CN105505976A - 一种维吉尼亚链霉菌ibl14产青霉素重组菌株的构建方法 - Google Patents
一种维吉尼亚链霉菌ibl14产青霉素重组菌株的构建方法 Download PDFInfo
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Abstract
本发明公开了一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,包括它含有水解青霉素的β-内酰胺酶基因和产青霉素基因簇序列特征、及因此而建立的一种产青霉素重组菌株的构建方法全过程。应用维吉尼亚链霉菌IBL14中自身的产青霉素基因和自身的CRISPR-Cas?I-B型基因编辑系统对相关基因进行编辑,以此来实现提高青霉素产量的目的。该发明为增加生物医药种类、提高生产水平和改善产品品质提供了新的途径与方法。
Description
技术领域
本发明涉及生物医药领域,确切地说是一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法。
背景技术
青霉素是英国人AlexanderFleming1928年意外发现地一种毒性低、疗效高的抗生素(许钟炜.(2005)发现青霉素.健康博览,29)。1939年,Fleming将历时10年培养的菌种提供给牛津大学澳大利亚病理学家弗洛里(HowardFlorey)和英国生物化学家钱恩(EarnestChain)。1940年,他们完成了制备青霉素结晶体和动物实验(林荫.(2007)青霉素的辉煌往事.科学24小时,40-42)。1941年,第二次世界大战爆发,美国政府下达了大规模量产青霉素,以供战时之需的艰巨任务,当时的辉瑞公司采用其特有的深罐发酵技术完成了任务,成为了世界上首个量产青霉素的公司(许钟炜.(2005)发现青霉素.健康博览,29)。从发现到量产历时14年,临床首选于G+球菌所致的感染。
β-内酰胺抗生素是具有四元内酰胺环的一大类抗生素,主要代表有青霉素和头孢菌素。青霉素具有与细菌细胞壁肽聚糖单体中D-丙氨酰-D-丙氨酸相似的结构,与其竞争转肽酶,阻碍肽聚糖的形成,造成细胞壁的缺损,进而起到杀菌作用(于海军.(2009)β-内酰胺类抗生素作用机制及头孢菌素发展.石家庄职业技术学院学报.21,12-16)。
β-内酰胺酶是一类破坏β-内酰胺环抗生素酶的总称。它使β-内酰胺环水解开环生成青霉素噻唑酸,该酶功能的缺失可使得维吉尼亚链霉菌IBL14中青霉素得到积累,同时β-内酰胺酶和青霉素酰胺酶水解基因和头孢菌素代谢支路中的异青霉素-N异构酶基因的缺失使得维吉尼亚链霉菌IBL14中青霉素的产量得到进一步的提高。
众所周知,当前青霉素生产的主要菌株是真菌(如:产黄青霉Penicilliumchrysogenum和点青霉Penicilliumnotatum),由细菌维吉尼亚链霉菌生产青霉素未见报道。
近年来,基于CRISPR-Cas系统发展而来的DNA编辑新技术,成功地应用于细胞染色体中基因组编辑与改造,在医药、食品、农业等领域中显示出了巨大的潜力(Doudna,J.A.andCharpentier,E.(2014)ThenewfrontierofgenomeengineeringwithCRISPR-Cas9.Science.346)。但到目前为止,广泛应用的CRISPR-Cas系统均为CRISPR-CasII型系统。
维吉尼亚链霉菌IBL-14(StreptomycesvirginiaeIBL14)是本实验室自行分离纯化得到的一株能降解多种甾体化合物的放线菌。对菌株IBL-14全基因组测序和数据库分析发现,菌株IBL-14中存在CRISPR-CasI-SV14B型系统。应用维吉尼亚链霉菌IBL-14自身的CasI-SV14B型系统对其生产青霉素的相关基因进行编辑,得到生产青霉素重组菌株未见报道。此外,该方法的建立对其他抗生素的生产也具有指导意义。
