CN111100801B - 一种糠醛等抑制物耐受酵母工程菌株及其构建方法与应用 - Google Patents
一种糠醛等抑制物耐受酵母工程菌株及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一种糠醛等抑制物耐受酵母工程菌株及其构建方法与应用,该糠醛等抑制物耐受酵母工程菌,命名为PYR,分类命名为酿酒酵母(Saccharomyces cerevisiae),于2019年6月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.17930。该工程菌可应用于制备第二代生物燃料乙醇以及以木质纤维素水解液为原料的其它生物基化学品。本发明通过转录调控基因联合过表达构建酿酒酵母工程菌,为从基因表达层面改造酵母菌株提供了技术手段。
Description
技术领域
本发明属于基因工程技术领域,具体地说,涉及一种糠醛等抑制物耐受酵母工程菌株及其构建方法与应用。
背景技术
第二代生物燃料乙醇被认为是交通能源石油的最佳替代品,其工业生产主要以秸秆、甘蔗渣等木质纤维素为原料,以酿酒酵母(Saccharomyces cerevisiae)作为乙醇发酵传统菌株。但是,木质纤维素在工业预处理过程中会产生大量以糠醛等抑制物为主的有毒副产物,对酿酒酵母的生长以及发酵有严重的抑制作用,是第二代生物燃料乙醇工业化生产的重要限制因素。虽然通过化学、物理等手段可以去除木质纤维素水解抑制物,但是会额外增加工业生产成本、降低原料利用率。研究发现,酿酒酵母自身对木质纤维素水解抑制物具有耐受能力。通过采用基因芯片技术、基因组和转录组测序技术、基因敲除与过表达技术等技术手段,进一步挖掘并分析了酿酒酵母中与糠醛等抑制物耐受相关的关键转录调控基因,这为实现转录调控基因联合过表达提高酿酒酵母对糠醛等抑制物的耐受能力提供了理论支撑。
与糠醛等抑制物耐受相关的转录调控基因,其编码的转录调控因子能够调控下游相应的功能基因群的表达,从而发挥其耐受能力,其中PDR1、YAP1、RPN4是最为关键转录调控基因。PDR1编码的转录调控因子Pdr1p,通过识别多效耐药响应元件pleiotropic drugresponse element(PDRE),激活与内、外源性有毒物质外排功能的基因群;YAP1编码的转录调控因子Yap1p,通过识别氧化应激响应元件Yap1p response element(YRE),激活下游的抗氧化功能基因群;RPN4编码的转录调控因子Rpn4p,通过识别蛋白酶体相关控制元件proteasome-associated control element(PACE),激活下游与受损蛋白质修饰与降解功能的基因群。
现有技术中,酿酒酵母表达载体容纳外源基因片段的能力有限,难以容纳核酸序列超过10kb长度的三个外源表达盒。
发明内容
有鉴于此,本发明针对上述的问题,提供了一种糠醛等抑制物耐受酵母工程菌株及其构建方法与应用,先将分子片段较大的pUG6-TEF1p-PDR1-CYC1t线性化重组质粒整合进染色体中;然后,剩下的两个表达盒依次整合进穿梭载体pRS42H中,该过程由于插入的两个片段合计大小(约为6kb)低于10kb,克服了现有技术中整合难度大的问题。
为了解决上述技术问题,本发明公开了一株糠醛等抑制物耐受酵母出发菌株,命名为YB-A-6-1(YBA_08),分类命名为酿酒酵母(Saccharomyces cerevisiae);于2019年6月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.17929。
本发明还公开了一株糠醛等抑制物耐受酵母工程菌,命名为PYR,分类命名为酿酒酵母(Saccharomyces cerevisiae),于2019年6月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.17930。
本发明还公开了一株糠醛等抑制物耐受酵母工程菌的构建方法,包括以下步骤:
步骤1、提取酵母单倍体菌株BY4742的基因组并扩增PDR1、YAP1、RPN4基因,进行胶回收;
步骤2、通过SpeI和SacII限制性内切酶对重组质粒pUG6-TEF1p-CYC1t进行双酶切,得到线性化的pUG6-TEF1p-CYC1片段,进行胶回收;同时通过SpeI和SacII限制性内切酶对YAP1片段进行双酶切以及纯化回收;
