CN116286421A - 一种用于生产麦角硫因的毕赤酵母菌株及其构建方法和应用 - Google Patents
一种用于生产麦角硫因的毕赤酵母菌株及其构建方法和应用 Download PDFInfo
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Abstract
本发明涉及麦角硫因合成技术领域,具体公开了一种用于生产麦角硫因的毕赤酵母菌株及其构建方法和应用。所述毕赤酵母菌株同时过表达egt1基因、egt2基因和XP_002490234基因。本发明首次在毕赤酵母中表达麦角硫因合成途径基因egt1和egt2,并同时过表达XP_002490234基因,实现麦角硫因的高效合成,并且毕赤酵母菌株可以高密度培养,调节氧化/过氧化状态,解决了毕赤酵母细胞氧化损伤削弱麦角硫因产量的问题。本发明在毕赤酵母GS115基础上转化egt1基因、egt2基因,同时过表达XP_002490234.1基因后,毕赤酵母菌株的麦角硫因产率达782mg/L,具备工业生产前景。
Description
技术领域
本发明涉及麦角硫因合成技术领域,尤其是一种用于生产麦角硫因的毕赤酵母菌株及其构建方法和应用。
背景技术
麦角硫因是一种硫醇化合物,又名疏基组氨酸三甲基内盐,是一种由组氨酸衍生而来的天然氨基酸衍生物,麦角硫因是一种的天然抗氧化剂,安全、无毒、高效,具有清除自由基、解毒、修复皮肤氧化损伤、调节免疫等多种生理功能,被广泛应用于食品、医药以及化妆品等领域,尤其是在美妆行业具有巨大需求。因此,麦角硫因的绿色制造具有广阔的市场前景。
利用微生物进行基因工程设计是量产各类生化产品的有效方法,目前已有利用大肠杆菌、酿酒酵母基因工程改造来生产麦角硫因的报道,均通过过表达麦角硫因合成途径基因。比如在大肠杆菌BL21(DE3)构建了两种不同来源的麦角硫因合成途径,通过异源表达耻垢分枝杆菌来源的麦角硫因合成基因簇egtBCDE,实现麦角硫因在大肠杆菌中的合成。但是大肠杆菌和酿酒酵母是较早开发用于外源基因表达的宿主系统,具有培养密度有限,分泌效率低等局限性。
迄今为止,尚无利用巴斯德毕赤酵母(Pichia pastoris,以下简称毕赤酵母)基因工程实现麦角硫因合成的报道,而毕赤酵母是重要的工业宿主菌,是一类能够利用甲醇作为唯一碳源和能源的酵母菌。1969年Koichi Ogata等人首次发现其利用甲醇为唯一碳源和能源生长(Ogata,et a1.1969),此后,广泛用于生产蛋白和药物抗体,如Cregg等人首次报道了用甲醇营养型酵母表达乙型肝炎表面抗原。但是毕赤酵母高密度发酵培养条件下,存在氧化损伤胁迫,甲醇代谢中产生的活性氧物质(ROS)会攻击胞内生物分子,造成细胞损伤,此外过量表达蛋白需要消耗大量资源,比如NADPH可用性降低,这对外源蛋白表达和酶催化反应造成不利影响。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种用于生产麦角硫因的毕赤酵母菌株及其构建方法和应用。本发明首次在毕赤酵母中表达麦角硫因合成途径基因egt1和egt2,并同时过表达毕赤酵母GS115来源的XP_002490234基因,实现麦角硫因的高效合成,并且获得的毕赤酵母菌株可以实现高密度培养,调节氧化/过氧化状态,解决了毕赤酵母细胞氧化损伤削弱麦角硫因产量的问题。
为实现上述目的,本发明采取的技术方案为:
第一目的,本发明提供了一种用于生产麦角硫因的毕赤酵母菌株,所述毕赤酵母菌株同时过表达egt1基因、egt2基因和XP_002490234基因。
本发明在毕赤酵母中引入麦角硫因合成途径,过量表达基因egt1和egt2,解决大肠杆菌和酿酒酵母细胞密度较低问题,并且同时过表达XP_002490234基因,可以调节氧化/过氧化状态,解决毕赤酵母细胞氧化损伤削弱麦角硫因产量问题。
本发明的毕赤酵母菌株同时过表达egt1基因、egt2基因和XP_002490234基因,使得制备的毕赤酵母菌株可以实现高密度培养,并且降低了毕赤酵母菌株高密度培养时细胞氧化损伤,从而有效提高生产麦角硫因的产量(最高可达到782mg/L),具备工业生产前景。
