CN116574162B - 一种里氏木霉转录激活因子Xyr1突变体及其在提高纤维素酶及木聚糖酶产量中的应用 - Google Patents
一种里氏木霉转录激活因子Xyr1突变体及其在提高纤维素酶及木聚糖酶产量中的应用 Download PDFInfo
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Abstract
本发明涉及一种里氏木霉转录激活因子Xyr1突变体及其在提高纤维素酶及木聚糖酶产量中的应用。其中里氏木霉转录激活因子突变体Xyr1R434L的氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4所示。本发明以里氏木霉Xyr1为基础，将第434位精氨酸突变为亮氨酸，得到里氏木霉转录激活因子突变体Xyr1R434L。该突变体提高了Xyr1的转录激活能力，能够有效促进里氏木霉纤维素酶及木聚糖酶的表达分泌。在过表达突变体Xyr1R434L后，发酵液中纤维素酶酶活及木聚糖酶酶活水平均有大幅度提高，比过表达Xyr1均分别提高了0.8倍。因而里氏木霉转录激活因子突变体Xyr1R434L可以有效提高纤维素酶及木聚糖酶产量，在降低酶的生产成本中具有很大的应用价值。
Description
技术领域
本发明涉及一种里氏木霉转录激活因子Xyr1突变体及其在提高纤维素酶及木聚糖酶产量中的应用，属于生物工程技术领域。
背景技术
木质纤维素生物质是地球上最丰富的可再生资源之一，可用于生产生物燃料以及丰富多样的生物基化学品，因此其开发利用对我国经济发展、环境保护等具有重要的战略意义。使用纤维素酶及木聚糖酶将木质纤维素生物质转化为可发酵糖是生物燃料生产的一个关键步骤。然而纤维素酶及木聚糖酶高昂的生产成本仍然是生物精炼过程中的瓶颈。
真菌里氏木霉已被广泛用于工业纤维素酶及木聚糖酶生产。里氏木霉能够分泌丰富的纤维素降解酶系，包括纤维素外切酶CBHI、CBHII，纤维素内切酶EGI、EGII、EGIII、EGV以及β-葡萄糖苷酶BGLI等。然而里氏木霉中木聚糖酶的分泌较低。在自然界中，里氏木霉纤维素酶及木聚糖酶基因通常需要诱导物的存在才能够表达，并且受到葡萄糖的阻遏，这提高了酶的生产成本。此外，里氏木霉纤维素酶及木聚糖酶的表达主要在转录水平上受到大量转录因子的调控。其中，Xyr1是调控纤维素酶及木聚糖酶基因转录最为关键的转录激活因子，纤维素酶基因的表达水平通常与Xyr1的表达水平呈正相关。xyr1基因的缺失几乎消除了所有纤维素酶及木聚糖酶的表达。而使用组成型启动子过表达Xyr1可直接提高纤维素酶表达水平，并克服葡萄糖碳阻遏代谢。因此，通过改造Xyr1转录因子，使里氏木霉在廉价且易操作的葡萄糖碳源下也能提高纤维素酶及木聚糖酶产量，从而有望实现经济可行的工业化生物精炼过程。
发明内容
针对现有技术存在的问题，本发明提供了一种里氏木霉转录激活因子Xyr1突变体及其在提高纤维素酶及木聚糖酶产量中的应用。
本发明技术方案如下：
一种里氏木霉转录激活因子突变体Xyr1R434L，其氨基酸序列相对于里氏木霉转录激活因子Xyr1的氨基酸序列仅存在下述突变：将第434位精氨酸突变为亮氨酸。
根据本发明优选的，所述里氏木霉转录激活因子突变体Xyr1R434L的氨基酸序列如SEQ ID NO.3所示。
根据本发明优选的，所述里氏木霉转录激活因子突变体Xyr1R434L的核苷酸序列如SEQ ID NO.4所示。
本发明的第二个方面，提供含有所述里氏木霉转录激活因子突变体Xyr1R434L的编码基因的表达盒、重组载体。所述重组载体对出发载体并没有特别的限制，可为本领域中已知的任何载体，只要它能够在宿主中进行复制即可。例如，所述载体包括但不限于质粒，噬菌体。一旦转化入合适的宿主之后，所述载体可以复制并独立于宿主基因组发挥功能，或者在某些情况下整合入基因组本身。
