CN112553099A - 一种重组酿酒酵母及其应用 - Google Patents

一种重组酿酒酵母及其应用 Download PDF

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CN112553099A
CN112553099A CN202011451952.7A CN202011451952A CN112553099A CN 112553099 A CN112553099 A CN 112553099A CN 202011451952 A CN202011451952 A CN 202011451952A CN 112553099 A CN112553099 A CN 112553099A
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诸葛斌
任全路
贺亚曦
陆信曜
宗红
马腾飞
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Abstract

本发明公开了一种重组酿酒酵母及其应用，属于基因工程技术领域。本发明首先构建了一株可以产蒎烯的菌株，通过基因操作同时敲除YPL062W和BTS1之后，菌株的生物量增加明显，对蒎烯的生物合成具有积极的作用，并通过融合表达蒎烯合酶基因Pt1和ERG20ww，使单位菌体产量达到最高1.15mg/L*OD。

Description

一种重组酿酒酵母及其应用
技术领域
本发明涉及一种重组酿酒酵母及其应用，属于基因工程技术领域。
背景技术
蒎烯是一种天然活性单萜烯，广泛用作调味剂、化学品和医药，特别是蒎烯二聚体可作为喷气燃料替代品的良好候选者，也是一种耐高温润滑剂。目前，在工业上大量获取蒎烯的方法还是利用高效精馏塔从脂松节油或粗硫酸盐松节油中进行分离提取。这类传统的获取蒎烯的方法存在着效率低、操作复杂繁琐、能源消耗大和设备技术要求高等缺点，同时破坏环境、消耗大量自然资源。因此，蒎烯的生物技术生产受到越来越多的关注。
酿酒酵母在生产萜类物质具有得天独厚的优势，酵母细胞在细胞色素P450酶的功能性表达方面比原核宿主具有优势，而细胞色素P450酶是合成更复杂的单萜类化合物所必需的。有文献报道酿酒酵母在生产倍半萜和多萜时的产量要高于大肠杆菌，证明了酿酒酵母更大的生产前景。到目前为止，虽然使用细菌宿主时,单萜生物生产更有效率。然而，在细菌底盘中设计单萜生物合成的下游步骤是具有挑战性的，因为其中几个步骤需要细胞色素p450酶的功能性表达，而这在细菌中是不容易实现的。但是到目前为止,低的单蒎烯产量阻碍了酵母细胞工厂有效的p450的进一步反应，并且现有的产率过低，导致生物法生产蒎烯在工业上的应用受限，所以实现酿酒酵母更高的蒎烯产量及更高的产率具有极大的意义。
发明内容
为了得到一种提高重组酿酒酵母生物量、蒎烯产量及催化效率的方法，本发明首先提供一种重组酿酒酵母，所述重组酿酒酵母强化表达了tHMG1，ERG20ww和IDI基因，敲除了YPL062W和BTS1基因，并融合表达了蒎烯合酶tPt1和ERG20ww。
所述ERG20ww是在氨基酸序列如SEQ ID NO.1所示的ERG20的基础上，将第96位的苯丙氨酸和第127位的天冬氨酸突变得到的。
在本发明的一种实施方式中，所述ERG20ww是在核氨酸序列如SEQ ID NO.1所示的ERG20的基础上，将第96位的苯丙氨酸突变为色氨酸，且将第127位的天冬氨酸突变为色氨酸得到的。
在本发明的一种实施方式中，所述融合表达为通过linker进行融合表达，所述linker序列为GSGSGSGS。
在本发明的一种实施方式中，所述蒎烯合酶tPt1来自火炬松，其为蒎烯合酶基因Pt的截短序列；所述蒎烯合酶tPt1核苷酸序列如SEQ ID NO.2所示，所述蒎烯合酶tPt1氨基酸序列如SEQ ID NO.3所示。在本发明的一种实施方式中，本发明提供的来自火炬松的蒎烯合酶基因Pt1由苏州金唯智公司合成，过表达来自火炬松的截短的tPt1，多拷贝位点整合过表达了酿酒酵母MVA通路的限制酶基因tHMG1、IDI和ERG20的突变体ERG20ww(F96W/N127W)，并通过敲除YPL062W和BTS1，以及融合表达ERG20ww和tPt1实现蒎烯的生产提高。
在本发明的一种实施方式中，所述ERG20的核苷酸序列如SEQ ID NO.7所示。
在本发明的一种实施方式中，所述ERG20ww的核苷酸序列如SEQ ID NO.4所示。
在本发明的一种实施方式中，所述tHMG1的氨基酸序列如SEQ ID NO.8所示。
在本发明的一种实施方式中，所述tHMG1的核苷酸序列如SEQ ID NO.9所示。
在本发明的一种实施方式中，所述IDI的氨基酸序列如SEQ ID NO.10所示。
在本发明的一种实施方式中，所述IDI的核苷酸序列如SEQ ID NO.11所示。
在本发明的一种实施方式中，所述YPL062W的氨基酸序列如SEQ ID NO.12所示。
在本发明的一种实施方式中，所述YPL062W的核苷酸序列如SEQ ID NO.13所示。
在本发明的一种实施方式中，所述BTS1的氨基酸序列如SEQ ID NO.6所示。
在本发明的一种实施方式中，所述BTS1的核苷酸序列如SEQ ID NO.5所示。
在本发明的一种实施方式中，以p424为表达载体。
在本发明的一种实施方式中，以S.cerevisiae w303、S.cerevisiae CEN.PK2-1C为宿主细胞。
本发明还提供了一种构建上述重组酿酒酵母的方法，包括以下步骤：
(1)化学合成蒎烯合酶基因，并将蒎烯合酶基因连接到质粒上，得到重组质粒；
(2)将编码核苷酸序列如SEQ ID NO.16所示的ERG20ww的基因，编码核苷酸序列如SEQ ID NO.9所示的tHMG1的基因和编码核苷酸序列如SEQ ID NO.11所示的IDI的基因通过同源重组的方法整合到酿酒酵母多拷贝整合位点德尔塔位点如SEQ ID NO.14所示，同时通过同源重组的方法将酿酒酵母染色体上的编码核苷酸序列如SEQ ID NO.13所示YPL062W的基因和编码核苷酸序列如SEQ ID NO.15所示BTS1的基因的敲除，得到表达宿主；
(3)将步骤(1)得到重组质粒转入步骤(2)得到的表达宿主中，构建得到重组酿酒酵母。
在本发明的一种实施方式中，所述蒎烯合酶来自火炬松，其为蒎烯合酶基因Pt的截短序列；所述蒎烯合酶tPt1核苷酸序列如SEQ ID NO.2所示，所述蒎烯合酶tPt1氨基酸序列如SEQ ID NO.3所示。
本发明还提供了一种合成蒎烯的方法，所述方法为，将上述重组酿酒酵母添加至培养基中培养，得到种子液，将种子液接种至培养基中，发酵制备蒎烯。
在本发明的一种实施方式中，所述方法包括以下步骤：
