CN114107357B - 一种磷酸果糖激酶2突变体生产乙醇的重组丝状真菌及其构建和产乙醇的应用 - Google Patents
一种磷酸果糖激酶2突变体生产乙醇的重组丝状真菌及其构建和产乙醇的应用 Download PDFInfo
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Abstract
本发明公开一种磷酸果糖激酶2突变和/或敲除用于乙醇生产的重组丝状真菌及其构建和产乙醇的应用，其是在所述丝状真菌中敲除内源的磷酸果糖激酶2基因和/或表达突变的磷酸果糖激酶2基因，其中所述突变的磷酸果糖激酶2是指突变后仅保留激酶活性而丧失或降低磷酸酶活性。所获得的基因工程菌株与其出发菌株相比，乙醇合成能力得到提高，且葡萄糖代谢速率得到加快。
Description
技术领域
本发明属于基因工程和生物技术领域。具体地，本发明涉及一种磷酸果糖激酶2基因敲除或者突变生产乙醇的重组丝状真菌及其构建和产乙醇的应用。本发明还涉及所述重组工程菌生产乙醇的方法，以及加快葡萄糖代谢速率的应用。
背景技术
随着世界经济的快速发展，人类对能源的需求日益增加，目前使用的石油等化石能源一方面储量有限，并且在燃烧过程中会产生大量污染。能源和环境问题已经成为人类发展面临的一个重大挑战。交通运输行业是能源消耗的主要部门，并且也是温室气体排放的重要途径，所以迫切需要发展清洁的，可再生的替代燃料。其中燃料乙醇具有原料丰富、清洁可再生以及与现有燃料体系融和度好等特点，成为目前最主要的替代能源(Zabed,H.,Sahu,J.N.,Suely,A.,Boyce,A.N.and Faruq,G.(2017)Bioethanol production fromrenewable sources:Current perspectives and technological progress.Renewableand Sustainable Energy Reviews,71,475-501.)。在燃料乙醇的生产中，第一代燃料乙醇生产所使用的原料主要是以甘蔗和玉米为主，随着燃料乙醇的消耗量快速增长，第一代燃料乙醇“与人争粮，与粮争地”的弊端日益凸显。而以非粮木质纤维素为原料的第二代燃料乙醇能够很好地解决这一问题，并且符合我国能源经济可持续发展的战略方针。我国木质纤维素资源储量丰富，每年产生的农作物秸秆总产量约为9亿吨(田芳,李凡,袁敬伟,许克家,王康,王灿,叔谋,李义,佟毅,崔兆宁.纤维素乙醇产业现状及关键过程技术难点[J].当代化工,2019,48(09):2051-2056)，其中大部分通过焚烧或粗略还田的方式进行处理，不仅造成了资源的巨大浪费，还造成严重的环境污染。充分利用这一数量巨大的生物质资源生产燃料乙醇，将会为我国能源安全提供重要保障，同时有利于优化能源结构，改善生态环境并能促进农村地区经济发展。
目前在燃料乙醇的生产过程中主要使用的发酵微生物是酿酒酵母(Saccharomyces cerevisiae)和运动发酵单胞菌(Zymomonas mobilis)(Sarris,D.andPapanikolaou,S.(2016)Biotechnological production of ethanol:Biochemistry,processes and technologies.Engineering in Life Sciences,16,307-329)，它们在第一代的粮食乙醇生产过程中展现出优良的生产性能。然而在第二代燃料乙醇的生产中，由于缺乏纤维素酶分泌能力及戊糖代谢能力(尤其是木糖和阿拉伯糖)，酿酒酵母和运动发酵单胞菌已经不能够满足生产要求。而生物质降解丝状真菌能够天然分泌纤维素酶，其中，纤维素降解嗜热真菌是耐高温纤维素酶的优良生产者，天然能够分泌大量生物质降解酶类，活性高同时具有更高的温度耐受性，并能充分利用降解产生的纤维二糖、葡萄糖和木糖等多种糖类(Karnaouri A,Topakas E,Antonopoulou I,Christakopoulos P.Genomicinsights into the fungal lignocellulolytic system of Myceliophthorathermophila.Front Microbiol.2014；5:281)。此外，这类微生物最适生长温度较高，可以节约发酵过程中的冷却水用量，降低生产成本，已被证实可以用来大规模工业发酵(VisserH,Joosten V,Punt PJ,Gusakov AV,Olson PT,Joosten R,Bartels J,VisserJ.Development of a mature fungal technology and production platform forindustrial enzymes based on a Myceliophthora thermophila isolate,previouslyknown as Chrysosporium lucknowense C1.Ind Biotechnol.2011；7:214-23)。如果能够通过遗传改造提高丝状真菌的乙醇产量，将有希望实现纤维素到乙醇的一步生产，能够大大简化生产过程，节约成本，把秸秆变废为宝。但是野生型丝状真菌(包括嗜热毁丝霉)的乙醇产量均较低，尚不能直接应用于乙醇的发酵生产，因而对纤维素降解真菌进行遗传改造，实现生物质原料到乙醇的一步高效生产，是目前第二代燃料乙醇生产中一个重要的方向，同时也具有巨大的应用潜力。
要实现丝状真菌纤维素到乙醇的一步发酵，就必须克服丝状真菌乙醇产量低，代谢速率慢的特点。乙醇产量的高低和代谢速率的快慢，很大程度上取决于糖酵解途径的强弱，而糖酵解途径中最重要的调节酶为磷酸果糖激酶，其活性受到ATP，NADH，柠檬酸和长链脂肪酸的抑制，而ADP，AMP和2,6-二磷酸果糖对该酶有激活作用(Lee,J.H.,Liu,R.,Li,J.,Zhang,C.,Wang,Y.,Cai,Q.,Qian,X.,Xia,Y.,Zheng,Y.,Piao,Y.et al.(2017)Stabilization of phosphofructokinase 1platelet isoform by AKT promotestumorigenesis.Nat Commun,8,949.)。其中，2,6-二磷酸果糖是磷酸果糖激酶已知的最强激活剂(Rider,M.H.,Bertrand,L.,Vertommen,D.,Michels,P.A.,Rousseau,G.G.and Hue,L.(2004)6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase:Head-to-headwith a bifunctional enzyme that controls glycolysis.Biochemical Journal,381,561-579.)。因此，提高细胞内2,6-二磷酸果糖的含量，就可以激活磷酸果糖激酶的活性，从而加快糖酵解的速率。然而，2,6-二磷酸果糖这个强有力的变构激活剂的含量受到严格的调控，其合成与降解是由一个多功能酶：磷酸果糖激酶2(Fructose-6-phosphate2-kinase/fructose-2,6-biphosphatase)催化完成。因此，想要提高2,6-二磷酸果糖的含量，加快糖酵解速率，就需要对磷酸果糖激酶2进行改造。
发明内容
为了攻克丝状真菌乙醇产量低，代谢速率慢的瓶颈，本发明通过对嗜热毁丝霉代谢途径的分析和多个基因的改造，找到一个能够同时显著加快葡萄糖代谢速率和提高乙醇产量的基因。所述基因是：磷酸果糖激酶2(Fructose-6-phosphate 2-kinase/fructose-2,6-biphosphatase,Mycth_71484)基因，其编码的蛋白质是调控糖酵解速率的一个关键步骤，负责催化可逆反应： 该反应的产物2,6-二磷酸果糖(D-fructose 2,6-bisphosphate)能够激活磷酸果糖激酶，加快糖酵解速率。为了能够促进葡萄糖代谢速率、提高乙醇产量，本发明对磷酸果糖激酶2基因进行了突变，获得了能够促进葡萄糖代谢速率，提高乙醇产量的突变体基因。同时，为了更进一步验证敲除磷酸果糖激酶2，以及过表达突变的磷酸果糖激酶2是否也能促进葡萄糖代谢速率和提高乙醇产量，本发明对磷酸果糖激酶2敲除菌株和过表达该基因突变体的菌株同样进行了实验验证。此外，为了验证本发明改造策略的有效性和通用性，本发明在嗜热毁丝霉野生型和工程菌株中均进行了验证，并在五种不同碳源(葡萄糖，纤维二糖，纤维素，蔗糖和木聚糖)中进行了乙醇产量的测定。
因此，本发明提供一种丝状真菌的基因工程菌的构建方法，其在所述丝状真菌中敲除其内源的磷酸果糖激酶2基因和/或表达突变的磷酸果糖激酶2基因，其中所述突变的磷酸果糖激酶2基因是指突变后保留激酶活性而丧失或降低磷酸酶活性的突变体基因。
优选地，所述丝状真菌是纤维素降解丝状真菌，优选地，所述丝状真菌选自脉孢菌(Neurospora)、曲霉(Aspergillus)、木霉(Trichoderma)、青霉(Penicillium)、毁丝霉(Myceliophthora)、侧孢霉(Sporotrichum)、镰孢菌(Fusarium)、根霉(Rhizopus)、毛霉(Mucor)和拟青霉(Paecilomyces)，更优选地，所述毁丝霉菌选自嗜热毁丝霉(Myceliophthora thermophila)、异梭毁丝霉(Myceliophthora heterothallica)，最优选为过表达酿酒酵母来源的Scadh1基因的嗜热毁丝霉。
在具体实施方式中，所述敲除包括截短，突变，删除，替换，插入等降低酶活或者完全失活酶活的方法，优选的采用基因编辑的方法敲除所述内源的磷酸果糖激酶2基因。
优选地，所述突变的磷酸果糖激酶2是指如SEQ ID NO.2所述的磷酸果糖激酶2的H233，E306和H371中的一个或两个或三个位点进行了突变，更具体的为三个位点中的一个或两个或三个突变为甘氨酸或丙氨酸。在具体实施方式中，所述导入是指将所要转化的DNA转入宿主细胞中，优选启动子为tef、gpdA、trpC、cbh1、glaA启动子。
本发明还提供一种利用如上述的构建方法所获得的基因重组菌在生产乙醇和/或提高葡萄糖代谢速率中的用途。
在具体实施方式中，其采用单糖或/和聚糖，或者含有单糖或/和聚糖的物质为底物，优选地，所述单糖为葡萄糖、木糖、阿拉伯糖或其组合；所述聚糖包括纤维二糖、木二糖、蔗糖、麦芽糖、木寡糖、纤维寡糖、纤维素、结晶纤维素、半纤维素、淀粉、植物木质生物质或其组合更优选地，所述的植物木质生物质选自农作物秸秆、林业废弃物、能源植物或其部分或全部分解产物；进一步优选地，所述农作物秸秆选自玉米秸秆，小麦秸秆，水稻秸秆，高粱秸秆，大豆秸秆，棉花秸秆，甘蔗渣，玉米芯；所述林业废弃物选自枝叶，锯末；所述能源植物选自甜高粱，柳枝稷，芒草，芦苇或其组合。
在一个优选实施方式中，所述重组菌为嗜热毁丝霉(Myceliophthorathermophila)，优选地为过表达酿酒酵母来源的Scadh1基因的嗜热毁丝霉。
本发明还提供一种生产乙醇的方法，其是在含有单糖或/和聚糖的培养基质中，培养本发明上述的构建方法所获得的基因工程菌，从培养物中收集乙醇。
在优先实施方式中，所述丝状真菌为毁丝霉菌或木霉；优选地，所述毁丝霉菌选自嗜热毁丝霉(Myceliophthora thermophila)、异梭毁丝霉(Myceliophthoraheterothallica)；最优选为所述毁丝霉菌选自嗜热毁丝霉(Myceliophthorathermophila)，进而优选地所述发酵温度为40-60℃，较佳地，为45-52℃，更佳地，为48-50℃。
本发明对丝状真菌尤其是毁丝霉属菌株中磷酸果糖激酶2基因进行改造，因而增强菌株的产乙醇能力和葡萄糖代谢能力，尤其是具备能够有效地利用聚糖或植物生物质这些普通菌株无法利用的发酵底物合成乙醇，将原本不用于生产乙醇的丝状真菌能够有效用于生产乙醇，尤其是采用嗜热毁丝霉菌，通过基因改造，可在普通菌株无法耐受的高温下高产率地合成出乙醇。利用嗜热毁丝霉能够实现生物质原料到乙醇的一步转化，可以降低纤维素乙醇的生产成本，因而具有突出的应用潜力和广泛的现实应用前景。
附图说明