根据本发明优选的，所述重组载体是将里氏木霉转录激活因子突变体Xyr1R434L的编码基因与表达载体连接后获得，所述表达载体为质粒。
进一步优选的，所述质粒为pUC19-Ppdc1-pyr4site。
本发明的第三个方面，提供含有所述里氏木霉转录激活因子突变体Xyr1R434L编码基因的重组宿主细胞。其中所述“宿主细胞”是具有本领域通常理解的含义，其能够导入本发明的突变体的编码基因的宿主细胞，导入之后称为重组宿主细胞。本发明的菌株可以是里氏木霉(Trichoderma reesei)QM9414。
本发明的第四个方面，提供上述里氏木霉转录激活因子突变体Xyr1R434L或里氏木霉转录激活因子突变体Xyr1R434L的编码基因在提高纤维素酶及木聚糖酶产量中的应用。
本发明中未详细描述的实验操作均可按照本技术领域的常规实验操作进行。
有益效果
本发明以里氏木霉转录激活因子Xyr1为基础，将第434位精氨酸突变为亮氨酸，得到里氏木霉转录激活因子突变体Xyr1R434L。相较于Xyr1，该突变体Xyr1R434L提高了转录激活能力，能够有效促进里氏木霉纤维素酶及木聚糖酶的表达分泌。在组成型表达突变体Xyr1R434L后，发酵液中纤维素酶酶活及木聚糖酶酶活水平均有大幅度提高，是组成型表达野生型Xyr1的1.8倍。因而里氏木霉转录激活因子突变体Xyr1R434L可以有效提高纤维素酶及木聚糖酶产量，在降低酶的生产成本中具有很大的应用价值。
附图说明
图1为组成型表达Xyr1及突变体Xyr1R434L提高纤维素酶及木聚糖酶产量的菌株构建示意图。
图2为组成型表达Xyr1及突变体Xyr1R434L提高纤维素酶及木聚糖酶产量比较图。
其中，a图为胞外发酵液中纤维素酶酶活水平分析；b图为胞外发酵液中木聚糖酶酶活水平分析；c图为胞外发酵液Western blot分析。
具体实施方式
下面结合实施例和附图对本发明作进一步说明，但是本发明的保护范围并不仅限于此。实施例中涉及的药品及试剂，若无特殊说明，均为普通市售产品；实施例中涉及的实验操作，若无特殊说明，均为本领域常规操作。实施例中的引物均由北京擎科生物科技有限公司合成。
实施例中的出发菌株里氏木霉(Trichoderma reesei)QM9414为现有菌种，可购自美国菌种保藏中心，其保藏号为ATCC 26921。
里氏木霉QM9414-Δxyr1是将里氏木霉QM9414中的xyr1基因敲除后得到的，构建方法可参考文献：Xyr1(xylanase regulator 1)regulates both the hydrolytic enzymesystem and D-xylose metabolism in Hypocrea jecorina。
为了获得具有更高转录激活能力的里氏木霉转录激活因子Xyr1突变体，我们首先在酿酒酵母中建立了一种基于酵母单杂交方法的Xyr1突变体定量筛选体系。该筛选体系包括一个里氏木霉cbh1启动子驱动的LacZ表达盒及一个酿酒酵母adh1启动子驱动的Xyr1突变体表达盒。通过检测β-葡萄糖苷酶活性的变化，从而筛选出转录激活活性提高的Xyr1突变体。使用该筛选体系对Xyr1的C端转录激活结构域及中间MHR区域的碱性氨基酸进行丙氨酸突变，发现了Xyr1第434位氨基酸对其活性非常重要，进一步通过饱和突变获得了一个具有更高转录激活能力的Xyr1R434L突变体。
实施例1：组成型表达Xyr1及突变体Xyr1R434L的载体构建