(1)挑取转化成功的上述重组菌单菌落接种到装有10mL培养基的50mL的摇瓶中，在30℃，200rpm摇床中培养24h，得到种子液；
(2)将步骤(1)得到的种子液以1％(v/v)的接种量，接种至装有50mL培养基的250mL的摇瓶中，在30℃，200rpm条件下发酵12h后，添加十二烷，接着摇瓶发酵72h；
(3)测定发酵的生物量并通过气相岛津GC2014检测上层十二烷中的蒎烯浓度。
在本发明的一种实施方式中，所述培养基为：SD-ura-leu-his液体培养基，或SD-ura–his液体培养基，或SD-ura液体培养基，或MM液体培养基。
本发明还提供了上述重组酿酒酵母，或上述方法在制备含有蒎烯的产品中的应用。
有益效果
本发明取得的有益效果如下：本发明表达来自火炬松的蒎烯合酶基因，并通过MVA通路的ERG20ww，tHMG1和IDI强化，YPL062W和BTS1的敲除，融合表达在S.cerevisiaeCEN.PK2-1C中实现更高的蒎烯生产及最高的单位菌体产量。其中YPL062W和BTS1的双敲除在YPD培养基中表现出很高的生物量生长，但是在蒎烯发酵过程中蒎烯最终的产量只增加了15％，在加入融合表达的蒎烯合酶表达质粒后，产量具有一个明显的提高，增加到7.09mg/L，单位菌体产量为1.15mg/L*OD。相对于仅仅敲除YPL062W和BTS1产率提高了11倍；相对于仅仅融合表达的2.2倍。
附图说明
图1：发酵十二烷层气相分析结果显示蒎烯出峰时间。
图2：发酵样品气相质谱分析检测蒎烯检测谱图。
图3：蒎烯标准品气相质谱分析检测谱图。
图4：ERG20ww，tHMG1和IDI整合到基因组的表达元件的构建。
图5：YPL062W和BTS1的敲除元件的构建。
图6：YPL062w和BTS1单敲除和双敲除在YPD培养基中的生长情况。
图7：蒎烯合成工程菌株的发酵生产蒎烯的结果图。
具体实施方式
下述实施例中所涉及的S.cerevisiae CEN.PK2-1C公开于论文：Identificationand characterization from Candida glycerinogenes of hexose transportershaving high efficiency at high glucose concentrations中。
下述实施例中所涉及的培养基如下：
YPD培养基：酵母膏10g/L，蛋白胨20g/L，葡萄糖20g/L。
SD-ura-leu-his固体培养基：YNB：6.7g/L，葡萄糖20g/L，ura20mg/L,leu60mg/L,his20mg/L,琼脂20g/L。
SD-ura-leu-his液体培养基：YNB：6.7g/L，葡萄糖20g/L，ura20mg/L,leu60mg/L,his20mg/L。
SD-ura-his固体培养基：YNB：6.7g/L，葡萄糖20g/L，ura20mg/L,his20mg/L,琼脂20g/L。
SD-ura固体培养基：YNB：6.7g/L，葡萄糖20g/L，ura20mg/L，琼脂20g/L。
MM固体培养基：YNB：6.7g/L，葡萄糖20g/L，琼脂20g/L。
SD-ura–his液体培养基：YNB：6.7g/L，葡萄糖20g/L，ura20mg/L，his20mg/L。
SD-ura液体培养基：YNB：6.7g/L，葡萄糖20g/L，ura20mg/L。
MM液体培养基：YNB：6.7g/L，葡萄糖20g/L。
下述实施例中所涉及的检测方法如下：
蒎烯含量的检测方法：
取上层十二烷层加入无水硫酸钠除水4-5h，过有机膜后进行气相质谱分析。气相分析条件：色谱条件为HP-5ms毛细管色谱柱(Agilent公司)，进样量1μL；柱温起始温度70℃，保持3min；然后以5℃·min-1升至100℃，保持3min；最后以40℃·min-1升至250℃，保持5min；进样口温度与色谱-质谱接口温度均为260℃。
下述实施例中所涉及的引物序列如表1和表2所示：
表1引物
Figure BDA0002827380040000041
Figure BDA0002827380040000051
表2引物
Figure BDA0002827380040000052
Figure BDA0002827380040000061
实施例1：来自火炬松的蒎烯合酶在S.cerevisiae CEN.PK2-1C中表达
具体步骤如下：
(1)化学合成编码核苷酸序列如SEQ ID NO.2所示蒎烯合酶基因tPt1；
(2)将蒎烯合酶基因tPt1通过BamHⅠ酶酶切连接到p424-TEF1质粒上构建p424-tPt1重组质粒，其中p424的构建方法：p424的质粒是由p414质粒更换了复制序列CEN/ARS为2μ而来，p414质粒是由Dominik Mumberg等人在商业质粒pRS416的基础上添加TEF1启动子构建而来(Yeast vectors for the controlled expression of heterologous proteinsin different genetic backgrounds)。
将重组质粒利用醋酸锂转化法转化到S.cerevisiae CEN.PK2-1C，并涂布于SD-ura-leu-his固体培养基进行筛选，挑取单菌落进行验证，挑取验证阳性转化子转接含有SD-ura-leu-his液体培养基的50mL摇瓶培养，甘油管保存菌株。
(3)将步骤(2)得到的菌株接种至10mL种子培养基的50mL的摇瓶中，在30℃，200rpm摇床中培养24h，得到种子液；将得到的种子液以1％(v/v)的接种量，接种至装有50mL发酵培养基的250mL的摇瓶中，在30℃，200rpm条件下发酵12h后，添加十二烷，接着摇瓶发酵120h，通过气相岛津GC2014检测上层十二烷中的蒎烯浓度。并通过质谱分析确定蒎烯的成功合成，气相质谱确定蒎烯合酶在酿酒酵母菌中表达，合成了蒎烯，如图2-3所示。
实施例2：重组质粒的构建：
具体步骤如下：
1、p424-ERG20ww-(GS)*4-tPt1的构建方法：
在构建的p424-tPt1的基础上，先用BamHⅠ单切p424-tPt1，通过设计引物(ERG20ww-(GS)4-tPt1-F，ERG20ww-(GS)4-tPt1-R)克隆ERG20ww，将GS*4的Linker连接到ERG20ww和tPt1中间，用同源重组酶连接到用BamHⅠ单切好的p424-tPt1质粒上，转化大肠杆菌感受态JM109，生长过夜后，挑取生长菌株做菌落PCR，验证正确的菌株提取质粒做进一步酶切验证并测序，确定p424-ERG20ww-(GS)*4-tPt1构建成功。
2、p424-tPt1-(GS)*4-ERG20ww的构建方法：