图1为野生型菌株WT，工程菌E17、E18、E19在葡萄糖和纤维素条件下乙醇产量；
图2为野生型菌株WT，工程菌E17、E18、E19在纤维二糖，蔗糖和木聚糖条件下乙醇产量；
图3为工程菌E1，E9，E16在葡萄糖和纤维素条件下乙醇产量；
图4为工程菌E1，E9，E16在纤维二糖，蔗糖和木聚糖条件下乙醇产量；
图5为野生型菌株WT，工程菌E17、E18、E19在培养3，5，7天后培养基中剩余葡萄糖含量；
图6为工程菌E1，E9，E16在培养3，5，7天后培养基中剩余葡萄糖含量；
图7为E17菌株PCR鉴定结果；
图8为E18菌株PCR鉴定结果；
图9为E19菌株PCR鉴定结果；
图10为E9菌株PCR鉴定结果；
图11为E16菌株PCR鉴定结果。
无特殊说明，本申请中Glucose代表葡萄糖，Cellobiose代表纤维二糖，Avicel代表纤维素，Sucrose代表蔗糖，Xylan代表木聚糖，WT代表嗜热毁丝霉野生型菌株ATCC42464。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果，以下结合本发明的优选实施例来进一步说明本发明的技术方案，应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本发明的保护范围。
下述实施例中所用方法如无特别说明均为常规方法，例如Sambrook等人所著的《分子克隆：实验室手册》(New York:Cold SpringHarbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
本发明实施例中所出现的百分比浓度如无特别说明均为质量百分浓度。
实施例1敲除磷酸果糖激酶2的sgRNA表达框和供体DNA的构建
首先本实施例对磷酸果糖激酶2(phosphofructokinase2，PFK2，Mycth_71484)编码基因进行了敲除，采用的是基于CRISPR/Cas9的基因组编辑技术(Qian Liu,etal.Development of a genome-editing CRISPR/Cas9 system in thermophilic fungalMyceliophthora species and its application to hyper-cellulase productionstrain engineering.Biotechnology for Biofuels,volume 10,Article number:1(2017))将目标基因失活。其中，PFK2蛋白的编码核苷酸序列如SEQ ID No.1所示。PFK2蛋白的氨基酸序列如SEQ ID No.2所示。
1.sgRNA表达框构建
通过软件sgRNACas9 tool设计目标基因pfk2(Mycth_71484)的protospacer即靶标位点。采用融合PCR的方法将序列sgRNA启动子、protospacer及scaffold连接在一起，构建出sgRNA表达框U6p-pfk2-sgRNA，序列如SEQ ID No.3所示。具体操作为：首先用引物sgRNA-a及71484SgRNA-b，扩增出启动子和protospacer DNA片段；用引物71484SgRNA-c及sgRNA-d，扩增出protospacer及和scaffold片段，然后以上述扩增出的两个DNA片段同时为模板，用引物sgRNA-a及sgRNA-d扩增出sgRNA表达框全长。
PCR反应体系为：Max Buffer 25μL，10mM dNTPs 1μL，上游/下游引物各1.5μL，模板1μL，/>Max Super-Fidelity DNA Polymerase 1μl，水19μl。
PCR反应条件为：先95℃30s；然后98℃15s，58℃15s，72℃1min，34个循环；最后72℃5min。
2.供体DNA载体构建
本发明中供体DNA片段分别由目标基因上游同源臂，草铵膦抗性基因表达框PtrpC-bar片段，下游同源臂，三部分通过融合PCR的方法连接到一起构成，最终构建为供体DNA片段donor-pfk2，其核酸序列如SEQ ID No.4所示。构建过程为：首先用引物71484up-F和71484up-R扩增出上游同源臂DNA，用引物71484Bar-F和71484Bar-R扩增出草铵膦抗性基因表达框PtrpC-bar片段，用71484down-F和71484down-R扩增出下游同源臂。然后以上游同源臂和PtrpC-bar片段同时为模板，用引物71484up-F和71484Bar-R扩增出上游同源臂和草铵膦抗性基因表达框的融合DNA片段，再用这个融合DNA片段与下游同源臂同时为模板，以71484up-F和71484down-R为引物，扩增出全长的供体DNA片段。最后通过基因测序，证明供体DNA序列的正确。
PCR反应条件为：先95℃30s；然后98℃15s，58℃15s，72℃2min，34个循环；最后72℃5min。
Cas9表达框构建如本发明人已发表文章(Qian Liu,et al.Development of agenome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthoraspecies and its application to hyper-cellulase production strainengineering.Biotechnol Biofuels 2017,10:1.)所述，其核酸序列如SEQ ID No.5所示。
本实施例中载体构建所用引物如下：
实施例2突变的磷酸果糖激酶2表达载体的构建
本实施例中磷酸果糖激酶2(PFK2，Mycth_71484)突变位点为：H233G，E306G，H371G。突变位点基于其结构信息选取，这三个突变位点为该酶的磷酸酶活性关键氨基酸通过突变可以失活磷酸果糖激酶2的磷酸酶活性，而只保留激酶活性。突变后的核苷酸和氨基酸序列分别如SEQ ID NO 6和SEQ ID NO 7所示。以嗜热毁丝霉基因组DNA为模板，采用融合PCR的方法扩增出突变的pfk2基因。具体操作为：首先以基因组DNA为模板，分别用71484mut-a和71484mut-b，71484mut-c和71484mut-d，71484mut-e和71484mut-f，71484mut-g和71484mut-h四对引物扩增出突变的pfk2基因的四个片段，然后用前两个片段同时为模板，71484mut-a和71484mut-d为引物，扩增出前两个DNA片段的融合片段，同理，以71484mut-e和71484mut-h为引物，用后两个DNA片段同时为模板，扩增出后两个DNA片段的融合片段。最后，以上述两个融合的DNA片段为模板，用引物71484mut-a和71484mut-h扩增出全长的突变的pfk2基因。然后用上述融合的全长突变的pfk2基因为模板，用引物Pfk2m-F和Pfk2m-R扩增出用于连接到载体中的DNA片段，利用NEB的Gibson Assembly将上述片段重组入经BcuI和BamHI酶切后的线性化载体pAN52-TB-Intron中，该载体的启动子为Ptef启动子(核酸序列如SEQ ID NO.8所示)，得到突变的pfk2基因的重组表达质粒pAN52-tef-pfk2。
本实施例中载体构建所用引物如下：
| SEQ ID NO. | 引物 | 序列(5’-3’) |
| 9 | 71484mut-a | ATGGCTCCAAAGATTAACGGCA |
| 10 | 71484mut-b | TCAGACTCGCCGCCGCGAGACAGC |
| 11 | 71484mut-c | GCTGTCTCGCGGCGGCGAGTCTGA |
| 12 | 71484mut-d | CAGCGTCTAGGCCATCCAGCGCCT |
| 13 | 71484mut-e | AGGCGCTGGATGGCCTAGACGCTG |
| 14 | 71484mut-f | ATGACGGCCTGGCCCGAGATGATG |
| 15 | 71484mut-g | CATCATCTCGGGCCAGGCCGTCAT |
| 16 | 71484mut-h | TCAAATGCCGCCTTCTGGGGTTG |
| 17 | Pfk2m-F | gccagtttcgttcttcagaactagtATGGCTCCAAAGATTAACGG |
| 18 | Pfk2m-R | gatttcagtaacgttaagtggatccTCAAATGCCGCCTTCTGG |
实施例3将转化DNA导入嗜热毁丝霉
将嗜热毁丝霉ATCC 42464接种于MM斜面培养基中，45℃培养10天后待用。MM培养基配方为：[50×Vogel’s盐20mL，蔗糖20g，琼脂15g，组氨酸(50mg/mL)20mL，定容体积到1L，高压灭菌]。50×Vogel’s盐(1L)配方为：柠檬酸三钠(1/2H2O)150g，无水KH2PO4 250g，无水NH4NO3 100g，MgSO4·7H2O 10g，CaCl2·2H2O 5g，微量元素液5mL，生物素(0.1mg/mL)2.5mL，定容体积到1L。微量元素液配方(100mL)：5g C6H8O·7H2O，5g ZnSO4·7H2O，1g Fe(NH4)2(SO4)·6H2O，0.25g CuSO4·5H2O，0.05g MnSO4·H2O，0.05g H3BO3，0.05g NaMoO4·2H2O，溶于水中，定容至100mL，
1、嗜热毁丝霉原生质体转化
1)菌丝体准备
将成熟的嗜热毁丝霉孢子(用于敲除pfk2基因的出发菌株为野生型菌株，用于过表达pfk2突变体的出发菌株为：野生型嗜热毁丝霉野生型菌株ATCC42464以及野生型敲除了pfk2基因的菌株)，用0.05％吐温80灭菌水收集，经擦镜纸过滤除去菌丝后，涂布于铺有玻璃纸的MM平板，37℃培养16h-24h。
2)原生质体制备
将带有菌丝的玻璃纸放置于30mL裂解液(配方：0.15g裂解酶，无菌操作加入30mL溶液A，过滤除菌；溶液A配方为:1.036g磷酸二氢钾，21.864g山梨醇，溶于90mL去离子水，氢氧化钾调pH到5.6，定量至100mL，高温灭菌)中，28℃裂解2h，每隔15min轻轻摇动。
而后经过擦镜纸过滤后，2000rpm 4℃离心10min，弃上清，加入4mL溶液B(0.735g氯化钙，18.22g山梨醇，1mL Tris·HCl(1M，pH7.5)，溶于90mL去离子水，盐酸调pH到7.6，定量至100mL，高温灭菌)，2000rpm 4℃离心10min；弃上清，按200μL/5ug质粒加入一定体积溶液B。
3)原生质体转化
预冷的15ml离心管，依次加入50μL预冷PEG(12.5g PEG6000，0.368g氯化钙，500μLTris·HCl(1M pH 7.5))，10μL DNA(敲除所用的DNA为：donor-pfk2，Cas9表达框和U6p-pfk2-sgRNA三种DNA，由实施例1所制备；过表达所用的DNA为HindIII线性化的重组表达质粒pAN52-tef-pfk2，由实施例2所制备)，200μL原生质体。放置冰上20min后加入2mL预冷PEG，室温5min，加入4mL溶液B，轻轻混匀。取3mL上述溶液加入12mL融化的含相应抗生素MM培养基中，置于平板中，45℃培养，2d-4d后于体式显微镜下挑取单个菌丝体于相应抗性平板生长。
2、嗜热毁丝霉转化子验证
1)基因组提取
采用酚氯仿法从上述转化过程中挑选的转化子提取基因组DNA，具体包括以下操作：
1)2.0mL的无菌的DNA提取管中加入200mg的锆珠及1mL的裂解液(lysis buffer，配方：0.2M Tris·HCl(pH 7.5)，0.5M NaCl，10mM EDTA，1％SDS(w/v))，挑取平板中生长的嗜热毁丝霉菌丝于DNA提取管中。
2)将所有DNA提取管置于助磨器上，最大转速振荡30s，重复两次。
3)65℃水浴30分钟，在水浴过程中每个几分钟取出漩涡振荡。
4)水浴结束后取出，每管加入80μL pH 7.5的1M Tris·HCl中和。
5)加入400μL的酚：氯仿(1:1)，13000rpm离心5分钟。
6)取300μl上清液于新的1.5mLEP管中，加入600μl 95％的乙醇(DNA)。
7)冰上孵育一小时，随后4℃、13000rpm离心，可看到白色的DNA沉淀到EP管底部。
