CN116121094A - 一株高产神经酰胺的酿酒酵母工程菌株、构建方法及其应用 - Google Patents
一株高产神经酰胺的酿酒酵母工程菌株、构建方法及其应用 Download PDFInfo
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- CN116121094A CN116121094A CN202310197822.2A CN202310197822A CN116121094A CN 116121094 A CN116121094 A CN 116121094A CN 202310197822 A CN202310197822 A CN 202310197822A CN 116121094 A CN116121094 A CN 116121094A
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Abstract
本发明公开了一株高产神经酰胺的酿酒酵母工程菌株,所述酿酒酵母工程菌株是在酿酒酵母宿主菌中敲除SEQ ID NO.1所示的YML115C基因所得,YML115C基因所编码的的氨基酸序列如SEQ ID NO.2所示。本发明所构建的酿酒酵母工程菌株在发酵12h后,神经酰胺达到8.90mg/gDCW,是野生型菌株的4.05倍。本发明所述工程菌株不但神经酰胺产量得到了提高,发酵条件较为简单,并且本发明酿酒酵母工程菌株的细胞壁较为脆弱易于后续的神经酰胺提取,降低投资成本,具有广阔的发展前景。
Description
技术领域
本发明属于基因工程技术领域,涉及酿酒酵母工程菌株的构建及其应用,尤其是一株高产神经酰胺的酿酒酵母工程菌株、构建方法及其应用。
背景技术
神经酰胺(Ceramide)是由一个鞘氨醇分子和一个脂肪酸分子构成的。神经酰胺在真核细胞的细胞膜中有较高的浓度,因为其是磷脂双分子层的主要成分——鞘磷脂的组成成分。以前的假设是,神经酰胺和其他鞘脂细胞膜内的鞘脂都是纯粹的支持性的结构分子。相对而言,现在认为神经酰胺可以参加各种各样的细胞信号通路。相关的例子包括调节细胞的分化、增殖和细胞程序性死亡。
神经酰胺是人体皮肤表皮角质层的细胞外间质中的脂质的主要成分(约占40%)。神经酰胺与胆固醇和饱和脂肪酸一起,产生一个不透水的保护性结构,以防止过多的水分蒸发,同时也阻止微生物的进入。神经酰胺具有防止皮肤水分蒸发、抗衰老、抑制黑色素、抗肿瘤活性、促进胶原蛋白生成等作用。因此,神经酰胺可以作为一些局部皮肤药物的成分,用于补充治疗皮肤疾病,如湿疹。神经酰胺也被用于化妆品,如一些肥皂,洗发水,护肤霜和防晒霜。此外,神经酰胺作为潜在的癌症治疗手段的可能也在积极的探索中。由此可见,神经酰胺在化妆品、保健食品、医药等领域应用广泛。
在当前全球市场中,神经酰胺生产企业主要集中于欧美及日本等国家,而我国神经酰胺市场规模在全球占比不到10%,在神经酰胺的生产和技术研发等方面都亟待提升,以满足国民随生活水平不断提高而对高质量产品的更大的需求。
目前神经酰胺的获取主要有三种方式:一、天然提取法,主要来源于植物,但植物提取方法受植物生长周期和季节的限制,产量较低;二、化学合成法,化学合成的主要是拟神经酰胺,其结构与神经酰胺相似,功能相似,可应用于化妆品中,但化学法对环境十分不友好;三、微生物发酵法,应用雪氏毕赤酵母(Pichia ciferrii)或酿酒酵母(Saccharomyces cerevisiae)发酵得到四乙酰植物鞘氨醇(TAPS),再对其去乙酰化得到植物鞘氨醇,加入脂肪酸合成神经酰胺等物质,但该方法主要是获得中间产物,并对中间产物进行二次加工,因此造成成本进一步增加并且会造成环境影响。因此,本领域迫切需要开发一种高效生产神经酰胺的酵母菌株。
