CN115772476A - 一种丝状真菌重组菌株在纤维素酶生产领域的应用 - Google Patents
一种丝状真菌重组菌株在纤维素酶生产领域的应用 Download PDFInfo
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Abstract
本发明涉及一种丝状真菌重组菌株在纤维素酶生产领域的应用。本发明基于对来源于里氏木霉(Trichoderma reesei,CCTCC NO:M2015804)的胞内β‑葡萄糖苷酶进行基因改造,将其174和177位点进行突变,可以解除葡萄糖条件下CCR效应对纤维素酶表达的抑制,最终提高主要纤维素酶酶活及蛋白量,有利于在葡萄糖存在条件下纤维素酶的生产。该研究结果对纤维素酶工业生产过程中诱导物开发具有重要的指导意义。
Description
技术领域
本发明属于纤维素酶工程菌株技术领域,具体涉及一种里氏木霉的重组菌株、所述重组菌株在纤维素酶生产领域的应用,以及通过基因工程手段获得该重组菌株的试剂盒、方法。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
丝状真菌是自然界中降解木质纤维素类生物质的主要微生物,其所产的纤维素酶主要包括三种组分(纤维素内切酶、纤维素外切酶和β-葡萄糖苷酶),这三种酶相互协同对纤维素底物进行有效降解。β-葡萄糖苷酶能够将来源于纤维素内切酶以及外切酶降解得到的成分-低聚糖以及二糖等进一步降解为可发酵性单糖。因此,β-葡萄糖苷酶在生物质的降解中发挥着非常重要的作用。
里氏木霉(Trichoderma reesei)是公认的纤维素酶高产菌株,也是纤维素酶生产工业菌株的主要来源。纤维素酶的生产需要诱导物的存在,常见的并且效果比较好的诱导物包括纤维二糖或者槐糖等,但是其比较昂贵,在纤维素酶发酵过程中容易被降解为葡萄糖,引起CCR效应;纤维素是纤维素酶发酵生产中较为常见的诱导性碳源,但是由于其不溶性,会加剧纤维素酶深层发酵中的粘度,影响发酵中的溶氧以及增加能耗,并且诱导效果比较好的微晶纤维素价格也相对比较高。因此,现在比较好的策略是用价格比较低廉的碳源替代一定量的固体诱导物是比较好的方法。葡萄糖是微生物常用的速效且廉价的碳源,但是又易引起CCR效应,从而抑制纤维素酶的生产。
碳代谢阻遏(Carbon Catabolite Repression,CCR)效应是微生物中普遍存在的一种重要调控机制,当有葡萄糖等速效碳源存在的情况下,降解复杂碳源的酶基因表达会受到明显抑制。例如,在T.reesei纤维素酶基因表达***中,有较高浓度葡萄糖存在情况下,槐糖等诱导物无法诱导纤维素酶基因表达,同时向已表达纤维素酶的诱导培养条件下添加葡萄糖,主要的纤维素酶基因转录也会消失。因此,通过人工诱变、基因工程改造等方法改良菌株性状,选育去CCR效应的菌株,对纤维素酶工业化生产有重要意义。
胞内β-葡萄糖苷酶,具有一定的水解二糖活性(参考Biotechnol Lett,2017,39(11),1717-1723;Appl Environ Microbiol,2002,68(9),4546-4553;Biotechnology forBiofuels,2018,11,314.),它能够将通过转运蛋白CRT1或者MFS(参考Biotechnology forBiofuels,2020,13,158.)转运到体内的二糖诱导物降解为葡萄糖,一方面增加胞内的葡萄糖会加剧菌株的CCR效应,另一方面,胞内的诱导物被降解,从而导致纤维素酶诱导产量下降。研究发现,在单纯诱导物-乳糖或者纤维素作为碳源的条件下,胞内的β-葡萄糖苷酶的敲除导致在乳糖培养条件下纤维素酶的诱导延迟(参考Eukaryot Cell,2014,13(8),1001-1013.)。由此可见,里氏木霉胞内β-葡萄糖苷酶对于纤维素酶的诱导是一个非常复杂的过程。并且对于额外添加葡萄糖廉价碳源从而生产纤维素酶鲜见报道。因此,发明人认为,通过进行敲除达到纤维素酶在葡萄糖培养条件下的高表达的方法具有很大的盲目性,并且不一定可行。
