CN117264793B - 通过分子对接提高解脂亚罗酵母产β-胡萝卜素的方法、所得工程菌、其构建方法及应用 - Google Patents
通过分子对接提高解脂亚罗酵母产β-胡萝卜素的方法、所得工程菌、其构建方法及应用 Download PDFInfo
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Abstract
本发明提供一种高产β‑胡萝卜素的重组解脂亚罗酵母,所述重组解脂亚罗酵母是在解脂亚罗酵母polf菌株中过表达carB、carRP和GGPPSA124Y‑T132D基因、并表达ERG20、IDI、ERG12和tHMGR基因的工程菌株,所述GGPPSA124Y‑T132D基因是将GGPPS基因的A124突变为酪氨酸、T132突变为天冬氨酸得到的。本发明所得重组解脂亚罗酵母在摇瓶发酵中β‑胡萝卜素产量达到2.09g/L(95.3mg/g DCW);在50L发酵罐中β‑胡萝卜素产量达到16.71g/L(184.98mg/g DCW),显著领先现有技术。
Description
技术领域
本发明属于基因工程技术领域。具体涉及通过分子对接提高重组解脂亚罗酵母的产β-胡萝卜素的能力,还涉及通过分子对接技术选择出的最佳重组解脂亚罗酵母菌,还涉及所得重组菌的构建方法及在生产β-胡萝卜素中的应用。
背景技术
β-胡萝卜素是自然界中以全反式结构存在于胡萝卜、木瓜等许多绿色蔬菜中的一种脂溶性的天然类胡萝卜素,因其含有11个共轭碳碳双键,而具有良好的抑制单线态氧、抗氧化、淬灭自由基等作用,并具有延缓衰老、防癌和抗癌等功能,可应用于食品、化妆品、畜牧业等行业。
解脂亚罗酵母(Y.lipolytica)是遗传背景清晰的安全的产油微生物,其体内可以提供足够的前体化合物法尼基焦磷酸FPP和能源供应物质乙酰COA、NADPH和ATP。解脂亚罗酵母本身可以产β-胡萝卜素合成的前体化合物法尼基焦磷酸FPP,并且其本身作为产油微生物,可以为β-胡萝卜素提供足够的储存空间。屈玉玲等通过在解脂亚罗酵母中表达类胡萝卜素合成基因八氢番茄红素合成酶/番茄红素环化酶基因、八氢番茄红素脱氢酶基因构建具有合成β-胡萝卜素能力的解脂亚罗酵母重组菌株,并且过表达解脂亚罗酵母中产类胡萝卜素的前体基因,最终在摇瓶中可产β-胡萝卜素26.03mg/g DCW[屈玉玲.重组解脂亚罗酵母生物合成β-胡萝卜素的研究[D].陕西师范大学,2017]。香叶基香叶基焦磷酸合成酶GGPPS可以促进法尼基焦磷酸FPP流向GGPP通路,避免合成麦角甾醇通路的竞争。大量研究发现,所有含有GGPP合成酶天然拷贝的工程菌株中,GGPP含量都非常低,这表明FPP转化为GGPP是GGPP衍生化合物生物合成的限速步骤。解脂亚罗酵母内源香叶基香叶基焦磷酸合成酶GGPPS的活性较差,研究表明来源于源于嗜酸热硫化叶菌的香叶基香叶基焦磷酸合成酶GGPPS的活性较好。[Dai Zhubo,Liu Yi,Huang Luqi,Zhang Xueli.Production ofmiltiradiene by metabolically engineered Saccharomyces cerevisiae.[J].Biotechnology and bioengineering,2012,109(11).]
基于此,通过生物工程技术进一步提高重组解脂亚罗酵母的产β-胡萝卜素能力依然是本技术领域想要解决的问题。
发明内容
本发明的目的是构建一种高产β-胡萝卜素的重组解脂亚罗酵母。
本发明的思路是在解脂亚罗酵母中构建可产β-胡萝卜素的基础菌株,在基础菌株上优选GGPPS突变模块,然后表达多个拷贝的关键基因,并且表达前体基因,以增加β-胡萝卜素合成的前体物质香叶基香叶基焦磷酸GGPP含量,最终构建一种高产β-胡萝卜素的重组解脂亚罗酵母。
针对上述目的,本发明提供一种高产β-胡萝卜素的重组解脂亚罗酵母,所述重组解脂亚罗酵母是在解脂亚罗酵母polf菌株中过表达carB、carRP和GGPPSA124Y-T132D基因、并表达ERG20、IDI、ERG12和tHMGR基因的工程菌株,所述GGPPSA124Y-T132D基因是将GGPPS基因的A124突变为酪氨酸、T132突变为天冬氨酸得到的。
在本发明中,所述carB、carRP基因来源于卷枝毛霉(Mucor lusitanicus);ERG20、IDI、ERG12、tHMGR均来源于解脂亚罗酵母(Yarrowia lipolytica);香叶基香叶基焦磷酸合成酶GGPPS来源于嗜酸热硫化叶菌(Sulfolobus acidocaldarius)。
在本发明中,所述GGPPSP124Y-S132D基因的核酸序列如SEQ ID No.1所示。
基于此,本发明还提供一种高产β-胡萝卜素的重组解脂亚罗酵母(Yarrowialipolytica)的方法,以解脂亚罗酵母polf菌株为出发菌株,表达carB、carRP、ERG20、IDI、ERG12、tHMGR和GGPPSA124Y-T132D基因,所述GGPPSA124Y-T132D基因是将GGPPS基因的A124突变为酪氨酸、T132突变为天冬氨酸。
进一步地,本发明还提供高产β-胡萝卜素的重组解脂亚罗酵母的构建方法,所述方法包括以下步骤:
(1)构建GGPPS基因突变体
