CN113265340B - 产角鲨烯的裂殖壶菌基因工程菌株及其构建方法和应用 - Google Patents
产角鲨烯的裂殖壶菌基因工程菌株及其构建方法和应用 Download PDFInfo
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Abstract
本发明公开了产角鲨烯的裂殖壶菌基因工程菌株及其构建方法和应用。本发明采用裂殖壶菌Schizochytrium limacinum SR21为野生型菌株,在大肠杆菌中构建以延长酶基因(elogase,elo1)两端序列为同源臂、博来霉素为筛选标记的elo1基因敲除载体pBlue‑ZEO‑elo1和以18sRNA为同源臂、潮霉素为筛选标记的磷脂酸磷酸酶基因(phosphatidic acid phosphatase,pap)干扰载体pBlue‑HYG‑siRNA‑18s,将两个载体同源重组结构域线性化后电转进入裂殖壶菌内,得到一株elo1基因敲除结合pap基因干扰的高产角鲨烯的裂殖壶菌基因工程菌株,命名为MIX菌株。本发明通过调控相关基因的表达水平改变了碳代谢流向,抑制了裂殖壶菌油脂合成的能力,为角鲨烯合成提供了更多的碳通量。本发明为裂殖壶菌实现多基因改造提供了理论依据,同时为裂殖壶菌生产角鲨烯奠定了实验基础。
Description
技术领域
本发明属于遗传工程技术领域,具体涉及裂殖壶菌基因工程菌株。
背景技术
角鲨烯(Squalene)作为一种重要的萜类化合物,具有降血脂、防癌和抗氧化等功效,因此广泛应用于医疗保健领域。由于自然资源的限制和化学合成的困难,利用微生物发酵生产角鲨烯已经成为主要趋势。生物合成法具有易操作、周期短以及转化率高的优点,同时通过微生物发酵获得的产物易于分离纯化,具有良好的经济效益。生物合成法的技术重点是筛选出适合角鲨烯生产的并且具有经济价值的菌种。目前用于角鲨烯合成的菌种主要包括破囊壶菌、酵母、大肠杆菌和自养型微藻等。Nakazawa等人从海水中筛选出来的破囊壶菌18W-13a角鲨烯产量可以达到1.29g/L。Liu等人优化后的酿酒酵母角鲨烯产量达到11.00g/L,是目前已知基因工程菌株的最高产量。Li等从香港红树林中分离得到的自养型微藻Aurantiochytrium mangrovei FB1角鲨烯的产量极易受培养条件的影响,无法准确掌握角鲨烯产量最高的培养时间,产量仅有0.53mg/g。Xu等人通过基因改造将真菌甲羟戊酸途径相关的基因导入大肠杆菌中表达,角鲨烯产量达到52.1mg/L,实现了大肠杆菌基因工程菌株产角鲨烯的重大突破。
裂殖壶菌(Schizochytrium sp.)又名裂壶藻,是一种海洋真菌,隶属于真菌门、网粘菌纲、破囊壶菌目和破囊壶菌科。裂殖壶菌生长速度快,发酵周期短,耐机械搅拌,是一种食品安全菌。其代谢途径可以合成多种高值化合物,包括磷脂、多不饱和脂肪酸、角鲨烯、类胡萝卜素、叶黄素和虾青素等。裂殖壶菌是具有高产角鲨烯潜力的优良菌株,但目前关于裂殖壶菌生产角鲨烯的研究主要停留于培养条件的优化阶段,代谢途径调控研究报道较少。
发明内容
本发明的目的在于克服现有技术的不足之处,提供了产角鲨烯的裂殖壶菌基因工程菌株及其构建方法和应用。
本发明解决其技术问题所采用的技术方案之一是:
首先,本发明提供了一种产角鲨烯的裂殖壶菌基因工程菌株,所述菌株是选用产油Schizochytrium limacinum SR21为材料构建而成,所述菌株基因组中延长酶基因(elogase,elo1)被敲除,同时磷脂酸磷酸酶基因(phosphatidic acid phosphatase,pap)被干扰表达。
其中,所述elo1基因和pap基因克隆于野生型菌株Schizochytrium limacinumSR21的基因组。