发明内容
基于上述技术背景,本发明针对丝状真菌碳阻遏效应进行了进一步研究,为了优化纤维素酶的表达,本发明证实了,通过对CEL1B蛋白进行了突变,纤维素酶酶活和表达量都出现了显著的提升,和出发菌株相比,纤维素酶活性(FPase activity)及菌株pNPC活性分别提高了3.83和10.6倍。
基于上述技术效果,本发明提供以下技术方案:
本发明第一方面,提供一种丝状真菌的重组菌株,所述重组菌株与出发菌株相比,具有CEL1B蛋白突变体,所述突变体的氨基酸序列如下:
(1)如SEQ ID NO:3所示;
(2)SEQ ID NO:3所述氨基酸序列具有一个或多个氨基酸添加、取代或缺失之后,仍具有相似生理活性的氨基酸序列。
第一方面所述的技术方案中,所述“一个或多个氨基酸的添加、取代或缺失”是指SEQ ID NO:3所示氨基酸序列的多肽进行一个或多个氨基酸的添加、取代或缺失后仍然能够仍然表现出纤维素酶高表达的氨基酸序列。所述“一个或多个氨基酸”添加、改变或缺失获得的衍生序列,与SEQ ID NO:3所示氨基酸序列的相似性可能达到85%、90%或95%以上,所述相似性通过Blast方式确定。
上述技术方案中SEQ ID NO:3所示氨基酸序列即为cel1bI177S/I174S突变体,具体表示CEL1B蛋白、即SEQ ID NO:2所述氨基酸序列(NCBI Accession ID:XP_006964838.1)外显子第177和1741位的异亮氨酸I突变为丝氨酸S。
第一方面所述丝状真菌优选为工业酶,包括但不限于黑曲霉(Aspergillusniger)、米曲霉(Aspergillus oryzae)、草酸青霉(Penicillium oxalicum)、构巢曲霉(Aspergillus nidulans)、棘孢曲霉(Aspergillus aculeatus)、里氏木霉(Trichodermareesei)中的一种;本发明验证的一种可行的方式中,所述丝状真菌为里氏木霉(Trichoderma reesei)。
应当明确的是,只要出发菌株当中含有纤维素酶表达的代谢流及具有表达CEL1B蛋白的能力即可作为出发菌株,上述里氏木霉包括野生型里氏木霉或里氏木霉的突变株;所述突变株包括经物理、化学诱变或基因工程手段改造后的菌株。
发明人针对丝状真菌碳阻遏效应进行了进一步研究,将T.reesei中的胞内β-葡萄糖苷酶cel1b,进行了敲除,发现葡萄糖存在条件下,主要纤维素酶基因的转录水平敲除cel1b发生下降。但是,在cel1b基因缺失的里氏木霉中,通过对CEL1B蛋白进行了突变之后,在含有葡萄糖的培养基中生长良好,且纤维素酶的产量得到提高。另外,本发明还提供的一种可行的方式中,所述出发菌株为里氏木霉(Trichoderma reesei)T1,已经于2015年12月30日保藏于中国典型培养物保藏中心(CCTCC),菌种保藏编号CCTCCNO:M2015804,该菌株于专利CN105779301B中公开。
上述菌株里氏木霉(Trichoderma reesei)T1为发明人研究团队经过多次诱变处理得到一株具有纤维素酶高表达活性的菌株,本发明以该高产菌株为出发菌株进行改造,仍然获得了纤维素酶产量和活性显著提高,这也证明了本发明提供突变方式是一种适用于多种工程菌株的基因改造方法,对纤维素酶的生产具有重要意义。
