CN114585726A - 被修饰以用于生产复合酶的木霉属真菌菌株 - Google Patents

被修饰以用于生产复合酶的木霉属真菌菌株 Download PDF

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CN114585726A
CN114585726A CN201980098500.4A CN201980098500A CN114585726A CN 114585726 A CN114585726 A CN 114585726A CN 201980098500 A CN201980098500 A CN 201980098500A CN 114585726 A CN114585726 A CN 114585726A
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马里奥·泰亚戈·穆拉卡米
卢卡斯·米兰达·丰塞卡
卢卡斯·萨莱拉·帕雷拉斯
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Abstract

本发明涉及一种木霉属(Trichoderma)新菌株，包括能够提高复合酶的产量的基因修饰，至少涉及根据SEQ ID No.1的转录因子Xyr1的上调；根据SEQ ID No.2的ACE1基因的中断；根据SEQ ID No.3的SLP1基因的中断；以及根据SEQ ID No.4的来自埃默森罗萨氏菌(Rasamsonia emersonii)的Cel3a基因的表达。

Description

被修饰以用于生产复合酶的木霉属真菌菌株
技术领域
本发明涉及一种木霉属(Trichoderma)新菌株，包括能够提高复合酶的产量的基因修饰。
背景技术
纤维素酶或半纤维素酶是构成能够作用于纤维素物质并促进其水解的复合物的酶。此类酶是在糖的释放中作用的高特异性生物催化剂，由于葡萄糖有可能转化为乙醇，所以葡萄糖是引起最大工业兴趣的一种糖。纤维素酶的高效生产对于生物精炼的定义很重要，生物精炼使用可再生木质纤维素物质生产具有高附加值的燃料和化学品。
由于其工业重要性，近几十年来，已经公布了关于影响这些酶及其理想培养基的生产的因素的若干研究。这是因为在从木质纤维素物质生产乙醇的背景下，纤维素酶是影响该过程的输入，并且可以代表工厂的重大运营成本。
为了提高酶生产的水平，可以使用不同的技术，其中有关纤维素酶的最常见技术是通过经典(随机)突变或通过基因表达来选择真菌。几十年来，该经典突变一直应用于诸如木霉属(Trichoderma)的真菌菌株。
在这个意义上，里氏木霉(Trichoderma reesei)(Martinez D.等人(2008)，生物质降解真菌里氏木霉的基因组测序和分析(Genome sequencing and analysis of thebiomass-degrading fungus Trichoderma reesei)Nat Biotechnol.26,553-560)，一种嗜温丝状真菌，红褐肉座菌(Hypocrea jecorina)真菌的无性型，已经生成了若干具有特定性质的菌株，其中报道最多的是Rut C30菌株(Koike H.等人(2013)，里氏木霉菌株的比较基因组学分析(Comparative genomics analysis of Trichoderma reesei strains.)Ind.Biotech.9(6):352-367)。由于cre1基因的突变(使得在酶的表达期间，细胞对葡萄糖的分解代谢阻遏作用更小)和内质网数量的增加(促进蛋白质的O糖基化速率提高)，该菌株的纤维素分解潜力提高。
已经证明，木霉属可用作宿主细胞以用于重组生产具有生物活性的多肽。一些出版物教导了通过中断或过表达一个或多个特定基因进行基因修饰，以寻求提高其活性(国际出版物WO9823642、WO17177289、WO11075677、WO10022518等)。
然而，由此类真菌生产的复合酶的活性表现仍然是一个挑战，因此非常需要结合基因修饰的选择的新菌株来提高复合酶的产量。
发明内容
本发明涉及一种木霉属(Trichoderma)新菌株，包括能够提高复合酶的产量的基因修饰选择，至少包括：如SEQ ID No.1所定义的转录因子Xyr1的过表达；根据SEQ ID No.2的ACE1基因的中断；根据SEQ ID No.3的SLP1基因的中断；以及根据SEQ ID No.4的来自埃默森罗萨氏菌(Rasamsonia emersonii)的Cel3a基因的表达。
另外，根据本发明的所述木霉属新菌株可以包括：根据SEQ ID No.5的PEP1基因的中断；和/或根据SEQ ID No.6的来自黑曲霉(Aspergillus niger)的SucA基因的表达。
附图说明
图1示出了称为CTBE_R2(对比)和CTBE_R4(根据本发明)的真菌菌株通过在含有糖蜜、酵母提取物和硫酸铵的生物反应器中培养而进行的酶的生产。
图2示出了由称为CTBE_R2(对比)和CTBE_R4(根据本发明)的真菌通过在含有糖蜜、酵母提取物和硫酸铵的生物反应器中培养而生产的复合酶的β-葡糖苷酶活性。
具体实施方式
木霉属(Trichoderma)新菌株可以由基因缺失技术来进行工程改造，以消除或减少基因表达。基因缺失技术允许部分或完全移除基因，从而消除其表达。在此类方法中，基因缺失由同源重组通过使用质粒来执行，该质粒已经被工程改造以连续地包含基因侧翼的5'和3'区域。
这些木霉属新突变体也可以通过引入、替换、过表达和/或移除基因中的一个或多个(若干)核苷酸或其转录或翻译所需的控制序列来进行工程改造。例如，核苷酸可以被***或移除以用于引入终止密码子、移除起始密码子或从开放阅读矩阵中置换。
基因中断技术也是可用的。木霉属新菌株也可以通过将破坏性核酸结构***到基因中而被工程改造，该破坏性核酸结构包括与该基因同源的核酸片段，该核酸片段将产生同源区的复制品并且合并复制区域之间的DNA结构。
所有这些修饰都可以通过传统诱变技术执行(Botstein和Shortle，体外诱变的策略与应用(Strategies and applications of in vitro mutagenesis)，科学，1985年9月20日；229(4719):1193-201等)。
为了提供在复合酶生产中表现出改进性能的菌株，开发了一种木霉属新菌株，该菌株包括以下具体基因修饰：如SEQ ID No.1所定义的转录因子Xyr1的过表达；根据SEQ IDNo.2的ACE1基因的中断；根据SEQ ID No.3的SLP1基因的中断；以及根据SEQ ID No.4的来自埃默森罗萨氏菌(Rasamsonia emersoni)的Cel3a基因的表达。
根据本发明所述的木霉属菌株可以是任何木霉属菌株，诸如野生型木霉属菌株或其突变体。
根据本发明所述的新菌株可以无任何限制地通过本领域技术人员已知的传统诱变技术生产。
在特定实施例中，该菌株是被修饰以包含根据本发明的至少四种具体基因修饰的里氏木霉(Trichoderma reesei)。
在更特定的实施例中，该菌株是里氏木霉RutC30(Koike H.等人2013.里氏木霉菌株的比较基因组学分析(Comparative genomics analysis of Trichoderma reeseistrains.)Ind.Biotech.9(6):352-367)。由于cre1基因的突变(使得在酶的表达期间，细胞对葡萄糖的分解代谢阻遏作用更小)和内质网数量的增加(促进蛋白质的O糖基化速率提高)，该菌株的纤维素分解潜力提高。
里氏木霉Xyr1转录因子是木质纤维素酶(纤维素酶和半纤维素酶)——更具体地说是木聚糖酶的表达和分泌所必需的，并且该基因的组成型表达增加了真菌分泌的混合物的酶活性。在本发明的特定实施例中，xyr1基因在RUT-C30菌株中的pdc1基因的表达控制下被放置。