8)用75％的酒精(DNA级)400μL清洗，4℃、13000rpm离心，轻轻取出上清液。
9)将EP管置于真空浓缩仪中，真空干燥酒精。
10)加入50μL ddH2O溶解DNA，用NanoDrop测定DNA浓度，测完浓度后将提取的DNA置于-20℃冰箱保存，以备下一步进行PCR验证。
2)PCR验证嗜热毁丝霉转化子
对于敲除pfk2基因的转化子，以提取的所述转化子基因组DNA为模版，用引物71484up-F及71484down-R对敲除转化子进行PCR验证。PCR扩增产物进行1％琼脂糖凝胶电泳(110V电压，30分钟)，结果如附图7所示，在凝胶成像系统下看基因扩增条带，显示在引物71484up-F和71484down-R引导下，转化子经PCR扩增获得了～1380bp的目的条带，而野生型菌株用PCR扩增获得了～2000bp的目的条带(用该引物扩增供体DNA获得的长度为1380bp，扩增野生型基因组获得的DNA长度约为2000bp)，结果表明磷酸果糖激酶2基因(PFK2，Mycth_71484)已经从嗜热毁丝霉基因组中敲除，将所获得的敲除菌株命名为E17菌株。
在敲除pfk2基因的基础上(E17菌株)，进一步过表达突变的磷酸果糖激酶2基因的转化子，以提取的所述转化子基因组DNA为模版，用引物Pfk2m-YZ-F及Pfk2m-YZ-R对过表达突变体PFK2的转化子进行PCR验证。PCR扩增产物进行1％琼脂糖凝胶电泳(110V电压，30分钟)，结果如附图8所示：在凝胶成像系统下看基因扩增条带，显示转化子经PCR扩增获得了～592bp的目的条带，而出发菌株E17用PCR扩增没有扩增出任何条带(只有转入过表达突变的pfk2基因的菌株才能在上述引物下扩增出～592bp的目的条带)，结果表明突变的磷酸果糖激酶2基因已经转入嗜热毁丝霉基因组中，将所获得的菌株命名为E18菌株。
为了验证直接过表达突变的磷酸果糖激酶2基因的效果，本发明在野生型菌株中也过表达了突变的pfk2基因，用获得的转化子的基因组DNA为模版，用引物Pfk2m-YZ-F及Pfk2m-YZ-R对转化子进行PCR验证。PCR扩增产物进行1％琼脂糖凝胶电泳(110V电压，30分钟)，结果如图9所示：在凝胶成像系统下看基因扩增条带，显示转化子经PCR扩增获得了～592bp的目的条带，而出发菌株WT用PCR扩增没有扩增出任何条带(只有转入过表达突变的pfk2基因的菌株才能在上述引物下扩增出～592bp的目的条带)，结果表明突变的磷酸果糖激酶2基因已经转入嗜热毁丝霉基因组中，将所获得的菌株命名为E19菌株
实施例4嗜热毁丝霉转化子生产乙醇能力测定
将上述验证的转化子全部接种至250mL三角瓶中，培养基体积为100mL，葡萄糖为碳源培养基配方为：Glucose 75g，Yeast extract 10g,0.15g KH2PO4,0.15g K2HPO4,0.15gMgSO4·7H2O，0.1g CaCl2·2H2O，1mL Tace element，1mL 0.1mg/mL生物素溶液,定容体积到1L，高压蒸汽灭菌。纤维素(Avicel)为碳源培养基配方为：Avicel 75g，Yeast extract10g,0.15g KH2PO4,0.15g K2HPO4,0.15g MgSO4·7H2O，0.1g CaCl2·2H2O，1mL Taceelement，1mL 0.1mg/mL生物素溶液,定容体积到1L，高压蒸汽灭菌。纤维二糖、蔗糖、木聚糖等培养基和葡萄糖/纤维素培养基除碳源外，其余组分均相同，只需将碳源替换为相同质量的对应碳源(纤维二糖、蔗糖、木聚糖)。采用无菌水收集获得的转化子孢子，经2层无菌擦镜纸过滤后，计算孢子的数量，接种量均为2.5*105个/mL，培养基体积为100mL/瓶，45℃条件下培养7天，摇床转速150rpm，第7天取样测定乙醇含量。
1)样品处理：
取1mL发酵液于1.5mL离心管中，12000rpm离心10min，取上清液测定乙醇含量。
2)乙醇和葡萄糖含量测定
处理后的样品用高效液相色谱测定乙醇和葡萄糖含量，其中检测器为示差检测器，5mM H2SO4为流动相，流速为0.5mL/min。
将嗜热毁丝霉野生型菌株ATCC42464(WT)，野生型敲除pfk2基因菌株E17，E17过表达突变的pfk2基因菌株E18，以及野生型过表达突变的pfk2基因菌株E19，分别接种于75g/L葡萄糖(Glucose)、75g/L纤维素(Avicel)、75g/L纤维二糖(Cellobiose)、75g/L木聚糖(Xylan)和75g/L蔗糖(Sucrose)培养基中。培养7天后，样品离心取上清液，测定培养基中生成乙醇的含量。乙醇产量如图1(葡萄糖和纤维素条件)和图2(纤维二糖，蔗糖和木聚糖条件)所示：在以葡萄糖为碳源时，E17菌株的乙醇产量为4.724g/L，比出发菌株WT(3.168g/L)提高了49.1％；E18菌株的乙醇产量为7.371g/L，比出发菌株E17提高了56.03％；E19菌株的乙醇产量为5.839g/L，比出发菌株WT(3.168g/L)提高了84.3％。在以纤维素为碳源发酵7天时，E17菌株的乙醇产量为0.153g/L显著高于出发菌株WT(0.0305g/L)，提高400％；E18菌株的乙醇产量为0.259g/L，比出发菌E17提高69.5％；E19菌株的乙醇产量为0.168g/L，比出发菌株WT(0.0305g/L)提高了449％。在以纤维二糖为碳源时，E17菌株的乙醇产量为6.826g/L，比出发菌株WT(3.454g/L)提高了97.6％；而E18菌株的乙醇产量为8.504g/L，比出发菌株E17提高24.6％；E19菌株的乙醇产量为6.121g/L，比出发菌株WT(3.454g/L)提高了77.2％。在以蔗糖为碳源时，E17菌株的乙醇产量为2.0875g/L，比出发菌株WT(0.0395g/L)提高了51.8倍。而E18菌株的乙醇产量为2.633g/L，比出发菌株E17提高了26.1％；E19菌株的乙醇产量为1.924g/L，比出发菌株WT(0.0395g/L)提高了47.7倍。在以木聚糖为碳源时，E17菌株的乙醇产量为3.581g/L，比出发菌株WT(2.077g/L)提高了72.4％。而E18菌株的乙醇产量为3.962g/L，比出发菌株E17提高了10.6％；E19菌株的乙醇产量为3.002g/L，比出发菌株WT(2.077g/L)提高了44.5％。说明敲除pfk2基因以及过表达突变的pfk2基因，都能够有效提高嗜热毁丝霉在葡萄糖，纤维素，纤维二糖，蔗糖和木聚糖多种碳源条件下的乙醇产量，即在以不同碳源为底物的条件下具有通用性。
此外，对上述四个菌株的葡萄糖代谢速率也进行了比较，将4个菌株同一时间分别接种于75g/L葡萄糖为碳源的培养基中(配方与实施例4相同)，接种后第3，5，7天同时取样，测定培养基中剩余葡萄糖含量，结果如图5所示：在上述时间点，E18菌株培养基中剩余葡萄糖含量均少于E17菌株；E17菌株培养基中剩余葡萄糖含量均少于野生型菌株WT；E19菌株培养基中剩余葡萄糖含量也均少于出发菌野生型菌株WT。表明E18菌株代谢葡萄糖的速率快于E17菌株，E17菌株代谢葡萄糖的速率快于WT菌株，E19菌株代谢葡萄糖的速率也快于WT菌株。说明敲除pfk2基因、以及过表达突变的pfk2基因都能够加快葡萄糖的代谢速率。
实施例5嗜热毁丝霉工程菌株E1中敲除pfk2基因，以及过表达突变的pfk2基因
为了探究敲除pfk2基因，以及过表达突变的pfk2基因，能够提高嗜热毁丝霉乙醇产量和葡萄糖代谢速率这一方法是否通用，在工程菌株中同样进行了验证。选取的工程菌株为E1菌株(WT菌株中过表达酿酒酵母来源的Scadh1基因)，E1菌株具体构建过程如本发明人已发表文章(Li,J.,Zhang,Y.,Li,J.,Sun,T.and Tian,C.(2020)Metabolicengineering of the cellulolytic thermophilic fungus Myceliophthorathermophila to produce ethanol from cellobiose.Biotechnol Biofuels,13,23)所述(也见于中国专利申请201911349044.4中的实施例1)。
在E1菌株中敲除pfk2基因所用的sgRNA，供体DNA和Cas9表达框和实施例1完全相同，均来源于实施例1所构建；转化及验证过程同实施例3完全相同，具体操作详见实施例3；在E1中敲除pfk2基因(Mycth_71484)的转化子命名为E9菌株。在E9菌株中过表达突变的pfk2基因获得的菌株命名为E16菌株，E16菌株所过表达突变的pfk2基因序列、启动子同实施例2完全相同；转化及验证过程同实施例3完全相同，具体操作详见实施例3。E9菌株PCR鉴定结果如图10所示，用鉴定引物71484up-F和71484down-R对基因组扩增后，E9菌株扩增出～1380bp的目的条带，而出发菌株E1扩增获得了～2000bp的目的条带(用该鉴定引物扩增供体DNA获得的长度为1380bp，扩增野生型基因组获得的DNA长度约为2000bp)，表明pfk2基因(Mycth_71484)已经从E1菌株中敲除。E16菌株的鉴定结果为：用验证引物Pfk2m-YZ-F及Pfk2m-YZ-R对过表达PFK2的转化子进行PCR验证，结果如图11所示：在凝胶成像系统下看基因扩增条带，显示转化子E16经PCR扩增获得了～592bp的目的条带，而出发菌株E9用PCR扩增没有扩增出任何条带(只有转入过表达突变的pfk2基因的菌株才能在上述引物下扩增出～592bp的目的条带)，结果表明突变的pfk2基因已经转入E16菌株基因组中。
获得E9和E16菌株后，对菌株乙醇产量进行了测定，将E9，E16菌株与E1菌株分别接种于75g/L葡萄糖(Glucose)、75g/L纤维素(Avicel)、75g/L纤维二糖(Cellobiose)、75g/L木聚糖(Xylan)和75g/L蔗糖(Sucrose)培养基中。培养7天后，样品离心取上清液，测定培养基中生成乙醇的含量，样品处理和测定方法详见实施例4。测定结果如图3(葡萄糖和纤维素条件)和图4(纤维二糖，蔗糖和木聚糖条件)所示：在以葡萄糖为碳源时，E9菌株的乙醇产量为6.91g/L，比出发菌株E1(4.6g/L)提高了50.2％；而E16菌株的乙醇产量为8.07g/L，比出发菌株E9提高了16.8％。在以纤维素为碳源发酵7天时，E9菌株的乙醇产量为0.21g/L显著高于出发菌株E1(0.087g/L)，提高141.4％；E16菌株的乙醇产量为1.013g/L，比出发菌E9提高3.82倍。在以为纤维二糖为碳源时，E9菌株的乙醇产量为10.931g/L，比出发菌株E1(6.709g/L)提高了62.9％。而E16菌株的乙醇产量为13.618g/L，比出发菌株E9提高了24.58％。在以蔗糖为碳源时，E9菌株的乙醇产量为4.737g/L，比出发菌株E1(3.689g/L)提高了28.4％。而E16菌株的乙醇产量为6.9g/L，比出发菌株E9提高了45.69％。在以木聚糖为碳源时，E9菌株的乙醇产量为6.097g/L，比出发菌株E1(5.423g/L)提高了12.43％。而E16菌株的乙醇产量为7.678g/L，比出发菌株E9提高了25.94％。说明敲除以及过表达突变的pfk2基因，能够有效提高嗜热毁丝霉在葡萄糖，纤维素，纤维二糖，蔗糖和木聚糖多种碳源条件下的乙醇产量。
此外，对三个菌株葡萄糖代谢速率也进行了比较，在接种后第3，5，7天取样，测定培养基中剩余葡萄糖含量，结果如图6所示：在上述时间点，E16菌株培养基中剩余葡萄糖含量均少于E9菌株，而E9菌株培养基中剩余葡萄糖含量均少于E1菌株，表明E16菌株代谢葡萄糖的速率快于E9菌株，而E9菌株代谢葡萄糖的速率快于E1菌株。从而说明敲除pfk2基因，以及过表达突变的pfk2基因都能够加快葡萄糖的代谢速率。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 一种磷酸果糖激酶2突变体生产乙醇的重组丝状真菌及其构建和产乙醇的应用
<160> 30
<170> PatentIn Version 3.1
<210> 1
<211> 1332
<212> DNA
<213> Myceliophthora thermophila
<400> 1