在构建的p424-tPt1的基础上，先用NotⅠ单切p424-tPt1，通过设计引物(tPt1-(GS)4-ERG20ww-F，tPt1-(GS)4-ERG20ww-R)克隆ERG20ww，将GS*4的Linker连接到tPt1和ERG20ww中间，用同源重组酶连接到用NotⅠ单切好的p424-tPt1质粒上，转化大肠杆菌感受态JM109，生长过夜后，挑取生长菌株做菌落PCR，验证正确的菌株提取质粒做进一步酶切验证并测序，确定p424-tPt1-(GS)*4-ERG20ww构建成功。
3、p424-tPt1的构建方法：
设计引物(p414-tPt1-F，p414-Pt1-R)从合成的质粒pUC57-Pt1上克隆下tPt1，用同源重组酶连接到用BamHⅠ单切的p424质粒上，转化大肠杆菌感受态JM109，生长过夜后，挑取生长菌株做菌落PCR，验证正确的菌株提取质粒做进一步酶切验证并测序，确定p424-tPt1构建成功。
实施例3：宿主细胞的构建：
将S.cerevisiae CEN.PK2-1C在YPD培养基中培养至稳定期，按照参考文献《酵母遗传学技术手册》进行提取基因组，然后设计tHMG1引物(tHMG–F，tHMG–R)从基因组上PCR扩增下来，进行1％琼脂糖凝胶电泳验证，纯化后连接到BamHⅠ单切的p424质粒上。构建成功p424-tHMG1。设计引物(pGAP-F，pGAP-R；IDI-F，IDI-R)从基因组上克隆出pGAP和IDI，利用融合PCR连接pGAP-IDI；纯化后连接到KpnⅠ的单切的p424-tHMG1上，构建成功p424-TEF1-tHMG1-tCYC1-pGAP-IDI；接着设计引物(德尔塔1-F，德尔塔1-R；p424-t-i-F，p424-t-i-R；tI-leu-F，tI-leu-R；德尔塔2-F，德尔塔2-R)其中LEU来自于商业质粒p181质粒，从基因组上克隆下德尔塔同源臂，同源序列如SEQ ID NO.14所示，克隆下TEF1-tHMG1-tCYC1-pGAP-IDI表达原件和LEU筛选标记，利用醋酸锂转化法将纯化后的片段转化S.cerevisiaeCEN.PK2-1C菌株，涂布SD-ura-trp-his固体培养基，生长3天后筛选验证构建成功CEN，tI菌株，整合元件如图4所示。
在以上构建的菌株的基础上构建过表达ERG20ww和另一个拷贝的IDI的菌株，设计引物(NTS1-2a-F，NTS1-2a-R；pTDH3-F，pTDH3-R；ERG20ww-F，ERG20ww-R；tADH-F，tADH-R；pPGK1-F，pPGK1-R；IDI-F，IDI-R；tPGK-F，tPGK-R；HIS-F，HIS-R；NTS1-2b-F，NTS1-2b-R)从基因组上克隆出同源片段，pTDH3，tADH，pPGK1，IDI，tPGK，以及突变好的ERG20ww，HIS来自于商业质粒pY13，采用同源序列如SEQ ID NO.15所示；进行1％琼脂糖凝胶电泳验证；利用醋酸锂转化法将纯化后的片段转化CEN，tI菌株，涂布SD-ura-trp固体培养基，生长3天后筛选验证构建成功CEN，tI，Ew-I菌株，。
通过对S.cerevisiae CEN.PK2-1C基因组分析查找，找到YPL062W和BTS1基因的上下游同源臂各500bp如表2所示，设计引物(YPL-UP-F，YPL-UP-R；KanMX-F，KanMX-R；YPL-DOWN-F，YPL-DOWN-R；BTS1-UP-F，BTS1-UP-R；LOXP-URA3-F，LOXP-URA3-R；BTS1-DOWN-F，BTS1-DOWN-RPCR)从基因组克隆获得，KanMX和URA3的分别来自于质粒pUG6和pYX212。进行1％琼脂糖凝胶电泳验证。利用醋酸锂转化法将纯化后的片段转化CEN，tI菌株；CEN，tI，Ew-I菌株，分别涂布YPD培养基添加200mg/L的G418固体培养基，SD-trp-his固体培养基，SD-trp固体培养基，生长3天后筛选验证构建成功，CEN.tI,ΔBTS1菌株；CEN.Δypl，tI菌株；CEN.Δypl，tI,ΔBTS1菌株；Δypl，ΔBTS1，tI,Ew-I菌株敲除元件如图5所示，菌株的生长状况如图6所示。
实施例4：重组菌SCPS的构建
将实施例2中构建好的质粒p424-tPt1利用醋酸锂转化法转化到实施例3中构建的宿主菌CEN，tI菌株；CEN，tI，Ew-I菌株；Δypl，ΔBTS1，tI,Ew-I菌株分别涂布SD-ura–his、SD-ura、MM固体培养基生长3天，筛选验证构建成重组菌SCPS-1、SCPS-2、SCPS-3。
将实施例2中构建好的质粒p424-ERG20ww-(GS)*4-tPt1和p424-tPt1-(GS)*4-ERG20ww利用醋酸锂转化法转化到实施例3中构建的宿主菌CEN，tI，Ew-I涂布SD-ura固体培养基生长3天，筛选验证构建成重组菌SCPS-4、SCPS-5。
将实施例2中构建好的质粒p424-ERG20ww-(GS)*4-tPt1利用醋酸锂转化法转化到实施例3中构建的宿主菌Δypl，ΔBTS1，tI,Ew-I涂布MM固体培养基生长3天，筛选验证构建成重组菌SCPS-6。
实施例5：发酵生产蒎烯
具体步骤如下：
将实施例4得到的菌株SCPS-1～SCPS-6接种至10mL种子培养基的50mL的摇瓶中，在30℃，200rpm摇床中培养24h，得到种子液；将得到的种子液以1％(v/v)的接种量，接种至装有50mL发酵培养基的250mL的摇瓶中，在30℃，200rpm条件下发酵12h后，添加十二烷，接着摇瓶发酵120h，测定发酵过程生物量的变化(如表3所示)，发酵结束后，取上层十二烷层1mL10000r/min离心10分钟，加入无水硫酸钠除水4h，然后通过气相岛津GC2014检测上层十二烷中的蒎烯出峰时间如图1所示，测定蒎烯产量如表4、如图7所示；通过菌株的产量与生物量的比值计算产率，结果如表5所示。
表3菌株生物量
菌株 生物量(OD600)
SCPS-1 7.12
SCPS-2 6.32
SCPS-3 6.57
SCPS-4 5.82
SCPS-5 6.38
SCPS-6 6.16
表4蒎烯的产量
菌株 产量(mg/L)
SCPS-1 0.283
SCPS-2 0.507
SCPS-3 0.583
SCPS-4 0.83
SCPS-5 2.18
SCPS-6 7.088
表5蒎烯的产率
菌株 产率(mg/L*OD)
SCPS-1 0.04
SCPS-2 0.08
SCPS-3 0.09
SCPS-4 0.14
SCPS-5 0.34
SCPS-6 1.15
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种重组酿酒酵母及其应用
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aatgataatg aaaataagta tttacctgat ttggcttcgc attccgacac cgccaccaat 960