1、组成型表达Xyr1的载体构建
(1)设计引物xyr1-upF/xyr1-downR，以里氏木霉QM9414 cDNA为模板，经PCR扩增得到Xyr1的cDNA编码片段，长约2.8kb，所述转录激活因子Xyr1的氨基酸序列如SEQ IDNO.1所示，转录激活因子Xyr1的核苷酸序列如SEQ ID NO.2所示。
其中，所述引物的序列如下：
xyr1-upF:5’-TACAGCACAATCGGCGCGCCATGTTGTCCAATCCT-3’；
xyr1-downR:5’-GTTAAGTGGATCGCGGCCGCTTAGAGGGCCAGACC-3’。
PCR扩增体系为：10μM上游引物2μL，10μM下游引物2μL，基因组DNA10ng，2.5mMdNTP混合物5μL，5×TransStart FastPfu缓冲液10μL，TransStart FastPfu DNA聚合酶1μL，ddH2O补足至50μL；
PCR扩增程序如下：98℃预变性5min；95℃变性30sec，58℃退火30sec，72℃延伸3min，30个循环；72℃终延伸10min；PCR完成后进行1％琼脂糖凝胶电泳，并回收DNA片段。
(2)设计引物upF/upR和downF/downR，经PCR扩增得到里氏木霉pyr4基因上下游同源臂片段，分别约2kb和1.4kb。
将pyr4基因的上下游同源臂片段分别连入含有潮霉素抗性基因hph表达盒的载体pUC19-hph中，上下游同源臂片段分别位于hph表达盒的两侧；进而在上游同源臂和hph表达盒之间顺序引入里氏木霉组成型启动子pdc1和终止子TrpC，进而构建成功由启动子pdc1驱动、pyr4定点的重组表达载体pUC19-Ppdc1-pyr4site。最后，将上述扩增得到的片段xyr1连接进入该重组表达载体，位于启动子和终止子之间，得到重组表达载体pMDOEX，该表达载体的线性示意图见图1。
其中，所述引物的序列如下：
upF：5’-CCCAAGCTTGGGAGTTTTGGGGGAAAAAGATGAT-3’；
upR：5’-AGCTTTGTTTAAACGGCTGATGAGGCTGAGAGAGGCTGAG-3’；
downF：5’-CGGAATTCGGGAGGGAAGGGAAGAAAGAAGTAAAG-3’；
downR：5’-CATGCCATGGTCGCGATCGCACTGATTTGCGGATATG-3’。
PCR扩增体系和扩增程序同上。
2、组成型表达突变体Xyr1R434L的载体构建
设计引物Xyr1R434LF/Xyr1R434LR，该引物R434位点的精氨酸密码子突变为亮氨酸。以xyr1-upF/Xyr1R434LR和Xyr1R434LF/xyr1-downR为引物，以xyr1 cDNA为模板，经PCR扩增得到片段1和片段2，然后采用常规重叠延伸PCR方法将片段1和片段2连接，得到突变体xyr1R434L片段，长约2.8kb。
所述引物的序列如下：
Xyr1R434LF:5’-CTGCCGTCGGCGCTCAGCAAGGTCT-3’；
Xyr1R434LR:5’-AGACCTTGCTGAGCGCCGACGGCAG-3’。
将构建好的xyr1R434L片段连入重组表达载体pUC19-Pdc1-pyr4site，构建得到重组表达载体pMDOEXR434L。
本实施例中的突变体Xyr1R434L氨基酸序列如SEQ ID NO.3所示，突变体Xyr1R434L的核苷酸序列如SEQ ID NO.4所示。
实施例2：组成型表达Xyr1及突变体Xyr1R434L的菌株构建
1、里氏木霉原生质体制备