该ace1基因已经被标识为阻遏里氏木霉中的(半)纤维素酶的生产的转录因子。缺失该基因提高了酶产量。
另一方面，蛋白酶的缺乏是酶制剂稳定性的决定性因素。为此，已经研究了从(半)纤维素酶生产真菌的基因组中移除蛋白酶编码基因。来自里氏木霉的slp1基因是一种蛋白酶编码基因并且在酶的全局生产中表现出增益。在现有技术中尚未报道slp1基因缺失对真菌生产率的单独影响。
转而，来自埃默森罗萨氏菌的cel3a基因(β-葡糖苷酶)的表达具有对β-葡糖苷酶的活性的显著影响，这对于木质纤维素物质的有效降解至关重要。在现有技术中已经频繁报道了里氏木霉分泌的复合酶中的低β-葡糖苷酶活性，并且其仍然是要克服的技术挑战。
令人惊讶的是，本发明提供了木霉属新菌株，其中具体基因修饰的选择极大地提高了β-葡糖苷酶活性。
在另一实施例中，本发明额外地设想了以下基因修饰中的一种或多种：根据SEQID No.5的PEP1基因的中断；根据SEQ ID No.6的来自黑曲霉(Aspergillus niger)的SucA基因的表达。
该PEP1基因的中断编码了天冬氨酸蛋白酶及其失活，与里氏木霉中的木质纤维素酶和异源酶的分泌的增长有关。
关于黑曲霉的SucA基因的表达，该基因编码了转化酶，该转化酶是一种催化蔗糖水解为果糖加葡萄糖的酶，从而允许将甘蔗糖蜜用作用于生产酶的碳源。
在本发明的范围内，术语“修饰”指的是，通过本领域技术人员已知的任何技术，在基因或其转录或翻译所需的控制序列中引入、替换或移除一个或多个(若干)核苷酸，或在选定的木霉属菌株中的基因中断、基因缺失、基因过表达。
根据本发明的木霉属菌株可以在营养培养基中培养，以用于使用本领域已知的方法生产感兴趣的多肽。例如，在特定实施例中，在没有任何限制的情况下，培养基可包括酵母(例如未自溶的全酵母(酿酒酵母))、至少一种碳源，诸如甘蔗糖蜜和硫酸铵。
以下示例无任何限制地描述了本发明的一些特定实施例，并且旨在展示其优点。
实施例1.对酶产量和β-葡糖苷酶活性的影响的对比结果
实施例
通过本领域技术人员已知的传统诱变技术修饰里氏木霉Rut C30菌株，以缺失RUT-C30菌株中的ace1基因。该缺失与含有V821F突变的xyr1基因的组成型表达同时进行，从而生成称为CTBE_R2的真菌(对比)。执行两种修饰以增长真菌的酶产量。
根据本发明，CTBE_R2菌株中蛋白酶slp1的缺失与来自埃默森罗萨氏菌的β-葡糖苷酶Cel3a的***同时进行，从而生成称为CTBE_R4的真菌(本发明)。
两种菌株在含有糖蜜、酵母提取物和硫酸铵的生物反应器中分别培养，呈现出图1和图2中所示的结果。
如图1所示，真菌CTBE_R4(本发明)的酶产量与真菌CTBE_R2(对比)的酶产量相似，这表示所进行的修饰并未改变真菌的生产率。
然而，如图2所示，真菌CTBE_R4(本发明)生产的复合酶展示出比真菌CTBE_R2(对比)生产的复合酶高42倍的β-葡糖苷酶活性。
本领域技术人员将很容易知道如何通过文本中包含的教导和所呈现的示例来评估本发明的优势，并且在不脱离本发明的范围的情况下提出变型和等效替代实施例，如本文权利要求中所定义的。
SEQUENCE LISTING
<110> 能源与材料国家研究中心
<120> 被修饰以用于生产复合酶的木霉属真菌菌株
<130> PCT008CN1
<140> PCT/BR2019/050275
<141> 2019-07-16
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 920
<212> PRT
<213> 里氏木霉
<400> 1
Met Leu Ser Asn Pro Leu Arg Arg Tyr Ser Ala Tyr Pro Asp Ile Ser
1 5 10 15
Ser Ala Ser Phe Asp Pro Asn Tyr His Gly Ser Gln Ser His Leu His
20 25 30
Ser Ile Asn Val Asn Thr Phe Gly Asn Ser His Pro Tyr Pro Met Gln
35 40 45
His Leu Ala Gln His Ala Glu Leu Ser Ser Ser Arg Met Ile Arg Ala
50 55 60
Ser Pro Val Gln Pro Lys Gln Arg Gln Gly Ser Leu Ile Ala Ala Arg
65 70 75 80
Lys Asn Ser Thr Gly Thr Ala Gly Pro Ile Arg Arg Arg Ile Ser Arg
85 90 95
Ala Cys Asp Gln Cys Asn Gln Leu Arg Thr Lys Cys Asp Gly Leu His
100 105 110
Pro Cys Ala His Cys Ile Glu Phe Gly Leu Gly Cys Glu Tyr Val Arg
115 120 125
Glu Arg Lys Lys Arg Gly Lys Ala Ser Arg Lys Asp Ile Ala Ala Gln
130 135 140
Gln Ala Ala Ala Ala Ala Ala Gln His Ser Gly Gln Val Gln Asp Gly
145 150 155 160
Pro Glu Asp Gln His Arg Lys Leu Ser Arg Gln Gln Ser Glu Ser Ser
165 170 175
Arg Gly Ser Ala Glu Leu Ala Gln Pro Ala His Asp Pro Pro His Gly
180 185 190
His Ile Glu Gly Ser Val Ser Ser Phe Ser Asp Asn Gly Leu Ser Gln
195 200 205
His Ala Ala Met Gly Gly Met Asp Gly Leu Glu Asp His His Gly His
210 215 220
Val Gly Val Asp Pro Ala Leu Gly Arg Thr Gln Leu Glu Ala Ser Ser
225 230 235 240
Ala Met Gly Leu Gly Ala Tyr Gly Glu Val His Pro Gly Tyr Glu Ser
245 250 255
Pro Gly Met Asn Gly His Val Met Val Pro Pro Ser Tyr Gly Ala Gln
260 265 270
Thr Thr Met Ala Gly Tyr Ser Gly Ile Ser Tyr Ala Ala Gln Ala Pro
275 280 285
Ser Pro Ala Thr Tyr Ser Ser Asp Gly Asn Phe Arg Leu Thr Gly His
290 295 300
Ile His Asp Tyr Pro Leu Ala Asn Gly Ser Ser Pro Ser Trp Gly Gln
305 310 315 320
Ser Asp Leu Arg Tyr Pro Val Leu Glu Pro Leu Leu Pro His Leu Gly
325 330 335
Asn Ile Leu Pro Val Ser Leu Ala Cys Asp Leu Ile Asp Leu Tyr Phe
340 345 350
Ser Ser Ser Ser Ser Ala Gln Met His Pro Met Ser Pro Tyr Val Leu
355 360 365
Gly Phe Val Phe Arg Lys Arg Ser Phe Leu His Pro Thr Asn Pro Arg
370 375 380
