Ma et al.CurrentMicrobiology，2010，DOI 10.1007/s00284-010-9664-8]。但是，该菌种发酵单位很低，不利于进行规模化生产。本发明拟利用调节基因acyB2，改造必特螺旋霉素产生菌，以提高异戊酰螺旋霉素I组分的含量及产量。
已知来源于耐热链霉菌的异戊酰基转移酶基因ist与调节基因acyB2连锁，调节基因的存在可以激活ist基因的表达，含有完整ist和acyB2调节基因的变铅青链霉菌可以将67-79％泰洛菌素转化为4”异戊酰泰洛菌素，而仅含有单个ist或ist与不完整的acyB2基因的变铅青链霉菌对泰洛菌素的转化率仅为0-2.4％[Arisawa A et al：Biosci Biotechnol Biochem 1993，57(12)：2020-2025]。将ist和acyB2 基因转入泰洛菌素产生菌(Streptomyces fradiae)，其转化子经发酵可以产生4”异戊酰泰洛菌素[Arisawa A.et al J Antibiotics 1996，49(4)：349-354]。
acyB2调节基因与螺旋霉素产生菌(Streptomyces ambofaciens)中的转录调控蛋白Srm28c同源[Fatma Karray et al Microbiology 2007，153，4111-4122]，其一致性(identity)为69％，与泰洛菌素产生菌(Streptomyces fradiae)中的调控蛋白tylR的一致性为41％，tylR为泰洛菌素产生菌生物合成正调控蛋白，它调控泰洛菌素中一个聚酮合酶模块(tylGI)的表达，同时还对泰洛菌素糖基的合成和聚酮环的氧化起调控作用。tylR基因在产生菌的高表达，可以提高泰洛菌素的产量[GeorgeS.etal Mol.Microbiology 2004，54(5)：1326-1334]。
本实验室曾利用基因工程技术将ist基因进行串连，使ist基因在螺旋霉素产生菌中增加其拷贝数，通过增强其基因剂量提高产生菌异戊酰基化能力；同时也利用强启动活性的启动子如红霉素抗性基因ermE启动子序列，替换原ist基因启动子序列，以增强ist基因的表达，使基因工程菌产生异戊酰螺旋霉素主组分含量提高了62％[专利申请号200910148767.8]。
本发明的目的，旨在利用调节基因acyB2激活ist基因表达的特点，将ist与调节基因acyB2在螺旋霉素产生菌中共表达，以获得必特螺旋霉素单组分异戊酰螺旋霉素I的高含量及高产量菌株。
发明内容：
本发明提供了异戊酰螺旋霉素I组分高含量、高产量菌株。
本发明还提供了异戊酰螺旋霉素I组分高含量、高产量菌株的构建与制备，具体包括以下步骤：
1、构建含ist-acyB2双基因重组质粒pSET52-ia；
2、将重组质粒pSET52-ia转入异戊酰螺旋霉素I产生菌，转化子发酵经培养，微生物法检测产生菌发酵效价，并以HPLC定量分析异戊酰螺旋霉素I的含量；
3、以含ist单基因异戊酰螺旋霉素I产生菌作为对照，比较acyB2基因引入其中后发酵效价及产生异戊酰螺旋霉素I组分的比例，证明acyB2调节基因的引入能明显提高菌种产生异戊酰螺旋霉素I组分的含量及产量。
发明效果：
本发明的积极效果在于证明acyB2调节基因引入异戊酰螺旋霉素I产生菌，可 以提高产生菌发酵单位414％，提高异戊酰螺旋霉素组分I含量170.2％，不仅有利于简化单组份抗生素工业化生产的质量控制与检测标准，为注射剂型的制备创造了条件，而且可为进一步提高临床治疗效果增添一种手段。
附图说明：
图1  ist-acyB2基因重组质粒pSET52-ia的构建
图2  含ist-acyB2基因重组质粒pSET52-ia菌种WSJ-IA发酵产生异戊酰螺旋霉素I组分HPLC分析
其中：A WSJ-2为含单ist基因菌种对照；B WSJ-IA为含ist和调节基因acyB2的基因工程菌
实施方案：
以下实施例仅为帮助本领域技术人员更好地理解本发明，但不以任何方式限制本发明。
《实施例1》构建含ist-acyB2双基因重组质粒pSET52-ia
从本实验室原先构建的重组质粒pSW4中[尚广东等，生物工程学1999，15(2)：171]，用SmaI-BamHI酶切，获得含ist完整基因和部分acyB2基因1820bp片段，以中国微生物菌种保藏管理委员会普通微生物中心提供的耐热链霉菌(Streptomyces thermotolarences CGMCC4.1501)总DNA为模板，根据NCBI公布的acyB2序列，设计引物p1：5’-CGCTCAGGGACGCAAGACC-3’和
P2：5’-CCGGAATTCGCCCCGTGACCTCACCGTC-3’(EcoRI)，通过PCR反应获得acyB2部分基因1013bp片段，将PCR产物用BamHI-EcoRI酶切，获得934bp片段，将上述SmaI-BamHI(1820bp)、BamHI-EcoRI(934bp)片段连入pBluesript II KS(+)载体(Merck公司)的SmaI-EcoRI酶切位点，获得含ist和完整acyB2基因重组质粒pBKS-ia。通过XbaI-EcoRI酶切位点连入大肠杆菌/链霉菌接合转移质粒pSET152(来自基因酷共享资源www.genecool.com)，获得ist-acyB2双基因重组质粒pSET52-ia。构建流程见图1。
