CN114958627A - 一株高产生育酚的重组裂殖壶菌工程菌的构建方法及应用 - Google Patents
一株高产生育酚的重组裂殖壶菌工程菌的构建方法及应用 Download PDFInfo
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Abstract
本发明提供高产生育酚的重组裂殖壶菌,所述重组裂殖壶菌是在裂殖壶菌中通过表达乙酰辅酶A合酶基因(ACS)、乙醛脱氢酶6基因(ALD6)和GGPP合成酶基因(crtE)构建而成的。本发明通过过表达ACS、ALD6和crtE基因达到提高乙酰辅酶A含量和GGPP含量,同时对启动子进行突变,筛选有益突变体对关键基因进行表达,来提高生育酚的合成。采用本发明的裂殖壶菌重组菌株ATCC‑20890进行发酵合成的总生育酚含量达到611.7μg/g DCW,其中γ‑生育酚含量达到365.5μg/g DCW,α‑生育酚含量达到246.2μg/g DCW,在原始菌株ATCC20889的基础上提高了3.5倍。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种产生育酚的重组工程菌株,还涉及所述重组菌株的构建方法,及其在生产生育酚中应用。
技术背景
维生素E(Vitamin E)是一种脂溶性维生素,其水解产物为生育酚(tocopherol),是最主要的抗氧化剂之一。维生素E的应用领域非常广泛,可以添加在化妆品、护肤品中,还可以作为医疗保健品预防各种疾病,在动植生命中有着不可替代的重要地位,故而人类对维生素E的需求量巨大。但是目前的一些化学合成、生物提取的方法存在收率低、纯度不高、浪费较大、易污染环境、设备昂贵、操作复杂及质量差等问题。因此,通过生物技术手段调控一些关键酶基因的表达,成为天然生育酚合成的新途径。
裂殖壶菌(Schizochytrium)是一类海洋异养微生物,其单细胞内能够积累大量对人体有益的活性物质,这些活性物质合成所用到的共同前体物质乙酰辅酶A在裂殖壶菌中含量比较高,而乙酰辅酶A也是生育酚合成上游过程中的一个关键前体物质,所以我们尝试在已成功构建生育酚合成通路的裂殖壶菌中调控关键基因的表达来调节前体物质的合成,从而提高裂殖壶菌中生育酚含量。
本专利通过在已成功构建生育酚合成通路的裂殖壶菌中过表达乙酰辅酶A合酶(ACS)和乙醛脱氢酶6基因(ALD6)达到提高乙酰辅酶A含量的目的,同时过表达GGPP合成酶基因(crtE)以提高GGPP含量,从而提高生育酚含量,得到一株能高产生育酚的裂殖壶菌重组菌株。
发明内容
本发明的目的是提高裂殖壶菌中生育酚含量,通过对生育酚合成途径中关键基因的过表达,从而促进生育酚的合成,提供一种高产生育酚的重组工程菌株,以及该重组菌株的构建方法和应用。
本发明的思路是通过在已成功构建生育酚合成通路的裂殖壶菌中调控关键基因的表达来调节前体物质的合成,过表达乙酰辅酶A合酶(ACS)、乙醛脱氢酶6基因(ALD6)和GGPP合成酶基因(crtE)达到提高乙酰辅酶A含量和GGPP含量,同时对启动子进行突变,筛选有益突变体对关键基因进行表达,从而提高裂殖壶菌中生育酚含量,得到一株能高产生育酚的裂殖壶菌重组菌株,其合成路线如图1所示。
基于此,本发明提供高产生育酚的重组裂殖壶菌,所述重组裂殖壶菌是在裂殖壶菌Schizochytrium sp.ATCC20889中通过表达乙酰辅酶A合酶基因(ACS)、乙醛脱氢酶6基因(ALD6)和GGPP合成酶基因(crtE)的构建而成的。
在本发明中,所述乙酰辅酶A合酶基因(ACS)和乙醛脱氢酶6基因(ALD6)构建的表达盒中分别含有启动子TEFp-2,启动子TEFp-2的序列如SEQ ID No.5所示;所述GGPP合成酶基因(crtE)构建的表达盒中含有启动子TEFp-3,启动子TEFp-3的序列如SEQ ID No.6所示;乙酰辅酶A合酶基因(ACS)的序列如SEQ ID No.1所示,乙醛脱氢酶6基因(ALD6)的序列如SEQ ID No.2所示,GGPP合成酶基因(crtE)的序列如SEQ ID No.3所示。
本发明还提供上述重组裂殖壶菌的构建方法,所述方法包括以下步骤:
((1)分别构建ACS基因、ALD6基因和crtE基因过表达质粒
根据测序获得的酰基转移酶(AT)基因序列设计引物ATup-F和ATup-R以及ATdown-F和ATdown-R扩增得到裂殖壶菌(Schizochytrium sp.ATCC20889)的上下游同源臂并连接到pJN44载体上,分别得到重组质粒pJN44-ATup和pJN44-ATdown,然后通过酶切将胶回收的ATup片段和ATdown片段连接到PBS-Zeo载体上,得到重组质粒PBS-Zeo-AT;
以裂殖壶菌Schizochytrium sp.ATCC20889基因组为模板,分别扩增获得的ACS基因、ALD6基因和crtE基因序列片段,将目的片段分别连接到载体pJN44的多酶切位点上,构建得到含有ACS表达盒的质粒pJN44-ACS、含有ALD6表达盒的质粒pJN44-ALD6和含有crtE表达盒的质粒pJN44-crtE;将质粒pJN44-ALD6和pJN44-crtE中含目的基因的表达盒进行酶切,并依次连接至质粒pJN44-ACS,得到重组质粒pJN44-ACS/ALD6/crtE,然后将重组质粒pJN44-ACS/ALD6/crtE中含目的基因的表达盒酶切并连接至载体PBS-Zeo-AT,得到重组质粒PBS-Zeo-ACS/CrtE/ALD6-AT;
其中,酰基转移酶(AT)基因序列如SEQ ID No.7所示;
(2)TEF启动子的替换
然后将TEFp-2启动子连接至pJN44-ACS/ALD6/crtE载体,分别替换ACS表达盒和ALD6表达盒原有启动子,将TEFp-3启动子连接至pJN44-ACS/ALD6/crtE载体,替换crtE表达盒的原有启动子,得到重组质粒;
(3)构建重组裂殖壶菌ATCC-20890
按照裂殖壶菌电转化方式将步骤(2)得到的重组质粒转化进入裂殖壶菌,得到重组裂殖壶菌ATCC20890在本发明中,所述重组菌株是选用Schizochytrium sp.ATCC-20889为底盘细胞构建而成。
在本发明中,PBS-Zeo载体是根据中国发明专利申请CN201510417269.4公开的方法的构建得到的。
本发明中的乙酰辅酶A合酶、乙醛脱氢酶6和GGPP合成酶基因均从裂殖壶菌ATCC-20889中克隆所得。
在本发明中,裂殖壶菌ATCC-20889的构建方法是:
(1)构建含GGH基因、HPT基因以及γ-TMT基因的重组质粒
获取来源于拟南芥的香叶基香叶基二磷酸还原酶基因GGH、来源于小球藻的尿黑酸植基转移酶基因HPT和来源于镰孢菌的γ-生育酚甲基转移酶基因γ-TMT的序列,合成并添加酶切位点,并在序列末端连接EGFP基因;将合成片段与质粒pBluZeo-18S分别经酶切和连接构建重组质粒pBluZeo-18S-AtGGH、pBluZeo-GaHPT-18S和pBluZeo-Ftγ-TMT-18S,将所得重组质粒热激转化大肠杆菌感受态细胞,筛选阳性转化子并测序验证正确;