发明内容
本发明要解决的技术问题是提供一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,通过靶向基因与基因编辑模板的设计与质粒构建及靶向基因与基因编辑模板重组质粒在链霉菌中的转化操作,对染色体中与产青霉素相关的基因进行编辑。本发明通过敲除青霉素水解酶、青霉素酰胺酶及异青霉素-N异构酶的基因,从而实现提高青霉素产量之目的。
采用如下技术方案:
一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,其特征在于:包括以下步骤;
(1)根据维吉尼亚链霉菌IBL14基因组测序数据,确定β-内酰胺酶、青霉素酰胺酶及异青霉素-N异构酶三个靶向基因的DNA序列;
(2)使用PfuDNA聚合酶通过PCR反应分别扩增得到末端带有限制性内切酶识别切割位点的,且具有overlapPCR互补序列的靶向基因上、下同源臂的PCR片段;
(3)利用overlapPCR反应将上、下两个同源臂结合构建编辑基因模板;
(4)直接合成首、尾分别包含启动子J23119和RNA终止子的靶向基因片段;
(5)利用限制性酶切位点上的粘性末端和连接酶将步骤(3)、步骤(4)获得的DNA片段连接到pKC1139上得到基因编辑质粒;
(6)在含链霉菌的高渗溶液中添加溶菌酶制备维吉尼亚链霉菌IBL14原生质体;
(7)将步骤(5)中得到基因编辑质粒转化到维吉尼亚链霉菌IBL14原生质体中得到基因编辑后的重组子;
(8)对重组子染色体进行PCR和基因测序分析,确证重组子为基因编辑后的目的重组菌株,并根据抑菌圈实验结果筛选抗性能力强的目的重组菌株。
所述的一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,其特征在于:维吉尼亚链霉菌IBL14染色体中β-内酰胺酶基因及产青霉素基因具有如表1所描述的核苷酸序列,其青霉素合成途径中的异青霉素-N异构酶的功能缺失导致异青霉素-N不能转化为青霉素N,β-内酰胺酶和青霉素酰胺酶的功能缺失导致青霉素不能分别转化为青霉酸和6氨基青霉烷酸,进而共同导致青霉素的积累。
所述的一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,其特征在于:所述的构建青霉素重组菌株是应用维吉尼亚链霉菌IBL14中自身的CRISPR-CasI-SV14B型系统对维吉尼亚链霉菌IBL14中主要影响青霉素生产的β-内酰胺酶基因、青霉素酰胺酶基因及异青霉素-N异构酶基因进行敲除、插入、无痕点突变及任意组合。
所述的一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,其特征在于:该方法构建所得到的维吉尼亚链霉菌IBL14重组菌株可直接用于青霉素及其衍生物生产。
一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,其特征在于:维吉尼亚链霉菌IBL14染色体中含有水解青霉素的β-内酰胺酶(β-lactamase)基因和产青霉素基因簇、及因此而建立的一种产青霉素重组菌株的构建方法。本发明目的通过以下技术方案来实现:
所述的维吉尼亚链霉菌IBL14染色体中水解青霉素的基因和产青霉素基因簇具有如表1所描述的核苷酸序列、且酶生产青霉素过程具有如附图1所示的代谢途径。
有益效果:
本发明首创了维吉尼亚链霉菌生产青霉素的途径;提供了CRISPR-CASI-SV14B型系统在链霉菌抗生素生产中的应用;为CRISPR-CASI-SV14B型系统在其它抗生素生产菌株研究中提供了新的方法和技术选择。应用维吉尼亚链霉菌IBL14中的CRISPR-CasI-SV14B型基因编辑系统对链霉菌遗传基因进行编辑可方便、快速、高效地改变生物体的遗传特征。该基因编辑的方法可应用于生物制药、食品、农业及其它生物应用领域中。