本发明解决其技术问题所采用的技术方案之二是:
其次,本发明提供了提供一种产角鲨烯的裂殖壶菌基因工程菌株的构建方法,为双基因改造方法,该方法包括如下步骤:
(1)克隆来源于野生型菌株Schizochytrium limacinum SR21基因组中elo1基因的上下游同源臂,插入pBlue-ZEO-18s质粒的同源重组区域,构建以博来霉素为抗性的elo1基因敲除载体pBlue-ZEO-elo1;
(2)将来源于野生型菌株Schizochytrium limacinum SR21基因组的pap基因的siRNA连接到真菌干扰质粒p-Silent-HYG的多克隆区域,利用PCR的方式扩增出潮霉素抗性表达框和siRNA的表达框;将该含有潮霉素抗性表达框和siRNA的表达框的片段整合进pBlue-ZEO-18s的多克隆区域,替换博来霉素表达框和过表达基因表达框,构建出以潮霉素为抗性,以18s基因序列为同源臂的pap基因干扰载体pBlue-HYG-siRNA-18s;
(3)将构建好的两载体同源重组区域线性化,电转入裂殖壶菌内,通过博来霉素和潮霉素抗性筛选和鉴定,获得elo1基因敲除结合pap基因干扰的高产角鲨烯的裂殖壶菌基因工程菌株,命名为MIX菌株。
进一步地,所述步骤1)中,所述elo1基因上下游同源臂的构建方法包括:根据Schizochytrium limacinum SR21基因组的elo1基因的序列信息,设计如SEQ ID No.1和SEQ ID No.2所示的上游同源臂扩增引物P1、如SEQ ID No.3和SEQ ID No.4所示的下游同源臂扩增引物P2;以Schizochytrium limacinum SR21基因组为模版,用所述引物P1和所述引物P2通过PCR方式获得elo1基因上下游同源臂。
进一步地,所述步骤1)中,所述pBlue-ZEO-elo1的构建方法包括:用HindⅢ和KpnⅠ对pBlue-ZEO-18s和所述elo1基因上下游同源臂进行双酶切,连接,转化至大肠杆菌Trans110感受态细胞,获得所述pBlue-ZEO-elo1。
进一步地,所述步骤2)中,所述pBlue-HYG-siRNA-18s的构建方法包括:以如SEQID No.5和SEQ ID No.6所示的引物P3构建编码pap基因敲除siRNA的DNA片段;用XhoⅠ和HindⅢ对真菌沉默质粒p-Silent-HYG进行双酶切后,与所述DNA片段连接,转化至大肠杆菌Trans110感受态细胞,获得p-Silent-siRNA重组质粒;以所述p-Silent-siRNA为模板,以如SEQ ID No.7和SEQ ID No.8所示的引物P4扩增出p-Silent-siRNA质粒上的潮霉素抗性表达框和siRNA表达框;用SpeⅠ和HindⅢ对所述pBlue-ZEO-18s进行双酶切,与所述扩增的产物连接,转化至大肠杆菌Trans110感受态细胞,获得所述pBlue-HYG-siRNA-18s。
进一步地,所述步骤3)中,将所述pBlue-ZEO-elo1和所述pBlue-HYG-siRNA-18s分别酶切线性化后,加入到裂殖壶菌Schizochytrium limacinum SR21的感受态细胞,电转,培养获得所述产角鲨烯的裂殖壶菌基因工程菌株。
裂殖壶菌中油脂占细胞干重的50%以上,并且大部分是以甘油三酯为主的中性油脂。角鲨烯是裂殖壶菌甲羟戊酸途径中合成甾醇类化合物的中间产物,甘油三酯的合成是以3-磷酸甘油醛为骨架与脂酰辅酶A发生酯化反应而来,脂酰辅酶A是通过脂肪酸合成途径形成,与角鲨烯合成共用底物乙酰辅酶A。本发明在现有技术的基础上,经大量研究发现,角鲨烯的合成代谢与甘油三酯合成存在较强的竞争关系。本发明通过调控相关基因的表达水平改变了碳代谢流向,抑制了裂殖壶菌油脂合成的能力,为角鲨烯合成提供了更多的碳通量。因此本发明不仅提供了一种裂殖壶菌的双基因改造方法,实质还提供了一种促进裂殖壶菌角鲨烯合成的调控方法。