ttacatgacg aattgttata tataatagac cacttatccg aattgtga 1008
<210> 6
<211> 335
<212> PRT
<213> 人工序列
<400> 6
Met Glu Ala Lys Ile Asp Glu Leu Ile Asn Asn Asp Pro Val Trp Ser
1 5 10 15
Ser Gln Asn Glu Ser Leu Ile Ser Lys Pro Tyr Asn His Ile Leu Leu
20 25 30
Lys Pro Gly Lys Asn Phe Arg Leu Asn Leu Ile Val Gln Ile Asn Arg
35 40 45
Val Met Asn Leu Pro Lys Asp Gln Leu Ala Ile Val Ser Gln Ile Val
50 55 60
Glu Leu Leu His Asn Ser Ser Leu Leu Ile Asp Asp Ile Glu Asp Asn
65 70 75 80
Ala Pro Leu Arg Arg Gly Gln Thr Thr Ser His Leu Ile Phe Gly Val
85 90 95
Pro Ser Thr Ile Asn Thr Ala Asn Tyr Met Tyr Phe Arg Ala Met Gln
100 105 110
Leu Val Ser Gln Leu Thr Thr Lys Glu Pro Leu Tyr His Asn Leu Ile
115 120 125
Thr Ile Phe Asn Glu Glu Leu Ile Asn Leu His Arg Gly Gln Gly Leu
130 135 140
Asp Ile Tyr Trp Arg Asp Phe Leu Pro Glu Ile Ile Pro Thr Gln Glu
145 150 155 160
Met Tyr Leu Asn Met Val Met Asn Lys Thr Gly Gly Leu Phe Arg Leu
165 170 175
Thr Leu Arg Leu Met Glu Ala Leu Ser Pro Ser Ser His His Gly His
180 185 190
Ser Leu Val Pro Phe Ile Asn Leu Leu Gly Ile Ile Tyr Gln Ile Arg
195 200 205
Asp Asp Tyr Leu Asn Leu Lys Asp Phe Gln Met Ser Ser Glu Lys Gly
210 215 220
Phe Ala Glu Asp Ile Thr Glu Gly Lys Leu Ser Phe Pro Ile Val His
225 230 235 240
Ala Leu Asn Phe Thr Lys Thr Lys Gly Gln Thr Glu Gln His Asn Glu
245 250 255
Ile Leu Arg Ile Leu Leu Leu Arg Thr Ser Asp Lys Asp Ile Lys Leu
260 265 270
Lys Leu Ile Gln Ile Leu Glu Phe Asp Thr Asn Ser Leu Ala Tyr Thr
275 280 285
Lys Asn Phe Ile Asn Gln Leu Val Asn Met Ile Lys Asn Asp Asn Glu
290 295 300
Asn Lys Tyr Leu Pro Asp Leu Ala Ser His Ser Asp Thr Ala Thr Asn
305 310 315 320
Leu His Asp Glu Leu Leu Tyr Ile Ile Asp His Leu Ser Glu Leu
325 330 335
<210> 7
<211> 1059
<212> DNA
<213> 人工序列
<400> 7
atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60
gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120
gcccactcat tgaactacaa cactccaggc ggtaagttaa atagaggttt gtccgttgtg 180
gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240
aaggttgcta ttctaggttg gtgcattgag ttgttgcagg cttacttctt ggtcgccgat 300
gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360
gttggggaaa ttgccatcaa tgacgcattc atgttagagg ctgctatcta caagcttttg 420
aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaagtc 480
accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540
gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600
tattctttct acttgcctgt cgcattggct atgtacgttg ccggtatcac agatgaaaag 660
gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatatttcca aattcaagat 720
gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780
gataacaaat gttcttgggt aatcaacaag gcattagaac ttgcttccgc agaacaaaga 840
aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900
attttcaatg acttgaaaat cgaccagtta taccacgaat atgaagagtc tgttgccaag 960
gatttgaagg ccaagatctc ccaagtcgac gagtctcgtg gcttcaaagc cgacgtctta 1020