步骤3、采用T4 DNA连接酶,将双酶切的pUG6-TEF1p-CYC1t片段与双酶切的YAP1片段进行粘性末端连接体外构建重组质粒,转化到大肠杆菌DH5α感受态细胞,在含100mg/L氨苄青霉素的LB固体平板上挑选出含重组质粒pUG6-TEF1p-YAP1-CYC1t的阳性克隆子;将双酶切的pUG6-TEF1p-CYC1t片段与PDR1、RPN4片段按照分子质量比为2:6~2:8混合后,分别转化大肠杆菌DH5α感受态细胞体内重组构建重组质粒,在含100mg/L氨苄青霉素的LB固体平板上分别挑选出含重组质粒pUG6-TEF1p-PDR1-CYC1t与pUG6-TEF1p-RPN4-CYC1t的阳性克隆子;pUG6-TEF1p-PDR1-CYC1t用PsiI单酶切以及鉴定引物扩增进行鉴定,pUG6-TEF1p-YAP1-CYC1t用BamHI单酶切以及鉴定引物扩增进行鉴定,pUG6-TEF1p-RPN4-CYC1t用PsiI单酶切以及鉴定引物扩增进行鉴定;
步骤4、将PsiI酶切线性化的pUG6-TEF1p-PDR1-CYC1t通过同源重组方式整合进出发菌株YB-A-6-1染色体中,在含有200μg/mL G418的抗性YPD平板上挑选阳性酿酒酵母单菌落进行PCR鉴定,阳性菌株命名为P菌株;
步骤5、通过EcoRV单酶切克隆载体pRS42H并进行胶回收,线性化pRS42H片段大小为6217bp;用表达盒扩增引物分别扩增pUG6-TEF1p-YAP1-CYC1t与pUG6-TEF1p-RPN4-CYC1t重组质粒上的TEF1p-YAP1-CYC1t和TEF1p-RPN4-CYC1t过表达盒,并进行胶回收;
步骤6、将线性化pRS42H片段与TEF1p-YAP1-CYC1t过表达盒,按照分子质量比为2:6~2:8混合转化大肠杆菌DH5α细胞,体内重组构建重组质粒,在含100mg/L的氨苄青霉素的LB固体平板上挑选出含重组质粒pRS42H-TEF1p-YAP1-CYC1t的阳性克隆子,EcoRV单酶切鉴定以及用鉴定引物进行PCR扩增鉴定;
步骤7、将EcoRV线性化的pRS42H-TEF1p-YAP1-CYC1t与TEF1p-RPN4-CYC1t过表达盒,按照一定比例混合转化大肠杆菌DH5α细胞,体内重组构建重组质粒,在含100mg/L的氨苄青霉素的LB固体平板上挑选出含重组质粒pRS42H-TEF1p-YAP1-CYC1t-TEF1p-RPN4-CYC1t的阳性克隆子,EcoRV单酶切鉴定以及用鉴定引物进行PCR扩增鉴定;
步骤8、将最后所得的环状pRS42H-TEF1p-YAP1-CYC1t-TEF1p-RPN4-CYC1t重组质粒转化到P菌株细胞浆中,在含300μg/mL潮霉素的抗性平板上挑选阳性工程菌PYR,用鉴定引物进行PCR扩增鉴定,确定目标工程菌PYR是否含有3个过表达盒,获得最终的转录调控基因PDR1、YAP1、RPN4的联合过表达PYR菌株。
可选地,所述步骤1中的PDR1基因的扩增引物为ScPDR1_F和ScPDR1_R,其核苷酸序列如SEQ ID NO.1和2所示;RPN4基因的扩增引物为ScRPN4_F和ScRPN4_R,其核苷酸序列如SEQ ID NO.3和4所示;YAP1基因的扩增引物为ScYAP1_F和ScYAP1_R,其核苷酸序列如SEQID NO.5和6所示。
可选地,所述的步骤3中的pUG6-TEF1p-PDR1-CYC1t的鉴定引物为iTEF1p-PDR1_F和iTEF1p-PDR1_R,其核苷酸序列如SEQ ID NO.11和12所示;pUG6-TEF1p-YAP1-CYC1t的鉴定引物为iTEF1p-YAP1_F和iTEF1p-YAP1_R,其核苷酸序列如SEQ ID NO.13和14所示;pUG6-TEF1p-RPN4-CYC1t的鉴定引物为iTEF1p-RPN4_F和iTEF1p-RPN4_R,其核苷酸序列如SEQ IDNO.15和16所示。
可选地,所述的步骤5中的pUG6-TEF1p-YAP1-CYC1t表达盒扩增引物为hcYAP1_F和hcYAP1_R,其核苷酸序列如SEQ ID NO.7和8所示;pUG6-TEF1p-RPN4-CYC1t表达盒扩增引物为hcRPN4_F和hcRPN4_R,其核苷酸序列如SEQ ID NO.9和10所示。
可选地,所述的步骤6中的含重组质粒pRS42H-TEF1p-YAP1-CYC1t的阳性克隆子的鉴定引物为iTEF1p-YAP1_F和iTEF1p-YAP1_R,其核苷酸序列如SEQ ID NO.13和14所示。