(2)重组质粒转化裂殖壶菌
将上述重组质粒酶切线性化处理后,取10μl加入100μl裂殖壶菌ATCC 20888感受态细胞,混匀后转移至预冷的电转杯,冰上静置30min后进行电击,电击结束后立即加入1ml预冷的含1M山梨醇的种子培养基,轻轻混匀后转移至预冷的含1M山梨醇的4mL种子培养基中,28℃、200rpm培养2h;然后取300μL菌液均匀涂于含50μg/mL博来霉素的固体平板,置于28℃培养箱培养24h,所得单菌落提取基因组进行PCR验证并验证正确;
(3)构建重组裂殖壶菌ATCC-20889
将步骤(2)中验证成功的重组质粒pBluZeo-AtGGH-18S,pBluZeo-GaHPT-18S,pBluZeo-Ftγ-TMT-18S中的AtGGH,GaHPT和Ftγ_TMT片段通过酶切连接,然后连在质粒pBluZeo-18S上获得重组质粒pBluZeo-AtGGH-GaHPT-Ftγ_TMT-18S,酶切线性化处理后,电击法法转入裂殖壶菌中,获得重组菌株ATCC-20889(ATCC-pBl-AtGGH-GaHPT-Ftγ_TMT)。
其中,所述基因GGH的核酸序列如SEQ ID No.8所示,所述基因HPT的核酸序列如SEQ ID No.9所示,所述基因γ-TMT的核酸序列如SEQ ID No.10所示。
在本发明中,所述重组菌株采用突变后的有益突变体启动子过表达乙酰辅酶A合酶、乙醛脱氢酶6和GGPP合成酶基因。
在本发明中,所述重组菌株转化方式为:将重组质粒取10μl加入100μl裂殖壶菌ATCC 20889感受态细胞,混匀后转移至预冷的电转杯,冰上静置30min后进行电击,电击结束后立即加入1ml预冷的含1M山梨醇的种子培养基,轻轻混匀后转移至预冷的含1M山梨醇的4mL种子培养基中,28℃、200rpm培养2h。
在本发明中,所述重组菌株培养条件为:在种子培养基中200rpm、28℃恒温摇床里培养48h,然后接入发酵培养基中,200rpm、28℃恒温摇床里培养96h。
本发明通过调控关键基因的表达来提高生育酚合成前体物质的合成,过表达乙酰辅酶A合酶(ACS)、乙醛脱氢酶6基因(ALD6)和GGPP合成酶基因(crtE)达到提高乙酰辅酶A含量和GGPP含量,同时对启动子进行突变,筛选有益突变体对关键基因进行表达,来提高生育酚的合成。采用本发明的裂殖壶菌重组菌株ATCC-20890进行发酵合成的总生育酚含量达到611.7μg/g DCW,其中γ-生育酚含量达到365.5μg/g DCW,α-生育酚含量达到246.2μg/gDCW,在原始菌株ATCC20889的基础上提高了3.5倍。
【附图说明】
图1为裂殖壶菌的生育酚合成路线。
【具体实施方式】
下面结合附图和实施例非限制性地解释本发明的技术方案。
在本发明中,如无特殊说明,用于说明浓度的“%”为重量百分比,“:”为重量比。
本发明涉及以下培养基:
种子培养基含有:葡萄糖40g/L、酵母膏2g/L、谷氨酸钠10g/L、KH2PO4 4g/L、NaCl15g/L、MgCl2 3g/L、CaCl2·2H2O 1g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl3 0.1g/L;
发酵培养基含有:葡萄糖40g/L、酵母膏2g/L、谷氨酸钠10g/L、KH2PO4 4g/L、NaCl15g/L、MgCl2 3g/L、(NH4)2SO4 6g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl3 0.1g/L。
固体种子培养基:在种子培养基的基础上,添加1.5-2%琼脂,灭菌,在超净台内倒入灭菌的平板中。
博莱霉素种子筛选培养基:1.5%固体种子培养基灭菌后,当培养基降温至50℃,加入终浓度为100μg/ml的博莱霉素(Z-cin),立即倒平板,4℃冰箱避光保存。
本发明涉及以下引物:
表1引物信息
在本发明中,发酵液中总生育酚产量的检测方法是:检测样品的制备:取1m1SD发酵液于1.5ml离心管中,12000rpm离心5min,取上清于新的离心管,并加入40ml冰醋酸,再用0.22μm水系针孔式滤头过滤。
HPLC检测条件:流动相:0.01M KH2PO4溶液(A)和甲醇(B);比例:90%A/10%B;流速:0.8ml/min;检测波长:290nm;色谱柱:YMC-Pack ODS-AQ(4.6×250mm)。
标准曲线制备方法如下:
γ-生育酚标准曲线的制作:用万分之一天平称取10mgγ-生育酚,溶于10ml纯甲醇溶液中,浓度定为1g/L的母液,依次用纯甲醇溶液梯度稀释,稀释得到浓度分别为250mg/L,100mg/L,50mg/L,20mg/L,5mg/L的γ-生育酚溶液,并用相同的方法再稀释出两批同浓度的γ-生育酚溶液,对所有样品经0.22μm有机系针孔式滤头过滤,再进行HPLC分析。
α-生育酚的标准曲线制备方法同γ-生育酚标准曲线的制作。
实施例1构建工程菌株裂殖壶菌ATCC20889
表2裂殖壶菌ATCC20889中GGH、HPT和γ-TMT基因来源
表3实施例1涉及以下引物
1、GGH、HPT、γ-TMT基因重组表达质粒构建
从NCBI上获取来源于拟南芥(AtGGH)的GGH基因序列,来源于小球藻(GaHPT)的HPT基因序列,以及来源于镰孢菌(Ftγ-TMT)的γ-TMT基因序列,送生工合成,添加酶切位点,并在序列末端连EGFP基因。将合成片段与质粒pBluZeo-18S(质粒来源:购于上海生工生物工程(上海)股份有限公司)分别经酶切和连接获得重组质粒pBluZeo-AtGGH-18S,pBluZeo-GaHPT-18S,pBluZeo-Ftγ-TMT-18S。将获得的重组质粒热激转化大肠杆菌感受态细胞中,在含有100μg/ml氨苄抗性的LB平板上培养筛选阳性转化子,提取质粒进行测序验证。
2、制备裂殖壶菌感受态细胞
挑取平板上已活化好的Schizochytrium sp.ATCC20888裂殖壶菌单菌落至50ml种子培养基中28℃,200r/min培养24h;按4%的接种量再次转接至50ml种子培养基中28℃,200r/min培养24h取20ml菌液,4000rpm离心2min,弃上清收集菌体,用25ml的预处理剂(含25ml DTT的20mM PH6.5磷酸缓冲溶液)重悬菌体,150rpm震荡30min以松散细胞壁。然后分别用20ml已预冷的无菌水和1M的无菌预冷山梨醇溶液分别洗涤菌体两次,离心条件为4000rpm,4℃,2min,最后用200μl的1M的无菌预冷山梨醇溶液重悬菌体,分装于1.5ml无菌离心管中。
3、重组表达质粒的转化及荧光比较
将上述验证正确的重组质粒酶切线性化处理后,取10μl加入100μl裂殖壶菌ATCC20888感受态细胞,混匀后转移至预冷的电转杯,冰上静置30min,然后将电转杯擦干后置于电转仪,电压设置为2kV,电击时间为一个脉冲。电击结束后立即加入1ml预冷的含1M山梨醇的种子培养基,轻轻混匀后转移至预冷的含1M山梨醇的4mL种子培养基中(装于25mL锥形瓶),28℃,200rpm培养2h。取300μL菌液均匀涂于含50μg/mL博来霉素的固体平板,置于28℃培养箱培养24h。