说明书附图
图1维吉尼亚链霉菌IBL14青霉素代谢途径;异青霉素-N异构酶/EC5.1.1.17的功能缺失导致异青霉素-N不能转化为青霉素N(penicillinN),β-内酰胺酶/EC3.5.2.6的功能缺失导致青霉素不能转化为青霉酸(penicilloicacid),青霉素酰胺酶/EC3.5.1.11的功能缺失导致青霉素不能转化为6氨基青霉烷酸
(6-aminopenicillanicacid),进而共同导致青霉素(penicillin)的积累;注:红色虚线代表被敲除基因;
图2抑菌圈抗性检测,其中A表明野生型IBL14对大肠杆菌DH5α无抑制;B表明敲除后的重组子对大肠杆菌DH5α有抑制。
具体实施方式
为了更充分理解本发明的技术内容,下面结合具体实施例对本发明的技术方案作进一步介绍和说明,旨在更好的解释本发明的内容,以下实施例不限制本发明的保护范围。此外,在所列实施例中如无特别说明均采用如下材料:
1)菌种
宿主为StreptomycesvirginiaeIBL14,敲除辅助质粒pKC1139。
2)培养基
种子培养基(FM培养基)
NH4Cl3.0g,K2HPO4·3H2O1.55g,NaH2PO4·2H2O0.85g,MgSO4·7H2O0.2g,CaCl2·2H2O10.0mg,FeSO4·7H2O1.0mg,ZnSO40.1mg;加自来水至1000ml,灭菌前pH调为7.0,然后加葡萄糖3.0g,酵母粉3.0g玉米浆3.0g,β-环糊精3.0g。
转化培养基(R2YE培养基)
蔗糖103g/L,K2SO40.25g/L,MgCl2·6H2O10.12g/L,葡萄糖10g/L,水解酪蛋白0.1g/L,酵母粉5g/L,TES5.73g/L,微量元素(Fe2(SO4)·6H2O0.01g,CuSO4·5H2O0.001,ZnSO4·5H2O0.001,MnSO4·H2O0.001;加蒸馏水至1000ml)2ml,琼脂22g/L,pH7.0(使用前融化并加入0.54%KH2PO410ml/L,2.5MCaCl2·2H2O8ml/L,20%L-脯氨酸15ml/L)。
P缓冲液(原生质体转化用)
蔗糖103g,K2SO40.25g,MgCl2·6H2O2.02g,微量元素溶液2ml,蒸馏水800ml灭菌后每80ml上述溶液中加入:0.5%KH2PO41ml,3.68%CaCl2·2H2O10ml,5.73%TES缓冲液(pH7.2)10ml。
所用试剂均为市售品。
实施例1(菌株IBL-14中异青霉素-N异构酶基因sviIPI/GVGL005789敲除)
(1)基因sviIPI01的引物设计与DNA扩增
根据全基因组测序信息,设计基因sviIPI特异性引物sviIPI-F和sviIPI-R(表2)。提取维吉尼亚链霉菌IBL-14基因组DNA,使用上海生工生物工程股份有限公司生产的PfuDNAPolymerase进行sviIPI基因PCR扩增,反应条件:95℃5min,94℃30s,52℃30s,72℃2min,2.5U生工公司生产的PfuDNAPolymerase(50μl反应体系),30个循环,72℃10min。PCR产物经1%琼脂糖电泳检测,试剂盒回收,得到纯化的sviIPI全长基因片段备用。
(2)制备上、下游同源臂
根据sviIPI基因全序列(表1)设计sviIPI基因上游同源臂引物sviIPI-UF和sviIPI-UR、下游同源臂引物sviIPI-DF和sviIPI-DR(表2)(黑体加粗为overlapPCR互补序列),且上同源臂上游引物含BamHI限制性内切酶酶切位点,下同源臂下游引物含EcoRI限制性内切酶酶切位点(下划线为酶切位点)。