可选地,所述步骤7中的含重组质粒pRS42H-TEF1p-YAP1-CYC1t-TEF1p-RPN4-CYC1t的阳性克隆子的鉴定引物为iTEF1p-YAP1_F和iTEF1p-YAP1_R、iTEF1p-RPN4_F和iTEF1p-RPN4_R,其核苷酸序列如SEQ ID NO.13-16所示。
可选地,所述的步骤8中的鉴定引物为iTEF1p-PDR1_F和iTEF1p-PDR1_R、iTEF1p-YAP1_F和iTEF1p-YAP1_R,iTEF1p-RPN4_F和iTEF1p-RPN4_R,其核苷酸序列如SEQ IDNO.13-18所示。
本发明还公开了一种糠醛等抑制物耐受酵母工程菌在制备第二代生物燃料乙醇以及以木质纤维素水解液为原料的其它生物基化学品中的应用。
与现有技术相比,本发明可以获得包括以下技术效果:
1)本发明所构建的过表达PDR1、YAP1和RPN4工程菌PYR增强了其对糠醛的耐受能力,且对其正常生长未产生显著影响。
2)本发明工程菌对糠醛耐受能力的提高具体表现为细胞壁抗性的明显增强,依赖于NADH作为辅酶的醛还原酶比活力的显著提高,以及一系列与有毒物质外排、细胞壁和细胞膜成分合成、蛋白质折叠、核苷酸合成等相关基因的显著上调转录表达。
3)该工程菌对5-羟甲基糠醛、乙酸、苯酚、乙醇以及玉米秸秆水解液中的抑制物等的耐受能力均有所增强,可作为较良好的工业发酵底盘菌株,具有用于第二代生物燃料乙醇以及以木质纤维素水解液为原料的其它生物基化学品工业化生产的应用潜力。
4)本发明通过转录调控基因联合过表达构建酿酒酵母工程菌,为从基因表达层面改造酵母菌株提供了技术手段。
当然,实施本发明的任一产品并不一定需要同时达到以上所述的所有技术效果。
附图说明
此处所说明的附图用来提供对本发明的进一步理解,构成本发明的一部分,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1是本发明酿酒酵母种属及倍型鉴定电泳图;其中,从左到右依次为1:倍型鉴定二重PCR扩增电泳图(404bp和544bp);2:ITS序列PCR扩增电泳图(841bp);3:26S rDNA序列PCR扩增电泳图(615bp);M:DNA marker;
图2是本发明扩增基因及重组质粒酶切电泳图;其中,A:转录调控基因扩增电泳图;从左到右依次为1:PDR1(3207bp);2:YAP1(1953bp);3:RPN4(1596bp);B:重组质粒酶切电泳图,1:pUG6-TEF1p-CYC1t酶切图(4956bp);M:DNA marker;
图3是本发明重组质粒扩增及酶切鉴定电泳图;其中,A:重组质粒扩增鉴定电泳图,从左到右依次为1:TEF1p-PDR1(663bp);2:TEF1p-YAP1(622bp);3:TEF1p-RPN4(945bp);B:重组质粒单酶切图,从左到右依次为1:pUG6-TEF1p-PDR1-CYC1t单酶切(8163bp);2:pUG6-TEF1p-YAP1-CYC1t单酶切(6909bp);3:pUG6-TEF1p-RPN4-CYC1t单酶切(6552bp);M:DNA marker;
图4是本发明线性化质粒与酿酒酵母染色体DNA整合模式图;
图5是本发明过表达P菌株扩增鉴定电泳图;其中,1:P菌株过表达盒TEF1p-PDR1鉴定引物扩增条带(663bp);M:DNA marker;
图6是本发明克隆载体单酶切及表达盒扩增电泳图;其中,A:克隆载体pRS42H单酶切条带(6217bp);B:过表达盒扩增条带,从左到右依次为1:TEF1p-YAP1过表达盒(3137bp);2:TEF1p-RPN4过表达盒(2780bp);M:DNA marker;
图7是本发明pRS42H-TEF1p-YAP1-CYC1t-TEF1p-RPN4-CYC1t重组质粒的构建模式图;
图8是本发明PYR工程菌构建示意图;
图9是本发明重组质粒单酶切及扩增鉴定电泳图;其中,A:重组质粒pRS42H-TEF1p-YAP1-CYC1t单酶切条带(9354bp);B:过表达盒TEF1p-YAP1鉴定引物扩增条带(622bp);C:重组质粒pRS42H-TEF1p-YAP1-CYC1t-TEF1p-RPN4-CYC1t单酶切条带(12128bp);D:从左到右依次为1:过表达盒TEF1p-YAP1鉴定引物扩增条带(622bp);2:过表达盒TEF1p-RPN4鉴定引物扩增条带(945bp);M:DNA marker;