将平板上长出的单菌落接种至种子培养基,28℃,200rpm培养24h后提取基因组做PCR验证。结果显示抗性筛选标记已重组至裂殖壶菌的基因组,实现了外源基因的同源重组,获得重组菌株4(ATCC-pBl-AtGGH);7(ATCC-pBl-GaHPT),9(ATCC-pBl-Ftγ-TMT)。
然后通过荧光显微镜定性观察重组菌株荧光有无来初步确认该基因是否能够在宿主中表达,结果显示来源于拟南芥(Arabidopsis thaliana)的香叶基香叶基二磷酸还原酶基因,和来源于小球藻的GaHPT基因以及来源于镰孢菌的Ftγ-TMT基因能在宿主中表达。
4、重组菌株ATCC-20889构建
将上述获得的3个重组质粒pBluZeo-AtGGH-18S,pBluZeo-GaHPT-18S,pBluZeo-Ftγ-TMT-18S中的AtGGH,GaHPT和Ftγ_TMT片段通过酶切连接,然后连在pBluZeo-18S上获得重组质粒pBluZeo-AtGGH-GaHPT-Ftγ_TMT-18S,经同样的方式酶切线性化处理后,按上述方法转入裂殖壶菌ATCC 20888中,获得重组菌株ATCC-20889(ATCC-pBl-AtGGH-GaHPT-Ftγ_TMT)。
实施例2乙酰辅酶A合酶基因、乙醛脱氢酶6基因和GGPP合成酶基因过表达质粒构建
1、获取裂殖壶菌AT基因上下游同源臂
根据测序获得的酰基转移酶AT基因序列(如SEQ ID No.7所示),设计引物ATup-F和ATup-R扩增裂殖壶菌(Schizochytrium sp.ATCC20889)的上游同源臂,并将其连接到pJN44载体上,得到重组质粒pJN44-ATup。类似地,获取裂殖壶菌AT基因下游同源臂,根据获得的裂殖壶菌AT序列设计引物ATdown-F和ATdown-R扩增下游同源臂,并将其连接到pJN44载体上,得到重组质粒pJN44-ATdown。
2、构建质粒PBS-Zeo-ATup
利用NotI和XbaI两个限制性内切酶对质粒pJN44-ATup和PBS-Zeo分别进行双酶切,然后将胶回收的ATup片段连接到线性化的PBS-Zeo载体上,得到重组质粒PBS-Zeo-ATupe。
3、构建质粒PBS-Zeo-AT
利用EcoRI和KpnI两个限制性内切酶对质粒pJN44-ATdown和PBS-Zeo-ATup分别进行双酶切,然后将胶回收的ATdown片段连接到线性化的PBS-Zeo-ATup载体上,得到重组质粒PBS-Zeo-AT。
4、构建质粒PBS-Zeo-ACS/CrtE/ALD6-AT
提取实施例1的裂殖壶菌(Schizochytrium sp.ATCC20889)基因组,设计带酶切位点引物ACS-F与ACS-R、ALD6-F与ALD6-R、crtE-F与crtE-R,如表1,利用PCR技术,从裂殖壶菌ATCC 20889扩增获得乙酰辅酶A合酶基因(ACS)、乙醛脱氢酶6基因(ALD6)和GGPP合成酶基因(crtE)序列片段。
将含启动子PTEF和终止子TXPR2的表达载体pJN44用限制性内切酶HindIII和SalI进行酶切,纯化并胶回收目的基因和载体的DNA片段;将回收的两个DNA片段在NEB DNA连接酶(NEB公司,产品编号:M0367S)作用下进行连接反应,得到含ACS表达盒的质粒pJN44-ACS和含CrtE表达盒的质粒pJN44-CrtE。同理,将表达载体pJN44用限制性内切酶PstI和TspMI进行酶切,纯化并胶回收目的基因和载体的DNA片段;将回收的两个DNA片段在NEB DNA连接酶作用下进行连接反应,得到含ALD6表达盒的质粒pJN44-ALD6。
将所得的含有CrtE表达盒的质粒pJN44-CrtE和含有ACS表达盒的质粒pJN44-ACS分别用PfoI和KasI进行酶切,纯化并胶回收目的基因和载体的DNA片段;将回收的两个DNA片段在NEB DNA连接酶作用下进行连接反应,得到含ACS表达盒和CrtE表达盒的质粒pJN44-ACS/CrtE。同理,将质粒pJN44-ALD6中的ALD6表达盒连接至质粒pJN44-ACS/CrtE的BamHI单酶切位点处,得到重组质粒pJN44-ACS/CrtE/ALD6。然后将重组质粒pJN44-ACS/CrtE/ALD6中含有ACS表达盒、ALD6表达盒和crtE表达盒使用PfoI和DraIII进行酶切并连接至载体PBS-Zeo-AT,最终得到重组质粒PBS-Zeo-ACS/CrtE/ALD6-AT,按照裂殖壶菌电转化方式将重组质粒PBS-Zeo-ACS/CrtE/ALD6-AT转入裂殖壶菌ATCC20889中进行游离表达,待长出转化子,挑选单克隆,提取质粒进行验证,对验证正确的菌株命名为裂殖壶菌ATCC20889-1,对其培养后取发酵液经HPLC检测,总生育酚最终产量可达298.5mg/g。
实施例3TEF强启动子筛选
从NCBI上下载EGFP序列(GenBank:MN623123.1),送生工合成,连接在通用质粒pUC57上,设计带酶切位点引物EGFP-F/EGFP-R,以合成质粒pUC57为模板,扩增得到EGFP片段,分别以HindⅢ/SalⅠ为酶切位点双酶切pJN44载体和EGFP片段并连接上获得重组质粒pJN44-EGFP。
以质粒pJN44为模板,TEF-F/TEF-R为引物,利用易错PCR技术对TEF启动子进行突变。得到TEF启动子突变片段TEFp,用限制性内切酶BamHⅠ/HindⅢ双酶切突变过后的启动子,然后将这些突变的启动子混合片段与经相同酶切后的质粒pJN44-EGFP连接,获得重组质粒库pJN44-EGFP(TEFp)。
其中,易错PCR反应体系为:100μL,含8mmol/L MgCl2 0.6mmol/L MnCl2、50mmol/LKCl、10mol/L Tris-Cl,pH8.3(25℃),6U的Taq DNA聚合酶,dATP、dGTP,dCTP、dTTP浓度分别为0.2、0.2、1和1mmol/L。易错PCR反应条件为:96℃预变性5min,96℃变性2min,58℃退火2min,72℃延伸1min,进行30个循环
将重组质粒库pJN44-EGFP(TEFp)用电转化方式转入裂殖壶菌ATCC20889感受态细胞中,然后加入1ml预冷的含1M山梨醇的种子培养基,轻轻混匀后转移至预冷的含1M山梨醇的4mL种子培养基中(装于25mL锥形瓶),28℃,200rpm培养2h。取300μL菌液均匀涂于含50μg/mL氨苄青霉素的固体平板,置于28℃培养箱培养24h,待长出单菌落,采用菌落PCR鉴定转化子,挑选阳性克隆。利用96深孔板,通过高通量筛选方法筛选构建的突变体,并用荧光酶标仪检测重组菌的绿色荧光蛋白EGFP荧光强度。随机挑取200个突变子进行筛选,检测到荧光强度较对照重组菌(野生型TEF启动子)显著提高的3个重组菌,进而得到3个启动子强度高于野生型TEF启动子的有益突变体TEFp-1、TEFp-2和TEFp-3。为了验证以上重组菌的EGFP荧光强度改变确实是因为启动子序列的突变引起,将突变TEFp启动子序列克隆并测序,TEFp-1、TEFp-2和TEFp-3的基因序列依次见SEQ ID No.4、SEQ ID No.5和SEQ ID No.6。
