CN115976094B - 一种提高内源酶分泌的基因工程菌及其构建方法和应用 - Google Patents
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Abstract
本发明公开了一种提高内源酶分泌的基因工程菌及其构建方法和应用,属于基因工程技术领域。本发明以多聚半乳糖醛酸酶这一内源酶为切入点,通过对正常菌株和敲除多聚半乳糖醛酸酶的菌株进行转录组测序分析,筛选出与内源酶分泌相关的关键基因Km_10019,通过实验验证该基因调控内源酶的胞外分泌。本发明对马克思克鲁维酵母进行基因工程改造,获得Km_10019过表达的基因工程菌,该菌株分泌多聚半乳糖醛酸酶的量是对照的1.7倍,本发明为蛋白分泌系统优化提供理论基础和相关基因,对于蛋白酶制剂的开发具有重要意义。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及基于转录组分析结果构建的一种提高内源酶分泌的基因工程菌及其构建方法和应用。
背景技术
随着社会的发展,传统的化学制造所造成环境污染、高能耗和生态恶化等问题引起社会高度重视,因此需要对工业生产废弃物或废水进行处理以满足排放要求。水果罐头加工、果酱生产、果胶生产与使用企业产生含果胶的废水,称之为果胶废水,果胶废水中含有高浓度的果胶,如何去除果胶是该类废水处理的关键。
目前常规的处理方式是通过预处理分离大部分果胶,然后通过生化处理进一步降解去除果胶。主要包括复合絮凝剂处理、混凝-吸附法处理、果胶酶处理、微生物分解处理。通过絮凝剂与果胶分子形成絮体而去除果胶是最快捷的去除果胶的方法,但容易产生二次污染,且后续程序繁琐。酶属于生物催化剂,具有专一性强、作用条件温和以及可生物降解等特点,利用果胶酶处理可以达到很好的效果,不仅对能源的消耗,废弃物的排放有重要的作用,还可以给社会带来巨大的经济效益。
目前,酵母已经被证明在表达异源蛋白方面具有很大的优势,不仅生长速度快,可进行蛋白翻译后修饰、易于遗传操作、可发酵,高生物量浓度和安全的无病原体生产而且还能将蛋白分泌到培养基,因此具有广泛的应用价值。
马克斯克鲁维酵母(Kluyveromyces marxianus)广泛存在于酸奶、水果、酸乳酒等环境中,是一种食品安全级酵母,具有生长周期短、耐高温、等优点。马克斯克鲁维酵母具有内源性的多聚半乳糖醛酸水解酶(果胶酶),因此,马克斯克鲁维酵母是一种理想的生产果胶酶的宿主。然而马克斯克鲁维酵母内源性果胶酶的分泌表达被限制在相对较低的水平,因此,需要对马克斯克鲁维酵母进行分子工程改造以优化其分泌途径提高果胶酶的分泌量。
目前有很多基因被报道与蛋白分泌有关,有研究表明,过量表达ATPases Hsp70家族中的伴侣基因BiP可以提高酿酒酵母中的蛋白质分泌,例如人促红细胞生成素分泌增加5倍(Protein Disulfide Isomerase Overexpression Increases Secretion of ForeignProteins in Saccharomyces cerevisiae[J].Biotechnology,1994,12(4):381-384),牛凝乳酶原增加26倍(Overexpression of binding protein and disruption of the PMR1gene synergistically stimulate secretion of bovine prochymosin but not plantthaumatin in yeast[J].Applied Microbiology&Biotechnology,1996,46(4):365-70.),而且在一些情况下,BiP表达降低会导致外源蛋白减少,但是BiP的过表达作用是蛋白质或宿主特异的,不是在所有情况下都有益。
通过分析马克斯克鲁维酵母转录水平,挖掘与果胶酶蛋白分泌相关基因,探究酵母蛋白分泌机制,构建提高内源酶分泌的基因工程菌对蛋白酶开发具有重要意义。
发明内容
本发明的目的在于通过对马克斯克鲁维酵母转录水平分析,筛选与内源酶分泌相关基因,为优化蛋白分泌系统提供基础。通过基因工程改造构建一种提高内源酶分泌的基因工程菌,应用于蛋白酶的开发。
为实现上述目的,本发明采用如下技术方案:
本发明通过对敲除多聚半乳糖醛酸酶菌株和正常菌株的转录组测序结果进行分析,筛选了一个关键基因Km_10019,其核苷酸序列如SEQ IDNO.1所示。通过分子生物学手段将该基因克隆,构建载体并转入马克斯克鲁维酵母菌株中,结果显示转入该基因的菌株分泌的内源酶(多聚半乳糖醛酸酶)的量显著提升,表明Km_10019基因具有促进酶分泌的作用。