本发明解决其技术问题所采用的技术方案之三是:
最后,本发明提供一种应用上述的产角鲨烯的裂殖壶菌基因工程菌株生产角鲨烯的方法,将所述产角鲨烯的裂殖壶菌基因工程菌株接种于种子培养基,26~30℃逐级放大培养获得发酵用菌种;将所述发酵用菌种接种于发酵培养基中,26~30℃进行发酵培养;收集发酵培养产物,提取得到角鲨烯。
进一步地,所述种子培养基的pH值为6.4~6.6,且包括配方比例为28~32g:9~11g:48~52mL:1.5~2.5mL的葡萄糖、酵母粉、20×组分A和500×CaCl2;其中,所述20×组分A包括:Na2SO4 238~242g/L,MgSO4 38~42g/L,KH2PO4 19~21g/L,(NH4)2SO4 19~21g/L,K2SO4 12~14g/L,KCl 9~11g/L,溶剂为水;所述500×CaCl2包括:CaCl2·2H2O 84~86g/L或无水CaCl2 64~65g/L,溶剂为水。
进一步地,所述发酵培养基的pH值为6.4~6.6,且包括:葡萄糖88~92g/L、玉米浆粉4~6g/L、蛋白胨4~6g/L和20×组分A 48~52mL/L;其中,所述20×组分A包括:Na2SO4238~242g/L,MgSO4 38~42g/L,KH2PO4 19~21g/L,(NH4)2SO4 19~21g/L,K2SO4 12~14g/L,KCl 9~11g/L,溶剂为水。
本发明以裂殖壶菌Schizochytrium sp.SR21为野生型菌株,在大肠杆菌中构建以elo1基因两端序列为同源臂、博来霉素为筛选标记的elo1基因敲除载体pBlue-ZEO-elo1和以18sRNA为同源臂、潮霉素为筛选标记的pap基因干扰载体pBlue-HYG-siRNA-18s,将两个载体同源重组结构域线性化后电转进入裂殖壶菌内,得到一株elo1基因敲除结合pap基因干扰的高产角鲨烯的裂殖壶菌基因工程菌株,命名为MIX菌株。本发明结合角鲨烯调控的研究和裂殖壶菌自身的优势,通过敲除脂肪酸合成途径elo1基因,同时干扰甘油三酯合成途径pap基因的表达,来抑制脂肪酸和甘油三酯的合成,从而增强角鲨烯合成的碳代谢流量,为促进角鲨烯合成提供更多前体。
本发明所涉及的设备、试剂、工艺、参数等,除有特别说明外,均为常规设备、试剂、工艺、参数等,不再作实施例。
本发明所列举的所有范围包括该范围内的所有点值。
本发明中,除有特别说明外,%在表示比例时均为质量百分比;%在表示浓度时,溶质为液体时%代表体积百分比,溶质为固体时%代表g/100mL。
本发明中,所述“室温”即常规环境温度,可以为10~30℃。
本发明的有益效果是:
(1)在裂殖壶菌转化系统的基础上,采用电转的基因转化方式,构建了在裂殖壶菌中elo1基因敲除和pap基因干扰的组合改造的基因工程菌株。获得的MIX菌株具有多次传代的遗传稳定性。
(2)在裂殖壶菌甘油三酯代谢通路上,敲除了elo1基因同时干扰了pap基因的表达,削弱了油脂代谢途径,增强了甲羟戊酸途径的代谢,提高了裂殖壶菌生长期角鲨烯的积累量,这为该基因工程菌株工业化合成角鲨烯提供了基础。
附图说明
图1为基因敲除载体pBlue-ZEO-18s示意图。
图2为elo1基因敲除载体pBlue-ZEO-elo1示意图。
图3为pap基因干扰载体pBlue-HYG-siRNA-18s示意图。
图4为重组菌株鉴定的琼脂糖凝胶电泳图。琼脂糖凝胶电泳分析:M为标准核酸样品;条带1和条带2:MIX菌株elo1基因区域;条带3和条带4:野生型菌株elo1基因区域;条带5和条带6:MIX菌株pap基因干扰区域验证片段;条带7和条带8:野生型菌株pap基因干扰区域验证片段。