利用限制性内切酶BamHI和HindⅢ对质粒pJN44-ACS/ALD6/crtE上ACS表达盒的原有启动子进行双酶切,并在TEFp-1、TEFp-2和TEFp-3启动子突变体片段两端分别添加BamHI和HindⅢ酶切位点,然后将TEFp-1、TEFp-2和TEFp-3启动子突变体片段分别连接至pJN44-ACS/ALD6/crtE载体,替换ACS表达盒的原有启动子。再按照裂殖壶菌电转化方式分别转化进入裂殖壶菌中进行游离表达,对应得到重组菌ACS-T1,ACS-T2,ACS-T3,将重组菌进行培养,采用HPLC测定菌液中生育酚的含量,其产量如表4,分别为318.0,368.1,310.2μg/g,经比较可得,引入突变过后的TEFp-2启动子来驱动ACS基因表达,效果最佳。
表4各重组菌株发酵液中各种生育酚含量
同理,将TEFp-1、TEFp-2和TEFp-3启动子突变体片段分别连接至pJN44-ACS/ALD6/crtE载体,替换ALD6表达盒的原有启动子,转化得到对应重组菌,经相同条件发酵培养过后,测得其产量如表5,分别为323.7,420.6,357.8μg/g,经比较发现,引入突变过后的TEFp-2启动子来驱动ALD6基因表达,效果最佳。
表5各重组菌株发酵液中各种生育酚含量
同理,将TEFp-1、TEFp-2和TEFp-3启动子突变体片段分别连接至pJN44-ACS/ALD6/crtE载体,替换crtE表达盒的原有启动子,转化得到对应重组菌,经相同条件发酵培养过后,测得其产量如表6,分别为335.5,346.4,434.0μg/g,经比较发现,引入突变过后的TEFp-3启动子来驱动CrtE基因表达,效果最佳。
表6各重组菌株发酵液中各种生育酚含量
实施例4含强启动子重组质粒构建并转化裂殖壶菌
对质粒PBS-Zeo-ACS/CrtE/ALD6-AT上ACS表达盒、ALD6表达盒和crtE表达盒的原有启动子分别进行双酶切,在筛选得到的TEFp-2和TEFp-3启动子突变体片段两端分别添加相同酶切位点,然后将TEFp-2和TEFp-3启动子突变体片段连接至PBS-Zeo-ACS/CrtE/ALD6-AT载体,分别替换ACS、ALD6和crtE表达盒的原有启动子,再按照裂殖壶菌电转化方式将该重组质粒转化进入裂殖壶菌,利用同源重组作用,将含3个目的基因的表达盒整合至裂殖壶菌染色体,进而得到能够稳定高效合成生育酚的重组裂殖壶菌Schizochytriumsp.ATCC20890。
实施例5重组裂殖壶菌ATCC20890在产生育酚中的应用
通过检测重组裂殖壶菌ATCC20890中生育酚含量来验证此菌株是否能够高效产生育酚。
将得到的重组菌株ATCC20890在发酵培养基中,220rpm、28℃条件下恒温摇床里培养96h,然后取5ml发酵液离心并收集菌体,研磨破胞后,用2m1丙酮分两次萃取,离心取上清,过滤后,进HPLC分析。
结果如表7显示,比较各菌株的生育酚含量,发现重组菌株ATCC-20890的总生育酚含量达到611.7μg/g DCW,其中γ-生育酚含量达到365.5μg/g DCW,α-生育酚含量达到246.2μg/g DCW,在原始菌株ATCC20889的基础上提高了3.5倍。
表7各重组菌株发酵液中各种生育酚含量
序列表
<110> 陕西海斯夫生物工程有限公司
<120> 一株高产生育酚的重组裂殖壶菌工程菌的构建方法及应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1938
<212> DNA
<213> 乙酰辅酶A合酶基因ACS(Unknown)
<400> 1
tacaaagcca accatgccac ctccatccaa gacacttccg cttactggtc ggatcttgca 60
aagtccaaat tggactggtt cgctcctttc cctgctgcag tgacgggttc gttcaaagat 120
ggaagcgtct cttggtttgc tggtggaaaa attaacgtgt gccataacgc cgtggatcgt 180
tattgtcttg cgccgtacaa ccgaggagat gagatagcca tcatctggga gggcgacgag 240
ccctcgtcag taaagaatat cacgtaccgt gagctccagt tgagtgtatg ccgtattgca 300
aacgcactca agtctcaggg agtacaaaag ggagacgtcg tcacgcttta catgccgatg 360
gttccagagt tggccatgac gatgcttgca tgcgctagaa ttggagctgt acactctgtt 420
atctttgctg gattcagtgc tgatgccatc gcagacagaa tcgatgatgc caagtcaaaa 480
tgggttgtaa cagcgagtgc aggaagccgt ggaggaagaa acctccccct caagtcaatt 540
tgtgatgcag cgattatgaa ggaacgttgt gcaggagtgg tggaaaaggt gtttgtattt 600
gatggggctg gggcaaatgg caatgaatgg gagaagaaag atgttcatgt gaagtttaca 660
gagttggttg aggcacagag gcctgtgtgt ccttgtgaat ggatggatgc tgaggaccca 720
ttgttcatcc tgtacacgtc gggaagtact gggaggccaa agggactcct tcatactact 780
ggaggatact ctatttatgc aatgcataca actgccaaca gtttcggact ttcgcagcaa 840
actggacctt cctctcccaa cgatcctcac aaagatgtgt acgcgtgcgt tgccgatgca 900
ggatggatta ctggacacac ctacatcgtc tacggtcctc tgttgaacgg tatctctacc 960
ttcatgttcg agtccactcc aatgtatccg gatcacggtc gttactggga tatggtgcaa 1020
cgtcacaaga ttactgtctt ttataccgcc cccactgcga ttcgtagttt gatgaggttc 1080
ggtgatgatg ctcctaaaaa atacgatttg tcttcgttga aggtgttggg tacggttgga 1140
gagccaatca atcctgctgc ttggaagtgg tattttgatg tcatcggaaa gggcaactgt 1200