通过检索,尚未发现与本发明专利申请相关的专利公开文献。
发明内容
本发明的目的在于克服现有技术上存在的问题,提供一株高产神经酰胺的酿酒酵母工程菌株、构建方法及其应用。
本发明解决技术问题所采用的技术方案是:
一株高产神经酰胺的酿酒酵母工程菌株,所述酿酒酵母工程菌株是在酿酒酵母宿主菌中敲除SEQ ID NO.1所示的YML115C基因所得,YML115C基因所编码的氨基酸序列如SEQID NO.2所示。
进一步地,所述工程菌株是利用Cre/loxP重组***,通过同源重组的方法,敲除SEQ ID NO.1所示的YML115C基因构建获得的;
其中,所采用的宿主菌为酿酒酵母菌株Saccharomyces cerevisiae BY4741。
如上所述的高产神经酰胺的酿酒酵母工程菌株的构建方法,所述方法包括以下步骤:
(1)根据SEQ ID NO.1所示的YML115C基因序列和Cre/loxP重组***设计从酿酒酵母基因组敲除YML115C基因的引物及其序列,引物为SEQ ID NO.3、SEQ ID NO.4;
(2)以Cre/loxP重组***中携带有筛选标记的质粒为模版,使用步骤(1)中所设计引物,PCR扩增用于从酿酒酵母基因组敲除YML115C基因的DNA片段,并加以纯化;
(3)将制备好的用于从酿酒酵母基因组敲除YML115C基因的DNA片段,采用高效酵母细胞转化法,转入酿酒酵母Saccharomyces cerevisiae BY4741中;
(4)通过筛选标记对应的营养缺陷型固体酵母培养基筛选得到重组酵母菌株。
进一步地,具体步骤如下:
(1)根据SEQ ID NO.1所示的YML115C的核苷酸序列和Cre/loxP重组***设计扩增引物:
(2)以Cre/loxP重组***质粒为模板,进行PCR扩增获得用于敲除YML115C基因的DNA片段;
(3)PCR产物进行琼脂糖凝胶电泳分析后,切胶回收目的片段;
(4)将目的片段采用醋酸锂转化法转入酿酒酵母BY4741;
(5)通过组氨酸营养缺陷型固体培养基进行筛选,在组氨酸营养缺陷型固体培养基上生长出的单菌落为候选菌落;
(6)对候选菌落的基因组中YML115C的点位进行基因测序,以确定YML115C被筛选标记基因替换成功,即得。
进一步地,每1L组氨酸营养缺陷型固体培养基为:酵母氮源基础(Yeast NitrogenBase)6.5-7g/L,灭菌结束后加入组氨酸缺陷型氨基酸混合物(-Leu Do supplementpowder)0.72-0.84g/L和葡萄糖1.7-2.3%,琼脂2-3%,用ddH2O定容至1L;
其中,上述百分数均为质量浓度百分数。
如上所述的高产神经酰胺的酿酒酵母工程菌株在神经酰胺生产方面中的应用。
利用如上所述的高产神经酰胺的酿酒酵母工程菌株发酵生产神经酰胺的方法,步骤如下:
将高产神经酰胺的酿酒酵母工程菌株通过三区划线涂布于固体丰富培养基平板,25~30℃培养48-72h,将活化后的菌种,接1-2环于装有3-5mL液体丰富培养基中,25~30℃,180-220rpm条件下培养14-20h即为种子培养液,将种子培养液按8-15%接种量接入液体丰富培养基中,25~30℃,180-220rpm条件下培养12-24h,即得。
进一步地,每1L固体丰富培养基为:硫酸腺嘌呤0.055-0.06g/L,酵母浸粉8-12g/L,蛋白胨16-24g/L,琼脂18-22g/L,灭菌后加入葡萄糖1.8-2.2%,用ddH2O定容至1L;