(1.1)以含有启动子PTEF、终止子Txpr2、leu2标记的表达质粒pJN44为载体,人工合成carB、carRP、GGPPS基因并分别连接至质粒pJN44的HindIII及SmaI之间,得到质粒pJN44-carB、pJN44-carRP、pJN44-GGPPS;
以含有loxp及ura3选择标记的ploxpura3loxp表达质粒为载体,以解脂亚罗酵母polf菌株基因组DNA为模板PCR扩增启动子PGPD和终止子TLip2,分别以合成质粒pJN44-carB、pJN44-carRP为模板PCR扩增carB、PTEF-carRP-Txpr2;
将TLip2、carB、PGPD、PTEF-carRP-Txpr2依次无缝连接至ploxpura3loxp上,得到重组质粒ploxpura3loxp-carB-carRP;
将线性化的ploxpura3loxp-carB-carRP转入解脂亚罗酵母polf菌株,得到工程菌株YLCA1;
(1.2)以合成质粒pJN44-GGPPS为模板,利用定点突变试剂盒,将GGPPS基因的第124氨基酸的碱基突变为酪氨酸和将第132氨基酸的碱基突变为天冬氨酸,得到质粒pJN44-GGPPSA124Y-T132D,转入YLCA1菌株;
(2)重组解脂亚罗酵母菌株构建
(2.1)以pJN44-GGPPSA124Y-T132D质粒为模板PCR扩增PTEF-GGPPSA124Y-T132D-Txpr2,无缝连接至质粒ploxpura3loxp-carB-carRP的NdeI、MIuI位点之间,得到ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D;
将用内切酶MIuI线性化的ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D转入解脂亚罗酵母polf菌株,得到菌株YLCA2;
(2.2)利用cre-loxp***去除YLCA2菌株中的ura3标记,得到菌株YLCA2D;
将线性化的ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D再次转入YLCA2D菌株,得到菌株YLCA3;
(2.3)利用cre-loxp***去除YLCA3菌株中的ura3标记,得到菌株YLCA3D;
将线性化的ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D再次转入YLCA3D菌株,得到菌株YLCA4;
(2.3)以含有loxp及leu2选择标记的ploxpleu2loxp表达质粒为载体,以polf菌株基因组DNA为模板PCR扩增ERG20、IDI、ERG12、tHMGR、启动子PTEF、PGPD、PEXP1和终止子TLip2、Txpr2;
将将PTEF、ERG20和Txpr2,TLip2、IDI和PEXP1,PTEF、tHMGR和Txpr2,TLip2、ERG12和PGPD依次连接至质粒ploxpleu2loxp上,得到重组质粒ploxpleu2loxp-ERG20-IDI-ERG12-tHMGR;
将用内切酶MIuI线性化的ploxpleu2loxp-ERG20-IDI-ERG12-tHMGR转入所述YLCA4菌株,得到一种高产β-胡萝卜素的重组解脂亚罗酵母;
类似地,在上述构建方法中,步骤(1)(2)中carB、carRP基因来源于卷枝毛霉(Mucor lusitanicus);ERG20、IDI、ERG12、tHMGR均来源于解脂亚罗酵母(Yarrowialipolytica);香叶基香叶基焦磷酸合成酶GGPPS来源于嗜酸热硫化叶菌(Sulfolobusacidocaldarius)。
步骤(1)(2)中所述GGPPSP124Y-S132D基因的核酸序列如SEQ ID No.1所示。
本发明还提供上述重组解脂亚罗酵母在产β-胡萝卜素中的应用,在摇瓶发酵中,所述重组解脂亚罗酵母在YPD液体培养基中180~220rpm,28~30℃发酵4~5d。
经检测,本发明所得重组解脂亚罗酵母在摇瓶发酵中β-胡萝卜素产量达到2.09g/L(95.3mg/g DCW)。
本发明还提供上述重组解脂亚罗酵母在产β-胡萝卜素中的应用,在生物反应器中,所述重组解脂亚罗酵母在含3.5L发酵培养基的5L生物反应器中,温度28~30℃、通气30~50L/min、转速100~650rpm、氧气10~20%、PH为5.5~6.0,葡萄糖维持约5g/L,发酵6~8d。
经检测,本发明所得重组解脂亚罗酵母在5L发酵罐中β-胡萝卜素产量达到16.71g/L(184.98mg/g DCW)。
本发明通过在解脂亚罗酵母polf菌株中表达β-胡萝卜素合成途径基因carB、carRP、ERG20、IDI、ERG12、tHMGR、GGPPS基因,其中,通过分子对接优选GGPPS增强香叶基香叶基焦磷酸的GGPP的供应,筛选出最优的GGPPS突变。最终所得重组解脂亚罗酵母在摇瓶发酵中β-胡萝卜素产量达到2.09g/L(95.3mg/g DCW);在50L发酵罐中β-胡萝卜素产量达到16.71g/L(184.98mg/g DCW),显著领先现有技术。
附图说明
图1为本发明的解脂亚罗酵母产β-胡萝卜素合成通路示意图;
图2为各菌株的摇瓶发酵结果;
图3为各菌株的50L生物反应器发酵结果;