因此,本发明提供了Km_10019基因在促进马克思克鲁维酵母内源酶分泌中的应用,所述Km_10019基因的核苷酸序列如SEQ ID NO.1所示,其编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。
进一步的,所述应用包括:以马克思克鲁维酵母为出发菌,利用生物学技术手段在基因组中插入含有Km_10019基因的过表达模块或者在宿主菌中导入含有Km_10019基因的重组表达质粒,获得功能性获得的基因工程菌。
进一步的,重组表达质粒的原始质粒为pIW1146载体。pIW1146载体为公众可获得材料,参见文献(CRISPR-mediated multigene integration enables Shikimate pathwayrefactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromycesmarxianus[J].Biotechnology for Biofuels,2021,14(1).)。
进一步的,所述基因工程菌采用SD培养基进行发酵培养。研究发现,马克思克鲁维酵母在SD培养基中培养可以分泌多聚半乳糖醛酸水解酶到胞外。SD培养基的组成包括:酵母基础氮源(无氨基酸和硫酸铵),氨基酸核苷酸混合物,葡萄糖。
本发明还提供了一种提高内源酶分泌的基因工程菌,所述基因工程菌以马克思克鲁维酵母为出发菌,基因工程菌中具有包含核苷酸序列如SEQ ID NO.1所示的Km_10019基因的过表达模块。
所述过表达模块中Km_10019基因上游具有过表达启动子,可以采用PGPD、PTEF等启动子。
进一步的,所述基因工程菌中含有包含Km_10019基因的重组表达质粒,所述重组表达质粒的原始载体为pIW1146载体,所述Km_10019基因替换了原始载体中的GFP基因。
本发明还提供了一种构建上述基因工程菌的方法,包括:以马克思克鲁维酵母为出发菌,利用生物学技术手段在基因组中插入含有Km_10019基因的过表达模块或者在宿主菌中导入含有Km_10019基因的重组表达质粒。
进一步的,本发明提供了一种利用双酶切去除pIW1146载体中的GFP基因,再利用无缝克隆的方法将Km_10019基因片段与载体连接,构建获得重组表达载体pIW1146-Km_10019,再转入马克思克鲁维酵母构建基因工程菌的方法。
本发明还提供了所述的基因工程菌在制备多聚半乳糖醛酸酶制剂中的应用。
进一步的,所述应用包括:将扩大培养后的基因工程菌接种于SD液体培养基中发酵培养,收集发酵液,从发酵液中分离获得多聚半乳糖醛酸酶。针对包含重组表达质粒pIW1146-Km_10019的基因工程菌,培养采用缺乏组氨酸的SD培养基。
所述发酵培养包括:基因工程菌接种于SD培养基的初始OD600值约为0.05,在30℃、220rpm条件下培养至OD600值为6-7。
本发明具备的有益效果:
(1)本发明以多聚半乳糖醛酸酶这一内源酶为切入点,通过对正常菌株和敲除多聚半乳糖醛酸酶的菌株(Km_ΔPG)进行转录组测序,筛选出关键基因Km_10019,对Km_10019基因进行实验验证,表明该基因调控内源酶的胞外分泌,为蛋白分泌系统优化提供理论基础和相关基因。
(2)本发明对马克思克鲁维酵母进行基因工程改造,获得Km_10019过表达的基因工程菌,该菌株分泌多聚半乳糖醛酸酶的量是对照的1.7倍,该菌株具有促进多聚半乳糖醛酸酶分泌的作用,对于蛋白酶制剂的开发具有重要意义。
附图说明
图1为对照菌株和Km_ΔPG在对数期和平台期的差异表达基因。
图2为GO富集分析柱状图。
图3为KEGG富集分析柱状图。
图4为Km_10019的克隆结果,M为DNA marker,泳道1为PCR产物。
图5为SDS-PAGE结果,其中control为野生型菌株,Km_ΔPG为敲除多聚半乳糖醛酸酶菌株,Km_10019为转入Km_10019基因质粒的基因工程菌株,下同。
图6为imageJ的相对灰度值分析。
图7为多聚半乳糖醛酸酶(PG)的相对酶活。
具体实施方式
下面结合具体实施例对本发明做进一步说明。以下实施例仅用于说明本发明,不用来限制本发明的适用范围。在不背离本发明精神和本质的情况下,对本发明方法、步骤或条件所做的修改或替换,均属于本发明的范围。
下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
菌株马克斯克鲁维酵母CBS6556购自宁波明舟生物科技有限公司。