图5为野生型菌株与MIX菌株油脂产量对比图。
图6为野生型菌株与MIX菌株角鲨烯产量对比图。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
下述各实施例中的平板的培养基的pH为6.5,且该培养基由葡萄糖20g、酵母粉10g、琼脂15~20g、20×组分A50mL和500×CaCl2 2mL组成;
每250mL的20×组分A的组成为:Na2SO4 60g,MgSO4 10g,KH2PO4 5g,(NH4)2SO4 5g,K2SO4 3.25g,KCl 2.5g,去离子水定容至250mL;
每100mL的500×CaCl2的组成为:CaCl2·2H2O 8.5g或无水CaCl2 6.418g,去离子水定容至100mL;
一级和二级种子液的培养基的pH为6.5,且由葡萄糖30g、酵母粉10g、20×组分A50mL和500×CaCl2 2mL组成;
基础培养基(即发酵培养基)的pH为6.5,发酵培养基中含有葡萄糖90g/L、玉米浆粉5g/L、蛋白胨5g/L和20×组分A 50mL/L;
发酵培养的整个周期为168h,每隔24h取样检测。
表1本发明实施例采用的序列汇总
实施例1.elo1基因敲除载体pBlue-ZEO-elo1构建
1、Elo1基因上下游同源臂扩增
根据裂殖壶菌elo1基因的序列信息,设计上游同源臂扩增引物P1(如SEQ ID No.1和SEQ ID No.2所示)和下游同源臂扩增引物P2(如SEQ ID No.3和SEQ ID No.4所示),以野生型菌株基因组为模版,用引物P1和P2(如SEQ ID No.1~4所示)、PrimerStar高保真聚合酶,通过PCR方式获得elo1基因上下游同源臂。
PCR程序为:94℃30s,60℃30s,72℃1.5min,34个循环,并对PCR产物进行纯化,纯化产物经1%的琼脂糖凝胶电泳验证。
2、敲除载体pBlue-ZEO-elo1构建
(1)用限制性内切酶HindⅢ和KpnⅠ对裂殖壶菌过表达载体pBlue-ZEO-18s(图1)和elo1基因上下游同源臂PCR纯化产物片段进行双酶切反应。酶切体系(50μL):2μL HindⅢ,2μL KpnⅠ,5μL Loading Buffer,20μL质粒或PCR纯化产物,21μL ddH2O,37℃水浴反应2h。酶切产物经1%琼脂糖凝胶电泳回收。
(2)用T4连接酶将elo1基因上下游同源臂片段和载体pBlue-ZEO-18s片段连接,16℃连接12h。连接体系(25μL):2μL目的基因片段,1μL载体酶切后片段,2.5μL连接酶buffer,19.5μL ddH2O。
(3)连接产物转化大肠杆菌Trans110感受态细胞,转化方法如下:
i.无菌状态下取100μL感受态细胞,加入连接产物混匀,冰上放置30min。
ii.42℃热激90s,迅速放置冰上3~5min。
iii.加入900μL LB培养基,37℃,150r/min孵育1h。
iv.取200μL涂布于含100μg/mL氨苄抗性LB平板上,倒置37℃培养过夜。
挑去阳性转化子,提取质粒,测序验证结果表明连接成功,最终获得elo1基因敲除载体pBlue-ZEO-elo1(图2)。
实施例2.Pap基因干扰载体pBlue-HYG-siRNA-18s构建
1、Pap基因敲除siRNA序列设计
利用软件设计编码siRNA的DNA序列,以引物P3(如SEQ ID No.5和SEQ ID No.6所示)的形式合成两条单链DNA后,在退火缓冲液的条件下形成双链DNA。
2、Pap基因siRNA插入真菌沉默质粒p-Silent