图4载体质粒ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D图谱;
图5载体质粒ploxpleu2loxp-ERG20-IDI-ERG12-tHMGR图谱;
具体实施方式
以下实施例用于非限制性地解释本发明的技术方案。
本发明中,如无特殊说明,用于说明浓度“%”为重量百分比,“:”为重量比。
在本发明中,各菌株的β-胡萝卜素产量通过以下方法检测:
发酵结束后,收集细胞,通过HPLC法检测β-胡萝卜素的含量。
β-胡萝卜素提取:取1mL菌液,离心,收集菌体;加入500μL二甲基亚砜,55℃孵育15分钟;加入500μL丙酮,45℃孵育15分钟,每两分钟拿出来震荡;离心,取上清,用0.45μm的滤膜过滤于液相进样瓶中待测。HPLC检测方法:色谱柱为C18柱(Diamonsil SB-C18column,4.6mm×250mm);波长为:450nm;流动相为(v/v):甲醇:乙腈:异丙醇=30:50:20;流速为:1mL/min;柱温35℃,以等度洗脱方式进行洗脱。
本发明涉及以下培养基:
LB液体培养基:10g/L蛋白胨、10g/L NaCl、5g/L酵母粉。
LB固体培养基:10g/L蛋白胨、10g/L NaCl、5g/L酵母粉、20g/L琼脂粉。
YPD液体培养基:20g/L蛋白胨、10g/L酵母粉、50g/L葡萄糖
YPD固体培养基:20g/L蛋白胨、10g/L酵母粉、20g/L葡萄糖;20g/L琼脂粉;
SD-leu液体培养基:50g/L葡萄糖、1.7g/L YNB(不含氨基酸及硫酸铵)、5g/L(NH4)2SO4、2g/L SD-leu。
SD-leu固体培养基:20g/L葡萄糖、1.7g/L YNB(不含氨基酸及硫酸铵)、5g/L(NH4)2SO4、2g/L SD-leu、20g/L琼脂粉。
SD-ura固体培养基:20g/L葡萄糖、1.7g/L YNB(不含氨基酸及硫酸铵)、5g/L(NH4)2SO4、2g/L SD-ura、20g/L琼脂粉;
发酵培养基:50g/L酵母提取物、100g/L蛋白胨和100g/L葡萄糖。
实施例1:构建具有产β-胡萝卜素能力的基础菌株YLCA1
1、构建表达载体
(1)提取原始菌株解脂亚罗酵母polf基因组:
取1mL解脂亚罗酵母polf(ATCC细胞库)菌液,离心,弃上清;加入200μL 200mMLioAc,1% SDS溶液,震荡混匀;70℃金属浴15min;加入等体积(200μL)的饱和酚:氯仿:异戊醇(25:24:1,v/v)溶液,旋涡振荡至乳白色,离心10min;吸取水相至新的灭菌的1.5mL离心管中,再加入等体积的异丙醇,混匀;-20℃放置1-2h,12000×g离心10min,得到DNA;加入1mL 70体积%乙醇清洗DNA(重复2次),离心4min,弃上清;将含有DNA的离心管倒扣在干净的滤纸上15min,50℃金属浴2min,使乙醇彻底挥发;DNA溶解于50μL去离子水中,在-20℃保存备用。
(2)在NCBI上找到目的基因carB(AJ238028.1)、carRP(AJ250827.1),以pJN44([1]Guokun W,Xiaochao X,Rishikesh G,et al.Exploring fatty alcohol-producingcapability of Yarrowia lipolytica.[J].Biotechnology for biofuels,2016,9(1))为载体,送往南京金斯瑞生物科技有限公司合成,得到pJN44-carB、pJN44-carRP。设计表1所示引物,送往上海生工生物有限公司合成。
表1引物序列
(3)将载体ploxpura3loxp(Guokun W,Xiaochao X,Rishikesh G,etal.Exploring fatty alcohol-producing capability of Yarrowia lipolytica.[J].Biotechnology for biofuels,2016,9(1))用限制性内切酶SpeI在37℃下酶切2h,1%琼脂糖凝胶电泳,切下所需片段,利用胶回收试剂盒(AxyGen)进行胶回收;以合成质粒pJN44-carB为模板PCR扩增carB基因,以解脂亚罗酵母polf基因组DNA为模板PCR扩增启动子PGPD(Carina H,I M D,R K K,et al.EasyCloneYALI:CRISPR/Cas9-Based Synthetic Toolboxfor Engineering of the Yeast Yarrowia lipolytica.[J].Biotechnology journal,2018,13(9).)和终止子TLip2(Carina H,I M D,R K K,et al.EasyCloneYALI:CRISPR/Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowialipolytica.[J].Biotechnology journal,2018,13(9).),1%琼脂糖凝胶电泳,切下所需片段,进行胶回收;将回收的ploxpura3loxp与TLip2、carB、PGPD通过无缝连接试剂盒(全式金生物科技有限公司-Basic Seamless Cloning and Assembly Kit)在50℃连接15min,转化入E.coli Trans1 T1(全式金生物科技有限公司)中,均匀涂布于含100μg/mL氨苄青霉素的LB固体培养基上,37℃过夜培养,将单菌落挑取至含100μg/mL氨苄青霉素的LB液体培养基,37℃,200rpm过夜培养,用质粒提取试剂盒(AxyGen)提取质粒,得到质粒ploxpura3loxp-carBmc。
