CN115058443B - 转录抑制质粒pCRISPR-nadG及其在提高产溶剂梭菌胞内还原力中的应用 - Google Patents

转录抑制质粒pCRISPR-nadG及其在提高产溶剂梭菌胞内还原力中的应用 Download PDF

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CN115058443B
CN115058443B CN202210711390.8A CN202210711390A CN115058443B CN 115058443 B CN115058443 B CN 115058443B CN 202210711390 A CN202210711390 A CN 202210711390A CN 115058443 B CN115058443 B CN 115058443B
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白圣凯
张志乾
吴奕瑞
谢薇
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Guangzhou Qianxiang Biotechnology Co Ltd
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Abstract

本发明涉及一种转录抑制质粒pCRISPR‑nadG，为将特异性靶向nadG基因的gRNA连接到携带dCas9表达元件的质粒骨架载体而成。本发明还公开了该质粒在提高产溶剂梭菌胞内还原力中的应用，以及一种工程菌WK‑dCas9ΔnadG及其应用。基于CRISPR‑dCas9介导的基因编辑技术，本发明构建转录抑制质粒pCRISPR‑nadG，后经电转化、克隆子验证及菌株恢复等一系列操作，最终成功获得工程菌株WK‑dCas9ΔnadG。通过对比工程菌株与野生型菌株的发酵过程，工程菌株中nadG基因的转录表达受到明显抑制，并伴随菌株胞内还原力水平的明显提高，且与还原力水平密切相关的溶剂产量得到了显著提升。