敲除多聚半乳糖醛酸酶菌株(Km_ΔPG)构建:利用CRISPR-Cas9技术将多聚半乳糖醛酸酶基因从马克斯克鲁维酵母CBS6556基因组上完全敲除。多聚半乳糖醛酸酶基因的核苷酸序列如SEQ ID NO.3所示。
菌株培养条件:酵母菌株采用SD培养基:1.7g/L BD Difco TM酵母氮源基础无氨基酸和硫酸铵(BD公司),氨基酸混合物(包含核苷酸)(coolaber公司),20g/L葡萄糖(coolaber公司)。
SD-His培养基为上述SD培养基基础上缺少组氨酸。
YPD培养基:10g/L酵母提取物(oxoid公司),20g/L蛋白胨(oxoid公司),20g/L葡萄糖(coolaber公司)。
实施例1
分别对对照菌株(野生型菌株马克斯克鲁维酵母CBS6556)和敲除多聚半乳糖醛酸酶菌株(Km_ΔPG)进行转录组测序,通过分析转录组数据筛选出差异较大的基因。
转录组测序样本:用SD培养基培养对照菌株与敲除多聚半乳糖醛酸酶菌株,选取8h的酵母菌为对数期样本,14h的酵母菌为平台期样本。
转录组测序结果显示,与对照菌株相比,在对数期,共有449个基因表现为差异表达基因,其中有313个基因在Km_ΔPG中下调,有136个基因表现为上调。同时,在平台期,共有177个基因为差异基因,其中63个基因为下调基因,114个基因为上调基因,见图1。共表达韦恩图也显示在对照菌株中有72个基因在对数期特异表达,有50个基因在平台期特异表达,有4915个基因对数期和平台期都有表达;在Km_ΔPG中,有50个基因在对数期特异表达,52个基因在平台期特异表达,有4934个基因在对数期和平台期共表达;当对数期的对照菌株和Km_ΔPG比较时,有44个基因在对照菌株中表达,41个基因在Km_ΔPG中表达,4943个基因在对照菌株和Km_ΔPG中都有表达;当平台期的对照菌株和Km_ΔPG比较时,有38个基因在对照菌株中特异表达,59个基因在Km_ΔPG中特异表达,4927个基因在对照菌株和Km_ΔPG中都有表达。因为我们所研究的多聚半乳糖醛酸酶为分泌蛋白,所以我们主要针对平台期的差异表达基因进行筛选分析。
根据差异基因GO富集分析,分为分子功能(Molecular function)、生物过程(Biological Process)和细胞组成(Cellular Component)三个部分。我们主要挑选了富集最显著的30个GO term,每个部分都有10个term。在平台期,生物过程(BiologicalProcess)中有7个差异基因是被富集到碳水化合物代谢过程(carbohydrate metabolicprocess),3个差异基因被富集到核分裂过程(nuclear division),13个差异基因被富集到跨膜转运过程(transmembrane transport),3个基因被富集到细胞器裂变过程(organellefission),6个基因被富集到DNA代谢过程(DNA metabolic process),5个基因被富集到应激反应过程(response to stress),3个基因富集到细胞周期过程(cell cycle process),4个基因富集到DNA修复(DNA repair),16个基因富集到运输过程(transport),4个基因富集到细胞对DNA损伤刺激的反应(cellular response to DNA damage stimulus)。在细胞组成(Cellular Component)中有13个基因注释为与膜(membrane)相关的,2个基因与细胞外围(cell periphery)相关,6个基因与膜整体组分(integral component of membrane)相关,6个基因与膜的固有成分(intrinsic component of membrane)相关,1个基因与核膜(nuclear envelope)相关,7个基因与膜部分(membrane part)相关,1个基因与细胞膜(plasma membrane)相关,1个基因与细胞质囊(cytoplasmic vesicle)相关,1个基因与囊泡(vesicle)相关,1个基因与细胞质囊泡部分(cytoplasmic vesicle part)相关。