本文主要研究内容
作者孟庆山（2019）在《里氏木霉纤维素酶基因转录调控因子鉴定及纤维素酶高产菌株构建》一文中研究指出：作为可再生资源,木质纤维素类生物质分布广泛,储量丰富。这类生物质主要组分是纤维素、半纤维素和木质素,其中纤维素的含量最高。利用微生物发酵生产的纤维素酶将纤维素组分降解为葡萄糖作为微生物细胞培养和发酵的基础原料,生产生物燃料和生物基化学品,不仅能减轻对石油等不可再生资源的依赖,而且生物燃料和生物基化学品还具有环境友好的特点,是经济和社会可持续发展的重大需求。然而,纤维素酶生产成本高导致水解糖的成本高,限制了木质纤维素类生物质资源的开发利用。丝状真菌是自然界中降解木质纤维素的主要微生物,其中里氏木霉(Trichodermareesei)最具有代表性,很多纤维素酶高产菌株都来自里氏木霉。目前对里氏木霉产纤维素酶的研究主要集中在两个方面:一方面是从机理上阐明里氏木霉产酶调控机制,为菌株遗传改造育种提供理论支持;另一方面是对里氏木霉酶系组分及性能进行优化,提高酶系各组分水解纤维素的协同效果。T.reesei Rut-C30曾是纤维素酶生产工业菌株,也是研究最广泛的产纤维素酶模式菌株及目前工业菌株选育的出发菌株。本文研究工作从里氏木霉人工锌指蛋白转录因子突变体文库中筛选获得了高产纤维素酶突变株T.reeseiM1和M2;以孢子接种方式进行摇瓶发酵,突变株M1和M2的纤维素酶活较出发菌株分别提高100.8%和53.2%,且M1突变株外泌蛋白量提高69.1%,M2内切纤维素酶活提高64.2%;对突变株中人工锌指蛋白转录因子序列进行分析,发现人工锌指蛋白转录因子基因片段在染色体中整合位点位于Scaffold 1:TrireRUTC30:4597和TrireRUTC30:67627两个基因间隔区,且与上述两基因启动子或终止子距离较远;RT-qPCR分析结果显示,突变株M1和M2中主要纤维素酶基因转录均上调,且纤维素酶主要正调控转录因子基因xyr1在M1突变株中有明显上调,而纤维素酶抑制转录因子基因ace1在两株突变株中都明显下调。上述研究结果表明人工锌指蛋白对T.reesei Rut-C30纤维素酶活性的影响具有多样性。对突变株M2中人工锌指蛋白转录因子预测靶基因的转录分析发现,基因TrireRUTC30:10530(Trctf1)转录水平明显下调,而敲除基因Trctfl导致菌株在纤维素诱导条件下纤维素酶酶活较出发株提高了 43.8%,而使用组成型强启动子pdcl持续高效表达Trctf1后,突变株的纤维素酶生产受到明显抑制,转录组分析进一步发现敲除菌株中纤维素酶转录激活因子Vib1、Xyr1和Ace3的转录均明显上调,而转录抑制因子Rce1转录量则明显下调。推测转录因子Trctf1在T.resei Rut-C30中对纤维素酶合成起负调控作用。这一研究结果表明人工锌指蛋白转录因子技术可用于靶基因功能鉴定。纤维素酶高产菌M2中人工锌指蛋白转录因子由特异DNA结合域和酵母来源的Ga14激活域组成,而T.rresei中纤维素酶合成主要由与酵母Ga14相似的转录激活因子Xyr1调控。基于此,设计了一个新型人工嵌合转录因子AZFP-M2-Xyr1AD并研究其对T.reesei纤维素酶合成的影响。分别将菌株M2中人工锌指蛋白转录因子AZFP-M2-Gal4和嵌合转录因子AZFP-M2-Xyr1 AD定点插入到T.reesei TU-6菌株xyn3基因位点,构建菌株QS1和QS2。摇瓶发酵结果显示:QS1和QS2纤维素酶滤纸酶活分别较出发株提高39.4%和73.7%。转录分析发现QS1和QS2中编码主要纤维素酶和辅助蛋白的基因转录较出发株均明显上调,而编码主要纤维素酶调控因子基因的转录却有显著差异,揭示上述两个人工转录因子参与调控T.reesei纤维素酶合成的分子机制不同。此外,比较出发株TU-6、QS1和QS2菌株发酵获得的粗酶液对碱预处理后玉米秸秆和菊芋秸秆的酶解效果,发现QS2菌株粗酶液水解后葡萄糖释放量比TU-6粗酶液水解分别提高了97.9%和14.0%,比QS1粗酶液处理分别提高90.2%和8.2%。上述实验结果表明,利用T.reesei内源转录因子Xyr1的激活域所构建的人工转录因子AZFP-M2-Xyr1 AD比酵母来源Gal4激活域构建的人工锌指蛋白转录因子AZFP-M2-Gal4更能有效调控T.reesei纤维素酶生产。里氏木霉中纤维素酶系不全导致各酶比例不均衡,严重影响纤维素组分水解过程协同作用效果。之前更多研究偏向于对酶系进行体外复配,但这无疑造成了成本增加。通过基因工程手段对T.reesei纤维素酶系合成进行改造和优化,不仅可以降低产酶成本也可以减少纤维素降解所需酶量。因此,研究工作将主要内切酶基因egl1定点插入到纤维素酶转录抑制因子ace1基因位点,通过基因工程手段对T.reesei酶系进行优化,构建菌株QS305。实验结果表明:QS305在摇瓶发酵中总纤维素酶和内切酶酶活分别较出发株Rut-C30提高90.0%和132.7%;在5-L发酵罐中发酵108 h,QS305菌株纤维素酶产量可达10.7 FPU/mL,较出发株提高75.4%。此外,QS305所产粗酶液较出发株能有效对碱预处理后玉米秸秆和菊芋秸秆进行降解。本研究工作利用人工锌指蛋白技术对.T reesei Rut-C30中未知的纤维素酶调控因子进行挖掘,为深入理解T.reesei菌株纤维素酶调控机制提供了参考,并通过基因工程手段对酶系合成进行了调控,为进一步优化纤维素组分酶解性能奠定了基础。
Abstract