每1L液体丰富培养基为:硫酸腺嘌呤0.055-0.06g/L,酵母浸粉8-12g/L,蛋白胨16-24g/L,灭菌后加入葡萄糖1.8-2.2%,用ddH2O定容至1L;
其中,上述百分数均为质量浓度百分数。
一种如上所述的高产神经酰胺的酿酒酵母工程菌株细胞内的神经酰胺含量测定的方法,具体步骤如下:
(1)酿酒酵母工程菌株发酵结束后,收集全部菌体;
(2)将收集到的菌体进行破碎;
(3)使用有机溶剂萃取,合并溶剂层;
(4)使用有机溶剂萃取多次,合并溶剂层;
(5)氮气吹干有机溶剂层,并复溶提取物质;
(6)使用UPLC/MS对所得到的物质进行检测;
(7)计算细胞内神经酰胺含量。
一种如上所述的高产神经酰胺的酿酒酵母工程菌株的生长曲线的测定方法,具体步骤如下:
(1)将高产神经酰胺的酿酒酵母工程菌株和对照菌株分别接种于液体丰富培养基的试管中,25~30℃,200~220rpm/min的条件下培养12-18h;
(2)分别取30μL、40μL、50μL菌液依次加入含有1mL液体丰富培养基的EP管中,振荡混匀,取200μL加入100孔板中,并且取200μL液体丰富培养基作为对照;将100孔板放置于全自动生长曲线测定仪中,25~30℃培养,每隔1h测定波长600nm处的吸光度值;
(3)以培养时间X为横坐标,OD600值Y为纵坐标,绘制生长曲线。
本发明取得的优点和有益效果:
1、本发明酿酒酵母工程菌株是在酿酒酵母宿主菌中敲除SEQ ID NO.1所示的YML115C基因所得,本发明菌株通过Cre/loxP***制备基因敲除工具盒,化转至酿酒酵母宿主菌中,获得高产神经酰胺的酿酒酵母工程菌株。本发明所构建的酿酒酵母工程菌株在发酵12h后,神经酰胺达到8.90mg/gDCW,是野生型菌株的4.05倍。本发明所述工程菌株不但神经酰胺产量得到了提高,发酵条件较为简单,并且本发明酿酒酵母工程菌株的细胞壁较为脆弱易于后续的神经酰胺提取,降低投资成本,具有广阔的发展前景。
2、本发明提供的酿酒酵母工程菌株不但具有产量高的优点,并且可以通过相对简单的方法条件进行发酵,在工业上易于实现并控制,投资成本低,具有广阔的应用前景。
附图说明
图1为本发明中用于敲除YML115C基因的DNA片段扩增验证图;其中,M:DNA分子量标准;泳道1:经扩增后用于敲除YML115C基因的DNA片段;
图2为本发明中所构建酿酒酵母工程菌株中YML115C基因敲除PCR验证图;其中,M:DNA分子量标准;泳道1:YML115C基因敲除PCR验证;
图3为本发明中所构建的酿酒酵母工程菌株与野生型酿酒酵母菌株中神经酰胺产量的比较图;其中,1:野生型酿酒酵母菌株,2:酿酒酵母工程菌株;
图4为本发明中所构建的酿酒酵母工程菌株及其对照菌株的生长曲线图。
具体实施方式
为更好理解本发明,下面结合实施例对本发明做进一步地详细说明,但是本发明要求保护的范围并不局限于实施例所表示的范围。
本发明中所使用的原料,如无特殊说明,均为常规市售产品,本发明中所使用的方法,如无特殊说明,均为本领域常规方法,本发明所使用的各物质质量均为常规使用质量。
一株高产神经酰胺的酿酒酵母工程菌株,所述酿酒酵母工程菌株是在酿酒酵母宿主菌中敲除SEQ ID NO.1所示的YML115C基因所得,YML115C基因所编码的氨基酸序列如SEQID NO.2所示。
较优地,所述工程菌株是利用Cre/loxP重组***,通过同源重组的方法,敲除SEQID NO.1所示的YML115C基因构建获得的;
其中,所采用的宿主菌为酿酒酵母菌株Saccharomyces cerevisiae BY4741。
如上所述的高产神经酰胺的酿酒酵母工程菌株的构建方法,所述方法包括以下步骤:
(1)根据SEQ ID NO.1所示的YML115C基因序列和Cre/loxP重组***设计从酿酒酵母基因组敲除YML115C基因的引物及其序列,引物为SEQ ID NO.3、SEQ ID NO.4;
(2)以Cre/loxP重组***中携带有筛选标记的质粒为模版,使用步骤(1)中所设计引物,PCR扩增用于从酿酒酵母基因组敲除YML115C基因的DNA片段,并加以纯化;
(3)将制备好的用于从酿酒酵母基因组敲除YML115C基因的DNA片段,采用高效酵母细胞转化法,转入酿酒酵母Saccharomyces cerevisiae BY4741中;
(4)通过筛选标记对应的营养缺陷型固体酵母培养基筛选得到重组酵母菌株。
较优地,具体步骤如下:
(1)根据SEQ ID NO.1所示的YML115C的核苷酸序列和Cre/loxP重组***设计扩增引物:
(2)以Cre/loxP重组***质粒为模板,进行PCR扩增获得用于敲除YML115C基因的DNA片段;
(3)PCR产物进行琼脂糖凝胶电泳分析后,切胶回收目的片段;
(4)将目的片段采用醋酸锂转化法转入酿酒酵母BY4741;
(5)通过组氨酸营养缺陷型固体培养基进行筛选,在组氨酸营养缺陷型固体培养基上生长出的单菌落为候选菌落;
(6)对候选菌落的基因组中YML115C的点位进行基因测序,以确定YML115C被筛选标记基因替换成功,即得。
较优地,每1L组氨酸营养缺陷型固体培养基为:酵母氮源基础(Yeast NitrogenBase)6.5-7g/L,灭菌结束后加入组氨酸缺陷型氨基酸混合物(-Leu Dosupplementpowder)0.72-0.84g/L和葡萄糖1.7-2.3%,琼脂2-3%,用ddH2O定容至1L;
其中,上述百分数均为质量浓度百分数。
如上所述的高产神经酰胺的酿酒酵母工程菌株在神经酰胺生产方面中的应用。
利用如上所述的高产神经酰胺的酿酒酵母工程菌株发酵生产神经酰胺的方法,步骤如下:
将高产神经酰胺的酿酒酵母工程菌株通过三区划线涂布于固体丰富培养基平板,25~30℃培养48-72h,将活化后的菌种,接1-2环于装有3-5mL液体丰富培养基中,25~30℃,180-220rpm条件下培养14-20h即为种子培养液,将种子培养液按8-15%接种量接入液体丰富培养基中,25~30℃,180-220rpm条件下培养12-24h,即得。
较优地,每1L固体丰富培养基为:硫酸腺嘌呤0.055-0.06g/L,酵母浸粉8-12g/L,蛋白胨16-24g/L,琼脂18-22g/L,灭菌后加入葡萄糖1.8-2.2%,用ddH2O定容至1L;
每1L液体丰富培养基为:硫酸腺嘌呤0.055-0.06g/L,酵母浸粉8-12g/L,蛋白胨16-24g/L,灭菌后加入葡萄糖1.8-2.2%,用ddH2O定容至1L;
其中,上述百分数均为质量浓度百分数。
一种如上所述的高产神经酰胺的酿酒酵母工程菌株细胞内的神经酰胺含量测定的方法,具体步骤如下:
(1)酿酒酵母工程菌株发酵结束后,收集全部菌体;
(2)将收集到的菌体进行破碎;
(3)使用有机溶剂萃取,合并溶剂层;
(4)使用有机溶剂萃取多次,合并溶剂层;
(5)氮气吹干有机溶剂层,并复溶提取物质;
(6)使用UPLC/MS对所得到的物质进行检测;
(7)计算细胞内神经酰胺含量。
一种如上所述的高产神经酰胺的酿酒酵母工程菌株的生长曲线的测定方法,具体步骤如下:
(1)将高产神经酰胺的酿酒酵母工程菌株和对照菌株分别接种于液体丰富培养基的试管中,25~30℃,200~220rpm/min的条件下培养12-18h;
(2)分别取30μL、40μL、50μL菌液依次加入含有1mL液体丰富培养基的EP管中,振荡混匀,取200μL加入100孔板中,并且取200μL液体丰富培养基作为对照;将100孔板放置于全自动生长曲线测定仪中,25~30℃培养,每隔1h测定波长600nm处的吸光度值;