用限制性内切酶XhoⅠ和HindⅢ对真菌沉默质粒p-Silent(购自武汉淼灵生物有限公司)进行双酶切后,与退火形成的编码siRNA的DNA片段连接,转化进入大肠杆菌Trans110感受态细胞中筛选出p-Silent-siRNA重组质粒,测序验证拼接结果。
3、裂殖壶菌pap基因干扰质粒pBlue-HYG-siRNA-18s构建
以p-Silent-siRNA质粒为模板,以P4(如SEQ ID No.7和SEQ ID No.8所示)为引物扩增出质粒上的潮霉素表达框和siRNA表达框,将过表达载体pBlue-ZEO-18s分别利用限制性内切酶SpeⅠ和HindⅢ进行双酶切后,通过无缝克隆技术将扩增产物与酶切产物连接,转化进入大肠杆菌Trans110感受态细胞,筛选出pBlue-HYG-siRNA-18s重组质粒(图3)。
实施例3.MIX菌株构建
1、裂殖壶菌感受态细胞的制备
(1)挑取平板上已活化好的裂殖壶菌(产油Schizochytrium limacinum SR21)单菌落至50mL种子培养基,28℃,200r/min摇床培养24h。
(2)按4%的接种量转接至50mL种子培养基,28℃,200r/min摇床培养24h。
(3)取20mL菌液,4000rpm室温下离心2min,弃上清。
(4)用25mL的预处理剂(含25mL DTT的20mM pH6.5磷酸缓冲液)重悬菌体,150rpm震荡30min以松散细胞壁。
(5)用20mL已预冷的无菌水洗涤菌体两次,离心条件均为:4000rpm,4℃离心2min。
(6)用1M的无菌预冷山梨醇溶液洗涤菌体两次,离心条件均为:4000rpm,4℃离心2min。
(7)用200μL的1M的无菌预冷山梨醇溶液重悬菌体,分装于1.5mL的无菌离心管,每管100μL,冰上备用。
2、裂殖壶菌电转化
(1)将pBlue-ZEO-elo1和pBlue-HYG-siRNA-18s分别酶切线性化后,加入到100μL裂殖壶菌感受态细胞,混匀后转移至预冷的电转杯,冰上静置30min。
(2)电击,2KV,一个脉冲。
(3)立即往电转杯加入1mL预冷的含1M山梨醇的种子培养基,混匀后转移至含1M山梨醇的种子培养基。
(4)28℃,200rpm培养2~3h。
(5)取适量菌液涂布于含50mg/L博莱霉素和200mg/L潮霉素的种子培养基平板上,28℃避光培养2~4天。
3、MIX菌株筛选和鉴定
(1)挑取平板菌落接种至含50mg/L博莱霉素和200mg/L潮霉素的种子培养基中,28℃,200rpm避光培养24h。
(2)传代7次保证过表达载体稳定遗传,每一代都重复步骤(1)中描述实验。
(3)稳定遗传的菌株为elo1基因敲除结合pap基因干扰的高产角鲨烯的裂殖壶菌基因工程菌株表型,保藏于-80℃冰箱。
(4)提取野生型菌株和MIX菌株基因组,设计引物P5(如SEQ ID No.9和SEQ IDNo.10所示)和P6(如SEQ ID No.11和SEQ ID No.12所示)进行PCR验证:P5为elo1基因扩增引物,P6为pap干扰片段验证引物,验证片段如图3中“verification”部分所示。重组菌株鉴定的琼脂糖凝胶电泳分析结果见图4,野生型菌株基因组中可以扩增完整的elo1基因(768bp),而MIX菌株由于敲除片段的同源重组无法扩增出正常的elo1基因片段,这说明elo1基因被成功敲除;在MIX菌株中可以扩增出验证片段(1123bp),而野生型菌株无法扩增出对应大小的片段,说明pap基因干扰片段成功整合进裂殖壶菌基因组中。
实施例4.MIX菌株发酵
1、种子培养