以纯化的sviIPI基因DNA为模板,先分别扩增上、下游同源臂,反应条件为:95℃5min,94℃30s,55℃30s,72℃1min,2.5U生工公司生产的PfuDNAPolymerase(50μl反应体系),30个循环,72℃10min。PCR产物经1%琼脂糖电泳检测,试剂盒回收,得到纯化后的上、下游同源臂DNA片段备用。
(3)制备编辑模板片段
取上同源臂纯化产物与下同源臂纯化产物0.5μl混合作为模板,30μl反应体系进行overlapPCR,反应条件为:94℃预变性5min,94℃变性lmin,60℃退火1min,72℃延伸30s,一个循环后加人引物UF与DR各1μl,继续PCR,反应条件为:95℃预变性5min,94℃变性30s,65℃退火30s,72℃延伸2min,进行30个循环,72℃10min。1%琼脂糖凝胶电泳检测扩增产物并纯化备用。
(4)制备靶向基因片段
含启动子J23119及guideDNA-sviIPIgk01连接产物的靶向基因片段(表2)由滁州通用生物公司直接合成,首尾分别加上HindШ及XbaI酶切位点,中间依次是启动子J23119,spacer,repeat以及终止子(小写字母为酶切位点,单下划线为启动子J23119,斜体为spacer,黑体加粗为repeat,双下划线为终止子terminator)。
(5)构建基因编辑质粒pKCSV14-sviIPIgk01
将实施例1步骤(3)中获得的编辑模板片段通过BamHI限制性内切酶、EcoRI限制性内切酶切出粘性末端,然后通过全式金T4连接酶将其连接到pKC1139质粒上,得编辑模板载体;将实施例1步骤(4)中的靶向基因片段通过HindШ限制性内切酶、XbaI限制性内切酶切出粘性末端,然后通过全式金T4连接酶将其连接到编辑模板载体上得到基因编辑质粒pKCSV14-sviIPIgk01。
(6)维吉尼亚链霉菌IBL14原生质体制备
在装有不锈钢弹簧的三角瓶中加入30ml的FM培养基(加0.15gGlycine),接种100μl的孢子悬液,于30℃摇床中培养24-36h。将培养物倒入50ml已灭菌的离心管中,用无菌水涮洗三角瓶,收集洗液于同一离心管中,然后3000rpm离心10min。弃上清,将菌丝体悬浮于15ml的10.3%的蔗糖溶液中,3000rpm离心10min,弃上清。同此法洗两次。取1ml菌丝体,加入4ml的溶菌酶(上海生工生物工程股份有限公司生产)溶液(溶菌酶母液为50mg/mlPBuffer,终浓度为2mg/ml,用PBuffer稀释),于30℃水浴30-60min(间隔7-8分钟轻轻摇动)至上清呈乳状。加入5ml的PBuffer并用5ml的吸管吹吸几次,继续温浴10min(使大量的原生质体释放出来)。用装有脱脂棉的试管过滤,滤液转入无菌干净的离心管中,3000rpm离心7min。原生质体沉淀呈黄色。弃上清,轻柔打散原生质体,用PBuffer洗两次(洗去溶菌酶)。每次仍使用3000rpm离心7min。弃上清,用枪将原生质体打散,分装,于-70度保存备用。
(7)基因编辑质粒pKCSV14-sviIPIgk01转化
将基因编辑质粒pKCSV14-sviIPIgk01转化到维吉尼亚链霉菌IBL-14原生质体中,在R2YE固体培养基中30℃培养,待平板呈雾状后涂布1ml(含50mg/ml的安普霉素溶液30μl)无菌水溶液、覆盖,超净台吹干后30℃培养约30h得到sviIPI基因敲除重组子。
(8)重组子染色体PCR和基因测序分析及重组子抗性能力的检测
挑取转化株基因组作为模板,再以步骤(1)sviIPI基因引物sviIPI-F/sviIPI-R进行PCR扩增反应,反应条件:95℃5min,94℃30s,52℃30s,72℃2min,2.5U生工公司生产的PfuDNAPolymerase(25μl反应体系),30个循环,72℃10min。PCR产物经1%琼脂糖电泳检测,观察重组子染色体DNA扩增条带是否减少了预期的370bp,经通用生物系统(安徽)有限公司测序证明sviIPI基因是否已成功敲除掉,若成功则所得目的重组子为sviIPI基因敲除重组子。将野生型IBL14菌液和sviIPI基因敲除菌液分别涂布到野生型大肠杆菌DH5α中,若基因敲除成功则青霉素产量增加,表现为实验组有抑菌圈形成。