gctgtggttg atacttattg gcagacggag acgggaggcc atgtcattac caacttgcct 1260
ggtgtcacac caatgaaacc tggctcttgc actctcgcat gctacggtat tgaggtggtt 1320
gtcttggacg cggcaactgg acaggaaatc aacaccacgg aagcagaggg agttttggca 1380
atcaaacaac cttggcctgg aatggctcgt acgtgtttgg gagatcacca acgctatctc 1440
aatgtgtaca tgaaacctta tcctgggtac tactttgctg gagatggatg tcgccgtgat 1500
gaggatggtt tcatctggat cactgggcgt gtggacgacg tgttgaatgt ttctggacat 1560
cgcattggta ccgctgaggt tgaatctgct cttgtggctc accctgcggt tgctcaggct 1620
gcagttgtcg gtgtacccca cgatatcaaa ggacaaggta tctgctgctt cactactctt 1680
gcggaggggt atcctgagag tgatgagttg atcaaggagt tgcgcaatgc agtacgaact 1740
tctatcggtc cattcgctac tcctgatatg attatcccta cctctgctct tcccatgaca 1800
cgttcaggaa agattatgag gcgaatcttg cgaaaggtgg ctgcgggaga gctggatacc 1860
cttggagata cgacaactct tgctgatcct tctgtggtgg atgctcttat tgccaaagtt 1920
gaacagtcga gaaagtag 1938
<210> 2
<211> 1503
<212> DNA
<213> 乙醛脱氢酶6基因ALD6(Unknown)
<400> 2
atgactaagc tacactttga cactgctgaa ccagtcaaga tcacacttcc aaatggtttg 60
acatacgagc aaccaaccgg tctattcatt aacaacaagt ttatgaaagc tcaagacggt 120
aagacctatc ccgtcgaaga tccttccact gaaaacaccg tttgtgaggt ctcttctgcc 180
accactgaag atgttgaata tgctatcgaa tgtgccgacc gtgctttcca cgacactgaa 240
tgggctaccc aagacccaag agaaagaggc cgtctactaa gtaagttggc tgacgaattg 300
gaaagccaaa ttgacttggt ttcttccatt gaagctttgg acaatggtaa aactttggcc 360
ttagcccgtg gggatgttac cattgcaatc aactgtctaa gagatgctgc tgcctatgcc 420
gacaaagtca acggtagaac aatcaacacc ggtgacggct acatgaactt caccacctta 480
gagccaatcg gtgtctgtgg tcaaattatt ccatggaact ttccaataat gatgttggct 540
tggaagatcg ccccagcatt ggccatgggt aacgtctgta tcttgaaacc cgctgctgtc 600
acacctttaa atgccctata ctttgcttct ttatgtaaga aggttggtat tccagctggt 660
gtcgtcaaca tcgttccagg tcctggtaga actgttggtg ctgctttgac caacgaccca 720
agaatcagaa agctggcttt taccggttct acagaagtcg gtaagagtgt tgctgtcgac 780
tcttctgaat ctaacttgaa gaaaatcact ttggaactag gtggtaagtc cgcccatttg 840
gtctttgacg atgctaacat taagaagact ttaccaaatc tagtaaacgg tattttcaag 900
aacgctggtc aaatttgttc ctctggttct agaatttacg ttcaagaagg tatttacgac 960
gaactattgg ctgctttcaa ggcttacttg gaaaccgaaa tcaaagttgg taatccattt 1020
gacaaggcta acttccaagg tgctatcact aaccgtcaac aattcgacac aattatgaac 1080
tacatcgata tcggtaagaa agaaggcgcc aagatcttaa ctggtggcga aaaagttggt 1140
gacaagggtt acttcatcag accaaccgtt ttctacgatg ttaatgaaga catgagaatt 1200
gttaaggaag aaatttttgg accagttgtc actgtcgcaa agttcaagac tttagaagaa 1260
ggtgtcgaaa tggctaacag ctctgaattc ggtctaggtt ctggtatcga aacagaatct 1320
ttgagcacag gtttgaaggt ggccaagatg ttgaaggccg gtaccgtctg gatcaacaca 1380
tacaacgatt ttgactccag agttccattc ggtggtgtta agcaatctgg ttacggtaga 1440
gaaatgggtg aagaagtcta ccatgcatac actgaagtaa aagctgtcag aattaagttg 1500
taa 1503
<210> 3
<211> 963
<212> DNA
<213> GGPP合成酶基因crtE(Unknown)
<400> 3
ctcgtgggta cagaccatcc ggttctcaac agtgcagctc gatacttctt caacagtgaa 60
gatggaaaag gaaaagggaa acaggtccgt cctgtaatgg ttatgctttt ttctcgagca 120
atgatgcaaa tggggacagg ggaaggcaac ccatcaacag ctcatgctct caagtcccag 180
cgacgtttag ctgaaatcac ggaaatgatt cacacagctt ctctgttcca cgacgacgtc 240