zuo wei ke zai sheng zi yuan ,mu zhi qian wei su lei sheng wu zhi fen bu an fan ,chu liang feng fu 。zhe lei sheng wu zhi zhu yao zu fen shi qian wei su 、ban qian wei su he mu zhi su ,ji zhong qian wei su de han liang zui gao 。li yong wei sheng wu fa jiao sheng chan de qian wei su mei jiang qian wei su zu fen jiang jie wei pu tao tang zuo wei wei sheng wu xi bao pei yang he fa jiao de ji chu yuan liao ,sheng chan sheng wu ran liao he sheng wu ji hua xue pin ,bu jin neng jian qing dui dan you deng bu ke zai sheng zi yuan de yi lai ,er ju sheng wu ran liao he sheng wu ji hua xue pin hai ju you huan jing you hao de te dian ,shi jing ji he she hui ke chi xu fa zhan de chong da xu qiu 。ran er ,qian wei su mei sheng chan cheng ben gao dao zhi shui jie tang de cheng ben gao ,xian zhi le mu zhi qian wei su lei sheng wu zhi zi yuan de kai fa li yong 。si zhuang zhen jun shi zi ran jie zhong jiang jie mu zhi qian wei su de zhu yao wei sheng wu ,ji zhong li shi mu mei (Trichodermareesei)zui ju you dai biao xing ,hen duo qian wei su mei gao chan jun zhu dou lai zi li shi mu mei 。mu qian dui li shi mu mei chan qian wei su mei de yan jiu zhu yao ji zhong zai liang ge fang mian :yi fang mian shi cong ji li shang chan ming li shi mu mei chan mei diao kong ji zhi ,wei jun zhu wei chuan gai zao yo chong di gong li lun zhi chi ;ling yi fang mian shi dui li shi mu mei mei ji zu fen ji xing neng jin hang you hua ,di gao mei ji ge zu fen shui jie qian wei su de xie tong xiao guo 。T.reesei Rut-C30ceng shi qian wei su mei sheng chan gong ye jun zhu ,ye shi yan jiu zui an fan de chan qian wei su mei mo shi jun zhu ji mu qian gong ye jun zhu shua yo de chu fa jun zhu 。ben wen yan jiu gong zuo cong li shi mu mei ren gong xin zhi dan bai zhuai lu yin zi tu bian ti wen ku zhong shai shua huo de le gao chan qian wei su mei tu bian zhu T.reeseiM1he M2;yi bao zi jie chong fang shi jin hang yao ping fa jiao ,tu bian zhu M1he M2de qian wei su mei huo jiao chu fa jun zhu fen bie di gao 100.8%he 53.2%,ju M1tu bian zhu wai bi dan bai liang di gao 69.1%,M2nei qie qian wei su mei huo di gao 64.2%;dui tu bian zhu zhong ren gong xin zhi dan bai zhuai lu yin zi xu lie jin hang fen xi ,fa xian ren gong xin zhi dan bai zhuai lu yin zi ji yin pian duan zai ran se ti zhong zheng ge wei dian wei yu Scaffold 1:TrireRUTC30:4597he TrireRUTC30:67627liang ge ji yin jian ge ou ,ju yu shang shu liang ji yin qi dong zi huo zhong zhi zi ju li jiao yuan ;RT-qPCRfen xi jie guo xian shi ,tu bian zhu M1he M2zhong zhu yao qian wei su mei ji yin zhuai lu jun shang diao ,ju qian wei su mei zhu yao zheng diao kong zhuai lu yin zi ji yin xyr1zai M1tu bian zhu zhong you ming xian shang diao ,er qian wei su mei yi zhi zhuai lu yin zi ji yin ace1zai liang zhu tu bian zhu zhong dou ming xian xia diao 。