作为本发明所述毕赤酵母菌株的优选实施方式,所述egt1基因和egt2基因的来源于真菌粗糙脉胞菌(Neurospora crassa)、粟酒裂殖酵母(Schizosaccharomyces pombe)、皱纹土霉(Geotricum rugosum)、星状诺卡氏菌(Nocardia asteroides)和链霉菌(Streptomyces)中的一种。
作为本发明所述毕赤酵母菌株的优选实施方式,所述egt1基因和egt2基因的来源于真菌粗糙脉胞菌(Neurospora crassa)。
作为本发明所述毕赤酵母菌株的优选实施方式,所述egt1基因的氨基酸序列如SEQ ID NO:1所示;所述egt2基因的氨基酸序列如SEQ ID NO:2所示。
作为本发明所述毕赤酵母菌株的优选实施方式,所述毕赤酵母菌株为毕赤酵母GS115及其衍生菌株。
毕赤酵母出发菌株P.pastoris GS115不合成麦角硫因,但转化egt1基因、egt2基因后获得的菌株(P.pastoris GS115/pPIC3.5k-egt1-egt2)的麦角硫因达到536mg/L,同时过表达XP_002490234.1基因后,毕赤酵母菌株(P.pastoris GS115/pPIC3.5k-egt1-egt2-XP_002490234.1)的麦角硫因产率达782mg/L,具备工业生产前景。
第二目的,本发明提供了上述毕赤酵母菌株的构建方法,将过表达egt1基因、egt2基因和XP_002490234基因的表达质粒转化至毕赤酵母GS115进行诱导性表达或者组成性表达,获得用于生产麦角硫因的毕赤酵母菌株。
优选地,所述诱导性表达的具体条件为当毕赤酵母菌株的OD600达到50后,开始流加甲醇诱导,甲醇浓度通过气相色谱检测,使得甲醇浓度控制在0.5%以下。
作为本发明所述毕赤酵母菌株的构建方法的优选实施方式,所述egt1基因和egt2基因的来源于真菌粗糙脉胞菌(Neurospora crassa)。所述egt1基因和egt2基因还可以来源于粟酒裂殖酵母(Schizosaccharomyces pombe)、皱纹土霉(Geotricum rugosum)、星状诺卡氏菌(Nocardia asteroides)和链霉菌(Streptomyces)中的一种。
作为本发明所述毕赤酵母菌株的构建方法的优选实施方式,所述表达质粒包括pPIC3.5k或pGAPZ。本发明限定的表达质粒不仅仅包括pPIC3.5k或pGAPZ,包括了本领域常见的表达质粒。
第三目的,本发明提供了上述毕赤酵母菌株在强化麦角硫因合成中的应用。
本发明利用改造的毕赤酵母菌株代谢工程可以强化麦角硫因的合成,通过调节氧化/过氧化状态,降低细胞氧化损伤,有利于提高生产麦角硫因的含量;同时,本发明改造后的毕赤酵母菌株细胞培养的密度较高,分泌麦角硫因的效率也较高。
第四目的,本发明提供了上述毕赤酵母菌株在提高麦角硫因含量中的应用。
经过基因改造后的毕赤酵母菌株获得的麦角硫因总量较高,说明经过基因(egt1基因、egt2基因和XP_002490234基因)过表达,可以提高麦角硫因的总含量。
第五目的,本发明提供了上述一种提高麦角硫因含量的制备方法,采用上述毕赤酵母菌株作为生产菌株。采用本发明改造后的毕赤酵母菌株可以作为生产菌株,有效提高麦角硫因含量的生产。
与现有技术相比,本发明具有以下有益效果:
一种用于生产麦角硫因的毕赤酵母菌株及其构建方法和应用。本发明首次在毕赤酵母中表达麦角硫因合成途径基因egt1和egt2,并同时过表达毕赤酵母GS115来源的XP_002490234基因,实现麦角硫因的高效合成,并且获得的毕赤酵母菌株可以实现高密度培养,调节氧化/过氧化状态,解决了毕赤酵母细胞氧化损伤削弱麦角硫因产量的问题。本发明在毕赤酵母GS115基础上转化egt1基因、egt2基因后获得的菌株(P.pastoris GS115/pPIC3.5k-egt1-egt2)的麦角硫因达到536mg/L,同时过表达XP_002490234.1基因后,毕赤酵母菌株(P.pastoris GS115/pPIC3.5k-egt1-egt2-XP_002490234.1)的麦角硫因产率达782mg/L,具备工业生产前景。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。