attgacgaag cggatactcg tcgaggacag cctgctgctc atcgtgcttt tggaaataag 300
atggccattt tagcaggaga ctatctgctg gcacgcgctt ccatatgtct cgctcggctg 360
cgaaatgtag atgttgttga gtgcatgagt accgttattg aacatttagt acgaggggaa 420
gtgcttcaga ttaaggataa tcgaactggt gtgcctgata tggaaggata tttgaggaag 480
aacttttaca agacagctag tcttatggcg aactcttgca aaagtgcagc tttactgggc 540
gcaagtgcca atggtagcgg tatctctcca gtggttgtgg aagctgcata tctctacgga 600
aagcacatag gtgtcgcgtt tcaactgatt gatgatgccc tagacttcga agggagtgcc 660
gctagcttgg gaaaaccagc acttgccgat ttgaatgctg gactttccac agctccagtt 720
ttgtttgcag cggaaattca caaggagttg ataccggcga tggcacgaaa atttaaggaa 780
gctggtgaca tcaactttgc gttgcgttgc attgaagggg caggaggggt cagaaaaaca 840
aaagaacttg cagccataca tgccgaagtg gcgatgaacg cagtggcatc agcattggaa 900
ccaagtccgt tccgagactc attggttcat ttggcatgtc gtgttgtgga tcgttctaga 960
taa 963
<210> 4
<211> 206
<212> DNA
<213> 启动子TEFp-1(Unknown)
<400> 4
tagtttcttt gtctggccat ccgggtaagc catgccggac gcaatgtaga ctactgaaaa 60
tttacctgct ttgtggttgg gactttagcc aagggtataa aagaccaccg tccccgaatt 120
acctttcctc ttcttttctc tctctccttg tcaactcaca cccgaaatcg ttaagcattt 180
ccttctgagt ataagaatca ttcaaa 206
<210> 5
<211> 206
<212> DNA
<213> 启动子TEFp-2(Unknown)
<400> 5
tagtttcttt gtctggccat ccgggtaacc catcacggac gctaaataga ctactgaaac 60
actttttgct ttgtggttgg gactttagcc aagggtataa aagaccaccg tccccgaatt 120
acctttcctc ttcttttctc tctctccttg tcaactcaca cccgaaatcg ttaagcattt 180
ccttctgagt ataagaatca ttcaaa 206
<210> 6
<211> 206
<212> DNA
<213> 启动子TEFp-3(Unknown)
<400> 6
tagtttcttt gtctggccat ccgggtaact catgccggac gcaacataga ctactccaaa 60
ttttcatgct ttgtggttgg gactttagcc aagggtataa aagaccaccg tccccgaatt 120
acctttcctc ttcttttctc tctctccttg tcaactcaca cccgaaatcg ttaagcattt 180
ccttctgagt ataagaatca ttcaaa 206
<210> 7
<211> 912
<212> DNA
<213> 酰基转移酶基因AT(Unknown)
<400> 7
atgagtgtga taggtaggtt cttgtattac ttgaggtccg tgttggtcgt actggcgctt 60
gcaggctgtg gcttttacgg tgtaatcgcc tctatccttt gcacgttaat cggtaagcaa 120
catttggctc agtggattac tgcgcgttgt ttttaccatg tcatgaaatt gatgcttggc 180
cttgacgtca aggtcgttgg cgaggagaat ttggccaaga agccatatat tatgattgcc 240
aatcaccaat ccaccttgga tatcttcatg ttaggtagga ttttcccccc tggttgcaca 300
gttactgcca agaagtcttt gaaatacgtc ccctttctgg gtcggttcat ggctttgagt 360
ggtacatatt tcttagacag atctaaaagg caagaagcca ttgacacctt gaataaaggt 420
ttagaaaatg ttaagaaaaa caagcgtgct ctatgggttt ttcctgaggg taccaggtct 480
tacacgagtg agctgacaat gttgcctttc aagaagggtg ctttccattt ggcacaacag 540
ggtaagatcc ccattgttcc agtggttgtt tccaatacca gtactttagt aagtcccaaa 600
tatggggtct tcaacagagg ctgtatgatt gttagaattt taaaacctat ttcaaccgag 660
aacttaacaa aggacaaaat tggtgaattt gctgaaaaag ttagagatca aatggttgac 720
actttgaagg agattggcta ctctcccgcc atcaacgata caaccctccc accacaagct 780
attgagtatg ccgctcttca acatgacaag aaagtgaaca agaaaatcaa gaatgagcct 840
gtgccttctg tcagcattag caacgatgtc aatacccata acgaaggttc atctgtaaaa 900
aagatgcatt aa 912
<210> 8
<211> 1404
<212> DNA
<213> 来源于拟南芥的香叶基香叶基二磷酸还原酶基因GGH(Arabidopsis thaliana)
<400> 8
atggcgacga cggttacact caaatccttc accggacttc gtcaatcatc aacggagcaa 60