在分子功能(Molecular function)中,有3个基因与水解酶活性,水解O-糖基化合物(hydrolaseactivity,hydrolyzing O-glycosyl compounds)相关,有3个基因与水解酶活性,糖基键(hydrolase activity,acting on glycosyl bonds)有关,有6个基因与辅因子结合(cofactor binding)有关,5个基因与辅酶结合(coenzyme binding)有关,2个与NAD结合(NAD binding)有关,8个与跨膜转运蛋白活性(transmembrane transporter activity)有关,8个基因与转运活性(transporter activity)有关,2个基因与金属离子跨膜转运活性(metal ion transmembrane transporter activity)有关,3个基因与转移酶活性,转移糖基(transferase activity,transferring glycosyl groups)有关,7个基因与氧化还原酶(oxidoreductase activity)相关,见图2。
根据KEGG富集的结果中,选取了最显著的20个KEGG通路,其中有2个基因与苯丙氨酸代谢(Phenylalanine metabolism)有关,2个基因与一碳代谢(One carbon pool byfolate)有关,5个基因与碳代谢(Carbon metabolism)有关,2个基因与色氨酸代谢(Tryptophan metabolism)有关,2个基因与果糖和甘露糖代谢(Fructose and mannosemetabolism)有关,3个基因与过氧物酶体(Peroxisome)有关,11个基因与次生代谢物的生物合成(Biosynthesis of secondary metabolites)有关,3个基因与糖酵解/糖异生(Glycolysis/Gluconeogenesis)有关,4个基因与剪接体(Spliceosome)有关,5个基因与酵母的细胞周期(Cell cycle-yeast)有关,2个基因与淀粉和蔗糖的代谢(Starch andsucrose metabolism)有关,2个基因与乙醛酸酯和二羧酸酯代谢(Glyoxylate anddicarboxylate metabolism)有关,2个基因与精氨酸和脯氨酸代谢(Arginine andproline metabolism)有关,2个基因与N-聚糖生物合成(N-Glycan biosynthesis)有关,2个基因与甘氨酸,丝氨酸和苏氨酸代谢(Glycine,serine and threonine metabolism)有关,2个基因与半胱氨酸和蛋氨酸代谢(Cysteine and methionine metabolism)有关,2个基因与丙酮酸代谢(Pyruvate metabolism)有关,1个基因与赖氨酸生物合成(Lysinebiosynthesis)有关,2个基因与泛素介导的蛋白质水解(Ubiquitin mediatedproteolysis)有关,1个基因与类固醇生物合成(Steroid biosynthesis)有关,见图3。
结合GO富集和KEGG富集结果,我们主要选取Km_10019基因进行试验验证。Km_10019基因的核苷酸序列如SEQ ID NO.1所示,编码的氨基酸序列如SEQ ID NO.2所示。
实施例2
一、针对转录组数据结果筛选出的关键基因,我们提取了马克斯克鲁维酵母的基因组并克隆了Km_10019基因。
基因组提取使用天根酵母基因组DNA提取试剂盒,方法如下:
1.取酵母菌液,12000rpm离心1min,去除上清。
2.酵母细胞壁的破除:(酶法)向菌体中加入600μL山梨醇buffer,加入50ULyticase,充分混匀,30℃处理30min,4000rpm离心10min,弃上清,收集沉淀。
3.加入200μL的GA缓冲液重悬,充分混匀。
4.加入20μL Proteinase K溶液,混匀。
5.加入220μL GB缓冲液,充分颠倒混匀,70℃放置10min,溶液变清亮后离心以去除管盖内壁的水珠。
6.加220μL无水乙醇,充分颠倒混匀,此时可能有絮状沉淀的出现,离心。
7.将上一步所得溶液和沉淀都加入吸附柱CB3中,12000rpm离心30sec,倒掉废液,随后将吸附柱CB3放入收集管中。
8.向吸附柱中加入500μL缓冲液GD,12000rpm离心30sec,倒掉废液。
9.将吸附柱中加入600μL漂洗液PW,12000rpm离心30sec,倒掉废液。
10.重复步骤9。
11.将吸附柱放回收集管中,12000rpm离心2min,倒掉废液。将吸附柱置于室温数分钟,以彻底晾干吸附材料中残余的漂洗液。
12.将吸附柱转入一个干净的1.5mL离心管中,向吸附膜的中间滴加50-200μL缓冲液TE,室温放置2-5min,12000rpm离心2min,收集溶液。