因此,本发明第二方面,提供第一方面所述重组菌株在纤维素酶生产或发酵领域的应用。
本发明第三方面,提供一种纤维素酶的生产方法,所述生产方法包括采用第一方面所述重组菌株进行发酵。
优选的,所述发酵碳源包括速效碳源、二糖、低聚糖中的一种或几种的组合。
另外,针对上述重组菌株的制备,本发明提供一种通过基因工程手段实现里氏木霉改造的方法,因此,本发明第四方面,提供一种解除CCR效应的基因序列,所述基因序列编码如SEQ ID NO:3所示的氨基酸序列。
上述技术方案中,所述基因序列包括由于密码子简并性可编码获得所述氨基酸序列的。
本发明第五方面,提供一种表达盒,所述表达盒中,至少包括第四方面所述基因序列。
本发明第六方面,提供一种重组载体,所述重组载体中包括第五方面所述表达盒或第四方面所述基因序列的完整编码阅读框序列。
所述重组载体将第四方面所述编码核酸序列***表达载体得到。所述表达载体包括本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。总之,只要能在宿主体内复制和稳定,任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。具体的实例中,所述表达载体为质粒或病毒载体,所述病毒载体为慢病毒载体、腺相关病毒载体或腺病毒载体。
本发明第七方面,提供一种解除丝状真菌碳阻遏效应的试剂盒,所述试剂盒中包括第四方面所述解除CCR效应的基因序列、第五方面所述表达盒或第六方面所述重组载体。
优选的,所述试剂盒中,还包括转化试剂、染色体抽提试剂、扩增酶及电泳试剂。
本发明第八方面,提供一种解除丝状真菌碳阻遏效应的方法,所述方法包括所述方法包括采用第六方面所述重组载体或第七方面所述试剂盒对丝状真菌CEL1B的序列进行改造。
优选的,所述改造步骤如下:
采用原生质体转化法将所述第五方面所述重组载体转化到丝状真菌中;通过初步抗性筛选及测序验证,得到突变株。
以上一个或多个技术方案的有益效果是:
经本发明方法改造的菌株,在不同碳源的培养基中均能够良好的生长,且纤维素酶的产量大幅度提高。本发明的出发菌株为突变筛选后的纤维素酶高产菌株,本发明针对该高产菌株序列进行改造,突变株的纤维素酶产量和活性都获得了进一步的提高。将该改造方法应用于工作酶的改造,使其可以适应速效碳源培养基,降低培养成本的同时提高酶的产量,具有良好的经济意义。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
正如背景技术所介绍的,当速效碳源存在的情况下,微生物会产生碳代谢阻遏效应从而抑制纤维素酶基因的表达。为了解决如上的技术问题,本发明提出了一种将里氏木霉基因cel1b序列的特定位点突变的基因改造方法,从而实现提高纤维素酶活力的方法。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例详细说明本发明的技术方案。
实施例1
实验材料和试剂:
麸皮培养基:称取200g麸皮到1L自来水中,沸水煮30min,使用8-12层纱布过滤,并定容到1L,加入2%的琼脂粉,121℃灭菌30min。
MM培养基:配方为0.5%(NH4)2SO4,0.06%MgSO4,1.5%KH2PO4,0.08%CaCl2,0.00005%FeSO4.7H2O,0.00016%MnSO4.H2O,0.00014%ZnSO4.7H2O,0.00002%CoCl2。
PDA固体培养基(g/L):土豆去皮薄片200g,加一定水煮沸30min,双层纱布过滤后定容到1000mL,葡萄糖20,琼脂粉20。
转化上层培养基:2%葡萄糖,18.22g山梨醇,MM盐定容至100mL,调pH到5.5,加入2%琼脂糖,115℃灭菌30min。