Arg Cys Gln Pro Ala Leu Leu Ala Ser Met Leu Trp Val Ala Ala Gln
385 390 395 400
Thr Ser Glu Ala Ser Phe Leu Thr Ser Leu Pro Ser Ala Arg Ser Lys
405 410 415
Val Cys Gln Lys Leu Leu Glu Leu Thr Val Gly Leu Leu Gln Pro Leu
420 425 430
Ile His Thr Gly Thr Asn Ser Pro Ser Pro Lys Thr Ser Pro Val Val
435 440 445
Gly Ala Ala Ala Leu Gly Val Leu Gly Val Ala Met Pro Gly Ser Leu
450 455 460
Asn Met Asp Ser Leu Ala Gly Glu Thr Gly Ala Phe Gly Ala Ile Gly
465 470 475 480
Ser Leu Asp Asp Val Ile Thr Tyr Val His Leu Ala Thr Val Val Ser
485 490 495
Ala Ser Glu Tyr Lys Gly Ala Ser Leu Arg Trp Trp Gly Ala Ala Trp
500 505 510
Ser Leu Ala Arg Glu Leu Lys Leu Gly Arg Glu Leu Pro Pro Gly Asn
515 520 525
Pro Pro Ala Asn Gln Glu Asp Gly Glu Gly Leu Ser Glu Asp Val Asp
530 535 540
Glu His Asp Leu Asn Arg Asn Asn Thr Arg Phe Val Thr Glu Glu Glu
545 550 555 560
Arg Glu Glu Arg Arg Arg Ala Trp Trp Leu Val Tyr Ile Val Asp Arg
565 570 575
His Leu Ala Leu Cys Tyr Asn Arg Pro Leu Phe Leu Leu Asp Ser Glu
580 585 590
Cys Ser Asp Leu Tyr His Pro Met Asp Asp Ile Lys Trp Gln Ala Gly
595 600 605
Lys Phe Arg Ser His Asp Ala Gly Asn Ser Ser Ile Asn Ile Asp Ser
610 615 620
Ser Met Thr Asp Glu Phe Gly Asp Ser Pro Arg Ala Ala Arg Gly Ala
625 630 635 640
His Tyr Glu Cys Arg Gly Arg Ser Ile Phe Gly Tyr Phe Leu Ser Leu
645 650 655
Met Thr Ile Leu Gly Glu Ile Val Asp Val His His Ala Lys Ser His
660 665 670
Pro Arg Phe Gly Val Gly Phe Arg Ser Ala Arg Asp Trp Asp Glu Gln
675 680 685
Val Ala Glu Ile Thr Arg His Leu Asp Met Tyr Glu Glu Ser Leu Lys
690 695 700
Arg Phe Val Ala Lys His Leu Pro Leu Ser Ser Lys Asp Lys Glu Gln
705 710 715 720
His Glu Met His Asp Ser Gly Ala Val Thr Asp Met Gln Ser Pro Leu
725 730 735
Ser Val Arg Thr Asn Ala Ser Ser Arg Met Thr Glu Ser Glu Ile Gln
740 745 750
Ala Ser Ile Val Val Ala Tyr Ser Thr His Val Met His Val Leu His
755 760 765
Ile Leu Leu Ala Asp Lys Trp Asp Pro Ile Asn Leu Leu Asp Asp Asp
770 775 780
Asp Leu Trp Ile Ser Ser Glu Gly Phe Val Thr Ala Thr Ser His Ala
785 790 795 800
Val Ser Ala Ala Glu Ala Ile Ser Gln Ile Leu Glu Phe Asp Pro Gly
805 810 815
Leu Glu Phe Met Pro Phe Phe Tyr Gly Val Tyr Leu Leu Gln Gly Ser
820 825 830
Phe Leu Leu Leu Leu Ile Ala Asp Lys Leu Gln Ala Glu Ala Ser Pro
835 840 845
Ser Val Ile Lys Ala Cys Glu Thr Ile Val Arg Ala His Glu Ala Cys
850 855 860
Val Val Thr Leu Ser Thr Glu Tyr Gln Arg Asn Phe Ser Lys Val Met
865 870 875 880
Arg Ser Ala Leu Ala Leu Ile Arg Gly Arg Val Pro Glu Asp Leu Ala
885 890 895
Glu Gln Gln Gln Arg Arg Arg Glu Leu Leu Ala Leu Tyr Arg Trp Thr
900 905 910
Gly Asn Gly Thr Gly Leu Ala Leu
915 920
<210> 2
<211> 733
<212> PRT
<213> 里氏木霉
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Met Ser Phe Ser Asn Pro Arg Arg Arg Thr Pro Val Thr Arg Pro Gly
1 5 10 15
Thr Asp Cys Glu His Gly Leu Ser Leu Lys Thr Thr Met Thr Leu Arg
20 25 30
Lys Gly Ala Thr Phe His Ser Pro Thr Ser Pro Ser Ala Ser Ser Ala
35 40 45
Ala Gly Asp Phe Val Pro Pro Thr Leu Thr Arg Ser Gln Ser Ala Phe
50 55 60
Asp Asp Val Val Asp Ala Ser Arg Arg Arg Ile Ala Met Thr Leu Asn
65 70 75 80
Asp Ile Asp Glu Ala Leu Ser Lys Ala Ser Leu Ser Asp Lys Ser Pro
85 90 95
Arg Pro Lys Pro Leu Arg Asp Thr Ser Leu Pro Val Pro Arg Gly Phe
100 105 110
Leu Glu Pro Pro Val Val Asp Pro Ala Met Asn Lys Gln Glu Pro Glu
115 120 125
Arg Arg Val Leu Arg Pro Arg Ser Val Arg Arg Thr Arg Asn His Ala
130 135 140
Ser Asp Ser Gly Ile Gly Ser Ser Val Val Ser Thr Asn Asp Lys Ala