在以下实施例中,所使用的实验方法如无特殊说明,均为常规方法,所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
如本文所使用的术语“表达”根据本领域普通技术人员所理解的其普通和常规含义使用,并且在没有限制的情况下用于指源自本技术的核酸片段的有义(mRNA)或反义RNA的转录和稳定积累。“过表达”是指在转基因或重组生物体中产生基因产物,其超过正常或非转化生物体中的产生水平。
“转化”根据本领域普通技术人员所理解的其普通和常规含义使用,并且在没有限制的情况下用于指多核苷酸转移至靶细胞中。转移的多核苷酸可以并入靶细胞的基因组或染色体DNA中,导致基因上稳定的遗传(genetically stable inheritance),或者其可以独立于宿主染色体复制。含有转化的核酸片段的宿主生物体被称为“转基因的”或“重组的”或“转化的”生物体。
本文所使用的标准重组DNA和分子克隆技术是本领域众所周知的。
除非另有定义,本文所使用的所有技术和科学术语具有与本公开所属领域普通技术人员通常理解的相同含义。尽管类似于或等同于本文所述的那些方法和材料的任何方法和材料可以用于本公开的实践或测试中,但下面描述了优选的材料和方法。
根据本公开,已经开发了用于生产麦角硫因和具有编码可用于生产麦角硫因的egt1基因、egt2基因和XP_002490234基因的毕赤酵母菌株的方法。令人惊讶且出人意料地,采用改造后的毕赤酵母菌株可以提高麦角硫因的含量。
当egt1基因和egt2基因的来源于真菌粗糙脉胞菌(Neurospora crassa)时,所述egt1基因的氨基酸序列如SEQ ID NO:1所示;所述egt2基因的氨基酸序列如SEQ ID NO:2所示。
实施例1、一种用于生产麦角硫因的毕赤酵母菌株及其构建方法
本实施例提供了一种用于生产麦角硫因的毕赤酵母菌株的构建方法,包括以下步骤:
1)利用PCR技术原理扩增真菌来源麦角硫因合成途径的2个关键酶基因,以粗糙脉胞菌为例,这2个基因分别为egt1基因和egt2基因;克隆具体操作参照[J.萨姆布鲁克(JOSEPH SAMBROOK),分子克隆实验指南(精编版),2008年,化学工业出版社]。
2)利用PCR技术原理扩增毕赤酵母GS115来源的XP_002490234基因(NCBIReference Sequence:XP_002490234.1),克隆具体操作参照[J.萨姆布鲁克(JOSEPHSAMBROOK),分子克隆实验指南(精编版),2008年,化学工业出版社]。
3)将上述egt1基因、egt2基因和XP_002490234基因三个基因通过pPIC3.5k转化毕赤酵母进行诱导性表达,具体诱导性表达条件为:当毕赤酵母菌株的OD600达到50后,开始流加甲醇诱导,甲醇浓度通过气相色谱检测,使得甲醇浓度控制在0.5%以下。或者通过pGAPZ转化毕赤酵母进行组成性表达(无需添加甲醇诱导剂,在正常甘油或葡萄糖做碳源时,酵母一边生长一边表达),筛选阳性转化子。具体操作见《分子生物学实验手册》(马文丽,2011年6月1日,人民军医出版社出版)。
4)阳性转化子经过电泳验证后,在摇瓶和发酵罐进行培养,具体操作见《发酵工程实验》(李江华主编,2011年,高等教育出版社)。
麦角硫因分析通过高效色谱液相进行检测分析,测定细胞外麦角硫酮浓度,将1mL发酵液样品以3000×g离心5分钟,取上清液进行分析;剩余的细胞用无菌水洗涤,并重新悬浮于1mL水中,细胞内麦角硫因的提取如下:混合物在94℃下加热10分钟后离心,使用多管涡流器在1600rpm下涡旋30分钟,并在10000×g离心5分钟。