《实施例2》重组质粒pSET52-ia转化异戊酰螺旋霉素I产生菌
异戊酰螺旋霉素I产生菌WSJ-2为本实验室构建，发表在[Chunyan Ma et al.Current Microbiology 2010，DOI 10.1007/s00284-010-9664-8]。菌种在斜面培养基[黄豆饼粉2.0％，葡萄糖1.0％，淀粉3.0％，CaCO3 0.5％，NaCl 0.4％，琼脂2.0％] 上28℃培养7-10天，按照文献[D.A.Hopwood等Genetic manipulation ofStreptomyces，A Laboratory Manual，Norwich；John Innes Foundation UK，1985]描述的方法制备原生质体，具体来说是将菌种挖块接种至含蔗糖R2YE培养基[蔗糖10.3％，葡萄糖1.0％，酵母提取液0.4％，胰蛋白胨0.2％，蛋白胨0.4％，酪蛋白氨基酸0.4％，K2SO4 0.025％，CaCl2 0.216％，KH2PO4 0.005％，MgCl2·6H2O1.012％，在100ml培养基中加入NaOH(1mol/L)0.5ml，Tris-HCl(0.25mol/LpH7.2)10ml，微量元素溶液[ZnCl2 0.04％，FeCl3·6H2O 0.02％，CuCl2 0.001％，MnCl·4H2O 0.001％，Na2B4O7·10H2O 0.001％，(NH4)6MO7O2·4H2O 0.0012％]0.2ml，28℃培养2-4天，以10％接种量转种上述培养基，并加入0.5％甘氨酸，培养20小时，离心收集菌丝，用10.3％蔗糖溶液洗涤2-3次，在含溶菌酶2mg/ml的P溶液[Tris-HCl(pH8.0)1mol/L 0.31ml，CaCl2·2H2O 0.368％，MgCl2·6H2O 0.204％，蔗糖10.3％，葡萄糖0.1％，微量元素溶液0.2ml]中37℃溶菌15-60分钟，经棉花过滤获得原生质体。将重组质粒pSET52-ia在PEG介导下转入原生质体，并涂布于R2YE固体培养基，28℃培养24小时后，用含阿泊拉霉素溶液覆盖培养物，使阿泊拉霉素在培养基中的终浓度为50微克/毫升，继续培养7-10天挑取转化子。获得含重组质粒pSET52-ia异戊酰螺旋霉素I产生菌，参椐的生物材料为WSJ-IA，于2010年6月25日送交中国微生物菌种保藏管理委员会普通微生物中心保藏，地址：北京市朝阳区北辰西路1号院3号，保藏编号CGMCC 3942，分类命名为：螺旋霉素链霉菌Streptomyces spiramyceticus。
《实施例3》WSJ-IA菌种发酵效价检测
WSJ-IA菌种在上述斜面培养基28℃培养7-10天，挖块接种接种于种子培养基：黄豆饼粉1.5％，淀粉3.0％，NaCl 0.4％，CaCO3 0.5％，鱼蛋白胨0.3％，KH2PO4 0.05％。28℃培养48小时，转入发酵培养基配方：葡萄糖0.5％，淀粉6.0％，酵母粉0.5％，鱼粉2.0％，NH4NO3 0.6％，NaCl 1.0％，CaCO3 0.5％，KH2PO40.05％，MgSO4 0.1％。28℃发酵培养96小时。发酵液离心取上清液，用含3％NaCl的pH 7.8～8.0磷酸盐缓冲液进行稀释。采用芽孢杆菌[CMCC(B)63501]为检定菌，培养基[蛋白胨0.6％，牛肉浸出粉0.15％，酵母浸出膏0.6％，葡萄糖0.2％，琼脂1.5-2.0％]，按照中国药典2010版二部附录XI A所述标准曲线方法，以乙酰螺旋霉素为标准品，计算发酵效价，WSJ-IA菌种发酵效价为1160微克/毫升，见表一。
《实施例4》WSJ-IA菌种发酵产生异戊酰螺旋霉素I组分的检测
将上述发酵液调pH至8.5，用等量乙酸乙酯提取，浓缩乙酸乙酯提取液至干，溶于甲醇。采用Kromasil C18柱(4.5mm×150mm，5μm)；流动相：甲醇∶磷酸二氢钠溶液(体积比53∶47)；检测波长：231nm；流速：1.0mL·min-1；柱温25℃；进样量：10-20μL。HPLC检测以峰高法计算产物中异戊酰螺旋霉素I组分的含量，HPLC分析结果见图2B，结果表明，WSJ-IA菌种发酵产生异戊酰螺旋霉素I总含量为12.061％。
《实施例5》调节基因提高菌种发酵效价和产生异戊酰螺旋霉素组分I含量检测
WSJ-IA与含ist单基因的WSJ-2菌种对照，按实施例3进行发酵培养，微生物法测定两个菌种发酵效价，乙酸乙酯提取产物并进行HPLC分析，结果见图2。比较两者产生异戊酰螺旋霉素I组分的情况，分别计算两者产生异戊酰螺旋霉素I与螺旋霉素的比例，结果显示，调节基因的引入提高WSJ-IA菌种发酵效价414％，产生异戊酰螺旋霉素I含量提高170.2％，相应地降低了螺旋霉素的产生，使异戊酰螺旋霉素I组分与螺旋霉素比例提高了218％(表一)。
本发明提供了一个含acyB2调节基因产生异戊酰螺旋霉素I的菌种，该菌种在一定发酵条件下，比原来只含ist基因的菌种产生异戊酰螺旋霉素I组分含量提高了1.70倍，发酵效价提高了4.14倍。
表一.WSJ-IA与WSJ-2菌种性能比较
Figure BSA00000206192800061