145 150 155 160
Gly Ala Ala Asp Ser Thr Lys Lys Pro Gln Ala Ser Ala Leu Thr Arg
165 170 175
Ser Ala Ala Ser Ser Thr Thr Ala Met Leu Pro Ser Leu Ser His Arg
180 185 190
Ala Val Asn Arg Ile Arg Glu His Thr Leu Arg Pro Leu Leu Glu Lys
195 200 205
Pro Thr Leu Lys Glu Phe Glu Pro Ile Val Leu Asp Val Pro Arg Arg
210 215 220
Ile Arg Ser Lys Glu Ile Ile Cys Leu Arg Asp Leu Glu Lys Thr Leu
225 230 235 240
Ile Phe Met Ala Pro Glu Lys Ala Lys Ser Ala Ala Leu Tyr Leu Asp
245 250 255
Phe Cys Leu Thr Ser Val Arg Cys Ile Gln Ala Thr Val Glu Tyr Leu
260 265 270
Thr Asp Arg Glu Gln Val Arg Pro Gly Asp Arg Pro Tyr Thr Asn Gly
275 280 285
Tyr Phe Ile Asp Leu Lys Glu Gln Ile Tyr Gln Tyr Gly Lys Gln Leu
290 295 300
Ala Ala Ile Lys Glu Lys Gly Ser Leu Ala Asp Asp Met Asp Ile Asp
305 310 315 320
Pro Ser Asp Glu Val Arg Leu Tyr Gly Gly Val Ala Glu Asn Gly Arg
325 330 335
Pro Ala Glu Leu Ile Arg Val Lys Lys Asp Gly Thr Ala Tyr Ser Met
340 345 350
Ala Thr Gly Lys Ile Val Asp Met Thr Glu Ser Pro Thr Pro Leu Lys
355 360 365
Arg Ser Leu Ser Glu Gln Arg Glu Asp Glu Glu Glu Ile Met Arg Ser
370 375 380
Met Ala Arg Arg Lys Lys Asn Ala Thr Pro Glu Asp Val Ala Pro Lys
385 390 395 400
Lys Cys Arg Glu Pro Gly Cys Thr Lys Glu Phe Lys Arg Pro Cys Asp
405 410 415
Leu Thr Lys His Glu Lys Thr His Ser Arg Pro Trp Lys Cys Pro Ile
420 425 430
Pro Thr Cys Lys Tyr His Glu Tyr Gly Trp Pro Thr Glu Lys Glu Met
435 440 445
Asp Arg His Ile Asn Asp Lys His Ser Asp Ala Pro Ala Met Tyr Glu
450 455 460
Cys Leu Phe Lys Pro Cys Pro Tyr Lys Ser Lys Arg Glu Ser Asn Cys
465 470 475 480
Lys Gln His Met Glu Lys Ala His Gly Trp Thr Tyr Val Arg Thr Lys
485 490 495
Thr Asn Gly Lys Lys Ala Pro Ser Gln Asn Gly Ser Thr Ala Gln Gln
500 505 510
Thr Pro Pro Leu Ala Asn Val Ser Thr Pro Ser Ser Thr Pro Ser Tyr
515 520 525
Ser Val Pro Thr Pro Pro Gln Asp Gln Val Met Ser Thr Asp Phe Pro
530 535 540
Met Tyr Pro Ala Asp Asp Asp Trp Leu Ala Thr Tyr Gly Ala Gln Pro
545 550 555 560
Asn Thr Ile Asp Ala Met Asp Leu Gly Leu Glu Asn Leu Ser Pro Ala
565 570 575
Ser Ala Ala Ser Ser Tyr Glu Gln Tyr Pro Pro Tyr Gln Asn Gly Ser
580 585 590
Thr Phe Ile Ile Asn Asp Glu Asp Ile Tyr Ala Ala His Val Gln Ile
595 600 605
Pro Ala Gln Leu Pro Thr Pro Glu Gln Val Tyr Thr Lys Met Met Pro
610 615 620
Gln Gln Met Pro Val Tyr His Val Gln Gln Glu Pro Cys Thr Thr Val
625 630 635 640
Pro Ile Leu Gly Glu Pro Gln Phe Ser Pro Asn Ala Gln Gln Asn Ala
645 650 655
Val Leu Tyr Thr Pro Thr Ser Leu Arg Glu Val Asp Glu Gly Phe Asp
660 665 670
Glu Ser Tyr Ala Ala Asp Gly Ala Asp Phe Gln Leu Phe Pro Ala Thr
675 680 685
Val Asp Lys Thr Asp Val Phe Gln Ser Leu Phe Thr Asp Met Pro Ser
690 695 700
Ala Asn Leu Gly Phe Ser Gln Thr Thr Gln Pro Asp Ile Phe Asn Gln
705 710 715 720
Ile Asp Trp Ser Asn Leu Asp Tyr Gln Gly Phe Gln Glu
725 730
<210> 3
<211> 882
<212> PRT
<213> 里氏木霉
<400> 3
Met Val Arg Ser Ala Leu Phe Val Ser Leu Leu Ala Thr Phe Ser Gly
1 5 10 15
Val Ile Ala Arg Val Ser Gly His Gly Ser Lys Ile Val Pro Gly Ala
20 25 30
Tyr Ile Phe Glu Phe Glu Asp Ser Gln Asp Thr Ala Asp Phe Tyr Lys
35 40 45
Lys Leu Asn Gly Glu Gly Ser Thr Arg Leu Lys Phe Asp Tyr Lys Leu
50 55 60
Phe Lys Gly Val Ser Val Gln Leu Lys Asp Leu Asp Asn His Glu Ala
65 70 75 80
Lys Ala Gln Gln Met Ala Gln Leu Pro Ala Val Lys Asn Val Trp Pro
85 90 95
Val Thr Leu Ile Asp Ala Pro Asn Pro Lys Val Glu Trp Val Ala Gly
100 105 110
Ser Thr Ala Pro Thr Leu Glu Ser Arg Ala Ile Lys Lys Pro Pro Ile