结果如表1所示,由结果可知,毕赤酵母出发菌株P.pastoris GS115(毕赤酵母GS115)不合成麦角硫因,本发明在毕赤酵母GS115基础上转化egt1基因、egt2基因后获得的菌株(P.pastoris GS115/pPIC3.5k-egt1-egt2)的麦角硫因达到536mg/L,同时过表达XP_002490234.1基因后,毕赤酵母菌株(P.pastoris GS115/pPIC3.5k-egt1-egt2-XP_002490234.1)的麦角硫因产率达782mg/L,具备工业生产前景。
表1不同菌株麦角硫因产量
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
1.一种用于生产麦角硫因的毕赤酵母菌株,其特征在于,所述毕赤酵母菌株同时过表达egt1基因、egt2基因和XP_002490234基因。
2.如权利要求1所述的毕赤酵母菌株,其特征在于,所述egt1基因和egt2基因的来源于真菌粗糙脉胞菌(Neurospora crassa)、粟酒裂殖酵母(Schizosaccharomyces pombe)、皱纹土霉(Geotricum rugosum)、星状诺卡氏菌(Nocardia asteroides)和链霉菌(Streptomyces)中的一种。
3.如权利要求2所述的毕赤酵母菌株,其特征在于,所述egt1基因和egt2基因的来源于真菌粗糙脉胞菌(Neurospora crassa)。
4.如权利要求3所述的毕赤酵母菌株,其特征在于,所述egt1基因的氨基酸序列如SEQID NO:1所示;所述egt2基因的氨基酸序列如SEQ ID NO:2所示。
5.如权利要求1所述的毕赤酵母菌株,其特征在于,所述毕赤酵母菌株为毕赤酵母GS115及其衍生菌株。
6.如权利要求1~5任一项所述的毕赤酵母菌株的构建方法,其特征在于,将过表达egt1基因、egt2基因和XP_002490234基因的表达质粒转化至毕赤酵母GS115进行诱导性表达或者组成性表达,获得用于生产麦角硫因的毕赤酵母菌株。
7.如权利要求6所述的构建方法,其特征在于,所述表达质粒包括pPIC3.5k或pGAPZ。
8.如权利要求1~5任一项所述的毕赤酵母菌株在强化麦角硫因合成中的应用。
9.如权利要求1~5任一项所述的毕赤酵母菌株在提高麦角硫因含量中的应用。
10.一种提高麦角硫因含量的制备方法,其特征在于,采用如权利要求1~5任一项所述的毕赤酵母菌株作为生产菌株。
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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CN114277042A (zh) * | 2021-12-29 | 2022-04-05 | 内蒙古金达威药业有限公司 | 一种高产麦角硫因的圆红冬孢酵母重组表达菌株及其构建方法和应用 |
Non-Patent Citations (2)
Title |
---|
GENBANK XP_002490234: "Mitochondrial NADH kinase, phosphorylates NADH [Komagataellaphaffii GS115]", Retrieved from the Internet <URL:NCBI> * |
MÀRIUSTOMÀS-GAMISANS: "Redox Engineering by Ectopic Overexpression of NADH Kinase in Recombinant Pichia pastoris ( Komagataellaphaffii): Impact on Cell Physiology and Recombinant Production of Secreted Proteins", APPL ENVIRON MICROBIOL, vol. 86, no. 6, pages 1 - 15 * |
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