将所述基因工程菌株MIX与裂殖壶菌原始型菌株WT经抗性平板活化后,挑去单菌落接种于50mL锥形瓶(含种子培养基10mL)中,28℃,200r/min避光培养24h后为一级种子。取4mL一级种子培养液接种于500mL锥形瓶(含种子培养基100mL)中,28℃,200r/min避光培养24h为二级种子,作为发酵用菌种。
2、摇瓶发酵培养
取4mL二级种子培养液接种于500mL锥形瓶(含发酵培养基100mL)中,28℃,200r/min培养168h,每隔24h取样。
实施例5.MIX菌株油脂含量测定
准确吸取5mL发酵液至离心管中,加入5mL 12mol/L的浓盐酸于65℃加热50min至菌体溶解。冷却至室温后重复加入3次5mL正己烷萃取,充分混匀后静置5min,收集上层有机相与50mL离心管中。氮气吹干后置于65℃烘箱干燥1h,至正己烷挥发完全后称重,所得重量减去空离心管重量即得总油脂产量。
实施例6.MIX菌株角鲨烯含量测定
1、不皂化物提取
(1)发酵液中加入100mL 12mol/L盐酸,65℃水浴1h。
(2)冷却至室温,加入50mL正己烷,震荡10min,静置10min,收集上层有机相。
(3)重复步骤(2)两次,合并有机相65℃旋蒸10min至正己烷挥发完全得到总油脂。
(4)加入50mL 10%氢氧化钾乙醇溶液,65℃水浴皂化反应1.5h。
(5)冷却至室温,加入50mL正己烷,震荡10min,静置10min,收集上层有机相。
(6)重复步骤(5)3次,直至洗脱出来的有机相变为无色,合并有机相。
(7)加入10%乙醇水溶液洗脱有机相3次,pH试纸测定流出液为中性为止。
(8)将有机相65℃旋蒸10min至正己烷挥发完全得到总不皂化物。
2、角鲨烯含量测定
用5mL色谱级正己烷充分溶解所得不皂化物,转移至含有适量无水硫酸钠的5mL离心管中,再将5mL离心管中的溶液用0.22μm有机滤膜过滤装入气相瓶中,用于气相色谱分析。
结果表明,MIX菌株总油脂量较WT野生型菌株降低了18.50%(图5),角鲨烯积累的最大产量高于WT野生型菌株41.43%(图6),达到10.07mg/L。表明Pap基因干扰结合elo1基因敲除抑制了裂殖壶菌油脂合成的能力,为角鲨烯合成提供了更多的碳代谢通量。
以上所述,仅为本发明较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
序列表
<110> 厦门大学
<120> 产角鲨烯的裂殖壶菌基因工程菌株及其构建方法和应用
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Claims (5)
1.一种产角鲨烯的裂殖壶菌基因工程菌株,其特征在于:所述基因工程菌株选用Schizochytrium limacinum SR21为材料构建而成,所述基因工程菌株的基因组中elo1基因被敲除,同时pap基因被干扰表达;所述基因工程菌株通过以下构建方法得到:
1)克隆来源于Schizochytrium limacinum SR21基因组的elo1基因的上下游同源臂,插入pBlue-ZEO-18s质粒的同源重组区域,构建以博来霉素为抗性的elo1基因敲除载体pBlue-ZEO-elo1;
所述elo1基因上下游同源臂的构建方法包括:根据Schizochytrium limacinum SR21基因组的elo1基因的序列信息,设计如SEQ ID No.1和SEQ ID No.2所示的上游同源臂扩增引物P1、如SEQ ID No.3和SEQ ID No.4所示的下游同源臂扩增引物P2;以Schizochytriumlimacinum SR21基因组为模版,用所述引物P1和所述引物P2通过PCR方式获得elo1基因上下游同源臂;