115 120 125
Pro Asn Asp Ser Ser Asp Phe Pro Thr His Gln Met Thr Gln Ile Asp
130 135 140
Lys Leu Arg Ala Lys Gly Tyr Thr Gly Lys Gly Val Arg Val Ala Val
145 150 155 160
Ile Asp Thr Gly Ile Asp Tyr Thr His Pro Ala Leu Gly Gly Cys Phe
165 170 175
Gly Arg Gly Cys Leu Val Ser Phe Gly Thr Asp Leu Val Gly Asp Asp
180 185 190
Tyr Thr Gly Phe Asn Thr Pro Val Pro Asp Asp Asp Pro Val Asp Cys
195 200 205
Ala Gly His Gly Ser His Val Ala Gly Ile Ile Ala Ala Gln Glu Asn
210 215 220
Pro Tyr Gly Phe Thr Gly Gly Ala Pro Asp Val Thr Leu Gly Ala Tyr
225 230 235 240
Arg Val Phe Gly Cys Asp Gly Gln Ala Gly Asn Asp Val Leu Ile Ser
245 250 255
Ala Tyr Asn Gln Ala Phe Glu Asp Gly Ala Gln Ile Ile Thr Ala Ser
260 265 270
Ile Gly Gly Pro Ser Gly Trp Ala Glu Glu Pro Trp Ala Val Ala Val
275 280 285
Thr Arg Ile Val Glu Ala Gly Val Pro Cys Thr Val Ser Ala Gly Asn
290 295 300
Glu Gly Asp Ser Gly Leu Phe Phe Ala Ser Thr Ala Ala Asn Gly Lys
305 310 315 320
Lys Val Ile Ala Val Ala Ser Val Asp Asn Glu Asn Ile Pro Ser Val
325 330 335
Leu Ser Val Ala Ser Tyr Lys Ile Asp Ser Gly Ala Ala Gln Asp Phe
340 345 350
Gly Tyr Val Ser Ser Ser Lys Ala Trp Asp Gly Val Ser Lys Pro Leu
355 360 365
Tyr Ala Val Ser Phe Asp Thr Thr Ile Pro Asp Asp Gly Cys Ser Pro
370 375 380
Leu Pro Asp Ser Thr Pro Asp Leu Ser Asp Tyr Ile Val Leu Val Arg
385 390 395 400
Arg Gly Thr Cys Thr Phe Val Gln Lys Ala Gln Asn Val Ala Ala Lys
405 410 415
Gly Ala Lys Tyr Leu Leu Tyr Tyr Asn Asn Ile Pro Gly Ala Leu Ala
420 425 430
Val Asp Val Ser Ala Val Pro Glu Ile Glu Ala Val Gly Met Val Asp
435 440 445
Asp Lys Thr Gly Ala Thr Trp Ile Ala Ala Leu Lys Asp Gly Lys Thr
450 455 460
Val Thr Leu Thr Leu Thr Asp Pro Ile Glu Ser Glu Lys Gln Ile Gln
465 470 475 480
Phe Ser Asp Asn Pro Thr Thr Gly Gly Ala Leu Ser Gly Tyr Thr Thr
485 490 495
Trp Gly Pro Thr Trp Glu Leu Asp Val Lys Pro Gln Ile Ser Ser Pro
500 505 510
Gly Gly Asn Ile Leu Ser Thr Tyr Pro Val Ala Leu Gly Gly Tyr Ala
515 520 525
Thr Leu Ser Gly Thr Ser Met Ala Cys Pro Leu Thr Ala Ala Ala Val
530 535 540
Ala Leu Ile Gly Gln Ala Arg Gly Thr Phe Asp Pro Ala Leu Ile Asp
545 550 555 560
Asn Leu Leu Ala Thr Thr Ala Asn Pro Gln Leu Phe Asn Asp Gly Glu
565 570 575
Lys Phe Tyr Asp Phe Leu Ala Pro Val Pro Gln Gln Gly Gly Gly Leu
580 585 590
Ile Gln Ala Tyr Asp Ala Ala Phe Ala Thr Thr Leu Leu Ser Pro Ser
595 600 605
Ser Leu Ser Phe Asn Asp Thr Asp His Phe Ile Lys Lys Lys Gln Ile
610 615 620
Thr Leu Lys Asn Thr Ser Lys Gln Arg Val Thr Tyr Lys Leu Asn His
625 630 635 640
Val Pro Thr Asn Thr Phe Tyr Thr Leu Ala Pro Gly Asn Gly Tyr Pro
645 650 655
Ala Pro Phe Pro Asn Asp Ala Val Ala Ala His Ala Asn Leu Lys Phe
660 665 670
Asn Leu Gln Gln Val Thr Leu Pro Ala Gly Arg Ser Ile Thr Val Asp
675 680 685
Val Phe Pro Thr Pro Pro Arg Asp Val Asp Ala Lys Arg Leu Ala Leu
690 695 700
Trp Ser Gly Tyr Ile Thr Val Asn Gly Thr Asp Gly Thr Ser Leu Ser
705 710 715 720
Val Pro Tyr Gln Gly Leu Thr Gly Ser Leu His Lys Gln Lys Val Leu
725 730 735
Tyr Pro Glu Asp Ser Trp Ile Ala Asp Ser Thr Asp Glu Ser Leu Ala
740 745 750
Pro Val Glu Asn Gly Thr Val Phe Thr Ile Pro Ala Pro Gly Asn Ala
755 760 765
Gly Pro Asp Asp Lys Leu Pro Ser Leu Val Val Ser Pro Ala Leu Gly
770 775 780
Ser Arg Tyr Val Arg Val Asp Leu Val Leu Leu Ser Ala Pro Pro His
785 790 795 800
Gly Thr Lys Leu Lys Thr Val Lys Phe Leu Asp Thr Thr Ser Ile Gly
805 810 815