将ploxpura3loxp-carBmc用限制性内切酶SpeI酶切,用上述相同方法进行电泳,胶回收;以合成质粒pJN44-carRP为模板PCR扩增PTEF-carRP-Txpr2,电泳,胶回收;将回收的ploxpura3loxp-carBmc与PTEF-carRP-Txpr2用上述相同方法无缝连接,转化培养,提取质粒,得到质粒ploxpura3loxp-carBmc-carRP。
2、构建重组菌株YLCA1
将质粒ploxpura3loxp-carBmc-carRP用限制性内切酶MIuI在37℃下酶切2h,用1%琼脂糖凝胶电泳,切下所需片段,利用胶回收试剂盒回收,通过酵母转化试剂盒(ZymoFrozen-EZ Yeast Transformation II Kit)的方法转化至解脂亚罗酵母polf菌株中,涂布SD-ura固体培养基,30℃培养4天,得到重组解脂亚罗酵母,命名为菌株YLCA1。
3、重组菌株YLCA1在产β-胡萝卜素中的应用
将重组解脂亚罗酵母菌株YLCA1挑取单菌落接入含2mLYPD液体培养基的试管中,30℃、200r/min培养24h;以2%的接种量转接至50mL/250mL的YPD液体培养基中,30℃、200r/min条件下发酵96h,然后收集细胞,检测β-胡萝卜素的含量,YLCA1在摇瓶发酵中β-胡萝卜素产量达到0.11g/L(5.12mg/g DCW)。
实施例2:GGPPS突变体筛选
1、表达载体构建
(1)分子对接:在NCBI上找到目的基因GGPPS(D28748.1),利用Swiss Model对GGPPS的氨基酸序列进行建模,确定GGPPS最适模型。FPP底物模型从ZINC中搜索下载。以GGPPS酶分子模型为受体、FPP底物为配体,利用Autodock 4.0对GGPPS和FPP分别加氢并平衡电荷;grid box先覆盖所有GGPPS结构,根据Lamarckian GA的docking算法完成分子对接,从不同对接结果中找到GGPPS中活性位点频率出现最高的区域,精准确定grid box;基于精准确定的GGPPS酶grid box,再次对接GGPPS和FPP,确定最佳模型。根据对接结果利用pyMOL找到蛋白活性位点附近(6A范围内的残基)与FPP之间接合的氢键长度及位置(长度小于3.5),根据结合能越低可能性越大排列顺序,找到结合位置Q119、A124、E131和T132。
(2)目的基因GGPPS,以pJN44为载体,送往南京金斯瑞生物科技有限公司合成,得到pJN44-GGPPS。利用定点突变试剂盒,设计部分重叠引物(如表2),送往上海生工生物有限公司合成。将Q119、A124、E131、T132位置的氨基酸碱基分别通过设计引物突变为NNK,以pJN44-GGPPS质粒为模板,利用定点突变试剂盒(全式金生物科技有限公司FastMutagenesis System)PCR扩增出GGPPS119NNK、GGPPS124NNK、GGPPS131NNK、GGPPS132NNK,扩增结束后,取10μL PCR产物,1%琼脂糖凝胶电泳检测。剩下的PCR液体加入1μL DMT酶于PCR产物中消化甲基化模板,混匀,37℃孵育1h。随后将质粒转化至E.coli DMT中,均匀涂布于含100μg/mL氨苄青霉素的LB固体培养基上,37℃过夜培养,将单菌落挑取至含100μg/mL氨苄青霉素的LB液体培养基,37℃,200rpm过夜培养,用质粒提取试剂盒提取质粒,得到119、124、131、132四个位置已分别突变为NNK的质粒pJN44-GGPPS119NNK、pJN44-GGPPS124NNK、pJN44-GGPPS131NNK、pJN44-GGPPS132NNK。
表2引物序列
2、构建游离筛选菌株
将pJN44-GGPPS、pJN44-GGPPS119NNK、pJN44-GGPPS124NNK、pJN44-GGPPS131NNK、pJN44-GGPPS132NNK利用酵母转化试剂盒分别游离入重组菌株YLCA2D中,均匀涂布于SD-leu固体培养基上,30℃培养4天,将单菌落扎菌于SD-leu液体培养基中30℃,200rpm培养48小时,2%传代于新的SD-leu液体培养基中30℃,200rpm培养48小时,连续传代4次充分饱和突变,稀释涂布SD-leu固体平板,30℃培养4天,得到菌株YL1、YL2、YL3、YL4、YL5。
3、游离筛选菌株发酵及GGPPS突变筛选
(1)将菌株YL1、YL2、YL3、YL4、YL5分别每种菌挑取40个单菌落,每个单菌落分别接入含2mL SD-leu液体培养基的试管中并在SD-leu固体平板划线,试管中菌株在30℃、200r/min培养发酵96h,收集细胞,检测β-胡萝卜素的含量,YL1为0.26g/L(17.37mg/g DCW)、YL2为0.24g/L(16.23mg/g DCW)、YL3为0.41g/L(26.67mg/g DCW)、YL4为0.29g/L(18.92mg/gDCW)、YL5为0.46g/L(29.6mg/g DCW)。