Claims (2)

1.一株异戊酰螺旋霉素I组分高含量、高产量基因工程菌，其特征是，所述基因工程菌是异戊酰基转移酶基因ist与acyB2调节基因连锁基因，在异戊酰螺旋霉素I产生菌中共表达的克隆菌株。
2.构建制备权利要求1所述基因工程菌的方法，其主要步骤包括：
A)构建含ist-acyB2双基因重组质粒pSET52-ia；
B)将重组质粒pSET52-ia转入异戊酰螺旋霉素I产生菌，获得含重组质粒pSET52-ia的异戊酰螺旋霉素I产生菌，命名为WSJ-IA，菌种保藏编号CGMCCNo3942；
C)WSJ-IA菌种发酵培养、提取；
D)WSJ-IA菌种发酵液微生物法效价检测；
E)HPLC定量分析WSJ-IA菌种发酵产物主组分异戊酰螺旋霉素I的含量。
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Denomination of invention: Isovalerylspiramycin I component high-content high-yield genetic engineering bacteria

Effective date of registration: 20150318

Granted publication date: 20120704

Pledgee: Shengjing bank Yingkou branch of Limited by Share Ltd

Pledgor: Tonglian Group Co., Ltd., Shenyang

Registration number: 2015210000002

PLDC Enforcement, change and cancellation of contracts on pledge of patent right or utility model