Gln Pro Ala Gly Ser Pro Leu Leu Trp Ile Ser Arg Gly Ala Asn Pro
820 825 830
Ile Ala Trp Thr Gly Glu Leu Ser Asp Asn Lys Phe Ala Pro Pro Gly
835 840 845
Thr Tyr Lys Ala Val Phe His Ala Leu Arg Ile Phe Gly Asn Glu Lys
850 855 860
Lys Lys Glu Asp Trp Asp Val Ser Glu Ser Pro Ala Phe Thr Ile Lys
865 870 875 880
Tyr Ala
<210> 4
<211> 857
<212> PRT
<213> 里氏木霉
<400> 4
Met Arg Asn Gly Leu Leu Lys Val Ala Ala Leu Ala Ala Ala Ser Ala
1 5 10 15
Val Asn Gly Glu Asn Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro
20 25 30
Trp Ala Asn Gly Gln Gly Asp Trp Ala Glu Ala Tyr Gln Lys Ala Val
35 40 45
Gln Phe Val Ser Gln Leu Thr Leu Ala Glu Lys Val Asn Leu Thr Thr
50 55 60
Gly Thr Gly Trp Glu Gln Asp Arg Cys Val Gly Gln Val Gly Ser Ile
65 70 75 80
Pro Arg Leu Gly Phe Pro Gly Leu Cys Met Gln Asp Ser Pro Leu Gly
85 90 95
Val Arg Asp Thr Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val
100 105 110
Ala Ala Thr Trp Asp Arg Asn Leu Ala Tyr Arg Arg Gly Val Ala Met
115 120 125
Gly Glu Glu His Arg Gly Lys Gly Val Asp Val Gln Leu Gly Pro Val
130 135 140
Ala Gly Pro Leu Gly Arg Ser Pro Asp Ala Gly Arg Asn Trp Glu Gly
145 150 155 160
Phe Ala Pro Asp Pro Val Leu Thr Gly Asn Met Met Ala Ser Thr Ile
165 170 175
Gln Gly Ile Gln Asp Ala Gly Val Ile Ala Cys Ala Lys His Phe Ile
180 185 190
Leu Tyr Glu Gln Glu His Phe Arg Gln Gly Ala Gln Asp Gly Tyr Asp
195 200 205
Ile Ser Asp Ser Ile Ser Ala Asn Ala Asp Asp Lys Thr Met His Glu
210 215 220
Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ser
225 230 235 240
Val Met Cys Ser Tyr Asn Gln Val Asn Asn Ser Tyr Ala Cys Ser Asn
245 250 255
Ser Tyr Thr Met Asn Lys Leu Leu Lys Ser Glu Leu Gly Phe Gln Gly
260 265 270
Phe Val Met Thr Asp Trp Gly Gly His His Ser Gly Val Gly Ser Ala
275 280 285
Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile Ala Phe Asp Ser
290 295 300
Gly Thr Ser Phe Trp Gly Thr Asn Leu Thr Val Ala Val Leu Asn Gly
305 310 315 320
Ser Ile Pro Glu Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ser
325 330 335
Ala Tyr Tyr Lys Val Gly Arg Asp Arg Tyr Ser Val Pro Ile Asn Phe
340 345 350
Asp Ser Trp Thr Leu Asp Thr Tyr Gly Pro Glu His Tyr Ala Val Gly
355 360 365
Gln Gly Gln Thr Lys Ile Asn Glu His Val Asp Val Arg Gly Asn His
370 375 380
Ala Glu Ile Ile His Glu Ile Gly Ala Ala Ser Ala Val Leu Leu Lys
385 390 395 400
Asn Lys Gly Gly Leu Pro Leu Thr Gly Thr Glu Arg Phe Val Gly Val
405 410 415
Phe Gly Lys Asp Ala Gly Ser Asn Pro Trp Gly Val Asn Gly Cys Ser
420 425 430
Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly Trp Gly Ser Gly
435 440 445
Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln Arg
450 455 460
Glu Val Leu Ser Arg Asn Gly Thr Phe Thr Gly Ile Thr Asp Asn Gly
465 470 475 480
Ala Leu Ala Glu Met Ala Ala Ala Ala Ser Gln Ala Asp Thr Cys Leu
485 490 495
Val Phe Ala Asn Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Asp Gly
500 505 510
Asn Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp Gln Gly Ala Asp Gln
515 520 525
Val Ile His Asn Val Ser Ala Asn Cys Asn Asn Thr Val Val Val Leu
530 535 540
His Thr Val Gly Pro Val Leu Ile Asp Asp Trp Tyr Asp His Pro Asn
545 550 555 560
Val Thr Ala Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly Asn
565 570 575
Ser Leu Val Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Lys Thr Pro
580 585 590
Phe Thr Trp Gly Arg Ala Arg Asp Asp Tyr Gly Ala Pro Leu Ile Val
595 600 605
Lys Pro Asn Asn Gly Lys Gly Ala Pro Gln Gln Asp Phe Thr Glu Gly