制备浓度为5×107～5×108个/mL的里氏木霉QM9414-Δxyr1孢子悬液，取1mL接种于50mL的MM液体培养基中，28℃、160rpm培养16h，得菌丝培养物；将菌丝培养物于4000rpm离心5min，弃上清，然后加入细胞壁裂解酶液，重悬，并于30℃、60rpm酶解2.5～3h，通过显微镜观察，当有大量游离的原生质体出现时，使用G2漏斗过滤，滤液于4℃，3500rpm离心5min收集原生质体，收集到的原生质体用15mL预冷的STC溶液洗涤两次，然后加入预冷的STC溶液重悬，使原生质体浓度为5×107～5×108个/mL；以上操作均在冰上进行。
MM液体培养基组分如下(均为质量百分比)：1％葡萄糖、0.5％(NH4)2SO4、1.5％KH2PO4、0.1％蛋白胨，用NaOH调pH至5.0-5.5，使用前加入终浓度为0.6g/L MgSO4、终浓度为0.4g/L CaCl2，并加入微量元素(终浓度1×，母液1000×)、10mM尿苷；
所述1000×微量元素母液配方：FeSO4·7H2O 0.5g，MnSO4·H2O 0.16g，ZnSO4·7H2O 0.14g，CoCl2·2H2O 0.2g，用ddH2O定容至100mL，过滤除菌；
细胞壁裂解酶液的组分如下：0.24g细胞壁裂解酶，溶于5.7mL浓度为0.7M的NaCl溶液中，过滤除菌，加入0.3mL pH5.8的PBS缓冲液；
STC溶液的组分如下：0.7MD-山梨糖醇，0.05M CaCl2，10mM Tris-HCl，pH 7.5。
2、重组表达载体的转化及转化子再生
分别取150μL实施例1中得到的重组表达载体pMDOEX和pMDOEXR434L，使用限制性内切酶SpeⅠ对载体进行线性化，并纯化回收片段，与150μL步骤1制备的里氏木霉QM9414-Δxyr1原生质体混匀后加入到10mL离心管底部，冰浴20min后逐滴加入1.5mL预冷的PTC溶液，冰浴20min，再室温放置20min，再加入2mL STC溶液后混匀，得转化液；将转化液涂布于含有尿苷的再生培养基平板上，28℃培养5-7天。
其中，PTC溶液的组分如下：质量体积比(w/v)为60％ PEG 4000(聚乙二醇4000)，10mMTris-HCl，10mM CaCl2，pH 7.5；
再生培养基的组分如下(均为质量百分比)：1％葡萄糖、0.5％(NH4)2SO4、1.5％KH2PO4、1M D-山梨糖醇、1.5％琼脂粉，用NaOH调pH至5.0-5.5，使用前加入终浓度为0.6g/LMgSO4、终浓度为0.4g/LCaCl2，并加入微量元素(终浓度1×，母液1000×)。1000×微量元素母液配方同上。
将上述再生培养基上长出的菌落转接到含有尿苷及潮霉素的筛选培养基上，28℃培养5～7天得转化子。
其中，筛选培养基的组分如下(均为质量百分比)：1％葡萄糖、0.5％(NH4)2SO4、1.5％KH2PO4、1.5％琼脂粉，用NaOH调pH至5.0-5.5，使用前加入终浓度为0.6g/L MgSO4、终浓度为0.4g/L CaCl2，并加入微量元素(终浓度1×，母液1000×)。1000×微量元素母液配方同上。
将上述重组表达载体pMDOEX和pMDOEXR434L分别转化进入里氏木霉原生质体后，由于重组表达载体上携带pyr4基因的上游同源臂序列和下游同源臂序列，上下游同源臂与基因组中pyr4基因的上下游区域发生同源重组，将xyr1或其突变体表达盒以及筛选标记hph整合到pyr4上下游同源臂之间的位点上，从而达到获得相应表达菌株的目的。
本实施例中组成型表达Xyr1及Xyr1R434L突变体的菌株构建示意图见图1。
3、重组里氏木霉菌株的验证与获得