acaaacttcg tctctcatgt accgtcatca ctttctctcc ctcaacgacg gacctctctc 120
cgagtaaccg cagccagagc cactcccaaa ctctccaacc gtaaactccg tgtcgccgtc 180
atcggtggtg gaccagcagg cggggcagct gcagagactc tagcacaagg aggaatcgag 240
acgattctca tcgagcgtaa gatggacaat tgcaagcctt gcggtggcgc gattcctctc 300
tgtatggtcg gagaattcaa cttgccgttg gatattattg atcggagagt gacgaagatg 360
aagatgattt cgccgtcgaa cattgctgtt gatattggtc gtacgcttaa ggagcatgag 420
tatataggta tggtgagaag agaagttctt gatgcttatc tgagagagag agctgagaag 480
agtggagcca ctgtgattaa cggtctcttc cttaagatgg atcatccgga gaattgggac 540
tcgccgtaca ctttgcatta cactgagtac gatggtaaaa ctggagctac agggacgaag 600
aaaacaatgg aggttgatgc tgtcattgga gctgatggag ctaactctag ggttgctaaa 660
tctattgatg ctggtgatta cgactacgca attgcatttc aggagaggat taggattcct 720
gatgagaaaa tgacttacta tgaggattta gctgagatgt atgttggaga tgatgtgtcg 780
ccggatttct atggttgggt gttccctaag tgcgaccatg tagctgttgg aacaggtact 840
gtgactcaca aaggtgacat caagaagttc cagctcgcga ccagaaacag agctaaggac 900
aagattcttg gagggaagat catccgtgtg gaggctcatc cgattcctga acatccgaga 960
ccacgtaggc tctcgaaacg tgtggctctt gtaggtgatg ctgcagggta tgtgactaaa 1020
tgctctggtg aagggatcta ctttgctgct aagagtggaa gaatgtgtgc tgaagccatt 1080
gtcgaaggtt cacagaatgg taagaagatg attgacgaag gggacttgag gaagtacttg 1140
gagaaatggg ataagacata cttgcctacc tacagggtac ttgatgtgtt gcagaaagtg 1200
ttttacagat caaatccggc tagagaagcg tttgtggaga tgtgtaatga tgagtatgtt 1260
cagaagatga cattcgatag ctatctgtac aagcgggttg cgccgggtag tcctttggag 1320
gatatcaagt tggctgtgaa caccattgga agtttggtta gggctaatgc tctaaggaga 1380
gagattgaga agcttagtgt ttaa 1404
<210> 9
<211> 927
<212> DNA
<213> 来源于小球藻的尿黑酸植基转移酶基因HPT(Gomphosphaeria aponina)
<400> 9
atgagtcaaa gttccccttc tcgccccaaa aacttattgc aagaaccttt ttcctggctg 60
tacagtttct ggaaattttc ccgcccccat actattattg gtacaagtct cagtatcttt 120
gcattatatc tgataacttt tgacagtagg atcacaccag ccaattttgg acaactgatg 180
ggaagttggt tagcttgtct atgtggcaat atctatattg taggactaaa tcagttagaa 240
gatgtagcaa ttgatcaaat taataagccc catttgccca ttgctgcagg agaattttcc 300
ccacaacaag gaaaatggat tgtgggaatt acaggaattt tagcactatt aatcgcagga 360
gggcaaggca cttggttgtt agcaacagtg ggaataagtt tagttatagg tactgcttat 420
tctttgccac ctatgagatt aaaacgcttt cccttttggg ctgcaatttg tatctttact 480
gtgcggggag tagtggttaa tttgggcatc tttctccatt ttagccaaca gaattttatt 540
cctccagaag tgtgggcatt gacactattt attgtggtat ttaccgtggc gatcgccatt 600
tttaaagatg tgccagacat ggagggagat aagcagtatc aaattaccac ttttacccta 660
ttattaggca atcaagcagt ttttaacctc actctttcgg taattacttt ttgttattta 720
gccatgatat ttgcagggat tttttggcta ccaaaagtta actctgtatt tctggtaatt 780
gcccatttaa ttttgttagg attactttgg tggcggagtc gaagtgtaaa tttaggagca 840
aagcaagaaa tagccaactt ttatcaattc atttggaaac tctttttcct agaataccta 900
ctctttcccg ccgcttttct cctttaa 927
<210> 10
<211> 990
<212> DNA
<213> 来源于镰孢菌的γ-生育酚甲基转移酶基因γ-TMT(Fusarium tjaetaba)
<400> 10
atggctgcca cagcagtgtc tgaccaaact accgacctga gcaagcaata tgacactcct 60
ctcggcctcg ctcactcaac tatgcaagtc ctcaaggaca gaatcaagct ccattacgat 120
cttgcaagtg actactattt gagtttgtgg ggcgaacata tccatcacgg gtactggcca 180