610 615 620
Ile Phe Ile Asp Tyr Arg Arg Phe Asp Lys Tyr Asn Ile Thr Pro Ile
625 630 635 640
Tyr Glu Phe Gly Phe Gly Leu Ser Tyr Thr Thr Phe Glu Phe Ser Gln
645 650 655
Leu Asn Val Gln Pro Ile Asn Ala Pro Pro Tyr Thr Pro Ala Ser Gly
660 665 670
Phe Thr Lys Ala Ala Gln Ser Phe Gly Gln Pro Ser Asn Ala Ser Asp
675 680 685
Asn Leu Tyr Pro Ser Asp Ile Glu Arg Val Pro Leu Tyr Ile Tyr Pro
690 695 700
Trp Leu Asn Ser Thr Asp Leu Lys Ala Ser Ala Asn Asp Pro Asp Tyr
705 710 715 720
Gly Leu Pro Thr Glu Lys Tyr Val Pro Pro Asn Ala Thr Asn Gly Asp
725 730 735
Pro Gln Pro Ile Asp Pro Ala Gly Gly Ala Pro Gly Gly Asn Pro Ser
740 745 750
Leu Tyr Glu Pro Val Ala Arg Val Thr Thr Ile Ile Thr Asn Thr Gly
755 760 765
Lys Val Thr Gly Asp Glu Val Pro Gln Leu Tyr Val Ser Leu Gly Gly
770 775 780
Pro Asp Asp Ala Pro Lys Val Leu Arg Gly Phe Asp Arg Ile Thr Leu
785 790 795 800
Ala Pro Gly Gln Gln Tyr Leu Trp Thr Thr Thr Leu Thr Arg Arg Asp
805 810 815
Ile Ser Asn Trp Asp Pro Val Thr Gln Asn Trp Val Val Thr Asn Tyr
820 825 830
Thr Lys Thr Ile Tyr Val Gly Asn Ser Ser Arg Asn Leu Pro Leu Gln
835 840 845
Ala Pro Leu Lys Pro Tyr Pro Gly Ile
850 855
<210> 5
<211> 407
<212> PRT
<213> 里氏木霉
<400> 5
Met Gln Thr Phe Gly Ala Phe Leu Val Ser Phe Leu Ala Ala Ser Gly
1 5 10 15
Leu Ala Ala Ala Leu Pro Thr Glu Gly Gln Lys Thr Ala Ser Val Glu
20 25 30
Val Gln Tyr Asn Lys Asn Tyr Val Pro His Gly Pro Thr Ala Leu Phe
35 40 45
Lys Ala Lys Arg Lys Tyr Gly Ala Pro Ile Ser Asp Asn Leu Lys Ser
50 55 60
Leu Val Ala Ala Arg Gln Ala Lys Gln Ala Leu Ala Lys Arg Gln Thr
65 70 75 80
Gly Ser Ala Pro Asn His Pro Ser Asp Ser Ala Asp Ser Glu Tyr Ile
85 90 95
Thr Ser Val Ser Ile Gly Thr Pro Ala Gln Val Leu Pro Leu Asp Phe
100 105 110
Asp Thr Gly Ser Ser Asp Leu Trp Val Phe Ser Ser Glu Thr Pro Lys
115 120 125
Ser Ser Ala Thr Gly His Ala Ile Tyr Thr Pro Ser Lys Ser Ser Thr
130 135 140
Ser Lys Lys Val Ser Gly Ala Ser Trp Ser Ile Ser Tyr Gly Asp Gly
145 150 155 160
Ser Ser Ser Ser Gly Asp Val Tyr Thr Asp Lys Val Thr Ile Gly Gly
165 170 175
Phe Ser Val Asn Thr Gln Gly Val Glu Ser Ala Thr Arg Val Ser Thr
180 185 190
Glu Phe Val Gln Asp Thr Val Ile Ser Gly Leu Val Gly Leu Ala Phe
195 200 205
Asp Ser Gly Asn Gln Val Arg Pro His Pro Gln Lys Thr Trp Phe Ser
210 215 220
Asn Ala Ala Ser Ser Leu Ala Glu Pro Leu Phe Thr Ala Asp Leu Arg
225 230 235 240
His Gly Gln Asn Gly Ser Tyr Asn Phe Gly Tyr Ile Asp Thr Ser Val
245 250 255
Ala Lys Gly Pro Val Ala Tyr Thr Pro Val Asp Asn Ser Gln Gly Phe
260 265 270
Trp Glu Phe Thr Ala Ser Gly Tyr Ser Val Gly Gly Gly Lys Leu Asn
275 280 285
Arg Asn Ser Ile Asp Gly Ile Ala Asp Thr Gly Thr Thr Leu Leu Leu
290 295 300
Leu Asp Asp Asn Val Val Asp Ala Tyr Tyr Ala Asn Val Gln Ser Ala
305 310 315 320
Gln Tyr Asp Asn Gln Gln Glu Gly Val Val Phe Asp Cys Asp Glu Asp
325 330 335
Leu Pro Ser Phe Ser Phe Gly Val Gly Ser Ser Thr Ile Thr Ile Pro
340 345 350
Gly Asp Leu Leu Asn Leu Thr Pro Leu Glu Glu Gly Ser Ser Thr Cys
355 360 365
Phe Gly Gly Leu Gln Ser Ser Ser Gly Ile Gly Ile Asn Ile Phe Gly
370 375 380
Asp Val Ala Leu Lys Ala Ala Leu Val Val Phe Asp Leu Gly Asn Glu
385 390 395 400
Arg Leu Gly Trp Ala Gln Lys
405
<210> 6
<211> 628
<212> PRT
<213> 里氏木霉
<400> 6
Met Lys Leu Gln Thr Ala Ser Val Leu Leu Gly Ser Ala Ala Ala Ala
1 5 10 15
Ser Pro Ser Met Gln Thr Arg Ala Ser Val Val Ile Asp Tyr Asn Val
20 25 30
Ala Pro Pro Asn Leu Ser Thr Leu Pro Asn Gly Ser Leu Phe Glu Thr
35 40 45