实施例2(菌株IBL-14中β-内酰胺酶基因sviLT/GVGL000792的敲除)
(1)基因sviLT引物设计与DNA扩增
设计基因引物为sviLT-F和sviLT-R(表2)。以IBL-14基因组DNA为模板,扩增sviLT基因片段。反应条件:95℃5min,94℃30s,52℃30s,72℃3min,2.5U生工公司生产的PfuDNAPolymerase(50μl反应体系),30个循环,72℃10min。PCR产物经1%琼脂糖电泳检测,试剂盒回收,得到纯化的sviLT全长基因片段备用。具体同实施例1步骤(1)。
(2)制备上、下游同源臂
上下游同源臂扩增具体同实施例1步骤(2)。设计的上下同源臂引物依次是sviLT-UF、sviLT-UR、sviLT-DF、sviLT-DR(表2)。以纯化的sviLT基因DNA为模板,先分别扩增上、下游同源臂,反应条件为:95℃5min,94℃30s,55℃30s,72℃1min,2.5U生工公司生产的PfuDNAPolymerase(50μl反应体系),30个循环,72℃10min。PCR产物经1%琼脂糖电泳检测,试剂盒回收,得到纯化后的上、下游同源臂DNA片段备用。
(3)制备编辑模板片段
取上同源臂纯化产物与下同源臂纯化产物0.5μl混合作为模板,30μl反应体系进行overlapPCR,反应条件为:94℃预变性5min,94℃变性lmin,58℃退火1min,72℃延伸30s,一个循环后加人引物UF与DR各1μl,继续PCR,反应条件为:95℃预变性5min,94℃变性30s,62℃退火30s,72℃延伸2min,进行30个循环,72℃10min。1%琼脂糖凝胶电泳检测扩增产物并纯化备用。
(4)制备靶向基因片段
具体同实施例1步骤(4),序列见表2。
(5)构建基因编辑质粒pKCSV14-sviLTgk01
同实施例1步骤(5)。
(6)维吉尼亚链霉菌IBL14原生质体制备
同实施例1步骤(6)。
(7)基因编辑质粒pKCSV14-sviLTgk01转化
同实施例1步骤(7)。
(8)重组子染色体PCR和基因测序分析及重组子抗性能力的检测
方法同实施例1步骤(8)。PCR产物经1%琼脂糖电泳检测,观察重组子染色体DNA扩增条带是否减少了预期的770bp,经通用生物系统(安徽)有限公司测序证明sviLT基因是否已成功敲除掉,若成功即得目的重组子为sviLT基因敲除重组子。
实施例3(菌株IBL-14中青霉素酰胺酶基因sviPA/GVGL002963的敲除)
(1)基因sviPA引物设计与DNA扩增
设计基因引物为sviPA-F和sviPA-R(表2)。以IBL-14基因组DNA为模板,扩增sviPA基因片段。反应条件:95℃5min,94℃30s,55℃30s,72℃3.5min,2.5U生工公司生产的PfuDNAPolymerase(50μl反应体系),30个循环,72℃10min。PCR产物经1%琼脂糖电泳检测,试剂盒回收,得到纯化的sviPA全长基因片段备用。具体同实施例1步骤(1)。
(2)制备上、下游同源臂
上下游同源臂扩增具体同实施例1步骤(2)。设计的上下同源臂引物依次是sviPA-UF、sviPA-UR、sviPA-DF、sviPA-DR(表2)。以纯化的sviPA基因DNA为模板,先分别扩增上、下游同源臂,反应条件为:95℃5min,94℃30s,55℃30s,72℃1min,2.5U生工公司生产的PfuDNAPolymerase(50μl反应体系),30个循环,72℃10min。PCR产物经1%琼脂糖电泳检测,试剂盒回收,得到纯化后的上、下游同源臂DNA片段备用。
(3)制备编辑模板片段