(3)以培养时间X为横坐标,OD600值Y为纵坐标,绘制生长曲线。
具体地,相关制备及检测如下:
实施例1用于敲除YML115C基因的DNA片段的扩增与制备:
根据YML115C的核苷酸序列和Cre/loxP重组***设计以下扩增引物:
YML115C-CL-KO-F:
5’-TAAGGGTGCAGTAACAGTCATTTGAGCTTGTTGACCTACCAATTAACACAAAAAACAGCTGAAGCTTCGTACGC-3’;
YML115C-CL-KO-R:
5’-AAACAAAACAAAAAATTAAATTAAATTAAATTAATATTAAATGTCATCATGATTCGCATAGGCCACTAGTGGATCTG-3’;
PCR反应体系:FastPfu Buffer 10μL,2.5mM dNTPs 4μL,模板DNA终浓度<1μg,上下游引物(10μM)各2.5μL,FastPfu DNApolymerase 0.5-1μL,ddH2O补齐至总体积50μL;
以Cre/loxP重组***质粒为模板,进行PCR扩增获得用于敲除YML115C基因的DNA片段。PCR反应条件为:95℃2-3min,95℃20-30s,55-58℃30s,72℃1-2min,循环30-40次,72℃5-10min,4℃保温。PCR产物进行琼脂糖凝胶电泳分析后,切胶回收目的片段,如图1所示。
实施例2YML115C基因敲除酿酒酵母工程菌株的构建:
利用醋酸锂高效转化法,将获得的基因敲除目的片段转化至Saccharomycescerevisiae BY4741菌株中,然后通过组氨酸营养缺陷型固体酵母培养基筛选,并将组氨酸营养缺陷型固体酵母培养基筛选所得到的单菌落进行富集。将候选菌落送金唯智公司测序,测序正确的即为所需的YML115C基因敲除酿酒酵母工程菌株。PCR扩增所得送检测序DNA片段的琼脂糖凝胶电泳结果如图2所示。
实施例3酿酒酵母工程菌株发酵生产神经酰胺:
(1)活化菌种
将产神经酰胺的酿酒酵母工程菌株划线于固体完全培养基平板,25℃培养48h;
(2)种子培养
将活化后的菌种,接一环于装有5mL液体完全培养基的试管中,25℃220rpm培养12h即为种子培养液;
液体培养基为:硫酸腺嘌呤0.055g/L,酵母浸粉10g/L,蛋白胨20g/L,葡萄糖2%。
(3)摇瓶发酵培养
将种子培养液按2%接种量接入装有200mL液体完全培养基的三角瓶中,25℃,220rpm条件下发酵12h;
发酵培养基为:硫酸腺嘌呤0.055g/L,酵母浸粉10g/L,蛋白胨20g/L,葡萄糖2%。
实施例4酿酒酵母工程菌株中神经酰胺含量测定:
(1)量取一致的沉淀或者体积,置于2mL EP管中;
(2)加入两颗小钢珠,加入800μL预冷的二氯甲烷/甲醇(3:1)的缓冲液;
(3)TissueLyser研磨5min,-20℃冰箱中沉淀2h或过夜;
(4)25,000×g,4℃离心15min,取上清650μL,置于新的EP管中再次25,000×g,4℃离心15min;
(5)取600μL上清液冷冻抽干,用600μL脂质复溶液(异丙醇:乙腈:水=2:1:1)复溶;
(6)25,000×g,4℃离心20min;
(7)取60μL上清液转到96孔微孔板,封膜标识,进行UPLC-MS检测;
结果如图3所示,从图中可以看出,发酵至12h后,经测定神经酰胺达到8.90mg/gDCW,是野生型菌株的4.05倍。
实施例5酿酒酵母工程菌株的生长曲线:
(1)将产神经酰胺的酿酒酵母工程菌株和对照菌株分别接种于液体丰富培养基的试管中,28℃,220rpm/min的条件下培养12-18h;
(2)分别取30μL、40μL、50μL菌液依次加入含有1mL液体丰富培养基的EP管中,振荡混匀,取200μL加入100孔板中,并且取200μL液体丰富培养基作为对照;将100孔板放置于全自动生长曲线测定仪中,28℃培养,每隔1h测定波长600nm处的吸光度值;
(3)以培养时间(X)为横坐标,OD600值(Y)为纵坐标,绘制生长曲线,如图4所示。
本发明所涉及的YML115C基因如下(序列表SEQ ID NO.1):
ATGGGCATGTTTTTTAATTTAAGGTCAAATATAAAGAAGAAAGCCATGGACAATGGACTA
AGCCTGCCCATTTCAAGGAACGGTAGCTCGAACAACATCAAGGACAAACGCTCAGAGCA
TAACTCCAACTCATTAAAGGGCAAATACAGGTACCAGCCGCGCTCCACACCGTCTAAATT
CCAGCTTACGGTGAGTATCACATCTCTTATTATTATCGCCGTTCTGTCGTTATATCTCTTTAT
ATCATTTCTCTCCGGAATGGGCATTGGTGTATCCACGCAAAATGGTAGGTCGTTGTTGGGT
TCCTCAAAATCCTCCGAAAATTACAAGACTATCGACCTAGAAGATGAAGAATATTACGACT
ATGATTTTGAGGATATCGATCCTGAAGTGATTTCAAAATTTGATGATGGTGTGCAACATTAT
CTAATATCACAATTTGGTTCAGAAGTGTTGACTCCCAAGGATGATGAAAAATACCAAAGG
GAACTCAACATGCTTTTTGATTCCACTGTTGAGGAGTACGACCTGTCGAATTTTGAAGGT
GCTCCGAATGGATTGGAAACACGTGATCACATTCTTTTATGCATTCCACTAAGAAACGCTG
CGGATGTATTGCCATTAATGTTCAAGCATTTAATGAACCTAACCTATCCACACGAGCTGATT
GATCTGGCCTTTTTAGTCAGTGATTGCTCAGAAGGTGACACTACGTTGGATGCTTTAATAG
CGTATTCTAGGCACTTACAAAATGGCACGTTGTCTCAGATTTTTCAAGAGATTGACGCTGT
CATTGATTCGCAAACAAAAGGCACCGATAAATTATATCTTAAATATATGGACGAAGGTTATA
TCAACCGTGTCCACCAGGCATTTTCACCACCATTCCATGAAAATTATGACAAGCCATTTAG
ATCAGTACAAATTTTCCAAAAGGATTTTGGCCAAGTAATTGGACAAGGTTTTAGTGACAG
ACATGCCGTTAAGGTTCAAGGTATAAGACGAAAACTAATGGGGAGGGCAAGAAATTGGT
TAACTGCCAATGCTTTGAAACCTTACCACTCATGGGTTTATTGGAGAGATGCTGATGTAGA
GCTGTGCCCTGGTTCAGTCATTCAAGATTTGATGAGCAAAAACTACGATGTTATCGTCCCT
AACGTTTGGAGACCACTACCTACATTTTTGGGAACTGAACAACCATATGATTTGAATTCTT
GGATGGAATCTCAGGAGGCACTAGCATTGGCAAAGACTCTTGACGAAGATGATGTTATTG
