CN101914481B - 一株异戊酰螺旋霉素高含量主组分基因工程菌 - Google Patents
一株异戊酰螺旋霉素高含量主组分基因工程菌 Download PDFInfo
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Abstract
本发明涉及利用调节基因构建高含量抗生素主组分的基因工程菌，所述基因工程菌，是将acyB2调节基因与异戊酰基转移酶基因ist连锁，在螺旋霉素产生菌中共表达的产物；实验研究表明，该菌株可以明显提高主组分异戊酰螺旋霉素比例213-232％，为工业化生产和临床应用提供了重要保障。
Description
技术领域：
本发明涉及利用调节基因构建高含量抗生素主组分的基因工程菌。
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。螺旋霉素是一种大环内酯类抗生素，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。由于螺旋霉素内酯环3位R羟基可以被乙酰或丙酰基酯化，而形成相应的螺旋霉素I、II、III组分，所以必特螺旋霉素也至少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：
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
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。前已完成基因工程必特螺旋霉素新药的三期临床试验研究，结果显示了良好的疗效和安全性。目前正在申报国家一类新药证书。
必特螺旋霉素主组分异戊酰螺旋霉素，是由异戊酰基转移酶催化螺旋霉素分子中的碳霉糖4”位羟基，进行O-酰基化反应，使碳霉糖4”位羟基异戊酰酯化而形成的。因此，异戊酰基转移酶的活性直接影响螺旋霉素的转化率和异戊酰螺旋霉素的产率，而异戊酰转移酶的活性，又受基因剂量、基因启动子强度和环境因素(即发酵条件)的影响与调节。
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，以提高菌种产生必特螺旋霉素主组分异戊酰螺旋霉素的含量。
本发明所述将acyB2调节基因与异戊酰基转移酶基因ist连锁，在螺旋霉素产生菌中共表达，以构建高含量异戊酰螺旋霉素主组份菌株，迄今尚未见有相关报道。
发明内容：
本发明提供的技术方案主要包括以下方面：
1.构建含ist-acyB2双基因重组质粒pSET52-ia；
2.将重组质粒pSET52-ia转入螺旋霉素产生菌，转化子经发酵培养、提取，并以HPLC定量分析异戊酰螺旋霉素的含量；
3.以含ist单基因必特螺旋霉素菌株作为对照，比较ist-acyB2基因引入螺旋霉素菌株后获得必特螺旋霉素产生菌，产生主组份异戊酰螺旋霉素的比例，证明acyB2调节基因的引入能明显提高菌种产生主组分异戊酰螺旋霉素的含量。
发明效果：
本发明的积极效果在于，证明acyB2调节基因引入必特螺旋霉素产生菌，可以明显提高主组分异戊酰螺旋霉素比例213-232％，为工业化生产和临床应用提供了重要保障。
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-195-IA发酵产生异戊酰螺旋霉素I、II、III组分HPLC分析；
其中：A WSJ-195为含单ist基因菌种对照；B WSJ-195-IA为本发明含ist-acyB2基因重组质粒pSET52-ia菌种。
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报1999，15(2)：171]，利用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转化螺旋霉素产生菌
螺旋霉素产生菌(S.spiramyceticus)菌种来源于中国医学科学院医药生物技术研究所菌种保藏中心(CGMCC4.1118)，该菌种的描述已经公开发表[微生物学报1982，22(1)：13-16]。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 of Streptomyces，ALaboratory 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％，MgCl2·6H2O 1.012％]，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·4H2O0.001％，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的螺旋霉素产生菌，即：螺旋霉素链霉菌，分类命名为：streptomyces spiramyceticus，并于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 No.3943，参椐的生物材料：WSJ-195-IA。
《实施例3》：WSJ-195-IA菌种发酵提取产物鉴定
WSJ-195-IA菌种在上述斜面培养基28℃培养7-10天，挖块接种于种子培养基：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：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％，KH2PO4 0.05％，MgSO4 0.1％，28℃发酵培养96小时。发酵液过滤，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。WSJ-195-IA菌种发酵提取产物经HPLC分析，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。检测并计算产物中异戊酰螺旋霉素组分I、II、III，以此确定总异戊酰螺旋霉素的含量(图2B)，结果表明，WSJ-195-IA菌种发酵产生主组分异戊酰螺旋霉素总量为42％。
《实施例4》：调节基因提高菌种发酵产生异戊酰螺旋霉素组分的含量
WSJ-195-IA与含ist单基因的WSJ-195[专利号：ZL02148771.5]菌种对照，按照实施例3的方法发酵培养，用乙酸乙酯提取产物，进行HPLC分析(图2A)。比较两者产生异戊酰螺旋霉素I、II、III三组分的情况(表一)。结果表明，调节基因提高菌种产生异戊酰螺旋霉素总含量213％(按峰面积计算)或232％(按峰高计算)。
表一.WSJ-195与WSJ-195-IA菌种产生异戊酰螺旋霉素组分产量HPLC分析
以上实验研究结果证明，本发明提供了一株含acyB2调节基因的必特螺旋霉素产生菌种，所述菌种在一定发酵条件下，比原来只含ist基因的菌种产生主组分异戊酰螺旋霉素含量提高了213-232％。
Claims (1)
1.一株必特螺旋霉素主组分异戊酰螺旋霉素的基因工程菌，其特征是，所述菌株系含异戊酰基转移酶基因ist与acyB2调节基因重组质粒并能提高主组分含量的螺旋霉素产生菌克隆菌株，菌种保藏编号CGMCC No.3943。
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