转化下层培养基:2%葡萄糖,1.5%磷酸二氢钾,18.22g山梨醇,加水定容至100mL,调节pH到5.5,加入2%琼脂糖,115℃灭菌30min。
传代培养基:2%的葡萄糖,MM盐,加水定容至100mL,调节pH到5.5,加入2%琼脂糖,115℃灭菌30min。
种子培养基:1%葡萄糖,1%蛋白胨,0.2%硫酸铵,0.05%无水硫酸镁,1%麸皮,1%玉米芯,0.3%磷酸二氢钾,0.5%碳酸钙。
产酶培养基:2%葡萄糖,0.1%蛋白胨,0.14%硫酸铵,0.2%磷酸二氢钾,0.013%尿素,0.05%Tween 80。
转化用溶液:
转化溶液1:1.2M山梨醇(21.88g),0.1M磷酸二氢钾(1.36g),蒸馏水定容至100mL,调节pH到5.6。
转化溶液2:1M山梨醇(18.22g),50mM氯化钙(0.735g),10mM Tris-HCI,1mL1 MTris-HCI,蒸馏水定容至100mL,调节pH到7.5。
转化溶液3:25%聚乙二醇6000(25g),50mM氯化钙(0.735g),10mM Tris-HCI,1mL1M Tris-HCI,蒸馏水定容至100mL,调节pH到7.5。
真菌染色体抽提Buffer:200mM Tris-HCl(4.846g),250mM氯化钠(2.922g),25mMEDTA(1.8612g),2%SDS(0.4g),蒸馏水定容至200ml,室温保存。
以下实施例中的出发菌株为里氏木霉(Trichoderma reesei,CCTCC NO:M2015804),为本实验室诱导突变而来的纤维素酶高产菌株,其CEL1A氨基酸序列(NCBIAccession ID:XP_006963436.1)如SEQ No.1所示,CEL1B氨基酸序列(NCBI Accession ID:XP_006964838.1)如SEQ No.2所示。
一、里氏木霉cel1b突变株构建
1.使用Phanta高保真酶对目的基因进行扩增,体系如下:
PCR程序设定:
2.胶配置及其电泳:
①0.8%(质量体积分数)琼脂糖凝胶的配置:
0.4g琼脂糖加入50mL 1×TAE电泳缓冲液中,微波炉加热至完全溶解/完全透明液体状态,加入3-4μL Genegreen核酸染料,倒入制胶板中,等待胶凝固;
②样品准备:5μL样品与0.5μL蓝色的loading buffer混匀;
③点样:将凝固的琼脂糖凝胶将与loading buffer混匀的样品点入胶的样品孔。
④回收:使用蓝光切胶仪将目的条带切下,并使用Gel Extraction Kit(OMEGA)按照说明书操作,通过DNA吸附柱回收DNA,最终使用微量分光光度计对所回收DNA进行质量及浓度的检测。
3.表达盒的构建及GB-dir连接方法
①取少量大肠杆菌,接种于0.8mL含四环素LB中,900rpm 30℃过夜培养。②将上述菌液转接40μL上述菌液于1.4mL,含四环素LB中900rpm 30℃进行培养。
③加入40μL***糖,37℃诱导40min,同时,对片段进行除盐处理。
④将菌体离心1min。加入1mL无菌水,对菌体进行重悬,并且重复一次,弃掉上清,留下30~40μL菌体。
⑤向菌体中加入20μL除盐后片段,吹打混匀,转移至电转杯中进行电转化。
⑥加入1mL不含抗性LB,将电转杯中大肠杆菌吹打混匀,并且转移至新的离心管中,在37℃条件下孵育1h。
⑦将菌体进行离心,弃掉上清,剩余50~100μL菌液,划线或涂布平板,37℃过夜培养。
⑧待第二天转化子长出后,挑取单克隆进行PCR、酶切或者测序验证。
4.里氏木霉原生质体转化
(1)菌体准备
①取新鲜生孢的平板(生长3-4天),使用灭菌洗孢子液3-4mL,将孢子洗下。