acaccagagt ctgaagccac gcagacaaag gaggaagcac aggccaatct catccagctc 240
cttctcgaca tttcaaagat tccatccaac agctcggttc ttgatgttgg ctgtggtatc 300
ggcggaacat cgcggtatct cgcttccaaa catggtagct ctgtgacggg catcaccatc 360
tcaagcaaac aggtcgagat cgcaaatcgc ctaaccaaag ctgctctcga agacacctct 420
tcgtctgatg tgtcagatga caatggcttc gccaaactcg gcgagggaaa agtcaaattc 480
ctcgagcttg acgccgagaa gatgggcgac ttcttcagcg accagcaggg caacttcgat 540
gcagtatgga taagtgaagc tcttagccat tttccaaaca aggcattgtt ctttgagaat 600
gtgatgaaag tactcaagcc tgggggcaag ttggtgttag cagattggtt cagggatgaa 660
gacattgacg aaacaacttt catcaatgat atcaagccta tcgaagatgg catgctactc 720
ccacccctct gtacccagca agggtacgtc gatctcgcga agaacgccgg acttacagtc 780
ttgtctgagc caaaggacat cagtcaacaa gtccgaaaaa catgggacat tacttggtca 840
cttgtgcaga acccatccct ttgggcattt gcgtttactc aaaaccggga cggtattgct 900
ttcttgcagt catttcgcgc aatgagacga ggttatgcca atggctcctt tcgctacgct 960
gtcatggcat tttataagga aatggcatga 990
Claims (7)
1.高产生育酚的重组裂殖壶菌,其特征在于所述重组裂殖壶菌是在裂殖壶菌Schizochytrium sp.ATCC20889中通过表达乙酰辅酶A合酶基因(ACS)、乙醛脱氢酶6基因(ALD6)和GGPP合成酶基因(crtE)的构建而成的。
2.根据权利要求1所述的重组裂殖壶菌,其特征在于所述乙酰辅酶A合酶基因(ACS)和乙醛脱氢酶6基因(ALD6)构建的表达盒中分别含有启动子TEFp-2,启动子TEFp-2的序列如SEQ ID No.5所示;所述GGPP合成酶基因(crtE)构建的表达盒中含有启动子TEFp-3,启动子TEFp-3的序列如SEQ ID No.6所示;乙酰辅酶A合酶基因(ACS)的序列如SEQ ID No.1所示,乙醛脱氢酶6基因(ALD6)的序列如SEQ ID No.2所示,GGPP合成酶基因(crtE)的序列如SEQID No.3所示。
3.权利要求1或2所述的重组裂殖壶菌的构建方法,所述方法包括以下步骤:
(1)分别构建ACS基因、ALD6基因和crtE基因过表达质粒
根据测序获得的酰基转移酶(AT)基因序列设计引物ATup-F和ATup-R以及ATdown-F和ATdown-R扩增得到裂殖壶菌(Schizochytrium sp.ATCC20889)的上下游同源臂并连接到pJN44载体上,分别得到重组质粒pJN44-ATup和pJN44-ATdown,然后通过酶切将胶回收的ATup片段和ATdown片段连接到PBS-Zeo载体上,得到重组质粒PBS-Zeo-AT;其中,酰基转移酶(AT)基因序列如SEQ ID No.7所示;
以裂殖壶菌Schizochytrium sp.ATCC20889基因组为模板,分别扩增获得的ACS基因、ALD6基因和crtE基因序列片段,将目的片段分别连接到载体pJN44的多酶切位点上,构建得到含有ACS表达盒的质粒pJN44-ACS、含有ALD6表达盒的质粒pJN44-ALD6和含有crtE表达盒的质粒pJN44-crtE;将质粒pJN44-ALD6和pJN44-crtE中含目的基因的表达盒进行酶切,并依次连接至质粒pJN44-ACS,得到重组质粒pJN44-ACS/ALD6/crtE,然后将重组质粒pJN44-ACS/ALD6/crtE中含目的基因的表达盒酶切并连接至载体PBS-Zeo-AT,得到重组质粒PBS-Zeo-ACS/CrtE/ALD6-AT;
(2)TEF启动子的替换
然后将TEFp-2启动子连接至pJN44-ACS/ALD6/crtE载体,分别替换ACS表达盒和ALD6表达盒原有启动子,将TEFp-3启动子连接至pJN44-ACS/ALD6/crtE载体,替换crtE表达盒的原有启动子,得到重组质粒;
(3)构建重组裂殖壶菌ATCC-20890
按照裂殖壶菌电转化方式将步骤(2)得到的重组质粒转化进入裂殖壶菌,得到重组裂殖壶菌ATCC20890。
4.根据权利要求3所述的构建方法,其特征在于步骤(3)的裂殖壶菌是裂殖壶菌Schizochytrium sp.ATCC20889,所述裂殖壶菌Schizochytrium sp.ATCC20889同时整合了来源于拟南芥的香叶基香叶基二磷酸还原酶基因GGH,和来源于小球藻的尿黑酸植基转移酶基因HPT以及来源于镰孢菌的γ-生育酚甲基转移酶基因γ-TMT。
5.权利要求1或2所述的重组裂殖壶菌在生产生育酚中的应用。
6.根据权利要求5所述的应用,其特征在于所得重组裂殖壶菌在种子培养基中,180-220rpm、27-29℃恒温摇床里培养48-72h,然后接种至发酵培养基中,180-220rpm、27-29℃恒温摇床里培养3-5d。
7.根据权利要求2所述裂殖壶菌重组菌株培养基,其特征在于所述种子培养基含有:葡萄糖40g/L、酵母膏2g/L、谷氨酸钠10g/L、KH2PO4 4g/L、NaCl 15g/L、MgCl2 3g/L、CaCl2·2H2O 1g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl30.1g/L;
所述发酵培养基含有:葡萄糖40g/L、酵母膏2g/L、谷氨酸钠10g/L、KH2PO4 4g/L、NaCl15g/L、MgCl2 3g/L、(NH4)2SO4 6g/L、KCl 2g/L、MgSO4·7H2O 5g/L、FeCl3 0.1g/L。
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CN116555054A (zh) * | 2023-06-09 | 2023-08-08 | 陕西海斯夫生物工程有限公司 | 一株高产dha的重组裂殖壶菌、其构建方法及应用 |
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