(2)从SD-leu固体平板挑取β-胡萝卜素含量最高的YL3和YL5单菌落置于离心管中,加入200微升200mM LioAc,1% SDS溶液,震荡均匀后,100℃高温处理15min,离心后上清用作PCR模板进行PCR扩增出GGPPS片段,送往上海生工生物有限公司测序,确认YL3株的GGPPS基因的124氨基酸在重组菌株中突变为TAC,TL5株的GGPPS基因的132氨基酸在重组菌株中突变为GAT。
(3)利用定点突变试剂盒,将有显著效果的2个突变GGPPSA124Y与GGPPST132D组合为GGPPSA124Y-T132D,得到质粒pJN44-GGPPSA124Y-T132D,将其利用酵母转化试剂盒分别游离入重组菌株YLCA1中,均匀涂布于SD-leu固体培养基上,30℃培养4天,得到菌株YL6;
将YL6单菌落扎菌于含2mL SD-leu液体培养基的试管中,试管中菌株在30℃、200r/min培养发酵96h,收集细胞,检测β-胡萝卜素的含量,YL6产量为0.55g/L(36.37mg/gDCW)。
实施例3:构建重组解脂亚罗酵母菌株YLCA2
1、表达载体构建
(1)设计表3引物,送往上海生工合成。
表3引物序列
(2)ploxpura3loxp-carB-carRP载体用限制性内切酶NdeI,MIuI在37℃下酶切2h,电泳,胶回收;以质粒pJN44-GGPPSA124Y-T132D为模板PCR扩增PTEF-GGPPSA124Y-T132D-Txpr2,电泳,胶回收;将PTEF-GGPPSA124Y-T132D-Txpr2与ploxpura3loxp-carB-carRP无缝连接,得到ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D。
2、构建重组菌株YLCA2
将质粒ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D用限制性内切酶MIuI在37℃下酶切2h,用1%琼脂糖凝胶电泳,切下所需片段,利用胶回收试剂盒回收,通过酵母转化试剂盒的方法转化至重组解脂亚罗酵母菌株polf中,涂布SD-ura固体养基,30℃培养4天,得到重组菌株YLCA2,其基因型为YLCA2(polf,ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D)。
3、重组菌株YLCA2在产β-胡萝卜素中的应用
将重组解脂亚罗酵母菌株YLCA2挑取单菌落接入含2mL YPD液体培养基的试管中,30℃、200r/min培养24h;以2%的接种量转接至50mL/250mL的YPD液体培养基中,30℃、200r/min条件下发酵96h,然后收集细胞,检测β-胡萝卜素的含量,YLCA2在摇瓶发酵中β-胡萝卜素产量达到1.10mg/L(52.44mg/g DCW)。
实施例4:构建重组解脂亚罗酵母菌株YLCA3
1、YLCA2菌株去除ura3标记
将JN44-cre质粒([1]Guokun W,Xiaochao X,Rishikesh G,et al.Exploringfatty alcohol-producing capability of Yarrowia lipolytica.[J].Biotechnologyfor biofuels,2016,9(1))利用酵母转化试剂盒转入重组菌株YLCA2中,均匀涂布于SD-leu固体培养基上,30℃培养3天,将单菌落扎菌于10mLSD-leu液体培养基中,30℃,200rpm培养36小时,稀释涂布至SD-leu固体培养基上,30℃培养3天,单菌落在SD-leu、SD-ura、YPD固体培养基上划线筛选,在SD-ura培养基上不生长,在SD-leu、YPD培养基上生长的为去掉ura3标记的菌株。将正确菌株扎菌至10mL的YPD液体培养基中,30℃,200rpm培养24小时,2%接种量传代于10mL新的YPD液体培养基中30℃,200rpm培养12小时,传代3次后稀释涂布YPD固体培养基,30℃培养3天,单菌落在YPD、SD-leu固体培养基上划线筛选,在SD-leu固体培养基上不生长,在YPD固体培养基上生长的菌株成功消除JN44-cre质粒,得到已成功去除ura3标记的菌株YLCA2D。
2、构建重组菌株YLCA3
将质粒ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D用限制性内切酶MIuI在37℃下酶切2h,用1%琼脂糖凝胶电泳,切下所需片段,利用胶回收试剂盒回收,通过酵母转化试剂盒的方法转化至重组解脂亚罗酵母菌株YLCA2D中,涂布SD-ura固体养基,30℃培养4天,得到重组菌株YLCA3,其基因型为YLCA3(YLCA2D,ploxpura3loxp-carB-carRP-GGPPSA124Y -T132D)。
3、重组菌株YLCA3在产β-胡萝卜素中的应用
将重组解脂亚罗酵母菌株YLCA2D,YLCA3挑取单菌落接入含2mL YPD液体培养基的试管中,30℃、200r/min培养24h;以2%的接种量转接至50mL/250mL的YPD液体培养基中,30℃、200r/min条件下发酵96h,然后收集细胞,检测β-胡萝卜素的含量,YLCA2D在摇瓶发酵中β-胡萝卜素产量达到1.04mg/L(49.89mg/g DCW),YLCA3在摇瓶发酵中β-胡萝卜素产量达到1.54mg/L(73.95mg/g DCW)。