atggctccaa agattaacgg cactggcgtc atgcccgagg acacgcgcat ctgcgttgtc 60
atggtcggcc ttccggctcg gggcaagagt tacatagccc aaagagcgca acgctatctc 120
aagtggctgt caatcccggc gaagaccttc aacgtgggca actaccgccg gaaagatgcg 180
ccacatccat cagccgactt cttcgacacc aacaacgccg aaggcgagag acagcgccgc 240
gcagctgcca acgccgccgt gacagacatg atccagtggt tcaaaaccgg cggtgttgtc 300
ggaatccttg acgctacaaa cagcaccaaa gaacggcgga aatgggtact cgagcgactc 360
tctaaagagg ggatcgaggt cctctttgtc gagtccaagt gcgacgacga ggaactgata 420
atggccaaca tccgcgatgt caaaaccact tccccggatt accagggtca ggaccctgag 480
caggctgccc ttgacttccg cgaacgcatc cgccattacg agaaggtcta caagtcgatc 540
aacgacgata atgatgaaga ccacttgacg tacctcaaga taatgaatgt gggcaagaag 600
gtcttcatca accgcattca ggactaccta cagagccgcg tcgtctactt cctcatgaac 660
ctgcatatcc ggccccgctc ggtttggctg tctcgccatg gcgagtctga atacaatttg 720
gaagggcgga ttggcggtga ttcattgctc tctcaccgcg gcgaagagta cgcccgcaaa 780
ttgcccgaat tggttcgcca gtctgttggt agcgaccgac cattaacggt ctggacgtcg 840
acgctgaaac ggaccatcgc cacagcccgg cacttgccaa aacactacaa ccaactgcag 900
tggaaggcgc tggatgagct agacgctggt gtctgcgacg ggttgacgta ccaggagatt 960
gcagaccagt atccggagga ctttcaggcg cgcgacgaag ataaatacaa ctaccgatat 1020
cgcggcggcg agagctaccg cgacgtcgtc attcgcctcg aacccatcat catggaactc 1080
gagcgcagcg aaaacatcct catcatctcg caccaggccg tcatccgctg catctacgcc 1140
tacttcatgc aaaagcctca agaggaaagc ccgtgggtgc ctgtaccgct gcacacccta 1200
atcaagctca cgccccgcgc ctacgacacc ctggtggaga agtacgatgc gaaaatcaag 1260
gccgtgagca cctggcgcgg caaggggagc accgccaaac acgaagatcc aaccccagaa 1320
ggcggcattt ga 1332
<210> 2
<211> 443
<212> PRT
<213> Myceliophthora thermophila
<400> 2
MET Ala Pro Lys Ile Asn Gly Thr Gly Val MET Pro Glu Asp Thr Arg
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Ile Cys Val Val MET Val Gly Leu Pro Ala Arg Gly Lys Ser Tyr Ile
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Ala Gln Arg Ala Gln Arg Tyr Leu Lys Trp Leu Ser Ile Pro Ala Lys
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Thr Phe Asn Val Gly Asn Tyr Arg Arg Lys Asp Ala Pro His Pro Ser
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Ala Asp Phe Phe Asp Thr Asn Asn Ala Glu Gly Glu Arg Gln Arg Arg
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Ala Ala Ala Asn Ala Ala Val Thr Asp MET Ile Gln Trp Phe Lys Thr
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Gly Gly Val Val Gly Ile Leu Asp Ala Thr Asn Ser Thr Lys Glu Arg
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Arg Lys Trp Val Leu Glu Arg Leu Ser Lys Glu Gly Ile Glu Val Leu
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Phe Val Glu Ser Lys Cys Asp Asp Glu Glu Leu Ile MET Ala Asn Ile
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Arg Asp Val Lys Thr Thr Ser Pro Asp Tyr Gln Gly Gln Asp Pro Glu
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Gln Ala Ala Leu Asp Phe Arg Glu Arg Ile Arg His Tyr Glu Lys Val
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Tyr Lys Ser Ile Asn Asp Asp Asn Asp Glu Asp His Leu Thr Tyr Leu
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Lys Ile MET Asn Val Gly Lys Lys Val Phe Ile Asn Arg Ile Gln Asp
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Tyr Leu Gln Ser Arg Val Val Tyr Phe Leu MET Asn Leu His Ile Arg
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Pro Arg Ser Val Trp Leu Ser Arg His Gly Glu Ser Glu Tyr Asn Leu
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Glu Gly Arg Ile Gly Gly Asp Ser Leu Leu Ser His Arg Gly Glu Glu
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Tyr Ala Arg Lys Leu Pro Glu Leu Val Arg Gln Ser Val Gly Ser Asp
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Arg Pro Leu Thr Val Trp Thr Ser Thr Leu Lys Arg Thr Ile Ala Thr
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Ala Arg His Leu Pro Lys His Tyr Asn Gln Leu Gln Trp Lys Ala Leu
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Asp Glu Leu Asp Ala Gly Val Cys Asp Gly Leu Thr Tyr Gln Glu Ile
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Ala Asp Gln Tyr Pro Glu Asp Phe Gln Ala Arg Asp Glu Asp Lys Tyr
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Asn Tyr Arg Tyr Arg Gly Gly Glu Ser Tyr Arg Asp Val Val Ile Arg
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Leu Glu Pro Ile Ile MET Glu Leu Glu Arg Ser Glu Asn Ile Leu Ile
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Ile Ser His Gln Ala Val Ile Arg Cys Ile Tyr Ala Tyr Phe MET Gln
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Lys Pro Gln Glu Glu Ser Pro Trp Val Pro Val Pro Leu His Thr Leu
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Ala Lys Ile Lys Ala Val Ser Thr Trp Arg Gly Lys Gly Ser Thr Ala
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Lys His Glu Asp Pro Thr Pro Glu Gly Gly Ile
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<210> 3
<211> 567
<212> DNA
<213> Myceliophthora thermophila
<400> 3
aggatcggtg gagtgaagtt cggaatcgag gttcggcgat gggtcgtaag catggcgact 60
tcgaacttac ttgcactggc aagcgttgcc agaacggcga gaaaaagaag ggtaagcgat 120
attcgcgtca tgatggactg ttccttttgg aacagtagtt gttgtgggaa gactatgtca 180
cacttgccca cctgcaaggc cagggtcgtg gtcgaacgag accagcctcg gcgctgctgg 240
gagctcaaga tgggcacgtt tgattcgtta gacgtcaaca aggctggagt tcctagtgac 300
agccaaaggc acagccacat taagtggcgc tttatctgtc cactaaggtt caattgtggc 360