shang shu yan jiu jie guo biao ming ren gong xin zhi dan bai dui T.reesei Rut-C30qian wei su mei huo xing de ying xiang ju you duo yang xing 。dui tu bian zhu M2zhong ren gong xin zhi dan bai zhuai lu yin zi yu ce ba ji yin de zhuai lu fen xi fa xian ,ji yin TrireRUTC30:10530(Trctf1)zhuai lu shui ping ming xian xia diao ,er qiao chu ji yin Trctfldao zhi jun zhu zai qian wei su you dao tiao jian xia qian wei su mei mei huo jiao chu fa zhu di gao le 43.8%,er shi yong zu cheng xing jiang qi dong zi pdclchi xu gao xiao biao da Trctf1hou ,tu bian zhu de qian wei su mei sheng chan shou dao ming xian yi zhi ,zhuai lu zu fen xi jin yi bu fa xian qiao chu jun zhu zhong qian wei su mei zhuai lu ji huo yin zi Vib1、Xyr1he Ace3de zhuai lu jun ming xian shang diao ,er zhuai lu yi zhi yin zi Rce1zhuai lu liang ze ming xian xia diao 。tui ce zhuai lu yin zi Trctf1zai T.resei Rut-C30zhong dui qian wei su mei ge cheng qi fu diao kong zuo yong 。zhe yi yan jiu jie guo biao ming ren gong xin zhi dan bai zhuai lu yin zi ji shu ke yong yu ba ji yin gong neng jian ding 。qian wei su mei gao chan jun M2zhong ren gong xin zhi dan bai zhuai lu yin zi you te yi DNAjie ge yu he jiao mu lai yuan de Ga14ji huo yu zu cheng ,er T.rreseizhong qian wei su mei ge cheng zhu yao you yu jiao mu Ga14xiang shi de zhuai lu ji huo yin zi Xyr1diao kong 。ji yu ci ,she ji le yi ge xin xing ren gong qian ge zhuai lu yin zi AZFP-M2-Xyr1ADbing yan jiu ji dui T.reeseiqian wei su mei ge cheng de ying xiang 。fen bie jiang jun zhu M2zhong ren gong xin zhi dan bai zhuai lu yin zi AZFP-M2-Gal4he qian ge zhuai lu yin zi AZFP-M2-Xyr1 ADding dian cha ru dao T.reesei TU-6jun zhu xyn3ji yin wei dian ,gou jian jun zhu QS1he QS2。yao ping fa jiao jie guo xian shi :QS1he QS2qian wei su mei lv zhi mei huo fen bie jiao chu fa zhu di gao 39.4%he 73.7%。zhuai lu fen xi fa xian QS1he QS2zhong bian ma zhu yao qian wei su mei he fu zhu dan bai de ji yin zhuai lu jiao chu fa zhu jun ming xian shang diao ,er bian ma zhu yao qian wei su mei diao kong yin zi ji yin de zhuai lu que you xian zhe cha yi ,jie shi shang shu liang ge ren gong zhuai lu yin zi can yu diao kong T.reeseiqian wei su mei ge cheng de fen zi ji zhi bu tong 。