实施例5:构建重组解脂亚罗酵母菌株YLCA4
1、YLCA3菌株去除ura3标记
将质粒JN44-cre质粒利用酵母转化试剂盒转入重组菌株YLCA3中,均匀涂布于SD-leu固体培养基上,30℃培养3天,将单菌落扎菌于10mLSD-leu液体培养基中,30℃,200rpm培养36小时,稀释涂布至SD-leu固体培养基上,30℃培养3天,单菌落在SD-leu、SD-ura、YPD固体培养基上划线筛选,在SD-ura培养基上不生长,在SD-leu、YPD培养基上生长的为去掉ura3标记的菌株。将正确菌株扎菌至10mL的YPD液体培养基中,30℃,200rpm培养24小时,2%接种量传代于10mL新的YPD液体培养基中30℃,200rpm培养12小时,传代3次后稀释涂布YPD固体培养基,30℃培养3天,单菌落在YPD、SD-leu固体培养基上划线筛选,在SD-leu固体培养基上不生长,在YPD固体培养基上生长的菌株成功消除JN44-cre质粒,得到已成功去除ura3标记的菌株YLCA3D。
2、构建重组菌株YLCA4
将质粒ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D用限制性内切酶MIuI在37℃下酶切2h,用1%琼脂糖凝胶电泳,切下所需片段,利用胶回收试剂盒回收,通过酵母转化试剂盒的方法转化至重组解脂亚罗酵母菌株YLCA3D中,涂布SD-ura固体养基,30℃培养4天,得到重组菌株YLCA4,其基因型为YLCA4(YLCA3D,ploxpura3loxp-carB-carRP-GGPPSA124Y -T132D)。
3、重组菌株YLCA4在产β-胡萝卜素中的应用
将重组解脂亚罗酵母菌株YLCA3D,YLCA4挑取单菌落接入含2mL YPD液体培养基的试管中,30℃、200r/min培养24h;以2%的接种量转接至50mL/250mL的YPD液体培养基中,30℃、200r/min条件下发酵96h,然后收集细胞,检测β-胡萝卜素的含量,YLCA3D在摇瓶发酵中β-胡萝卜素产量达到1.45mg/L(70.15mg/g DCW),YLCA4在摇瓶发酵中β-胡萝卜素产量达到1.6mg/L(76.16mg/g DCW)。
实施例6:高产β-胡萝卜素的重组解脂亚罗酵母菌株YLCA5构建
1、构建表达载体
(1)在NCBI上找到目的基因ERG12(YALI0B16038g)、ERG20(YALI0E05753g)、HMG1(YALI0E04807g)、IDI(YALI0F04015g)、leu2(AF260230.1)、PEXP1(MP977616.1),其中将HMG1的前493个氨基酸序列删除能够避免其N端结构域介导的自降解,得到截断的tHMGR。设计表4引物,送往上海生工生物有限公司合成。
表4引物序列
(2)以ploxpura3loxp质粒为模板PCR扩增出除ura3标记外的其它基因,电泳,胶回收;以解脂亚罗酵母polf基因组DNA为模板PCR扩增leu2基因,电泳,胶回收;将回收的无ura3标记的其它基因与leu2基因用无缝连接试剂盒连接,转化培养,提取质粒,得到质粒ploxpleu2loxp。ploxpleu2loxp表达前体基因ERG20、IDI、ERG12、tHMGR并含有leu2基因。
以polf菌株基因组DNA为模板PCR扩增启动子PTEF、ERG20、终止子Txpr2,电泳,胶回收;将质粒载体ploxpleu2loxp用限制性内切酶pstI酶切,电泳,胶回收;将回收的ploxpleu2loxp与启动子PTEF、ERG20、终止子Txpr2用无缝连接试剂盒连接,转化培养,提取质粒,得到质粒ploxpleu2loxp-ERG20。
以polf菌株基因组DNA为模板PCR扩增终止子TLip2、IDI、启动子PEXP1,电泳,胶回收;将质粒载体ploxpleu2loxp-ERG20用限制性内切酶NdeI酶切,电泳,胶回收;将回收的ploxpleu2loxp-ERG20与终止子TLip2、IDI、启动子PEXP1用无缝连接试剂盒连接,转化培养,提取质粒,得到质粒ploxpleu2loxp-ERG20-IDI。
以polf菌株基因组DNA为模板PCR扩增启动子PTEF、tHMGR、终止子Txpr2,电泳,胶回收;将质粒载体ploxpleu2loxp-ERG20-IDI用限制性内切酶NdeI酶切,电泳,胶回收;将回收的ploxpleu2loxp-ERG20-IDI与启动子PTEF、tHMGR、终止子Txpr2用无缝连接试剂盒连接,转化培养,提取质粒,得到质粒ploxpleu2loxp-ERG20-IDI-tHMGR。
以polf菌株基因组DNA为模板PCR扩增终止子TLip2、ERG12、启动子PGPD,电泳,胶回收;将质粒载体ploxpleu2loxp-ERG20-IDI-tHMGR用限制性内切酶MIuI酶切,电泳,胶回收;将回收的ploxpleu2loxp-ERG20-IDI-tHMGR与终止子TLip2、ERG12、启动子PGPD用无缝连接试剂盒连接,转化培养,提取质粒,得到质粒ploxpleu2loxp-ERG20-IDI-tHMGR-ERG12。