tttgagccgc gcagtgtgca gtcgtgcatt ggccacctag ctagcagtat ttaagatcct 420
cttctctccc gagatcttcc tcctcttctt ttctttcttt cctcggagat tgcagaccag 480
tatcgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 540
aagtggcacc gagtcggtgc ttttttt 567
<210> 4
<211> 2954
<212> DNA
<213> Myceliophthora thermophila
<400> 4
ggctcgggtc ggttaatggg aactgctcgc ttcccggttt cagccggccg agtatctaga 60
aggcactctg actggactgg aagttgtgca tatttggaaa tatgaggagc aattattgat 120
tcggcagagg gctcgcaatc agacagagcg tggggggagg cgatttgtgg ttggcattgc 180
tagatggcgg tcagacgggt ccactggcca aaggctgcca gctttcgttc ctgcagggag 240
aaagcgaaaa cccatcgcgt cacgggcgct cgaacccagt cgaacccagc ctgagcgccc 300
ccacttttag tctgcctctt cgggttcgtc cggggaagaa cggctggagc caaaaacccg 360
ctgttggcca ggctctggct gaacttggcg gcgccatgat gcccaggaac caggatcttc 420
gcccgctggc ccgccttact tgccgtccct gggaatcccg atgcaacaag acaagtgatg 480
tagctcgatg ctaccgaggt gcagaaagca aaattgcggt tgctcttctt cctttcgacc 540
taaaatttcc acaccgacga cgtcgccgtc gtccctccga gattttgcag ccgttggagc 600
ccattgtttc attcccgccg tgcctcgctg cttcgacgcg cggcgacagc gccaaaggtg 660
tgacgtcgga agtgggagag ctcccctctc aatagcccac tatcgccaac cgccttctcg 720
gcccgcagcc ggtggcccac gaagcgctca cgccatcaac atctacgcac gctccccgct 780
gacatctccg gctctgcgct gttcctcggc cccaatcagg ctgctacatg tctcgatcac 840
ctatcctgcc ctcctccgcc tccagaacgt tgctacagcg gctgtcgctc agcaaagata 900
ttgaaggagc actttttggg cttggctgga gctagtggag gtcaacaatg aatgcctatt 960
ttggtttagt cgtccaggcg gtgagcacaa aatttgtgtc gtttgacaag atggttcatt 1020
taggcaactg gtcagatcag ccccacttgt agcagtagcg gcggcgctcg aagtgtgact 1080
cttattagca gacaggaacg aggacattat tatcatctgc tgcttggtgc acgataactt 1140
ggtgcgtttg tcaagcaagg taagtgaacg acccggtcat accttcttaa gttcgccctt 1200
cctcccttta tttcagattc aatctgactt acctattcta cccaagcaaa gcttcgatta 1260
ggaagtaacc atgagcccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga 1320
catgccggcg gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg 1380
taccgagccg caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta 1440
tccctggctc gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg 1500
gaaggcacgc aacgcctacg actggacggc cgagtcgacc gtgtacgtct ccccccgcca 1560
ccagcggacg ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca 1620
gggcttcaag agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca 1680
cgaggcgctc ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa 1740
ctggcatgac gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt 1800
cctgcccgtc accgagatct gacatctgcc tcacggggtg aacacgggag gcgtgccaac 1860
gtgatttgcc gtattgtctg ttctcttctc tcagcatatc tagacacgat gtttcgtttc 1920
ctatttttgg cgacaggctg tgccttgcat ttctgggata acatcatatg agaagtccgt 1980
ggttgaaact gggcggaaac ctctgggccg gcgttttgcg gttgggtgtc agcgttggct 2040
tttgctcacg gactgagctt ttgagtccga caaggaaatg atacgctact ggtaccatct 2100
gggagcggtt acttggctgg tttagacggc aaaggaaaac aaaaaacggg ccaactggac 2160
gcatggaatg gacctaaatc gacggctcgc acggaagcac gtatggacgc tgcgtgctct 2220
gccaccgggc agtggcatta tctacatgcc caattgggcc aacatttggc attataaaga 2280
ctaagcattg ccgcttttat ctcttgacca agtttccgat gattctgtca cataccatcc 2340
acgtgtcgag gaaacagcct ttggtcaatg ggagtgtggg cgccatctcc ttattgcgtt 2400
tcgttccttg ataccgcgac aaagtcttct gtccgtggcc taactgtgca gggaggattc 2460
aacttcccaa atatttgaaa cgcatgcttt ctacaggcta gcattgggca ttcggctccg 2520
tgtggatgtc tcagaagaga aaacatggtg ctgctcacat tatacaatga atgccagaga 2580
gtatcagggg caatgacagt gcttaagatg ctaggctaaa gcgtccgttt tgtcgtgcaa 2640
ccatccgccg agattgacac acttgcttct gcaccaaatt gccccttcta gcggtaaacg 2700
ctgaccatga tagcttgaga gcaatacatg gcaaaggcac agcagcgcct atcaataatg 2760
ttggcggggg atgaaacgca tctgggcttg accaacgtcg aagatgacca gcgcgttttg 2820
catgaatgcg tccccaagaa tatatgggcc cgagcctcca tccgcaatgg ccgtcatgca 2880
aagccccgtc accggatcaa ccaaggtacg ataaaggagg tccaaagggc tgatgttgaa 2940
ctttacgcca ttga 2954
<210> 291380
<211> 61341440
<212> DNA1500
<213> 人工合成1560
<220>1620
<221> misc_feature1680
<222> (5718)..(5718)1740
<223> n is a, c, g, or t1800
<220>
<221> misc_feature
<222> (5926)..(5926)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (6017)..(6017)
<223> n is a, c, g, or t
<400> 5
tcctccgagg ttcgacatca gggttcgtca tagggagtga aacacccgcc atgattccgt 60
agccgcgcgc gaagatacga agcagatatt tcacggacat ggcggagata cttgtttccc 120
gtactaaggt agtcatgtcg gagacatctg aacgacagag ctggccaaga gaaccgacca 180
gttgccccag gacgatctag acaaaaaaaa agagagatga gtgggccact tttgccacaa 240
catcgacggc cctgcgaccg cccccaggca aacaaacaaa ccgccgaaca ataatacttt 300
tgtcatttta ggaggagcgt tgtatggata aaaacaacat ctcgttgctg cagaatgtgg 360
acttcaaact tgcagaaaat gggaggcgga tttgcatgat cggagggtag ttgactcacg 420
ccgcaggctg caaatccgtc ctccattatt ccatgaacaa cttcgtaagg ttgggctgag 480
cgccaatgcc taacggaccg ggggccacag cgcaacgtcc cacttaaagg ccagcgtgac 540
atgccagttc cataccaagt agtggcacca gaggcggcca atgctcagta agggcaggga 600
gggaggctca aacgattggc aaaaagaggg gcttgccagt tcagttccct gtgcgagcgc 660
gagaggggca gtttcaaatc tggaggggtg tgttgcgctg gtctgaagag aaagagaaga 720
ctgtacttaa taattgttca aagagtccat catcgcgttg cggactcctc tagctgtatt 780
tagagcccta tcattacttg tcgggtgcga atcaaaatac cgggatgcag ccctctggcg 840
atttgcatgc ggttgtggag gaagtgaagc ctgaatcgcg gggctgggcg gcaaagcacg 900
acgtgaaatt cctggcgaaa ttcgagggct tgccccaccg tggttgaagt ttttgtgctg 960
cgtaacccca ccaacccgcc ttgcccctcc cgcctgccca taaaaacttc gacccctcct 1020