ci wai ,bi jiao chu fa zhu TU-6、QS1he QS2jun zhu fa jiao huo de de cu mei ye dui jian yu chu li hou yu mi jie gan he ju yu jie gan de mei jie xiao guo ,fa xian QS2jun zhu cu mei ye shui jie hou pu tao tang shi fang liang bi TU-6cu mei ye shui jie fen bie di gao le 97.9%he 14.0%,bi QS1cu mei ye chu li fen bie di gao 90.2%he 8.2%。shang shu shi yan jie guo biao ming ,li yong T.reeseinei yuan zhuai lu yin zi Xyr1de ji huo yu suo gou jian de ren gong zhuai lu yin zi AZFP-M2-Xyr1 ADbi jiao mu lai yuan Gal4ji huo yu gou jian de ren gong xin zhi dan bai zhuai lu yin zi AZFP-M2-Gal4geng neng you xiao diao kong T.reeseiqian wei su mei sheng chan 。li shi mu mei zhong qian wei su mei ji bu quan dao zhi ge mei bi li bu jun heng ,yan chong ying xiang qian wei su zu fen shui jie guo cheng xie tong zuo yong xiao guo 。zhi qian geng duo yan jiu pian xiang yu dui mei ji jin hang ti wai fu pei ,dan zhe mo yi zao cheng le cheng ben zeng jia 。tong guo ji yin gong cheng shou duan dui T.reeseiqian wei su mei ji ge cheng jin hang gai zao he you hua ,bu jin ke yi jiang di chan mei cheng ben ye ke yi jian shao qian wei su jiang jie suo xu mei liang 。yin ci ,yan jiu gong zuo jiang zhu yao nei qie mei ji yin egl1ding dian cha ru dao qian wei su mei zhuai lu yi zhi yin zi ace1ji yin wei dian ,tong guo ji yin gong cheng shou duan dui T.reeseimei ji jin hang you hua ,gou jian jun zhu QS305。shi yan jie guo biao ming :QS305zai yao ping fa jiao zhong zong qian wei su mei he nei qie mei mei huo fen bie jiao chu fa zhu Rut-C30di gao 90.0%he 132.7%;zai 5-Lfa jiao guan zhong fa jiao 108 h,QS305jun zhu qian wei su mei chan liang ke da 10.7 FPU/mL,jiao chu fa zhu di gao 75.4%。ci wai ,QS305suo chan cu mei ye jiao chu fa zhu neng you xiao dui jian yu chu li hou yu mi jie gan he ju yu jie gan jin hang jiang jie 。ben yan jiu gong zuo li yong ren gong xin zhi dan bai ji shu dui .T reesei Rut-C30zhong wei zhi de qian wei su mei diao kong yin zi jin hang wa jue ,wei shen ru li jie T.reeseijun zhu qian wei su mei diao kong ji zhi di gong le can kao ,bing tong guo ji yin gong cheng shou duan dui mei ji ge cheng jin hang le diao kong ,wei jin yi bu you hua qian wei su zu fen mei jie xing neng dian ding le ji chu 。
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论文作者分别是来自大连理工大学的孟庆山，发表于刊物大连理工大学2019-04-22论文，是一篇关于里氏木霉论文,纤维素酶合成论文,人工锌指蛋白论文,转录调控因子论文,转录组分析论文,基因工程论文,纤维素水解酶系优化论文,大连理工大学2019-04-22论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自大连理工大学2019-04-22论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：里氏木霉论文; 纤维素酶合成论文; 人工锌指蛋白论文; 转录调控因子论文; 转录组分析论文; 基因工程论文; 纤维素水解酶系优化论文; 大连理工大学2019-04-22论文;