图10是本发明PYR工程菌PCR扩增鉴定电泳图;其中,从左到右依次为1:过表达盒TEF1p-PDR1鉴定引物扩增条带(663bp);2:过表达盒TEF1p-YAP1鉴定引物扩增条带(622bp);3:过表达盒TEF1p-RPN4鉴定引物扩增条带(945bp);M:DNA marker;
图11是本发明糠醛应激条件下的生长曲线图;其中,A:0mM糠醛;B:25mM糠醛;C:35mM糠醛;D:45mM糠醛;CK:出发菌YB-A-6-1;
图12是本发明糠醛应激条件下菌株的醛还原酶比活力;*表示差异显著(p<0.05);**表示差异性极显著(p<0.01);CK:出发菌YB-A-6-1;
图13是本发明其它抑制物应激条件下的生长曲线图,其中,A:85mM 5-羟甲基糠醛;B:5.2g/L乙酸;C:2.0g/L苯酚;D:10.5%无水乙醇;E:13%玉米秸秆水解液;CK:出发菌YB-A-6-1。
具体实施方式
以下将配合实施例来详细说明本发明的实施方式,藉此对本发明如何应用技术手段来解决技术问题并达成技术功效的实现过程能充分理解并据以实施。
实施例1出发菌株YB-A-6-1的分离与鉴定:
一、出发菌株YB-A-6-1的分离
2011年,采集四川省宜宾白酒厂的酒醅样品,置于封口塑料膜,-20℃低温保存。用移液器取1.0mL菌液(稀释度为10-5、10-6、10-7)涂布于丙酸钠、硫酸链霉素-YEPD固体培养基,于28℃恒温培养箱培养48h,直至长出乳白色菌落,挑取具有典型酵母菌落特征的菌落,划线分离2~3次,经镜检为纯种后用接种环接入YEPD液体培养基,于28℃摇床恒温培养24h后与30%灭菌甘油按1:1体积注入冻存管混匀,在-80℃冰箱保存。
二、出发菌株YB-A-6-1的鉴定:
出发菌株YB-A-6-1(YBA_08)经26S rDNA及ITS DNA扩增及序列测定,其种属被鉴定为酿酒酵母;经倍型鉴定引物(MATα_F和MATα_R、MATa_F和MATa_R)二重PCR扩增,鉴定为二倍体型酵母菌,命名为YB-A-6-1,分类命名为酿酒酵母(Saccharomyces cerevisiae);于2019年6月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCCNo.17929。琼脂糖凝胶电泳结果见图1所示,通过电泳图中片段大小能够鉴定该菌为二倍体酿酒酵母。引物序列信息见表1所示(26S rDNA_F和26S rDNA_R、ITS_F和ITS_R),26S rDNA序列测定结果见GenBank no.KF141699.1,ITS序列测定结果见GenBank no.MN158119.1。
表1引物信息
注:双横线序列为同源臂序列;ACTAGT:SpeI;CCGCGG:SacII.
实施例2PYR的构建过程:
1、提取酵母单倍体菌株BY4742的基因组并扩增PDR1、YAP1、RPN4基因,进行胶回收,PDR1片段大小为3207bp;YAP1片段大小为1953bp;RPN4片段大小为1596bp。琼脂糖凝胶电泳结果见图2A所示,引物序列信息见表1所示中的ScPDR1_F和ScPDR1_R;ScYAP1_F和ScYAP1_R;ScRPN4_F和ScRPN4_R所示。
2、通过SpeI和SacII限制性内切酶对重组质粒pUG6-TEF1p-CYC1t进行双酶切,得到线性化的pUG6-TEF1p-CYC1片段(4956bp),进行胶回收。电泳结果见图2B所示,通过电泳图中片段大小验证该质粒片段大小正确。同时通过SpeI和SacII限制性内切酶对YAP1片段进行双酶切以及纯化回收。
3、采用T4 DNA连接酶,将双酶切的pUG6-TEF1p-CYC1t片段与双酶切的YAP1片段进行粘性末端连接体外构建重组质粒,转化到大肠杆菌DH5α感受态细胞,在含氨苄青霉素(100mg/L)的LB固体平板上挑选出含重组质粒pUG6-TEF1p-YAP1-CYC1t的阳性克隆子。将双酶切的pUG6-TEF1p-CYC1t片段分别与PDR1、RPN4片段按照分子质量比为2:6~2:8混合(按照此比例融合,能够有效地提高重组连接效率)后,分别转化大肠杆菌DH5α感受态细胞体内重组构建重组质粒,在含氨苄青霉素(100mg/L)的LB固体平板上分别挑选出含重组质粒pUG6-TEF1p-PDR1-CYC1t与pUG6-TEF1p-RPN4-CYC1t的阳性克隆子。