caaatcttct tcgattcttc ctcttcactt ccttcgtcgg catacctgat tcaagcaatc 1080
acctgccact ttcaagtgcg tataccatca tcgatacact ggttcttgac aagtacatcg 1140
tctctaactt tcctttttgc agttttcatt aagcgcaagt cgccagtttc gttcttcaga 1200
aagcttatgg actacaagga ccatgatggc gattacaagg accacgacat cgattataag 1260
gatgatgatg acaagcctcc gaggaaacgt gccaaaacag aagatgagat ggataagaag 1320
tactccatcg gcctcgacat cggcaccaac tccgtcggct gggccgtcat caccgatgag 1380
tacaaggtcc cttccaagaa gttcaaggtc ctcggcaaca ccgatcgcca ttccatcaag 1440
aagaacctga tcggcgccct cctgttcgat tccggcgaaa ccgccgaggc cacccgcctt 1500
aaacgcaccg cccgtcgccg ctacacccgc cgcaagaacc gcatctgcta cctccaagaa 1560
atcttctcca acgagatggc caaggtcgat gatagcttct tccaccgcct cgaagagtcc 1620
ttcctggtcg aagaggataa gaagcacgag cgccatccta tcttcggcaa catcgtcgat 1680
gaggtcgcct accatgagaa gtaccctacc atctaccatc tccgcaagaa gctcgtcgat 1740
tccaccgata aggccgatct ccgcctcatc tacctcgccc tcgcccatat gatcaagttc 1800
cgcggccatt tcctcatcga gggcgatctc aaccctgata actccgatgt cgataagctg 1860
ttcatccagc tcgtccagac ctacaaccag ctgttcgagg aaaaccctat caacgcctcc 1920
ggcgtcgatg ccaaggccat cctctccgct cgcctctcca agtctcgccg ccttgagaac 1980
cttatcgccc agctccctgg cgagaagaag aacggcctct tcggcaacct gatcgccctc 2040
tccctcggcc tcacccctaa cttcaagtcc aacttcgatc tcgccgagga tgccaagctc 2100
cagctctcca aggataccta cgatgatgat ctcgataacc tcctcgccca gatcggcgat 2160
cagtacgccg atctgttcct cgccgccaag aacctctccg atgccatcct cctctccgac 2220
atcctccgcg tcaacaccga gatcaccaag gcccctctgt ccgcctccat gatcaagcgc 2280
tacgatgagc atcatcagga cctcaccctg ctcaaggccc tcgtccgcca gcagctccct 2340
gagaagtaca aagagatttt cttcgatcag tccaagaacg gctacgccgg ctacatcgat 2400
ggcggcgctt cccaagaaga gttctacaag ttcatcaagc ctatccttga gaagatggat 2460
ggcaccgagg aactcctcgt caagctcaac cgcgaggacc tcctccgcaa gcagcgcacc 2520
ttcgataacg gctccatccc tcatcaaatc catctcggcg agctgcatgc catcttgcgc 2580
cgccaagagg atttctaccc attcctcaag gataaccgcg agaagatcga aaagattctc 2640
accttccgca tcccttacta cgtcggccct ctcgctcgcg gcaactcccg cttcgcctgg 2700
atgacccgca agtccgagga aaccatcacc ccttggaact tcgaggaagt cgtcgataag 2760
ggcgcctccg cccagtcctt catcgagcgc atgaccaact tcgataagaa cctccctaac 2820
gagaaggtcc tccctaagca ctccctgctc tacgagtact tcaccgtcta caacgagctg 2880
accaaggtca agtacgtcac cgagggtatg cgcaagcctg ccttcctgtc cggcgagcag 2940
aagaaggcca tcgtcgatct gctgttcaag accaaccgca aggtcaccgt caagcagctc 3000
aaagaggatt acttcaagaa aatcgagtgc ttcgattccg tcgagatcag cggcgtcgag 3060
gaccgcttca acgcctccct cggaacctac catgatctcc tcaagattat caaggataag 3120
gatttcctcg acaacgagga aaacgaggac atccttgagg acatcgtcct caccctcacc 3180
ctcttcgagg accgcgaaat gatcgaggaa cgcctcaaga cctacgccca tctcttcgat 3240
gataaggtca tgaagcagct caagcgccgt cgctacaccg gctggggtcg cctctcccgc 3300
aagctcatca acggcatccg cgataagcag tccggcaaga ctatcctcga tttcctcaag 3360
tccgatggct tcgccaaccg caacttcatg cagctcatcc atgatgattc cctcaccttc 3420
aaagaggaca tccagaaggc ccaggtcagc ggccagggcg attccctcca tgagcatatc 3480
gccaacctcg ccggctcccc tgccatcaag aagggcatcc tccagaccgt caaggtcgtc 3540
gatgagctgg tcaaggtcat gggccgccat aagcctgaga acatcgtcat cgagatggcc 3600
cgcgagaacc agaccaccca gaagggccag aagaactccc gcgagcgcat gaagcgcatc 3660
gaggaaggca tcaaagagct gggcagccaa atcctcaaag agcatcctgt cgagaacacc 3720
cagctccaga acgagaagct ctacctctac tacctccaga acggccgcga tatgtacgtc 3780
gatcaagagc tggacatcaa ccgcctctcc gattacgatg tcgatcatat cgtccctcag 3840
tccttcctga aggatgattc catcgataac aaggtcctca cccgctccga taagaaccgc 3900
ggcaagtccg ataacgtccc ttccgaagag gtcgtcaaga agatgaagaa ctactggcgc 3960
cagctcctca acgccaagct catcacccag cgcaagttcg ataacctcac caaggccgag 4020
cgcggtggcc tctccgagct ggataaggcc ggcttcatca agcgccagct cgtcgaaacc 4080
cgccagatca ccaagcacgt cgcccaaatc ctcgattccc gcatgaacac caagtacgat 4140
gagaacgata agctcatccg cgaagtcaag gtcatcaccc tcaagtccaa gctcgtcagc 4200
gatttccgca aggatttcca gttctacaag gtccgcgaga tcaacaacta ccatcatgcc 4260
catgatgcct acctcaacgc cgtcgtcggc accgccctca tcaagaagta ccccaagctc 4320
gaatccgagt tcgtctacgg tgattacaag gtctacgatg tccgcaagat gatcgccaag 4380
tccgagcaag agatcggcaa ggctaccgcc aagtacttct tctactccaa catcatgaat 4440
ttcttcaaga ccgaaatcac cctcgccaac ggcgaaatcc gcaagcgccc tctcatcgag 4500
actaacggcg agactggcga gatcgtctgg gataagggcc gcgatttcgc caccgtccgc 4560
aaggtcctct ccatgcctca ggtcaacatc gtcaagaaaa ccgaggtcca gaccggcggc 4620
ttctccaaag agtccatcct ccccaagcgc aactccgata agctgatcgc ccgcaagaag 4680
gattgggacc ctaagaagta cggcggcttc gattccccta ccgtcgccta ctccgtcctc 4740
gtcgtcgcca aggtcgagaa gggcaagtcc aagaagctca agtccgtcaa agagctgctc 4800
ggcatcacta ttatggaacg ctccagcttc gagaagaacc ctatcgattt ccttgaggcc 4860
aagggctaca aagaggtcaa gaaggacctc atcatcaagc tccccaagta ctccctgttc 4920
gagcttgaga acggccgcaa gcgcatgctc gcctccgccg gtgagcttca gaagggcaac 4980
gagctggccc tgccttccaa gtacgtcaac ttcctctacc tcgcctccca ttacgagaag 5040
ctcaagggct cccctgagga taacgagcag aagcagctgt tcgtcgagca gcataagcac 5100
tacctcgatg agatcatcga gcagatcagc gagttctcca agcgcgtcat cctcgccgat 5160
gccaacctcg ataaggtcct gtccgcctac aacaagcacc gcgataagcc tatccgcgag 5220
caggccgaga acatcatcca tctcttcacc ctcaccaacc tcggtgcccc tgccgccttc 5280
aagtacttcg ataccaccat cgatcgcaag cgctacacct ccaccaaaga ggtcctggac 5340
gccaccctca tccatcagtc catcaccggc ctctacgaaa cccgcatcga tctctcccag 5400
ctcggcggcg accctccgag gaaacgtgcc aaaacagaag atgagtgatg aggatccact 5460