2、构建重组菌株YLCA5
将质粒ploxpleu2loxp-ERG20-IDI-tHMGR-ERG12用限制性内切酶MIuI在37℃下酶切2h,用1%琼脂糖凝胶电泳,切下所需片段,利用胶回收试剂盒回收,通过酵母转化试剂盒的方法转化至重组解脂亚罗酵母菌株YLCA4中,涂布SD-leu固体养基,30℃培养4天,得到重组菌株YLCA5,其基因型为YLCA5(YLCA4,ploxpleu2loxp-ERG20-IDI-tHMGR-ERG12)。
3、重组菌株YLCA5在产β-胡萝卜素中的应用
将重组解脂亚罗酵母菌株YLCA5挑取单菌落接入含2mLYPD液体培养基的试管中,30℃、200r/min培养24h;以2%的接种量转接至50mL/250mL的YPD液体培养基中,30℃、200r/min条件下发酵96h,然后收集细胞,检测β-胡萝卜素的含量,YLCA5在摇瓶发酵中β-胡萝卜素产量达到2.09g/L(95.30mg/g DCW)。
以上结果可以看出,与YLCA2在摇瓶发酵中β-胡萝卜素产量1.10mg/L(52.44mg/gDCW)相比,多次表达carB,carRP,GGPPSA124Y-T132D基因,可以提高重组解脂亚罗酵母的β-胡萝卜素产量,但表达次数增多,效果逐渐减弱,ERG20、IDI、tHMGR和ERG12基因的表达能够一定程度提高重组解脂亚罗酵母的β-胡萝卜素产量,而去除ura3或leu2标记则会导致β-胡萝卜素产量稍微降低,所以最优选择是补齐polf菌株原有的ura3及leu2缺陷的重组菌,实现了高产β-胡萝卜素。
实施例7:重组菌株YLCA5在产β-胡萝卜素中的应用
将重组解脂亚罗酵母菌株YLCA5挑取单菌落接入含50mL/250YPD液体培养基的摇瓶中,30℃、200r/min培养24h,得到种子液;最终以2%的接种量将种子液接入含3.5L发酵培养基的5L生物反应器中,在温度28~30℃、通气30~50L/min、转速100~650rpm、氧气10~20%、添加5M氢氧化钠及盐酸调节PH为5.5~6.0,葡萄糖维持约5g/L,发酵6~8d。(每2h收集细胞,检测β-胡萝卜素的含量及糖含量)。
在5L发酵罐中β-胡萝卜素产量达到16.71mg/L(184.98mg/g DCW)。
对照例1:构建重组解脂亚罗酵母菌株YLCA2-CR
1、表达载体构建
(1)设计表5中引物,送往上海生工合成。
表5引物序列
(2)ploxpura3loxp-carB-carRP载体用限制性内切酶NdeI,MIuI在37℃下酶切2h,电泳,胶回收;以质粒pJN44-GGPPS为模板PCR扩增PTEF-GGPPS-Txpr2,电泳,胶回收;将PTEF-GGPPS-Txpr2与ploxpura3loxp-carB-carRP无缝连接,得到ploxpura3loxp-carB-carRP-GGPPS。
2、构建重组菌株YLCA2-CR
将质粒ploxpura3loxp-carB-carRP-GGPPS用限制性内切酶MIuI在37℃下酶切2h,用1%琼脂糖凝胶电泳,切下所需片段,利用胶回收试剂盒回收,通过酵母转化试剂盒的方法转化至重组解脂亚罗酵母菌株polf中,涂布SD-ura固体培养基,30℃培养4天,得到重组菌株YLCA2-CR,其基因型为YLCA2-CR(polf,ploxpura3loxp-carB-carRP-GGPPS)。
3、重组菌株YLCA2-CR在产β-胡萝卜素中的应用
将重组解脂亚罗酵母菌株YLCA2-CR挑取单菌落接入含2mL YPD液体培养基的试管中,30℃、200r/min培养24h;以2%的接种量转接至50mL/250mL的YPD液体培养基中,30℃、200r/min条件下发酵96h,然后收集细胞,检测β-胡萝卜素的含量,YLCA2-CR在摇瓶发酵中β-胡萝卜素产量达到0.54mg/L(25.72mg/g DCW)。
与YLCA2的β-胡萝卜素产量1.10mg/L(52.44mg/g DCW)相比,YLCA2的产量约为YLCA2-CR的2倍。
由此可见,突变体GGPPSA124Y和T132突变体GGPPST132D、特别是GGPPSA124Y-T132D在提高重组解脂亚罗酵母发酵产β-胡萝卜素的产量中具有显著效果。
Claims (7)
1.一种高产β-胡萝卜素的重组解脂亚罗酵母,其特征在于所述重组解脂亚罗酵母是在解脂亚罗酵母polf菌株中过表达carB、carRP和GGPPSA124Y-T132D基因、并表达ERG20、IDI、ERG12和tHMGR基因的工程菌株,所述GGPPSA124Y-T132D基因是将GGPPS基因的A124突变为酪氨酸、T132突变为天冬氨酸得到的,所述GGPPSA124Y-S132D基因的核酸序列如SEQ ID No.1所示;所述carB、carRP基因来源于卷枝毛霉(Mucor lusitanicus);ERG20、IDI、ERG12、tHMGR均来源于解脂亚罗酵母(Yarrowia lipolytica);香叶基香叶基焦磷酸合成酶GGPPS来源于嗜酸热硫化叶菌(Sulfolobus acidocaldarius)。