actgcgtttt tgaacaaggt ttacaagaga agtaaataa 1059
<210> 8
<211> 525
<212> PRT
<213> 人工序列
<400> 8
Met Asp Gln Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr Ala
1 5 10 15
Pro Val Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val Ile
20 25 30
Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser Ser
35 40 45
Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu Ser
50 55 60
Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu Ser
65 70 75 80
Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu Val
85 90 95
Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly Asp
100 105 110
Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser Ile Leu Ala
115 120 125
Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn Tyr Asp
130 135 140
Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr Met
145 150 155 160
Pro Leu Pro Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr Ser
165 170 175
Tyr His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser Ala
180 185 190
Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr Val
195 200 205
Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro Thr
210 215 220
Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu Glu Gly
225 230 235 240
Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala Arg
245 250 255
Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met Arg
260 265 270
Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser Lys
275 280 285
Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp Glu
290 295 300
Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys Lys
305 310 315 320
Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val Ala
325 330 335
Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys Ser Asp
340 345 350
Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu Val Gly Ser
355 360 365
Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn Leu
370 375 380
Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn Val
385 390 395 400
Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp Leu
405 410 415
Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly Gly
420 425 430
Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly Val
435 440 445
Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu Ala
450 455 460
Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu Cys Ala
465 470 475 480
Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His Asn Arg
485 490 495
Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr Asp Ile
500 505 510
Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser
515 520 525
<210> 9
<211> 1578
<212> DNA
<213> 人工序列
<400> 9
atggaccaat tggtgaaaac tgaagtcacc aagaagtctt ttactgctcc tgtacaaaag 60
gcttctacac cagttttaac caataaaaca gtcatttctg gatcgaaagt caaaagttta 120
tcatctgcgc aatcgagctc atcaggacct tcatcatcta gtgaggaaga tgattcccgc 180
gatattgaaa gcttggataa gaaaatacgt cctttagaag aattagaagc attattaagt 240
agtggaaata caaaacaatt gaagaacaaa gaggtcgctg ccttggttat tcacggtaag 300