taacgttact gaaatcatca aacagcttga cgaatctgga tataagatcg ttggtgtcga 5520
tgtcagctcc ggagttgaga caaatggtgt tcaggatctc gataagatac gttcatttgt 5580
ccaagcagca aagagtgcct tctagtgatt taatagctcc atgtcaacaa gaataaaacg 5640
cgttttcggg tttacctctt ccagatacag ctcatctgca atgcattaat gcattgactg 5700
caacctagta acgccttnca ggctccggcg aagagaagaa tagcttagca gagctatttt 5760
cattttcggg agacgagatc aagcagatca acggtcgtca agagacctac gagactgagg 5820
aatccgctct tggctccacg cgactatata tttgtctcta attgtacttt gacatgctcc 5880
tcttctttac tctgatagct tgactatgaa aattccgtca ccagcncctg ggttcgcaaa 5940
gataattgca tgtttcttcc ttgaactctc aagcctacag gacacacatt catcgtaggt 6000
ataaacctcg aaatcanttc ctactaagat ggtatacaat agtaaccatg catggttgcc 6060
tagtgaatgc tccgtaacac ccaatacgcc ggccgaaact tttttacaac tctcctatga 6120
gtcgtttacc caga 6134
<210> 6
<211> 1332
<212> DNA
<213> Myceliophthora thermophila
<400> 6
atggctccaa agattaacgg cactggcgtc atgcccgagg acacgcgcat ctgcgttgtc 60
atggtcggcc ttccggctcg gggcaagagt tacatagccc aaagagcgca acgctatctc 120
aagtggctgt caatcccggc gaagaccttc aacgtgggca actaccgccg gaaagatgcg 180
ccacatccat cagccgactt cttcgacacc aacaacgccg aaggcgagag acagcgccgc 240
gcagctgcca acgccgccgt gacagacatg atccagtggt tcaaaaccgg cggtgttgtc 300
ggaatccttg acgctacaaa cagcaccaaa gaacggcgga aatgggtact cgagcgactc 360
tctaaagagg ggatcgaggt cctctttgtc gagtccaagt gcgacgacga ggaactgata 420
atggccaaca tccgcgatgt caaaaccact tccccggatt accagggtca ggaccctgag 480
caggctgccc ttgacttccg cgaacgcatc cgccattacg agaaggtcta caagtcgatc 540
aacgacgata atgatgaaga ccacttgacg tacctcaaga taatgaatgt gggcaagaag 600
gtcttcatca accgcattca ggactaccta cagagccgcg tcgtctactt cctcatgaac 660
ctgcatatcc ggccccgctc ggtttggctg tctcgcggcg gcgagtctga atacaatttg 720
gaagggcgga ttggcggtga ttcattgctc tctcaccgcg gcgaagagta cgcccgcaaa 780
ttgcccgaat tggttcgcca gtctgttggt agcgaccgac cattaacggt ctggacgtcg 840
acgctgaaac ggaccatcgc cacagcccgg cacttgccaa aacactacaa ccaactgcag 900
tggaaggcgc tggatggcct agacgctggt gtctgcgacg ggttgacgta ccaggagatt 960
gcagaccagt atccggagga ctttcaggcg cgcgacgaag ataaatacaa ctaccgatat 1020
cgcggcggcg agagctaccg cgacgtcgtc attcgcctcg aacccatcat catggaactc 1080
gagcgcagcg aaaacatcct catcatctcg ggccaggccg tcatccgctg catctacgcc 1140
tacttcatgc aaaagcctca agaggaaagc ccgtgggtgc ctgtaccgct gcacacccta 1200
atcaagctca cgccccgcgc ctacgacacc ctggtggaga agtacgatgc gaaaatcaag 1260
gccgtgagca cctggcgcgg caaggggagc accgccaaac acgaagatcc aaccccagaa 1320
ggcggcattt ga 1332
<210> 7
<211> 443
<212> PRT
<213> Myceliophthora thermophila
<400> 7
Met Ala Pro Lys Ile Asn Gly Thr Gly Val Met Pro Glu Asp Thr Arg
1 5 10 15
Ile Cys Val Val Met Val Gly Leu Pro Ala Arg Gly Lys Ser Tyr Ile
20 25 30
Ala Gln Arg Ala Gln Arg Tyr Leu Lys Trp Leu Ser Ile Pro Ala Lys
35 40 45
Thr Phe Asn Val Gly Asn Tyr Arg Arg Lys Asp Ala Pro His Pro Ser
50 55 60
Ala Asp Phe Phe Asp Thr Asn Asn Ala Glu Gly Glu Arg Gln Arg Arg
65 70 75 80
Ala Ala Ala Asn Ala Ala Val Thr Asp Met Ile Gln Trp Phe Lys Thr
85 90 95
Gly Gly Val Val Gly Ile Leu Asp Ala Thr Asn Ser Thr Lys Glu Arg
100 105 110
Arg Lys Trp Val Leu Glu Arg Leu Ser Lys Glu Gly Ile Glu Val Leu
115 120 125
Phe Val Glu Ser Lys Cys Asp Asp Glu Glu Leu Ile Met Ala Asn Ile
130 135 140
Arg Asp Val Lys Thr Thr Ser Pro Asp Tyr Gln Gly Gln Asp Pro Glu
145 150 155 160
Gln Ala Ala Leu Asp Phe Arg Glu Arg Ile Arg His Tyr Glu Lys Val
165 170 175
Tyr Lys Ser Ile Asn Asp Asp Asn Asp Glu Asp His Leu Thr Tyr Leu
180 185 190
Lys Ile Met Asn Val Gly Lys Lys Val Phe Ile Asn Arg Ile Gln Asp
195 200 205
Tyr Leu Gln Ser Arg Val Val Tyr Phe Leu Met Asn Leu His Ile Arg
210 215 220
Pro Arg Ser Val Trp Leu Ser Arg Gly Gly Glu Ser Glu Tyr Asn Leu
225 230 235 240
Glu Gly Arg Ile Gly Gly Asp Ser Leu Leu Ser His Arg Gly Glu Glu
245 250 255
Tyr Ala Arg Lys Leu Pro Glu Leu Val Arg Gln Ser Val Gly Ser Asp
260 265 270
Arg Pro Leu Thr Val Trp Thr Ser Thr Leu Lys Arg Thr Ile Ala Thr
275 280 285
Ala Arg His Leu Pro Lys His Tyr Asn Gln Leu Gln Trp Lys Ala Leu
290 295 300
Asp Gly Leu Asp Ala Gly Val Cys Asp Gly Leu Thr Tyr Gln Glu Ile
305 310 315 320
Ala Asp Gln Tyr Pro Glu Asp Phe Gln Ala Arg Asp Glu Asp Lys Tyr
325 330 335
Asn Tyr Arg Tyr Arg Gly Gly Glu Ser Tyr Arg Asp Val Val Ile Arg
340 345 350
Leu Glu Pro Ile Ile Met Glu Leu Glu Arg Ser Glu Asn Ile Leu Ile
355 360 365
Ile Ser Gly Gln Ala Val Ile Arg Cys Ile Tyr Ala Tyr Phe Met Gln
370 375 380
Lys Pro Gln Glu Glu Ser Pro Trp Val Pro Val Pro Leu His Thr Leu
385 390 395 400
Ile Lys Leu Thr Pro Arg Ala Tyr Asp Thr Leu Val Glu Lys Tyr Asp
405 410 415
Ala Lys Ile Lys Ala Val Ser Thr Trp Arg Gly Lys Gly Ser Thr Ala
420 425 430
Lys His Glu Asp Pro Thr Pro Glu Gly Gly Ile
435 440
<210> 8
<211> 1216
<212> DNA
<213> Myceliophthora thermophila
<400> 8
CCTCCGAGGT TCGACATCAG GGTTCGTCAT AGGGAGTGAA ACACCCGCCA TGATTCCGTA 60
GCCGCGCGCG AAGATACGAA GCAGATATTT CACGGACATG GCGGAGATAC TTGTTTCCCG 120
TACTAAGGTA GTCATGTCGG AGACATCTGA ACGACAGAGC TGGCCAAGAG AACCGACCAG 180
TTGCCCCAGG ACGATCTAGA CAAAAAAAAA GAGAGATGAG TGGGCCACTT TTGCCACAAC 240
ATCGACGGCC CTGCGACCGC CCCCAGGCAA ACAAACAAAC CGCCGAACAA TAATACTTTT 300
GTCATTTTAG GAGGAGCGTT GTATGGATAA AAACAACATC TCGTTGCTGC AGAATGTGGA 360
CTTCAAACTT GCAGAAAATG GGAGGCGGAT TTGCATGATC GGAGGGTAGT TGACTCACGC 420
CGCAGGCTGC AAATCCGTCC TCCATTATTC CATGAACAAC TTCGTAAGGT TGGGCTGAGC 480