pUG6-TEF1p-PDR1-CYC1t用PsiI单酶切以及鉴定引物(iTEF1p-PDR1_F和iTEF1p-PDR1_R)扩增进行鉴定,pUG6-TEF1p-YAP1-CYC1t用BamHI单酶切以及鉴定引物(iTEF1p-YAP1_F和iTEF1p-YAP1_R)扩增进行鉴定,pUG6-TEF1p-RPN4-CYC1t用PsiI单酶切以及鉴定引物(iTEF1p-RPN4_F和iTEF1p-RPN4_R)扩增进行鉴定。鉴定引物扩增出的片段大小分别为663bp、622bp、945bp;单酶切片段大小依次为8163bp、6909bp、6552bp。电泳结果见图3A和图3B所示。
4、将PsiI酶切线性化的pUG6-TEF1p-PDR1-CYC1t通过同源重组方式整合进出发菌株YB-A-6-1染色体中(整合模式过程见图4所示),在含有G418(200μg/mL)的抗性YPD平板上挑选阳性酿酒酵母单菌落进行PCR鉴定,扩增片段大小为663bp,电泳结果见图5所示。阳性菌株命名为P菌株。
5、通过EcoRV单酶切克隆载体pRS42H并进行胶回收,线性化pRS42H片段大小为6217bp;用表达盒扩增引物(hcYAP1_F和hcYAP1_R、hcRPN4_F和hcRPN4_R)分别扩增pUG6-TEF1p-YAP1-CYC1t与pUG6-TEF1p-RPN4-CYC1t重组质粒上的TEF1p-YAP1-CYC1t和TEF1p-RPN4-CYC1t过表达盒,并进行胶回收,扩增片段大小分别为3137bp和2780bp。上述电泳结果见图6A和图6B所示。
6、将线性化pRS42H片段与TEF1p-YAP1-CYC1t过表达盒,按照分子质量比为2:6~2:8混合(按照此比例融合,能够有效地提高重组连接效率)转化大肠杆菌DH5α细胞,体内重组构建重组质粒,在含氨苄青霉素(100mg/L)的LB固体平板上挑选出含重组质粒pRS42H-TEF1p-YAP1-CYC1t的阳性克隆子,EcoRV单酶切鉴定以及用鉴定引物(iTEF1p-YAP1_F和iTEF1p-YAP1_R)进行PCR扩增鉴定,单酶切的片段大小为9354bp,鉴定引物扩增出的片段大小为622bp。电泳结果见图9A和图9B所示。
7、将EcoRV线性化的pRS42H-TEF1p-YAP1-CYC1t与TEF1p-RPN4-CYC1t过表达盒,按照分子质量比为2:6~2:8混合(按照此比例融合,能够有效地提高重组连接效率)转化大肠杆菌DH5α细胞,体内重组构建重组质粒,在含氨苄青霉素(100mg/L)的LB固体平板上挑选出含重组质粒pRS42H-TEF1p-YAP1-CYC1t-TEF1p-RPN4-CYC1t的阳性克隆子(其构建模式图如图7所示),EcoRV单酶切鉴定以及用鉴定引物(iTEF1p-YAP1_F、iTEF1p-YAP1_R、iTEF1p-RPN4_F和iTEF1p-RPN4_R)进行PCR扩增鉴定,单酶切的片段为12128bp,鉴定引物扩增出的片段大小分别为622bp和945bp。电泳结果见图9C和图9D所示。
8、将最后所得的环状pRS42H-TEF1p-YAP1-CYC1t-TEF1p-RPN4-CYC1t重组质粒转化到P菌株细胞浆中,在含潮霉素(300μg/mL)的抗性平板上挑选阳性工程菌PYR(PYR工程菌构建示意图见图8),用鉴定引物(iTEF1p-PDR1_F和iTEF1p-PDR1_R、iTEF1p-YAP1_F和iTEF1p-YAP1_R、iTEF1p-RPN4_F和iTEF1p-RPN4_R)进行PCR扩增鉴定,确定目标工程菌PYR是否含有3个过表达盒,鉴定引物扩增出的片段大小分别为663bp、622bp、945bp。电泳结果见图10所示。获得最终的转录调控基因PDR1、YAP1、RPN4的联合过表达PYR菌株。
基于以上特征,将菌株PYR鉴定为酿酒酵母(Saccharomyces cerevisiae),于2019年6月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCCNo.17930。该PYR工程菌是在出发菌YB-A-6-1的基础上,通过基因重组技术构建的一株转录调控基因(PDR1、YAP1、RPN4)联合过表达菌株。