ttacctttgt acgctttgga gaaaaaatta ggtgatacta cgagagcggt tgcggtacgt 360
aggaaggctc tttcaatttt ggcagaagct cctgtattag catctgatcg tttaccatat 420
aaaaattatg actacgaccg cgtatttggc gcttgttgtg aaaatgttat aggttacatg 480
cctttgcccg ttggtgttat aggccccttg gttatcgatg gtacatctta tcatatacca 540
atggcaacta cagagggttg tttggtagct tctgccatgc gtggctgtaa ggcaatcaat 600
gctggcggtg gtgcaacaac tgttttaact aaggatggta tgacaagagg cccagtagtc 660
cgtttcccaa ctttgaaaag atctggtgcc tgtaagatat ggttagactc agaagaggga 720
caaaacgcaa ttaaaaaagc ttttaactct acatcaagat ttgcacgtct gcaacatatt 780
caaacttgtc tagcaggaga tttactcttc atgagattta gaacaactac tggtgacgca 840
atgggtatga atatgatttc taaaggtgtc gaatactcat taaagcaaat ggtagaagag 900
tatggctggg aagatatgga ggttgtctcc gtttctggta actactgtac cgacaaaaaa 960
ccagctgcca tcaactggat cgaaggtcgt ggtaagagtg tcgtcgcaga agctactatt 1020
cctggtgatg ttgtcagaaa agtgttaaaa agtgatgttt ccgcattggt tgagttgaac 1080
attgctaaga atttggttgg atctgcaatg gctgggtctg ttggtggatt taacgcacat 1140
gcagctaatt tagtgacagc tgttttcttg gcattaggac aagatcctgc acaaaatgtt 1200
gaaagttcca actgtataac attgatgaaa gaagtggacg gtgatttgag aatttccgta 1260
tccatgccat ccatcgaagt aggtaccatc ggtggtggta ctgttctaga accacaaggt 1320
gccatgttgg acttattagg tgtaagaggc ccgcatgcta ccgctcctgg taccaacgca 1380
cgtcaattag caagaatagt tgcctgtgcc gtcttggcag gtgaattatc cttatgtgct 1440
gccctagcag ccggccattt ggttcaaagt catatgaccc acaacaggaa acctgctgaa 1500
ccaacaaaac ctaacaattt ggacgccact gatataaatc gtttgaaaga tgggtccgtc 1560
acctgcatta aatcctaa 1578
<210> 10
<211> 288
<212> PRT
<213> 人工序列
<400> 10
Met Thr Ala Asp Asn Asn Ser Met Pro His Gly Ala Val Ser Ser Tyr
1 5 10 15
Ala Lys Leu Val Gln Asn Gln Thr Pro Glu Asp Ile Leu Glu Glu Phe
20 25 30
Pro Glu Ile Ile Pro Leu Gln Gln Arg Pro Asn Thr Arg Ser Ser Glu
35 40 45
Thr Ser Asn Asp Glu Ser Gly Glu Thr Cys Phe Ser Gly His Asp Glu
50 55 60
Glu Gln Ile Lys Leu Met Asn Glu Asn Cys Ile Val Leu Asp Trp Asp
65 70 75 80
Asp Asn Ala Ile Gly Ala Gly Thr Lys Lys Val Cys His Leu Met Glu
85 90 95
Asn Ile Glu Lys Gly Leu Leu His Arg Ala Phe Ser Val Phe Ile Phe
100 105 110
Asn Glu Gln Gly Glu Leu Leu Leu Gln Gln Arg Ala Thr Glu Lys Ile
115 120 125
Thr Phe Pro Asp Leu Trp Thr Asn Thr Cys Cys Ser His Pro Leu Cys
130 135 140
Ile Asp Asp Glu Leu Gly Leu Lys Gly Lys Leu Asp Asp Lys Ile Lys
145 150 155 160
Gly Ala Ile Thr Ala Ala Val Arg Lys Leu Asp His Glu Leu Gly Ile
165 170 175
Pro Glu Asp Glu Thr Lys Thr Arg Gly Lys Phe His Phe Leu Asn Arg
180 185 190
Ile His Tyr Met Ala Pro Ser Asn Glu Pro Trp Gly Glu His Glu Ile
195 200 205
Asp Tyr Ile Leu Phe Tyr Lys Ile Asn Ala Lys Glu Asn Leu Thr Val
210 215 220
Asn Pro Asn Val Asn Glu Val Arg Asp Phe Lys Trp Val Ser Pro Asn
225 230 235 240
Asp Leu Lys Thr Met Phe Ala Asp Pro Ser Tyr Lys Phe Thr Pro Trp
245 250 255
Phe Lys Ile Ile Cys Glu Asn Tyr Leu Phe Asn Trp Trp Glu Gln Leu
260 265 270
Asp Asp Leu Ser Glu Val Glu Asn Asp Arg Gln Ile His Arg Met Leu