TAGAAGGCTATGCAGAATATCCTACGTGGAGAGTTCACTTAGCCTATATCAGAGATGCAGA
AGGTGATCCAAATGAAGCGGTGGACTTGGACGGTGTAGGGGGAGTTTCTATTTTGGCGA
AGGCCAAAATATTTAGAAACGGGGTACAGTTTCCTGCATTTACTTTTGAAAATCATGCAG
AAACAGAAGCATTTGGTAAAATGGCAAAGAAAATGGGGTACAGGGTTGGTGGTTTACCG
CATTATACTATTTGGCATATTTATGAACCGAGCGATGACGATTTGAAGGAAATTGCGTCAA
GGGAAAGAGAGAAGAGAAGACAATCAGAGTAA
SEQ ID NO.1所编码的Yml115cp的氨基酸序列如SEQ ID NO.2所示:MGMFFNLRSNIKKKAMDNGLSLPISRNGSSNNIKDKRSEHNSNSLKGKYRYQPRSTPSKFQLTVSITSLIIIAVLSLYLFISFLSGMGIGVSTQNGRSLLGSSKSSENYKTIDLEDEEYYDYDFEDIDPEVISKFDDGVQHYLISQFGSEVLTPKDDEKYQRELNMLFDSTVEEYDLSNFEGAPNGLETRDHILLCIPLRNAADVLPLMFKHLMNLTYPHELIDLAFLVSDCSEGDTTLDALIAYSRHLQNGTLSQIFQEIDAVIDSQTKGTDKLYLKYMDEGYINRVHQAFSPPFHENYDKPFRSVQIFQKDFGQVIGQGFSDRHAVKVQGIRRKLMGRARNWLTANALKPYHSWVYWRDADVELCPGSVIQDLMSKNYDVIVPNVWRPLPTFLGTEQPYDLNSWMESQEALALAKTLDEDDVIVEGYAEYPTWRVHLAYIRDAEGDPNEAVDLDGVGGVSILAKAKIFRNGVQFPAFTFENHAETEAFGKMAKKMGYRVGGLPHYTIWHIYEPSDDDLKEIASREREKRRQSE。
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例所公开的内容。
Claims (10)
1.一株高产神经酰胺的酿酒酵母工程菌株,其特征在于:所述酿酒酵母工程菌株是在酿酒酵母宿主菌中敲除SEQ ID NO.1所示的YML115C基因所得,YML115C基因所编码的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的高产神经酰胺的酿酒酵母工程菌株,其特征在于:所述工程菌株是利用Cre/loxP重组***,通过同源重组的方法,敲除SEQ ID NO.1所示的YML115C基因构建获得的;
其中,所采用的宿主菌为酿酒酵母菌株Saccharomyces cerevisiae BY4741。
3.如权利要求1或2所述的高产神经酰胺的酿酒酵母工程菌株的构建方法,其特征在于:所述方法包括以下步骤:
(1)根据SEQ ID NO.1所示的YML115C基因序列和Cre/loxP重组***设计从酿酒酵母基因组敲除YML115C基因的引物及其序列,引物为SEQ ID NO.3、SEQ ID NO.4;
(2)以Cre/loxP重组***中携带有筛选标记的质粒为模版,使用步骤(1)中所设计引物,PCR扩增用于从酿酒酵母基因组敲除YML115C基因的DNA片段,并加以纯化;
(3)将制备好的用于从酿酒酵母基因组敲除YML115C基因的DNA片段,采用高效酵母细胞转化法,转入酿酒酵母Saccharomyces cerevisiae BY4741中;