本发明基于PDR1、YAP1、RPN4这3个关键转录调控基因的不同功能,通过基因重组技术实现这3个关键转录调控基因在持续表达的强启动子TEF1p控制下的联合过表达,从而提高酿酒酵母对于糠醛等抑制物的耐受能力,更好地实现第二代生物燃料乙醇工业生产,降低其生产成本;此外构建的工程菌,还可以作为底盘菌株用于其它生物基化学品的生产。
实施例3糠醛应激条件下的迟滞期测定及抗性测定:
1、糠醛应激条件下的迟滞期测定:为了测定构建的工程菌PYR是否在出发菌YB-A-6-1的基础上进一步提高对糠醛的耐受能力,采取光密度法测定比较两株菌在糠醛应激压力下的摇瓶液体培养生长状况,通过生长曲线的迟滞期长短反映其耐受性。选取25mM、35mM、45mM糠醛作为实验的3个梯度浓度值,进行糠醛耐受性测定。具体操作步骤如下:
步骤1、将工程菌PYR和出发菌(CK)分别接种到含有30mL YPD液体的锥形瓶中,30℃、200r/min摇床振荡过夜预培养;
步骤2、使用分光光度计测定菌液OD600值,用同批次的YPD培养液调零。待菌液OD600值到达1.0,以10%的接种量离心收集细胞,接种到预先准备好的含有0mM、25mM、35mM、45mM浓度糠醛的YPD培养液中,使菌液OD600值约为0.1,设置3个生物学重复;
步骤3、放入摇床进行30℃、200r/min生长培养,间隔6h取样测定OD600值。整理数据绘制生长曲线图。
如图11所示,在无糠醛应激条件下,工程菌PYR与出发菌CK均未出现生长迟滞期,并且生长曲线几乎重合,表明通过转录调控基因联合过表达方式构建的工程菌不会对正常的生长造成显著影响。在不同浓度糠醛抑制物下,工程菌与出发菌均出现相应的迟滞期,工程菌的生长迟滞期明显比出发菌短。在25mM糠醛浓度下,工程菌的迟滞期比出发菌缩短了约6h;在35mM糠醛浓度下,工程菌的迟滞期比出发菌缩短了约12h;而在45mM糠醛浓度下,工程菌的迟滞期比出发菌缩短了约24h。试验结果表明,工程菌能够明显缩短糠醛应激条件下的迟滞期,从而更快地恢复生长进入对数期,并且随着糠醛浓度的增加效果更加明显。因此,PDR1、YAP1和RPN4 3个转录调控基因的联合过表达提高了工程菌株PYR对糠醛的耐受能力。
2、细胞壁抗性测定
细胞壁是酵母细胞直接接触外界环境的关键结构组分,酿酒酵母细胞壁完整性对糠醛耐受以及维持自身特定形态方面均有重要作用。以出发菌作为对照,选取55mM糠醛浓度作为该测定的浓度值,通过检测细胞对细胞壁裂解酶lyticase的敏感程度,间接反映工程菌PYR细胞壁的抗性能力。具体操作步骤如下:
步骤1、分别活化工程菌PYR和出发菌(CK),30℃、200r/min摇床振荡过夜预培养;
步骤2、将处于生长对数期的菌液分别接种到含有55mM浓度糠醛的体积为30mL新鲜YPD培养液中,调节各菌液的OD600为1.0,设置3个生物学重复;
步骤3、将两株菌在30℃、200r/min摇床振荡培养,分别于0h和8h吸取2mL的菌液于离心管中,离心收集菌体;无菌水洗涤,离心收集菌体;
步骤4、向菌体加入2mL磷酸缓冲液(0.1mM,pH=7.5)重悬,然后添加50μL浓度为0.6mg/mL的lyticase于2mL离心管中,30℃孵育1h;
步骤5、测定酶裂解处理后的细胞液在两个时间点的OD600值,整理数据得出工程菌PYR和出发菌CK之间细胞壁抗性的结果差异。
见表2所示,在未经糠醛应激处理下的酵母细胞,lyticase裂解作用1h后,出发菌CK细胞OD600的变化值约0.629,工程菌PYR细胞OD600的变化值约0.610;在经过55mM糠醛应激处理下的酵母细胞,lyticase裂解作用1h后,出发菌细胞OD600的变化值约1.044,工程菌细胞OD600的变化值约0.902。出发菌OD600的变化值在糠醛处理与未处理情况下差值约0.415,而工程菌OD600的变化值在糠醛处理与未处理情况下差值约0.293,二者之间差异极显著(p<0.01)。试验结果表明,出发菌细胞壁对lyticase的敏感程度相比于出发菌强,即出发菌细胞壁相比工程菌的裂解程度较严重,工程菌细胞壁的抗性能力相比于出发菌提高了约29.4%。说明工程菌PYR的细胞壁抗性能力明显强于出发菌YB-A-6-1。
表2细胞壁抗性测定
注:CK为出发菌株YB-A-6-1;Δ0h指糠醛处理细胞0h,用lyticase处理细胞壁1h前后,OD600的变化值;Δ8h指糠醛处理细胞8h,用lyticase处理细胞壁1h前后,OD600的变化值;Δ8h-Δ0h指对细胞壁进行损伤处理前后,OD600的变化值.**表示工程菌株与出发菌株之间的差异性极显著(p<0.01).