275 280 285
<210> 11
<211> 867
<212> DNA
<213> 人工序列
<400> 11
atgactgccg acaacaatag tatgccccat ggtgcagtat ctagttacgc caaattagtg 60
caaaaccaaa cacctgaaga cattttggaa gagtttcctg aaattattcc attacaacaa 120
agacctaata cccgatctag tgagacgtca aatgacgaaa gcggagaaac atgtttttct 180
ggtcatgatg aggagcaaat taagttaatg aatgaaaatt gtattgtttt ggattgggac 240
gataatgcta ttggtgccgg taccaagaaa gtttgtcatt taatggaaaa tattgaaaag 300
ggtttactac atcgtgcatt ctccgtcttt attttcaatg aacaaggtga attactttta 360
caacaaagag ccactgaaaa aataactttc cctgatcttt ggactaacac atgctgctct 420
catccactat gtattgatga cgaattaggt ttgaagggta agctagacga taagattaag 480
ggcgctatta ctgcggcggt gagaaaacta gatcatgaat taggtattcc agaagatgaa 540
actaagacaa ggggtaagtt tcacttttta aacagaatcc attacatggc accaagcaat 600
gaaccatggg gtgaacatga aattgattac atcctatttt ataagatcaa cgctaaagaa 660
aacttgactg tcaacccaaa cgtcaatgaa gttagagact tcaaatgggt ttcaccaaat 720
gatttgaaaa ctatgtttgc tgacccaagt tacaagttta cgccttggtt taagattatt 780
tgcgagaatt acttattcaa ctggtgggag caattagatg acctttctga agtggaaaat 840
gacaggcaaa ttcatagaat gctataa 867
<210> 12
<211> 134
<212> PRT
<213> 人工序列
<400> 12
Met Ile Glu Leu Asp Tyr Val Lys Gly Glu Asp Thr Ile Val Glu Ala
1 5 10 15
Thr Ser Thr Ser Pro Trp Leu Met Arg Ser Pro Leu Ala Arg Ala Ala
20 25 30
Glu Lys Arg Gly Ser Gly Leu Phe Phe Asp Ile Asn Glu Gly His Gly
35 40 45
Gln His Arg Asp Val Ile Val Ala Tyr Gly Val Ser Lys Pro Lys Arg
50 55 60
Arg Ser Pro His Pro His Gly Asn Lys Ala Ala Asp Lys Arg Lys Thr
65 70 75 80
Thr Glu Lys Glu Pro Glu Arg Lys Lys Arg Val Gly Ala Pro Arg Thr
85 90 95
Cys Lys Lys Ile Cys Ile Gln Leu Leu Tyr Arg Phe Asn Phe Thr Val
100 105 110
Leu Gly Ile Gly Asn Val Cys Asn Ile Asp Leu Leu Leu Gly Thr Val
115 120 125
Ser Ala Thr Asn Ala Ile
130
<210> 13
<211> 405
<212> DNA
<213> 人工序列
<400> 13
atgatagaat tggattatgt aaaaggtgaa gataccattg tagaagcaac cagcacgtcg 60
ccgtggctga tgaggtctcc tcttgcccgg gccgcagaaa agaggggcag tggcctgttt 120
ttcgacataa atgaggggca tggccagcac cgagacgtca ttgttgcata tggcgtatcc 180
aagccgaaac ggcgctcgcc tcatccccac gggaataagg cagccgacaa aagaaaaacg 240
accgaaaagg aaccagaaag aaaaaagagg gtgggcgcgc cgcggacgtg taaaaagata 300
tgcatccagc ttctatatcg ctttaacttt accgttttgg gcatcgggaa cgtatgtaac 360
attgatctcc tcttgggaac ggtgagtgca acgaatgcga tatag 405
<210> 14
<211> 688
<212> DNA
<213> 人工序列
<400> 14
ggaacagctg atgaagcagg tgttgttgtc tgttgagagt tagccttagt ggaagccttc 60
tcacattctt ctgttttgga agctgaaacg tctaacggat cttgatttgt gtggacttcc 120
ttagaagtaa ccgaagcaca ggcgctacca tgagaaatgg gtgaatgttg agataattgt 180
tgggattcca ttgttgataa aggctataat attaggtata cagaatatac tagaagttct 240
cctcgaggat ataggaatcc tcaaaatgga atctatattt ctacatacta atattacgat 300
tattcctcat tccgttttat atgtttatat tcattgatcc tattacatta tcaatccttg 360
cgtttcagct tccactaatt tagatgacta tttctcatca tttgcgtcat cttctaacac 420
cgtatatgat aatatactag taatgtaaat actagttagt agatgatagt tgatttctat 480