设计PCR引物,序列(5’-3’)如下:Km_10019_F:agaactagtggatcccccgggATGTTTGCAAGTATTCTGGTCATATCA;Km_10019_R:taactaattacatgactcgagTCAAAAAACCGCCAAAAAGAG。
PCR反应体系:
PCR反应程序:95℃3min,95℃15sec,56-72℃15sec,72℃1min/kb,35个循环,72℃5min。
PCR产物进行凝胶电泳检测,结果如图4所示。
二、构建重组表达质粒,质粒载体采用pIW1146载体框架(Li M,Lang X,Cabrera MM,et al.CRISPR-mediated multigene integration enables Shikimate pathwayrefactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromycesmarxianus[J].Biotechnology for Biofuels,2021,14(1).),该载体由pIW 578改造得到,其中含有GPD启动子序列、GFP编码基因、CYC1终止子序列。
先将载体用酶切的方法将GFP基因切除,后利用无缝克隆的方法将Km_10019构建至pIW1146载体,空载质粒的构建则是将GFP去除。随后将质粒转染至酵母菌中,构建成新的菌株。其中转染方法如下:将原始菌株于2mL YPD培养基中在30℃培养箱摇16h,取1mL菌体5000×g离心1min,倒掉上清液,用1mL ddH2O洗涤两次后,加入500μL的转染buffer(40%3350PEG,0.1M LiAc,10mM pH7.5的Tris-HCl,1mM EDTA,10mM DTT),10μL salmon spermDNA,1μg质粒,常温放置15min,47℃热激15min,5000×g离心1min,用500μL选择培养基(SD-His培养基)重悬,随后吸50μL于2mL的选择培养基振荡培养24-48h,最后涂布于选择培养基固体培养基板上,获得阳性克隆。阳性克隆送公司测序,测序正确的即为转入Km_10019基因质粒的基因工程菌株,保存。
实施例3
将实施例2构建的基因工程菌株活化后接种于2mL SD-His液体培养基中,30℃,220rpm条件下培养17h后测OD600,再接种于25mL SD-His液体培养基的摇瓶中(初始OD600都为0.05),30℃,220rpm条件下培养20h,取2mL菌液离心,取上清液至浓缩管中,离心最后浓缩至180μL(期间用PBS缓冲液洗两次)。利用SDS-PAGE进行蛋白分析,并用imageJ分析其灰度值。为了验证此分泌到胞外的蛋白为多聚半乳糖醛酸水解酶,将敲除多聚半乳糖醛酸酶菌株(Km_ΔPG)同样进行上述操作,结果如图5所示。
结果显示敲除多聚半乳糖醛酸酶菌株(Km_ΔPG)没有目的蛋白条带,表明野生型菌株和重组菌株分泌到胞外的蛋白为多聚半乳糖醛酸酶。与对照(野生型菌株)相比,重组菌株分泌的多聚半乳糖醛酸酶的量是对照的1.7倍,见图6。
酶活的测定:取上述浓缩管中蛋白溶液100μL,与900μL果胶溶液(2g/L)混合,在50℃培养5min,加入4mL DNS溶液(100mL DNS溶液包含80mL 0.5M NaOH,1g DNS,30g酒石酸钾钠),100℃反应10min,立即放于冰上,测540nm下的吸光值。结果如图7所示,实验组的相对酶活是对照的1.6倍,进一步证明过表达Km_10019基因对多聚半乳糖醛酸酶的分泌起促进作用。
Claims (5)
1.核苷酸序列如SEQ ID NO.1所示的Km_10019基因在促进马克思克鲁维酵母内源酶分泌中的应用。
2.如权利要求1所述的应用,其特征在于,所述Km_10019基因编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。
3.如权利要求1所述的应用,其特征在于,所述应用包括:以马克思克鲁维酵母为出发菌,利用生物学技术手段在基因组中插入含有Km_10019基因的过表达模块或者在宿主菌中导入含有Km_10019基因的重组表达质粒,获得功能性获得的基因工程菌。
4.如权利要求3所述的应用,其特征在于,重组表达质粒的原始质粒为pIW1146载体。
5.如权利要求3所述的应用,其特征在于,所述基因工程菌采用SD培养基进行发酵培养。
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