②准备10个麸皮平板,并在培养基表面铺匀一层玻璃纸,在表面均匀涂布150-200μL新鲜孢子液,30℃过夜培养,大约12-15h。
(2)原生质体的制备
①加入0.1g裂解酶到20mL溶液1中,轻轻摇匀。
②待平板的玻璃纸上长出新鲜生长的菌丝后,吸取2~3mL酶液到一个新的平皿中,加上一层带有萌发孢子的玻璃纸,再加入2~3mL酶液,依次叠放8~10层。将此培养皿放置于30℃培养箱中。
③大约酶解2h后,用镊子将玻璃纸挑出,同时使用溶液1洗净上边的菌丝。
④准备带有4层擦镜纸的玻璃漏斗,用此漏斗过滤原生质体悬液到提前冰上预冷的50mL离心管中。
注意:接下来步骤均在冰上进行。
④使用2000rpm预冷水平离心机离心10min,小心去掉上清液,并用4mL溶液2小心重悬原生质体。再次离心。
⑥小心去掉上清液,并用0.5-1mL溶液2重悬原生质体,放置在冰上。
⑦使用显微镜进行镜检。
(3)原生质体转化(在冰上进行)
①将200μL原生质体悬液,10μL纯化DNA片段,50μL PEG轻轻混匀,置于冰上20min。
②加入室温状态下2mL PEG,轻轻混匀,20℃放置5min,加入4mL溶液2轻轻混匀。
③将全部的得到的原生质体,加入预保温的上层选择培养基(含抗性)中,轻轻混匀,倒在铺有下层培养基的平板上,等培养基凝固后置于30℃进行培养。④在筛选培养基上培养3~4天后,用枪尖挑取转化子到传代培养基中,一个平板上可接种多个转化子。
5.里氏木霉转化子验证
通过抗性筛选获得转化子,提取转化子基因组(详细步骤参见《分子克隆实验指南》第三版),进行PCR验证,获得定点***表达盒的纯合子菌株。构建菌株见表1。
表1本实施例中所构建的Cre1突变的菌株
二、里氏木霉转化子在葡萄糖条件下主要纤维素酶基因酶活及蛋白检测
采用2%葡萄糖作为碳源,接种方式采用种子培养基2-3天→转接1%到2%葡萄糖+1%到2%微晶纤维素为碳源的产酶培养基,在10天进行FPA,pNPC和pNPG的测定。
实验平行及重复:3次技术平行,2次生物学重复。
(1)滤纸酶活(FPA):
底物使用1*6.4cm的Whatman快速定性滤纸条,将滤纸条折叠,放入25mL比色管找那个,加入1.5mL柠檬酸缓冲液(pH为4.8),实验组加入0.5mL纤维素酶酶液,并混匀(对照组不加),50℃水浴反应1h,对照组加入0.5mL酶液,实验组及对照组均立即加入3mL新鲜配置DNS溶液将反应终止,接下来沸水浴煮沸10min,后加入ddH2O将比色管定容到25mL,盖上盖子,来回颠倒数次混匀,使用分光光度计测量OD540时的吸光值。
(2)pNPC酶活测定:
在1.5ml的离心管中,加入50μL的pNPC,实验组加入100μL纤维素酶酶液,吹打混匀,50℃水浴反应30min,对照组加入100μL纤维素酶酶液。接下来加入150μL 10%Na2CO3将反应终止,使用分光光度计测量OD420时的吸光值。
(3)pNPG酶活测定:
在1.5ml的离心管中,加入50μL的pNPG,实验组加入100μL纤维素酶酶液,吹打混匀,50℃水浴反应30min,对照组加入100μL纤维素酶酶液。接下来加入150μL 10%Na2CO3将反应终止,使用分光光度计测量OD420时的吸光值。
蛋白测定方法
结果发现:如表2所示,当使用含有葡萄糖的混合碳源作为培养基的碳源时,cel1b基因缺失的里氏木霉,纤维素酶活性(FPase activity)及菌株pNPC活性以及pNPG活性不仅没有提高,而且还有所降低;同时将Cel1b突变为Cel1bI177S/I174S后,和出发菌株相比,纤维素酶活性(FPase activity),pNPC活性以及pNPG活性分别提高了约2-4倍左右;进一步,过表达含量Cel1bI177S/I174S后,和出发菌株相比,纤维素酶活性(FPase activity),pNPC活性以及pNPG活性分别提高了3-10倍左右。