取上同源臂纯化产物与下同源臂纯化产物0.5μl混合作为模板,30μl反应体系进行overlapPCR,反应条件为:94℃预变性5min,94℃变性lmin,58℃退火1min,72℃延伸30s,一个循环后加人引物UF与DR各1μl,继续PCR,反应条件为:95℃预变性5min,94℃变性30s,65℃退火30s,72℃延伸2min,进行30个循环,72℃10min。1%琼脂糖凝胶电泳检测扩增产物并纯化备用。
(4)制备靶向基因片段
具体同实施例1步骤(4),序列见表2。
(5)构建基因编辑质粒pKCSV14-sviPAgk01
同实施例1步骤(5)。
(6)维吉尼亚链霉菌IBL14原生质体制备
同实施例1步骤(6)。
(7)基因编辑质粒pKCSV14-sviPAgk01转化
同实施例1步骤(7)。
(8)重组子染色体PCR和基因测序分析及重组子抗性能力的检测
方法同实施例1步骤(8)。PCR产物经1%琼脂糖电泳检测,观察重组子染色体DNA扩增条带是否减少了预期的940bp,经通用生物系统(安徽)有限公司测序证明sviPA基因是否已成功敲除掉,若成功即得目的重组子为sviPA基因敲除重组子。
以上所述仅以实施例来进一步说明本发明的技术内容,以便于读者更容易理解,但不代表本发明的实施方式仅限于此,任何依本发明所做的技术延伸或再创造,均受本发明的保护。
Claims (4)
1.一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,其特征在于:包括以下步骤;
(1)根据维吉尼亚链霉菌IBL14基因组测序数据,确定β-内酰胺酶、青霉素酰胺酶及异青霉素-N异构酶三个靶向基因的DNA序列;
(2)使用PfuDNA聚合酶通过PCR反应分别扩增得到末端带有限制性内切酶识别切割位点的,且具有overlapPCR互补序列的靶向基因上、下同源臂的PCR片段;
(3)利用overlapPCR反应将上、下两个同源臂结合构建编辑基因模板;
(4)直接合成首、尾分别包含启动子J23119和RNA终止子的靶向基因片段;
(5)利用限制性酶切位点上的粘性末端和连接酶将步骤(3)、步骤(4)获得的DNA片段连接到pKC1139上得到基因编辑质粒;
(6)在含链霉菌的高渗溶液中添加溶菌酶制备维吉尼亚链霉菌IBL14原生质体;
(7)将步骤(5)中得到基因编辑质粒转化到维吉尼亚链霉菌IBL14原生质体中得到基因编辑后的重组子;
(8)对重组子染色体进行PCR和基因测序分析,确证重组子为基因编辑后的目的重组菌株,并根据抑菌圈实验结果筛选抗性能力强的目的重组菌株。
2.根据权利要求1所述的一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,其特征在于:维吉尼亚链霉菌IBL14染色体中β-内酰胺酶基因及产青霉素基因具有如表1所描述的核苷酸序列,其青霉素合成途径中的异青霉素-N异构酶的功能缺失导致异青霉素-N不能转化为青霉素N,β-内酰胺酶和青霉素酰胺酶的功能缺失导致青霉素不能分别转化为青霉酸和6氨基青霉烷酸,进而共同导致青霉素的积累。
3.根据权利要求1所述的一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,其特征在于:所述的构建青霉素重组菌株是应用维吉尼亚链霉菌IBL14中自身的CRISPR-CasI-SV14B型系统对维吉尼亚链霉菌IBL14中主要影响青霉素生产的β-内酰胺酶基因、青霉素酰胺酶基因及异青霉素-N异构酶基因进行敲除、插入、无痕点突变及任意组合。
4.根据权利要求1所述的一种维吉尼亚链霉菌IBL14产青霉素重组菌株的构建方法,其特征在于:该方法构建所得到的维吉尼亚链霉菌IBL14重组菌株可直接用于青霉素及其衍生物生产。
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