tccaacacat ctactaacta gtatttacgt tactagtata ttatcatata cggtgttaga 540
agatgacgca aatgatgaga aatagtcatc taaattagtg gaagctgaaa cgcaaggatt 600
gataatgtaa taggatcaat gaatattaac atataaaacg atgataataa tatttataga 660
attgtgtaga attgcagatt ccctttta 688
<210> 15
<211> 875
<212> DNA
<213> 人工序列
<400> 15
ttggtctttt tattttttat tttttttttc gttgcaaaga tgggttgaaa gagaagggct 60
ttcacaaagc ttcccgagcg tgaaaggatt tgcccggaca gtttgcttca tggagcagtt 120
ttttccgcac catcagagcg gcaaacatga gtgcttgtat aagtttagag aattgagaaa 180
agctcatttc ctatagttaa caggacatgc ctttgatatg aaaaaaaata ctacgaacta 240
cgattttacc aagaaagatg taagagacaa gtgaacagtg aacagtgaac agtggggaca 300
tttttttttt aagtaaatgg cagtttctag ggaatgatga tggcaagttc cagagaggca 360
gcgtaaaagg atgaggctac tgggaagaag aaagaggaaa agtgcaagat gaatagccag 420
tgcaatatat acatgtatac ttaacagata tggaatggtt ggcgaagtaa atttttggcg 480
acgcggcgac gcggcgacgc ggtatgcgga gttgtaagat gtactacgat catatagtgt 540
aatgcggcaa aatgttagtg caggaaagcg ggaaggaata agaagcaact aaacgagggt 600
gtagaaaaag acgaagagga aagtattaac cgtagaagag agggaaatgg agggaagaga 660
acaaaaccgg agtgtttttt tttttttttt tgtaggaata tcggaggaga atattgtttg 720
tacatatcaa gtagtagcaa cccaatgagc ataatggagt gcttaactct tcagaagaag 780
agtgcagctg gatagtgcga atttttctga atcgaatggt aggttagtta tgggatttag 840
cataggaagc caagaaacta gaaaaaagaa ataaa 875

Claims (10)

1.一种重组酿酒酵母，其特征在于，强化表达了tHMG1，ERG20ww和IDI基因，敲除了YPL062W和BTS1基因，并融合表达了蒎烯合酶tPt1和ERG20ww；
所述ERG20ww是在氨基酸序列如SEQ ID NO.1所示的ERG20的基础上，将第96位的苯丙氨酸和第127位的天冬氨酸突变得到的。
2.如权利要求1所述的一种重组酿酒酵母，其特征在于，所述ERG20ww是在氨基酸序列如SEQ ID NO.1所示的ERG20的基础上，将第96位的苯丙氨酸突变为色氨酸，并将第127位的天冬氨酸突变为色氨酸得到的。
3.如权利要求2所述的一种重组酿酒酵母，其特征在于，所述融合表达是将蒎烯合酶tPt1和ERG20ww通过linker融合连接后表达，所述linker序列为GSGSGSGS。
4.如权利要求1-3任一所述的一种重组酿酒酵母，其特征在于，所述蒎烯合酶tPt1的氨基酸序列如SEQ ID NO.2所示；优选的，所述tHMG1的氨基酸序列如SEQ ID NO.8所示；优选的，所述IDI的氨基酸序列如SEQ ID NO.10所示。
5.如权利要求1-4任一所述的一种重组酿酒酵母，其特征在于，所述YPL062W的氨基酸序列如SEQ ID NO.12所示；优选的，所述BTS1的氨基酸序列如SEQ ID NO.6所示。
6.如权利要求1-5任一所述的一种重组酿酒酵母，其特征在于，以S.cerevisiae w303或S.cerevisiae CEN.PK2-1C为宿主细胞。
7.一种构建权利要求1-6任一所述的重组酿酒酵母的方法，其特征在于，包括以下步骤：
(1)化学合成蒎烯合酶基因，并将蒎烯合酶基因连接到质粒上，得到重组质粒；
(2)将编码核苷酸序列如SEQ ID NO.4所示的ERG20ww的基因，编码核苷酸序列如SEQID NO.9所示的tHMG1的基因和编码核苷酸序列如SEQ ID NO.11所示的IDI的基因通过同源重组的方法整合到酿酒酵母多拷贝整合位点德尔塔位点如SEQ ID NO.14所示，同时通过同源重组的方法将酿酒酵母染色体上的编码核苷酸序列如SEQ ID NO.13所示YPL062W的基因和编码核苷酸序列如SEQ ID NO.5所示BTS1的基因的敲除，得到表达宿主；
(3)将步骤(1)得到重组质粒转入步骤(2)得到的表达宿主中，构建得到重组酿酒酵母。
8.一种合成蒎烯的方法，其特征在于，将权利要求1-6任一所述的重组酿酒酵母添加至培养基中培养，得到种子液，再将种子液接种至培养基中，发酵制备蒎烯。
9.如权利要求8所述的方法，其特征在于，所述发酵是在30℃，200rpm条件下发酵10-12h后，添加十二烷，接着摇瓶发酵70-120h。
10.权利要求1-6任一所述的重组酿酒酵母，或权利要求8或9所述的方法在制备含有蒎烯的产品中的应用。
CN202011451952.7A 2020-12-09 2020-12-09 一种重组酿酒酵母及其应用 Pending CN112553099A (zh)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
孙明雪等: "调控酿酒酵母类异戊二烯合成途径强化芳樟醇合成", 《生物工程学报》 *
贠小芸: "CRISPR/Cas9改造酿酒酵母萜类合成通路以提高α-香树脂醇的产量", 《硕士电子期刊》 *
郭睿等: "产青蒿二烯的人工酵母细胞的构建及发酵优化", 《化工学报》 *
陈天华等: "产蒎烯人工酵母细胞的构建", 《化工学报》 *
陈艳: "高产番茄红素酿酒酵母的设计构建与发酵过程优化", 《博士电子期刊》 *

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