提取得到的转化子基因组DNA，以基因组DNA为模板，采用PCR技术进行上下游同源臂序列锚定验证，其中上游同源臂序列锚定验证引物为AnUp-F和An-R，目的条带的长度约为3.5kb；下游同源臂序列锚定验证引物为An-F/AnDown-R，目的条带的长度约为2.7kb；所述引物序列如下：
AnUp-F:5’-TGTACAGCAAGGAGCAGGTGGATCAG-3’；
An-R:5’-CTCACGCCTCTGTCTGTAAT-3’；
An-F:5’-TCGTTATGTTTATCGGCACTTTGC-3’；
AnDown-R:5’-TTCTGCAGGAAGCTCAGCGTCGAGG-3’。
采用单孢分离的方法对上下游同源臂均正确锚定的转化子进行纯化，采用PCR技术并以Pyr4-in-F和Pyr4-in-R引物进行pyr4上下游同源臂之间的内部基因验证，无目的条带，即内部基因成功被表达盒和筛选标记hph取代，表达菌株构建成功。
其中，所述引物序列如下：
Pyr4-in-F:5’-GGCGACGCACCCGCTGACGGCTTAC-3’，
Pyr4-in-R:5’-CGGTGTAGGCTTTCCACGCTGCTGA-3’。
上述验证PCR反应体系均为20μL，具体反应体系和反应条件参见GenStar2×TaqPCR StarMix(购自武汉飞羿科技有限公司)说明书。
经过鉴定成功获得目标重组菌株OEX和OEXR434L。说明Xyr1的组成型表达菌株和突变体Xyr1R434L的组成型表达菌株均成功构建，分别命名为重组里氏木霉OEX和OEXR434L。
实施例3：组成型表达Xyr1及突变体Xyr1R434L引起的纤维素酶及木聚糖酶生产水平分析
将实施例2中得到的重组里氏木霉OEX和OEXR434L的孢子分别接种于含有0.2％蛋白胨和1％甘油的MA液体培养基中，置于30℃、200rpm的摇床中培养36h，取等量湿重菌丝(约3g)再转接于含有3％葡萄糖的MA液体培养基中，于24h、48h、72h和96h分别取胞外发酵液。
其中，MA液体培养基配方为：每1L培养基中加入Na2HPO4·12H2O 17.907g、(NH4)2SO41.4g、KH2PO4 2.0g、尿素0.3g、Tween-800.5mL，用无水柠檬酸调pH至5.0。使用前需要添加终浓度为0.6g/L MgSO4、终浓度为0.4g/L CaCl2，并加入微量元素(终浓度1×，母液1000×)、10mM尿苷；1000×微量元素母液配方：FeSO4·7H2O 0.5g，MnSO4·H2O 0.16g，ZnSO4·7H2O0.14g，CoCl2·2H2O 0.2g，用ddH2O定容至100mL，过滤除菌。
1、发酵液中滤纸酶活分析
滤纸酶活分析方法如下：将Whatman number 1定量滤纸裁剪成0.5cm*0.5cm大小的滤纸片，毎个反应管(2ml离心管)中放1片该大小的滤纸片。往上述离心管中加入60μL醋酸-醋酸钠缓冲液(pH 4.8)，加入60μL稀释10倍的发酵液，在50℃下反应30min后，加入120μL DNS(3,5-dinnitrosalicylic acid)，混合后煮沸10min以终止反应。冷却后取反应液于550nm波长处读取产物吸光值，结果如图2a所示。
滤纸酶活的一个酶活力单位被定义为每分钟产生1μmol还原糖所需要的酶量。
由图2a可知，首先OEX及OEXR434L菌株在萄萄糖碳源培养条件下能完全恢复Δxyr1产生纤维素酶的能力，并克服碳代谢阻遏，从而大量表达纤维素酶。与OEX相比，OEXR434L的纤维素酶活性显著提高，其FPase活性提高了0.8倍。表明组成型表达Xyr1R434L突变体可极大促进纤维素酶的生产。