实施例4醛还原酶比活力测定
醛还原酶能够将糠醛还原为低毒的2-呋喃甲醇从而达到脱毒作用。醛还原酶比活力大小是衡量酵母细胞对糠醛脱毒及耐受能力的重要指标。该催化反应需要辅酶NADH或NADPH的参与,其中NADH在糠醛的脱毒中发挥着更为重要的作用。本测定选用NADH作为醛还原酶的辅酶,酶活力通过反应所需NADH在340nm吸光度的下降来进行测定,以每分钟氧化1μmol NADH为1个酶活力单位。本试验在45mM糠醛浓度应激条件下,选取0h、4h、8h三个时间点收集细胞进行醛还原酶比活力测定。
1、粗酶提取:
步骤1、分别活化工程菌PYR和出发菌(CK),30℃、200r/min摇床过夜培养;
步骤2、将处于生长对数期的菌液分别接种到含有45mM浓度糠醛的体积为30mL新鲜YPD培养液中,调节各菌液的OD600为1.0,设置3个生物学重复;
步骤3、将两株菌在30℃、200r/min摇床振荡培养,分别于0h、4h、8h吸取2mL的菌液于离心管中,5000r/min室温离心5min,去上清液,称取酵母菌体湿重;
步骤4、按照约每50mg的菌体湿重加入500μL的蛋白质提取试剂,每毫升的提取试剂加入1μL的蛋白酶抑制剂,80μL的DTT以及10μL的PMSF,充分混匀;
步骤5、盖上离心管,30℃、200r/min摇床振荡大约20min;
步骤6、13000r/min离心15min,将上清液转移到新的离心管中备用,此上清液即为粗酶溶液。
2、比活测定:
步骤1、将待测粗酶溶液样品放置于冰上,所有试剂均在所需实验温度下放置;
步骤2、利用考马斯亮蓝法在595nm波长测定粗酶溶液的蛋白质浓度;
步骤3、向30℃、pH=7.0的450μL的100mM PBS缓冲液中按照顺序加入10μL 0.5M糠醛,10μL 5mM辅酶NADH;
步骤4、最后加入30μL的粗酶溶液,迅速混匀后转入比色皿中读取OD340的吸光度值;在30℃环境下反应2min后,再次读取OD340的吸光度值;计算醛还原酶的比活力值。
见图12所示,以NADH作为辅酶,45mM糠醛处理0h时,出发菌PYR与工程菌CK之间的醛还原酶比活力值几乎无差异,它们的醛还原酶比活力约为0.024U/mg;处理4h后,工程菌的醛还原酶比活力强于出发菌,出发菌和工程菌的醛还原酶比活力分别约为0.038U/mg,0.044U/mg,工程菌的醛还原酶比活力相比于出发菌提高了约1.4倍,二者之间表现出显著差异(p<0.05);处理8h后,出发菌和工程菌的醛还原酶比活力分别约为0.035U/mg,0.057U/mg,工程菌的醛还原酶比活力相比于出发菌提高了约1.6倍,二者之间表现出极显著差异(p<0.01)。说明工程菌在糠醛应激条件下,依赖于NADH作为辅酶的醛还原酶表达水平相比出发菌得到显著提高。
实施例5基因转录表达分析
通过转录组测序数据结果,进一步从基因表达层面挖掘工程菌发挥糠醛耐受性的功能基因群。其具体操作步骤如下:
步骤1、分别活化工程菌PYR和出发菌(CK)进行过夜预培养,调整菌液OD600值为1.0,以10%的接种量收集细胞接种到含有45mM糠醛的YPD培养液中,在30℃、200r/min条件下进行摇床震荡培养。实验设置3个生物学重复;
步骤2、在3h时间点收集细胞,迅速经液氮冻存处理送至北京诺禾致源公司进行建库测序;
步骤3、提取样品的RNA并检验其浓度、纯度以及完整性;
步骤4、进行文库构建,通过稀释调整浓度并对文库的有效浓度进行准确定量以保证文库质量;
步骤5、检测合格后,按照特定的浓度值进行Illumina测序得到序列数据信息;
步骤6、对数据信息进行统计学分析,筛选样本在糠醛应激压力下表达水平显著差异的基因,显著差异基因筛选标准为log2(Fold change)>1&p值<0.05;
步骤7、通过对差异基因集进行富集分析,找到糠醛应激条件下工程菌糠醛耐受生物学功能相关的差异基因并进行功能注释。