(4)通过筛选标记对应的营养缺陷型固体酵母培养基筛选得到重组酵母菌株。
4.根据权利要求3所述的构建方法,其特征在于:具体步骤如下:
(1)根据SEQ ID NO.1所示的YML115C的核苷酸序列和Cre/loxP重组***设计扩增引物:
(2)以Cre/loxP重组***质粒为模板,进行PCR扩增获得用于敲除YML115C基因的DNA片段;
(3)PCR产物进行琼脂糖凝胶电泳分析后,切胶回收目的片段;
(4)将目的片段采用醋酸锂转化法转入酿酒酵母BY4741;
(5)通过组氨酸营养缺陷型固体培养基进行筛选,在组氨酸营养缺陷型固体培养基上生长出的单菌落为候选菌落;
(6)对候选菌落的基因组中YML115C的点位进行基因测序,以确定YML115C被筛选标记基因替换成功,即得。
5.根据权利要求4所述的构建方法,其特征在于:每1L组氨酸营养缺陷型固体培养基为:酵母氮源基础6.5-7g/L,灭菌结束后加入组氨酸缺陷型氨基酸混合物0.72-0.84g/L和葡萄糖1.7-2.3%,琼脂2-3%,用ddH2O定容至1L;
其中,上述百分数均为质量浓度百分数。
6.如权利要求1或2所述的高产神经酰胺的酿酒酵母工程菌株在神经酰胺生产方面中的应用。
7.利用如权利要求1或2所述的高产神经酰胺的酿酒酵母工程菌株发酵生产神经酰胺的方法,其特征在于:步骤如下:
将高产神经酰胺的酿酒酵母工程菌株通过三区划线涂布于固体丰富培养基平板,25~30℃培养48-72h,将活化后的菌种,接1-2环于装有3-5mL液体丰富培养基中,25~30℃,180-220rpm条件下培养14-20h即为种子培养液,将种子培养液按8-15%接种量接入液体丰富培养基中,25~30℃,180-220rpm条件下培养12-24h,即得。
8.根据权利要求7所述的方法,其特征在于:每1L固体丰富培养基为:硫酸腺嘌呤0.055-0.06g/L,酵母浸粉8-12g/L,蛋白胨16-24g/L,琼脂18-22g/L,灭菌后加入葡萄糖1.8-2.2%,用ddH2O定容至1L;
每1L液体丰富培养基为:硫酸腺嘌呤0.055-0.06g/L,酵母浸粉8-12g/L,蛋白胨16-24g/L,灭菌后加入葡萄糖1.8-2.2%,用ddH2O定容至1L;
其中,上述百分数均为质量浓度百分数。
9.一种如权利要求1或2所述的高产神经酰胺的酿酒酵母工程菌株细胞内的神经酰胺含量测定的方法,其特征在于:具体步骤如下:
(1)酿酒酵母工程菌株发酵结束后,收集全部菌体;
(2)将收集到的菌体进行破碎;
(3)使用有机溶剂萃取,合并溶剂层;
(4)使用有机溶剂萃取多次,合并溶剂层;
(5)氮气吹干有机溶剂层,并复溶提取物质;
(6)使用UPLC/MS对所得到的物质进行检测;
(7)计算细胞内神经酰胺含量。
10.一种如权利要求1或2所述的高产神经酰胺的酿酒酵母工程菌株的生长曲线的测定方法,其特征在于:具体步骤如下:
(1)将高产神经酰胺的酿酒酵母工程菌株和对照菌株分别接种于液体丰富培养基的试管中,25~30℃,200~220rpm/min的条件下培养12-18h;
(2)分别取30μL、40μL、50μL菌液依次加入含有1mL液体丰富培养基的EP管中,振荡混匀,取200μL加入100孔板中,并且取200μL液体丰富培养基作为对照;将100孔板放置于全自动生长曲线测定仪中,25~30℃培养,每隔1h测定波长600nm处的吸光度值;
(3)以培养时间X为横坐标,OD600值Y为纵坐标,绘制生长曲线。
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