2、发酵液中木聚糖酶酶活分析
木聚糖酶酶活分析方法如下：取60μL稀释100倍的发酵液和60μL溶于醋酸-醋酸钠缓冲液(pH 4.8)的木聚糖底物(0.5％，w/v)混合均匀后，在50℃下反应30min后，加入120μLDNS(3,5-dinnitrosalicylic acid)，混合后煮沸10min以终止反应。冷却后取反应液于550nm波长处读取产物吸光值，结果如图2b所示。
木聚糖酶的一个酶活力单位被定义为每分钟产生1μmol还原糖所需要的酶量。
由图2b可知，组成型表达Xyr1的重组里氏木霉OEX的发酵液中木聚糖酶的酶活较低。而在组成型表达突变体Xyr1R434L的重组里氏木霉OEXR434L的发酵液中，木聚糖酶酶活水平得到显著提升，组成型表达突变体Xyr1R434L后木聚糖酶酶活是组成型表达野生型Xyr1的1.8倍，表明组成型表达突变体Xyr1R434L可极大促进木聚糖酶的生产。
3、胞外发酵液的Western blot分析
为了进一步验证上述酶活测定结果，采取了灵敏的Western blot方法对重组里氏木霉OEX和OEXR434L的的胞外发酵液中纤维素酶及木聚糖酶的分泌水平进行定量。使用CBHI、CBHII、EGI、XYNII及XYNI抗体进行Western blot分析，结果如图2c所示。
由图2c可知，杂交条带的亮度分析表明组成型表达突变体Xyr1R434L的重组里氏木霉OEXR434L的胞外发酵液中的CBHI、CBHII、EGI、XYNII及XYNI表达分泌水平明显高于重组里氏木霉OEX的水平，进一步验证了组成型表达突变体Xyr1R434L大幅度提高纤维素酶及木聚糖酶产量，降低酶的生产成本，在经济高效的木质纤维素生物精炼中具有很大的应用价值。
Claims (6)
1.一种里氏木霉转录激活因子突变体Xyr1R434L，其特征在于，其氨基酸序列相对于里氏木霉Xyr1的氨基酸序列SEQ ID NO.1仅存在下述突变：在第434位精氨酸突变为亮氨酸；所述里氏木霉转录激活因子突变体Xyr1R434L的氨基酸序列如SEQ ID NO.3所示。
2.权利要求1所述的里氏木霉转录激活因子突变体Xyr1R434L的编码基因。
3.如权利要求2所述的编码基因，其特征在于，核苷酸序列如SEQ ID NO.4所示。
4.含有权利要求2所述的里氏木霉转录激活因子突变体Xyr1R434L编码基因的重组载体。
5.含有权利要求2所述的里氏木霉转录激活因子突变体Xyr1R434L的编码基因的重组宿主细胞。
6.如权利要求1所述的里氏木霉转录激活因子突变体Xyr1R434L、或权利要求3所述的里氏木霉转录激活因子突变体Xyr1R434L编码基因在提高纤维素酶及木聚糖酶产量中的应用。
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| CN112961788A (zh) * | 2021-02-24 | 2021-06-15 | 江南大学 | 一种在里氏木霉中高产木聚糖酶的方法及其应用 |
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| CN105802854A (zh) * | 2014-12-30 | 2016-07-27 | 中国科学院上海生命科学研究院 | 一种纤维素酶高产菌株及其应用 |
| CN112961788A (zh) * | 2021-02-24 | 2021-06-15 | 江南大学 | 一种在里氏木霉中高产木聚糖酶的方法及其应用 |
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