通过工程菌与出发菌在糠醛应激条件下转录组水平的显著差异表达基因的功能注释,挖掘到了工程菌发挥糠醛耐受性的重要基因,见表3所示。主要是膜外排蛋白基因、细胞壁和细胞膜成分合成基因、蛋白质折叠分子伴侣基因以及磷酸戊糖途径相关的基因。膜外排蛋白基因AZR1具有将细胞内有毒物质(进入细胞内的过量糠醛和细胞代谢产生的有毒物质)外排到细胞外的功能。细胞壁和细胞膜成分合成基因主要涉及几丁质、麦角固醇、脂肪酸和磷脂等脂类的合成。几丁质在细胞壁重塑和拯救途径中能起到弥补受损细胞壁的作用,麦角固醇、脂肪酸、磷脂等脂类成分能起到维持细胞膜的完整性、流动性等重要作用。蛋白质分子伴侣基因SSB1和SSB2能够帮助新生蛋白质多肽链在糠醛应激条件下的正常折叠,从而发挥其正常的功能。磷酸戊糖途径相关的基因SOL3、GND2、RKI1、PRS3在磷酸戊糖途径构成了一条链式催化反应,能将6-磷酸葡糖酸通过上述基因所对应的代谢途径酶转变为5-磷酸核糖-1-焦磷酸(PRPP),而PRPP参与嘌呤核苷酸和嘧啶核苷酸的补救途径以及从头合成,工程菌能通过更多地合成核苷酸替代DNA链中受损的核苷酸分子从而维持正常的生物学功能。
表3糠醛耐受相关的重要基因
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注:CK为出发菌株YB-A-6-1.
实施例6其它抑制物应激条件下的迟滞期测定
木质纤维素生物质通过预处理产生的有毒抑制物除了主要抑制物糠醛外,还含有其它抑制物:5-羟甲基糠醛、乙酸、苯酚等,这些抑制物也会对酿酒酵母的生长与发酵产生严重影响,此外酿酒酵母的发酵产物乙醇也会对其生长与发酵造成影响。因此,除了测定比较工程菌与出发菌对于糠醛的耐受性外,有必要测定比较在这些单一抑制物(5-羟甲基糠醛、乙酸、苯酚、乙醇)下的耐受性以及复合抑制物(木质纤维素水解液)下的耐受性。按照实施例3方法所述,本测定选取85mM 5-羟甲基糠醛、5.2g/L乙酸、2.0g/L苯酚、10.5%无水乙醇以及13%玉米秸秆水解液进行测定。
如图13所示,在85mM的5-羟甲基糠醛应激的液体摇瓶培养下,工程菌PYR的生长迟滞期明显短于出发菌CK,工程菌比出发菌提前了约32h恢复生长;在5.2g/L的乙酸应激条件下,工程菌比出发菌提前了约6h先恢复生长;在2.0g/L的苯酚应激条件下,两株菌均未出现明显的生长迟滞期,但是工程菌的生长速率明显大于出发菌,并且工程菌最后到达稳定期的细胞总量也明显多于出发菌;在10.5%的无水乙醇应激条件下,出发菌在测定的72h内,没有出现生长,而工程菌在经过约12h的迟滞期后进入对数生长期;在13%的玉米秸秆水解液应激条件下,工程菌的迟滞期约为12h,出发菌的迟滞期约为24h,工程菌比出发菌提前了约12h恢复生长。综上,工程菌PYR对于其它有毒抑制物也具有较好的耐受性。
上述说明示出并描述了发明的若干优选实施例,但如前所述,应当理解发明并非局限于本文所披露的形式,不应看作是对其他实施例的排除,而可用于各种其他组合、修改和环境,并能够在本文所述发明构想范围内,通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离发明的精神和范围,则都应在发明所附权利要求的保护范围内。
序列表
<110> 四川农业大学
<120> 一种糠醛等抑制物耐受酵母工程菌株及其构建方法与应用
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Claims (3)
1.一株抑制物耐受酵母工程菌,其特征在于,命名为PYR,分类命名为酿酒酵母(Saccharomyces cerevisiae),于2019年6月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.17930;所述抑制物为糠醛、5-羟甲基糠醛、乙酸、苯酚、乙醇或者木质纤维素水解液。
2.权利要求1所述的抑制物耐受酵母工程菌在以木质纤维素水解液为原料发酵制备生物基化学品中的应用。
3.根据权利要求2所述的应用,其特征在于,所述生物基化学品为第二代生物燃料乙醇。
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