GCCAATGCCT AACGGACCGG GGGCCACAGC GCAACGTCCC ACTTAAAGGC CAGCGTGACA 540
TGCCAGTTCC ATACCAAGTA GTGGCACCAG AGGCGGCCAA TGCTCAGTAA GGGCAGGGAG 600
GGAGGCTCAA ACGATTGGCA AAAAGAGGGG CTTGCCAGTT CAGTTCCCTG TGCGAGCGCG 660
AGAGGGGCAG TTTCAAATCT GGAGGGGTGT GTTGCGCTGG TCTGAAGAGA AAGAGAAGAC 720
TGTACTTAAT AATTGTTCAA AGAGTCCATC ATCGCGTTGC GGACTCCTCT AGCTGTATTT 780
AGAGCCCTAT CATTACTTGT CGGGTGCGAA TCAAAATACC GGGATGCAGC CCTCTGGCGA 840
TTTGCATGCG GTTGTGGAGG AAGTGAAGCC TGAATCGCGG GGCTGGGCGG CAAAGCACGA 900
CGTGAAATTC CTGGCGAAAT TCGAGGGCTT GCCCCACCGT GGTTGAAGTT TTTGTGCTGC 960
GTAACCCCAC CAACCCGCCT TGCCCCTCCC GCCTGCCCAT AAAAACTTCG ACCCCTCCTC 1020
AAATCTTCTT CGATTCTTCC TCTTCACTTC CTTCGTCGGC ATACCTGATT CAAGCAATCA 1080
CCTGCCACTT TCAAGTGCGT ATACCATCAT CGATACACTG GTTCTTGACA AGTACATCGT 1140
CTCTAACTTT CCTTTTTGCA GTTTTCATTA AGCGCAAGTC GCCAGTTTCG TTCTTCAGAA 1200
CACAAATACC GTCAAA 1216
<210> 9
<211> 22
<212> DNA
<213> 引物
<400> 9
ATGGCTCCAA AGATTAACGG CA 22
<210> 10
<211> 24
<212> DNA
<213> 引物
<400> 10
TCAGACTCGC CGCCGCGAGA CAGC 24
<210> 11
<211> 24
<212> DNA
<213> 引物
<400> 11
GCTGTCTCGC GGCGGCGAGT CTGA 24
<210> 12
<211> 24
<212> DNA
<213> 引物
<400> 12
CAGCGTCTAG GCCATCCAGC GCCT 24
<210> 13
<211> 24
<212> DNA
<213> 引物
<400> 13
AGGCGCTGG ATGGCCTAGAC GCTG 24
<210> 14
<211> 24
<212> DNA
<213> 引物
<400> 14
ATGACGGCC TGGCCCGAGAT GATG 24
<210> 15
<211> 24
<212> DNA
<213> 引物
<400> 15
CATCATCTCG GGCCAGGCCG TCAT 24
<210> 16
<211> 23
<212> DNA
<213> 引物
<400> 16
TCAAATGCC GCCTTCTGGGG TTG 23
<210> 17
<211> 45
<212> DNA
<213> 引物
<400> 17
gccagtttcg ttcttcagaa ctagtatggc tccaaagatt aacgg 45
<210> 18
<211> 43
<212> DNA
<213> 引物
<400> 18
gatttcagta acgttaagtg gatcctcaaa tgccgccttc tgg 43
<210> 19
<211> 22
<212> DNA
<213> 引物
<400> 19
aggatcggtg gagtgaagtt cg 22
<210> 20
<211> 40
<212> DNA
<213> 引物
<400> 20
atactggtct gcaatctccg aggaaagaaa gaaaagaaga 40
<210> 21
<211> 42
<212> DNA
<213> 引物
<400> 21
agattgcaga ccagtatcgt tttagagcta gaaatagcaa gt 42
<210> 22
<211> 23
<212> DNA
<213> 引物
<400> 22
aaaaaaagca ccgactcggt gcc 23
<210> 23
<211> 43
<212> DNA
<213> 引物
<400> 23
tacacagtac acgaggactt ctagaggctc gggtcggtta atg 43
<210> 24
<211> 28
<212> DNA
<213> 引物
<400> 24
ccttcaatat ctttgctgag cgacagcc 28
<210> 25
<211> 33
<212> DNA
<213> 引物
<400> 25
ctcagcaaag atattgaagg agcacttttt ggg 33
<210> 26
<211> 28
<212> DNA
<213> 引物
<400> 26
gaggcagatg tcagatctcg gtgacggg 28
<210> 27
<211> 28
<212> DNA
<213> 引物
<400> 27
cgagatctga catctgcctc acggggtg 28
<210> 28
<211> 50
<212> DNA
<213> 引物
<400> 28
aatatcatct tctgtcgagg aattctcaat ggcgtaaagt tcaacatcag 50
<210> 29
<211> 27
<212> DNA
<213> 引物
<400> 29
catcatcgat acactggttc ttgacaa 27
<210> 30
<211> 25
<212> DNA
<213> 引物
<400> 30
ctcagggtcc tgaccctggt aatcc 25
Claims (11)
1.一种丝状真菌的基因工程菌的构建方法，其特征在于，在所述丝状真菌中敲除其内源的磷酸果糖激酶2基因，并且进一步还导入并表达突变的磷酸果糖激酶2基因，其中所述突变的磷酸果糖激酶2是由同时突变 H233G，E306G，H371G后的获得；
所述丝状真菌为嗜热毁丝霉(Myceliophthora thermophila)；
所述的磷酸果糖激酶2基因是指编码氨基酸序列如SEQ ID No.2 所示的基因。
2.如权利要求1所述的构建方法，其特征在于，所述敲除是通过截短，突变，删除，替换，插入实现降低酶活性或者完全失活酶活性的方法。
3.如权利要求2所述的构建方法，其特征在于，采用基因编辑方法敲除所述内源的磷酸果糖激酶2基因。
4.如权利要求1所述的构建方法，其特征在于，所述导入是将启动子控制下的突变的磷酸果糖激酶2基因的表达载体转入宿主细胞中。
5.如权利要求4所述的构建方法，其特征在于，所述启动子选自tef、gpdA、trpC、cbh1、glaA启动子。
6.一种利用如权利要求1至5任一项所述的构建方法所获得的基因重组菌在生产乙醇和/或提高葡萄糖代谢速率中的用途。
7.如权利要求6所述的用途，其特征在于，其采用单糖或/和聚糖，或者含有单糖或/和聚糖的物质为底物，所述单糖为葡萄糖、木糖、阿拉伯糖或其组合；所述聚糖为纤维二糖、木二糖、蔗糖、麦芽糖、木寡糖、纤维寡糖、纤维素、半纤维素、淀粉或其组合；
所述含有单糖或/和聚糖的物质是植物木质生物质，所述植物木质生物质选自农作物秸秆、林业废弃物、能源植物或其部分或全部分解产物。
8.如权利要求7所述的用途，其特征在于，所述农作物秸秆选自玉米秸秆，小麦秸秆，水稻秸秆，高粱秸秆，大豆秸秆，棉花秸秆，甘蔗渣，玉米芯；所述林业废弃物选自枝叶，锯末；所述能源植物选自甜高粱，柳枝稷，芒草，芦苇或其组合。
9.一种生产乙醇的方法，其特征在于，在含有单糖或/和聚糖的培养基质中，培养如权利要求1至5任一项所述的构建方法所获得的基因工程菌，从培养物中收集乙醇；
所述单糖为葡萄糖、木糖、阿拉伯糖或其组合；所述聚糖为纤维二糖、木二糖、蔗糖、麦芽糖、木寡糖、纤维寡糖、纤维素、半纤维素、淀粉或其组合。
10.如权利要求9所述的生产乙醇的方法，其特征在于，所述发酵温度为40-60℃。
11.如权利要求10所述的生产乙醇的方法，其特征在于，所述发酵温度为45-52℃。
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| CN202010874879.8A CN114107357B (zh) | 2020-08-27 | 2020-08-27 | 一种磷酸果糖激酶2突变体生产乙醇的重组丝状真菌及其构建和产乙醇的应用 |
| BR112022012602A BR112022012602A2 (pt) | 2019-12-24 | 2020-12-09 | Método de construção de fungos geneticamente modificados de fungos filamentosos e método de produção de etanol |
| US17/786,997 US20230053729A1 (en) | 2019-12-24 | 2020-12-09 | A recombinant filamentous fungus for producing ethanol and its construction and application |
| PCT/CN2020/134922 WO2021129396A1 (zh) | 2019-12-24 | 2020-12-09 | 一种生产乙醇的重组丝状真菌及其构建和应用 |
| EP20904625.9A EP4063484A4 (en) | 2019-12-24 | 2020-12-09 | RECOMBINANT FILAMENTOUS FUNGI FOR THE PRODUCTION OF ETHANOL, AND CONSTRUCTION AND APPLICATION THEREOF |
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| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (3)
| Title |
|---|
| Carole H. Sellem等.Mutations in the phosphatase domain of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase result in the transcriptional activation of the alternative oxidase and gluconeogenic pathways in Podospora anserina.《Fungal Genetics and Biology》.2019,第130卷第7页第2-4段. * |
| Metabolic engineering of the cellulolytic thermophilic fungus Myceliophthora thermophila to produce ethanol from cellobiose;Jinyang Li等;《Biotechnol Biofuels.》;第13卷(第23期);第1-15页 * |
| Perspectives and new directions for the production of bioethanol using consolidated bioprocessing of lignocellulose;Qi Xu等;《Curr Opin Biotechnol. 》;第20卷(第3期);第364-371页 * |
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