Trp Arg Pro Arg Ala His Val Leu Pro Pro Asn Gly Gln Ile Gly Asp
50 55 60
Pro Cys Leu His Tyr Thr Asp Pro Ser Thr Gly Leu Phe His Val Gly
65 70 75 80
Phe Leu His Asp Gly Ser Gly Ile Ser Ser Ala Thr Thr Asp Asp Leu
85 90 95
Ala Thr Tyr Lys Asp Leu Asn Gln Gly Asn Gln Val Ile Val Pro Gly
100 105 110
Gly Ile Asn Asp Pro Val Ala Val Phe Asp Gly Ser Val Ile Pro Ser
115 120 125
Gly Ile Asn Gly Leu Pro Thr Leu Leu Tyr Thr Ser Val Ser Phe Leu
130 135 140
Pro Ile His Trp Ser Ile Pro Tyr Thr Arg Gly Ser Glu Thr Gln Ser
145 150 155 160
Leu Ala Val Ser Ser Asp Gly Gly Ser Asn Phe Thr Lys Leu Asp Gln
165 170 175
Gly Pro Val Ile Pro Gly Pro Pro Phe Ala Tyr Asn Val Thr Ala Phe
180 185 190
Arg Asp Pro Tyr Val Phe Gln Asn Pro Thr Leu Asp Ser Leu Leu His
195 200 205
Ser Lys Asn Asn Thr Trp Tyr Thr Val Ile Ser Gly Gly Leu His Gly
210 215 220
Lys Gly Pro Ala Gln Phe Leu Tyr Arg Gln Tyr Asp Pro Asp Phe Gln
225 230 235 240
Tyr Trp Glu Phe Leu Gly Gln Trp Trp His Glu Pro Thr Asn Ser Thr
245 250 255
Trp Gly Asn Gly Thr Trp Ala Gly Arg Trp Ala Phe Asn Phe Glu Thr
260 265 270
Gly Asn Val Phe Ser Leu Asp Glu Tyr Gly Tyr Asn Pro His Gly Gln
275 280 285
Ile Phe Ser Thr Ile Gly Thr Glu Gly Ser Asp Gln Pro Val Val Pro
290 295 300
Gln Leu Thr Ser Ile His Asp Met Leu Trp Val Ser Gly Asn Val Ser
305 310 315 320
Arg Asn Gly Ser Val Ser Phe Thr Pro Asn Met Ala Gly Phe Leu Asp
325 330 335
Trp Gly Phe Ser Ser Tyr Ala Ala Ala Gly Lys Val Leu Pro Ser Thr
340 345 350
Ser Leu Pro Ser Thr Lys Ser Gly Ala Pro Asp Arg Phe Ile Ser Tyr
355 360 365
Val Trp Leu Ser Gly Asp Leu Phe Glu Gln Ala Glu Gly Phe Pro Thr
370 375 380
Asn Gln Gln Asn Trp Thr Gly Thr Leu Leu Leu Pro Arg Glu Leu Arg
385 390 395 400
Val Leu Tyr Ile Pro Asn Val Val Asp Asn Ala Leu Ala Arg Glu Ser
405 410 415
Gly Ala Ser Trp Gln Val Val Ser Ser Asp Ser Ser Ala Gly Thr Val
420 425 430
Glu Leu Gln Thr Leu Gly Ile Ser Ile Ala Arg Glu Thr Lys Ala Ala
435 440 445
Leu Leu Ser Gly Thr Ser Phe Thr Glu Ser Asp Arg Thr Leu Asn Ser
450 455 460
Ser Gly Val Val Pro Phe Lys Arg Ser Pro Ser Glu Lys Phe Phe Val
465 470 475 480
Leu Ser Ala Gln Leu Ser Phe Pro Ala Ser Ala Arg Gly Ser Gly Leu
485 490 495
Lys Ser Gly Phe Gln Ile Leu Ser Ser Glu Leu Glu Ser Thr Thr Val
500 505 510
Tyr Tyr Gln Phe Ser Asn Glu Ser Ile Ile Val Asp Arg Ser Asn Thr
515 520 525
Ser Ala Ala Ala Arg Thr Thr Asp Gly Ile Asp Ser Ser Ala Glu Ala
530 535 540
Gly Lys Leu Arg Leu Phe Asp Val Leu Asn Gly Gly Glu Gln Ala Ile
545 550 555 560
Glu Thr Leu Asp Leu Thr Leu Val Val Asp Asn Ser Val Leu Glu Ile
565 570 575
Tyr Ala Asn Gly Arg Phe Ala Leu Ser Thr Trp Val Arg Ser Trp Tyr
580 585 590
Ala Asn Ser Thr Asn Ile Ser Phe Phe Gln Asn Gly Val Gly Gly Val
595 600 605
Ala Phe Ser Asn Val Thr Val Ser Glu Gly Leu Tyr Asp Ala Trp Pro
610 615 620
Asp Arg Gln Ser
625

Claims (5)

1.一种被修饰以用于生产复合酶的木霉属(Trichoderma)真菌菌株，其特征在于，至少包括：
如SEQ ID No.1所定义的转录因子Xyr1的过表达；
根据SEQ ID No.2的ACE1基因的中断；
根据SEQ ID No.3的SLP1基因的中断；以及
根据SEQ ID No.4的来自埃默森罗萨氏菌(Rasamsonia emersonii)的Cel3a基因的表达。
2.根据权利要求1所述的菌株，其特征在于，所述基因修饰的真菌优选地为里氏木霉(Trichoderma reesei)。
3.根据权利要求2所述的菌株，其特征在于，所述基因修饰的真菌优选地为里氏木霉(Trichoderma reesei)Rut C30菌株。
4.根据权利要求1所述的菌株，其特征在于，所述菌株包括根据SEQ ID No.5的PEP1基因的中断。
5.根据权利要求1所述的菌株，其特征在于，所述菌株包括根据SEQ ID No.6的黑曲霉(Aspergillus niger)的SucA基因的表达。
CN201980098500.4A 2019-07-16 2019-07-16 被修饰以用于生产复合酶的木霉属真菌菌株 Pending CN114585726A (zh)

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