2.一种提高解脂亚罗酵母(Yarrowia lipolytica)产β-胡萝卜素的方法,其特征在于以解脂亚罗酵母polf菌株为出发菌株,表达carB、carRP、ERG20、IDI、ERG12、tHMGR和GGPPSA124Y-T132D基因,所述GGPPSA124Y-T132D基因是将GGPPS基因的A124突变为酪氨酸、T132突变为天冬氨酸,所述GGPPSA124Y-S132D基因的核酸序列如SEQ ID No.1所示;所述carB、carRP基因来源于卷枝毛霉(Mucor lusitanicus);ERG20、IDI、ERG12、tHMGR均来源于解脂亚罗酵母(Yarrowia lipolytica);香叶基香叶基焦磷酸合成酶GGPPS来源于嗜酸热硫化叶菌(Sulfolobus acidocaldarius)。
3.GGPPS基因的突变体GGPPSA124Y-T132D在提高重组解脂亚罗酵母发酵产β-胡萝卜素的产量中的应用,所述GGPPSA124Y-S132D基因的核酸序列如SEQ ID No.1所示。
4.根据权利要求1所述的高产β-胡萝卜素的重组解脂亚罗酵母的构建方法,所述方法包括以下步骤:
(1)构建GGPPS基因突变体
(1.1)以含有启动子PTEF、终止子Txpr2、leu2标记的表达质粒pJN44为载体,人工合成carB、carRP基因;
以含有loxp及ura3选择标记的ploxpura3loxp表达质粒为载体,以解脂亚罗酵母polf菌株基因组DNA为模板PCR扩增启动子PGPD和终止子TLip2,以合成质粒为模板PCR扩增carB、PTEF-carRP-Txpr2;将TLip2、carB、PGPD、PTEF-carRP-Txpr2依次连接至ploxpura3loxp上,得到重组质粒ploxpura3loxp-carB-carRP;
将线性化的ploxpura3loxp-carB-carRP转入解脂亚罗酵母polf菌株,得到工程菌株YLCA1;
(1.2)以合成质粒pJN44-GGPPS为模板,利用定点突变试剂盒,将GGPPS基因的第124氨基酸的碱基突变为酪氨酸和将第132氨基酸的碱基突变为天冬氨酸得到突变体GGPPSA124Y -T132D,所述突变体的核酸序列如SEQ ID No.1所示,得到质粒pJN44-GGPPSA124Y-T132D,转入YLCA1菌株;
(2)重组解脂亚罗酵母菌株构建
(2.1)以pJN44-GGPPSA124Y-T132D为模板PCR扩增PTEF-GGPPSA124Y-T132D-Txpr2,无缝连接至质粒ploxpura3loxp-carB-carRP上,得到ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D;
将线性化的ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D转入解脂亚罗酵母polf菌株,得到菌株YLCA2;
(2.2)去除YLCA2菌株中的ura3标记,得到菌株YLCA2D;
将线性化的ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D再次转入YLCA2D菌株,得到菌株YLCA3;
(2.3)去除YLCA3菌株中的ura3标记,得到菌株YLCA3D;
将线性化的ploxpura3loxp-carB-carRP-GGPPSA124Y-T132D再次转入YLCA3D菌株,得到菌株YLCA4;
(2.4)以含有loxp及leu2选择标记的ploxpleu2loxp表达质粒为载体,以polf菌株基因组DNA为模板PCR扩增ERG20、IDI、ERG12、tHMGR、启动子PTEF、PGPD、PEXP1和终止子TLip2、Txpr2;
将PTEF、ERG20和Txpr2,TLip2、IDI和PEXP1,PTEF、tHMGR和Txpr2,TLip2、ERG12和PGPD依次连接至质粒ploxpleu2loxp上,得到重组质粒ploxpleu2loxp-ERG20-IDI-ERG12-tHMGR;
将线性化的ploxpleu2loxp-ERG20-IDI-ERG12-tHMGR转入所述YLCA4菌株,得到一种高产β-胡萝卜素的重组解脂亚罗酵母。
5.根据权利要求4所述的构建方法,其特征在于步骤(2.4)的质粒ploxpleu2loxp是以ploxpura3loxp质粒为模板PCR扩增出除ura3标记外的其它基因,电泳,胶回收;以基因组DNA为模板PCR扩增leu2基因,电泳,胶回收;将回收的无ura3标记的其它基因与leu2基因用无缝克隆连接酶连接,转化培养,提取质粒,得到质粒ploxpleu2loxp。
6.权利要求1所述的重组解脂亚罗酵母或根据权利要求4所述的构建方法得到的重组解脂亚罗酵母在产β-胡萝卜素中的应用,其特征在于所述重组解脂亚罗酵母在YPD液体培养基中180~220rpm,28~30℃发酵4~5d。
7.权利要求1所述的重组解脂亚罗酵母或根据权利要求4所述的构建方法得到的重组解脂亚罗酵母在产β-胡萝卜素中的应用,其特征在于所述重组解脂亚罗酵母在含发酵培养基的5L生物反应器中,温度28~30℃ 、通气30~50L/min、转速100~650rpm、氧气10~20%、PH为5.5~6.0 ,葡萄糖维持约5g/L,发酵6~8d。
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