所述pBlue-ZEO-elo1载体的构建方法包括:用HindⅢ和KpnⅠ对pBlue-ZEO-18s和所述elo1基因上下游同源臂进行双酶切,连接,转化至大肠杆菌Trans110感受态细胞,获得所述pBlue-ZEO-elo1;
2)将来源于野生型菌株Schizochytrium limacinum SR21基因组的pap基因的siRNA连接到真菌干扰质粒p-Silent-HYG的多克隆区域,利用PCR的方式扩增出潮霉素抗性表达框和siRNA的表达框;将该含有潮霉素抗性表达框和siRNA的表达框的片段整合进过表达质粒pBlue-ZEO-18s的多克隆区域,替换博来霉素表达框和过表达基因表达框,构建出以潮霉素为抗性,以18s-rRNA基因序列为同源臂的pap基因干扰载体pBlue-HYG-siRNA-18s;
所述pBlue-HYG-siRNA-18s的构建方法包括:以如SEQ ID No.5和SEQ ID No.6所示的引物P3构建编码pap基因敲除siRNA的DNA片段;用XhoⅠ和HindⅢ对真菌沉默质粒p-Silent-HYG进行双酶切后,与所述DNA片段连接,转化至大肠杆菌Trans110感受态细胞,获得p-Silent-siRNA重组质粒;以所述p-Silent-siRNA为模板,以如SEQ ID No.7和SEQ ID No.8所示的引物P4扩增出p-Silent-siRNA质粒上的潮霉素抗性表达框和siRNA表达框;用SpeⅠ和HindⅢ对所述pBlue-ZEO-18s进行双酶切,与所述扩增的产物连接,转化至大肠杆菌Trans110感受态细胞,获得所述pBlue-HYG-siRNA-18s重组载体;
3)制备裂殖壶菌感受态细胞,将构建好的两重组载体分别酶切线性化,并电转入裂殖壶菌内,通过博来霉素和潮霉素抗性筛选和鉴定,获得elo1基因敲除结合pap基因干扰的所述产角鲨烯的裂殖壶菌基因工程菌株。
2.根据权利要求1所述的基因工程菌株,其特征在于:所述步骤3)中,将所述pBlue-ZEO-elo1和所述pBlue-HYG-siRNA-18s分别酶切线性化后,加入到裂殖壶菌Schizochytrium limacinum SR21的感受态细胞,电转,培养获得所述产角鲨烯的裂殖壶菌基因工程菌株。
3.一种应用权利要求1或2所述的基因工程菌株生产角鲨烯的方法,其特征在于:将所述产角鲨烯的裂殖壶菌基因工程菌株接种于种子培养基,26~30℃逐级放大培养获得发酵用菌种;将所述发酵用菌种接种于发酵培养基中,26~30℃进行发酵培养;收集发酵培养产物,提取得到角鲨烯。
4.根据权利要求3所述的方法,其特征在于:所述种子培养基的pH值为6.4~6.6,且包括配方比例为28~32g:9~11g:48~52mL:1.5~2.5mL的葡萄糖、酵母粉、20×组分A和500×CaCl2;其中,所述20×组分A包括:Na2SO4 238~242g/L,MgSO4 38~42g/L,KH2PO4 19~21g/L,(NH4)2SO4 19~21g/L,K2SO4 12~14g/L,KCl 9~11g/L,溶剂为水;所述500×CaCl2包括:CaCl2·2H2O 84~86g/L或无水CaCl2 64~65g/L,溶剂为水。
5.根据权利要求3所述的方法,其特征在于:所述发酵培养基的pH值为6.4~6.6,且包括:葡萄糖88~92g/L、玉米浆粉4~6g/L、蛋白胨4~6g/L和20×组分A 48~52mL/L;其中,所述20×组分A包括:Na2SO4 238~242g/L,MgSO4 38~42g/L,KH2PO4 19~21g/L,(NH4)2SO419~21g/L,K2SO4 12~14g/L,KCl 9~11g/L,溶剂为水。
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