表2改造菌株与出发菌株酶活及蛋白比较相对值
注:表2是以出发菌株为100%,改造菌株纤维素酶活(FPase)、pNPC及pNPG的相对值。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
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Claims (10)
1.一种丝状真菌的重组菌株,其特征在于,所述重组菌株与出发菌株相比,具有CEL1B蛋白突变体,所述突变体的氨基酸序列如下:
(1)如SEQ ID NO:3所示;
(2)SEQ ID NO:3所述氨基酸序列具有一个或多个氨基酸添加、取代或缺失之后,仍具有相似生理活性的氨基酸序列。
2.如权利要求1所述丝状真菌的重组菌株,其特征在于,所述丝状真菌优选为工业酶,包括但不限于黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、草酸青霉(Penicillium oxalicum)、构巢曲霉(Aspergillus nidulans)、棘孢曲霉(Aspergillusaculeatus)、里氏木霉(Trichoderma reesei)中的一种;进一步的,所述丝状真菌为里氏木霉(Trichoderma reesei)。
3.如权利要求3所述丝状真菌的重组菌株,其特征在于,所述里氏木霉包括野生型里氏木霉或里氏木霉的突变株;所述突变株包括经物理、化学诱变或基因工程手段改造后的菌株;
优选的,所述出发菌株为通过基因工程手段敲除cel1a基因的里氏木霉;
优选的,所述出发菌株为里氏木霉(Trichoderma reesei)T1。
4.权利要求1-3任一项所述重组菌株在纤维素酶生产或发酵领域的应用。
5.一种纤维素酶的生产方法,其特征在于,所述生产方法包括采用权利要求1-3任一项所述重组菌株进行发酵;
优选的,所述发酵碳源包括速效碳源、二糖、低聚糖中的一种或几种的组合。
6.一种解除CCR效应的基因序列,其特征在于,所述基因序列编码如SEQ ID NO:3所示的氨基酸序列;
优选的,所述基因序列包括由于密码子简并性可编码获得所述氨基酸序列的。
7.一种表达盒,其特征在于,所述表达盒中,至少包括权利要求6所述基因序列。
8.一种重组载体,其特征在于,所述重组载体中包括权利要求7所述表达盒或权利要求6所述基因序列的完整编码阅读框序列;
优选的,所述重组载体将第四方面权利要求6所述核酸序列***表达载体得到;所述表达载体包括本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体;进一步的,所述表达载体为质粒或病毒载体,所述病毒载体为慢病毒载体、腺相关病毒载体或腺病毒载体。
9.一种解除丝状真菌碳阻遏效应的试剂盒,其特征在于,所述试剂盒中包括权利要求6所述解除CCR效应的基因序列、权利要求7所述表达盒或权利要求8所述重组载体;
优选的,所述试剂盒中,还包括转化试剂、染色体抽提试剂、扩增酶及电泳试剂。
10.一种解除丝状真菌碳阻遏效应的方法,其特征在于,所述方法包括所述方法包括采用权利要求8所述重组载体或权利要求9所述试剂盒对丝状真菌CEL1B的序列进行改造;
优选的,所述改造步骤如下:采用原生质体转化法将所述权利要求8所述重组载体转化到丝状真菌中;通过初步抗性筛选及测序验证,得到突变株。
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