Description

转录抑制质粒pCRISPR-nadG及其在提高产溶剂梭菌胞内还原 力中的应用
技术领域
本发明涉及一种转录抑制质粒pCRISPR-nadG，该质粒在提高产溶剂梭菌胞内还原力中的应用，以及一种工程菌WK-dCas9ΔnadG及其应用。
背景技术
现阶段，为了避免化石燃料供需不平衡引起的价格波动，及其开采过程中的环境污染问题，寻找具有绿色、环保且可替代化石能源的生物燃料已经成为全球发展可持续性经济的共同需求。生物燃料可以由产溶剂梭菌菌株通过利用糖类作为底物，经ABE(Acetone-Butanol-Ethanol，丙酮-丁醇-乙醇)途径发酵产生，目前传统的发酵工艺主要面临的阻碍之一是由于微生物细胞内的还原力水平较低引起的溶剂转化效率低，这也成为了生物溶剂生产的工业化进程一大阻碍。如果能通过基因编辑技术建立维持菌株胞内高还原力水平体系，将能有效缓解此代谢障碍，但迄今相关报道罕见。
近年来，CRISPR基因编辑技术迅速发展(Hsu et al.,2014)，CRISPR-dCas9系统作为衍生系统，同样由gRNA引导并基于dCas9蛋白实现对基因编辑或干预其转录及表达过程。dCas9 蛋白是Cas9蛋白的突变体，其内切酶活性全部消失，只保留由gRNA引导结合DNA的能力，可以阻止RNA聚合酶与启动子序列的结合，或作为转录终止子阻止RNA聚合酶的运行，实现有效的基因转录抑制过程(Wang et al.,2016)。
发明内容
本发明选择产溶剂梭菌Clostridium sp.WK进行CRISPR-dCas9的基因编辑改造，通过构建转录抑制质粒pCRISPR-nadG，后经电转化、克隆子验证及菌株恢复等一系列操作，最终成功获得工程菌株WK-dCas9ΔnadG。该菌株主要通过抑制nadG基因的转录表达来实现降低其对 NAD(P)H的消耗，进而维持产溶剂梭菌WK胞内的还原力水平，转录抑制后的菌株胞内还原力水平显著提高，在以30g/L葡萄糖为底物的培养基中发酵，工程菌株WK-dCas9ΔnadG的还原力表征指数NAD(P)H/NAD(P)+均明显高于野生型菌对照组，总溶剂产量在提高15.6％的同时，菌株整体维持与野生菌一致的良好发酵性能。
1.转录抑制质粒pCRISPR-nadG的构建及电转结果验证
根据野生型菌株WK的基因组序列文件，通过gRNA设计软件sgRNAcas9_3.0.5设计靶向nadG基因的20bp片段大小的gRNA，并利用PCR扩增获得上游带有Spe I酶切位点和下游带有Not I酶切位点的双链gRNA序列。质粒所用的骨架载体pdCASclos购自Addgene公司，经限制性核酸内切酶Spe I和Not I双酶切并纯化回收后与上述酶切完gRNA序列连接，最终实现转录抑制质粒pCRISPR-nadG的构建。构建完的质粒经电转化方法导入至菌株WK 中，经无抗性培养基复苏后将菌液涂布于含100μg/mL氨苄青霉素抗性的琼脂平板上，通过菌落PCR和测序双验证，最终获得工程菌株WK-dCas9ΔnadG。
2.菌株改造前后的胞内还原力水平、RT-qPCR转录水平差异分析及ABE发酵效果评价
以30g/L葡萄糖作为发酵底物，将工程菌株与野生型菌株分别置于在37℃和150rpm 条件下进行发酵，通过NAD(P)+/NAD(P)H检测试剂盒和增强型ATP检测试剂盒分别完成对菌株胞内还原力水平和ATP含量的测定，经比较菌株改造前后的胞内还原力水平指标 NADPH/NAD(P)+确定nadG基因是否在利用胞内还原力途径发挥关键作用。此外，在利用qPCR技术分析nadG基因在不同菌株中的表达差异的同时，也分析其产溶剂能力的区别，进一步验证了胞内还原力水平与梭菌菌株产溶剂能力的正相关关系。
本发明的有益效果：
本发明选择了产溶剂梭菌Clostridium sp.WK作为基因编辑对象，该菌株保存于广东省微生物菌种保藏中心(GDMCC)，保藏号为GDMCC61493，保藏时间为2021年2月1日，保藏地址为广东省广州市先烈中路100号大院59号楼5楼。该菌株具有丁醇转化率高、副产物低且无需 pH调控等特点，是一株具有工业应用潜力的微生物菌株。辅助因子NAD(P)H在产溶剂梭菌的生物溶剂合成过程中起到关键作用，它作为细胞内众多脱氢酶的关键辅酶，如果供应不足，会严重限制生物溶剂如丁醇的合成。因此提高产溶剂梭菌胞内NAD(P)H的供应是显著提高溶剂产量的有效途径之一(Xu et al.,2015)。通过对菌株WK基因组进行分析，找到编码NADH- 醌氧化还原酶亚基G(序列如SEQ ID No.2所示)的nadG基因，序列如SEQ IDNo.1所示，在基因组中只有一个拷贝，能够调节胞内NAD(P)H的水平，从而影响NAD(P)H依赖型的脱氢酶活性及胞内ATP水平，进一步调节目的产物——生物丁醇的合成。
本发明采用CRISPR-dCas9基因编辑技术构建基因编辑转录抑制质粒pCRISPR-nadG，经电转筛选成功获得工程菌株WK-dCas9ΔnadG，实现与消耗胞内还原力水平密切相关基因 nadG转录表达的靶向抑制。转录水平和还原力水平测定均显示了工程菌株中基因nadG转录水平受到明显抑制，其中最大转录抑制效率达到37％，其胞内的NAD(P)H、ATP含量均明显高于野生型对照组，即表现为更高的胞内还原力水平。此外，工程菌株WK-dCas9ΔnadG的生物溶剂ABE总产量较野生菌WK提升15.6％，其中生物丁醇提升了10.9％，达7.93g/L。相关结果证明了针对nadG基因转录表达的靶向抑制可以有效地维持菌株胞内的高还原力水平，保证菌株处于有利溶剂合成的发酵状态，也同时进一步验证菌株还原力水平与溶剂产量之间的相关性。
附图说明
图1为本发明具体流程图；
图2为本发明实施例1中构建完成的转录抑制质粒pCRISPR-nadG图谱；
图3为本发明实施例2中工程菌株WK-dCas9ΔnadG中nadG基因的抑制效率分析情况；
图4为本发明实施例3中菌株改造前后胞内各项还原力水平指标变化的对比图；
图5为本发明实施例3中野生型菌株WK与工程菌株WK-dCas9ΔnadG分别以30g/L葡萄糖为底物发酵时的生物溶剂合成对比图(**p＜0.01，***p<0.001)。
具体实施方式
下面结合具体的实施例，对本发明做进一步说明，这些具体实施例不应以任何方式被解释为限制本发明的应用范围。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。图1为本发明具体流程图。
实施例1：转录抑制质粒pCRISPR-nadG的构建及电转验证
通过对野生菌株WK基因组的注释分析，获取需要编辑的NADH-醌氧化还原酶亚基G基因 (nadG)。靶向nadG基因的gRNA序列设计采用软件sgRNAcas9_3.0.5，并由南京金斯瑞公司合成。骨架载体pdCASclos购自Addgene公司，携带dCas9表达元件。gRNA负责识别靶位点， dCas9负责对靶位点的包裹，以实现对nadG基因的转录抑制过程。
通过设计含有相应酶切位点的特异引物(gRNA-F和gRNA-R)，使用高保真DNA聚合酶从携带gRNA的pUC57质粒中扩增获取gRNA片段。gRNA的PCR扩增片段与骨架载体pdCASclos同经Spe I，Not I双酶切后纯化回收。通过酶连反应，转化TOP10菌株，检测获得阳性克隆子，即完成转录抑制质粒pCRISPR-nadG的构建。相关引物序列如下文所示：
gRNA-F：5’-ATACTAGTGATGTTTGGGGCTGT-3’(下划线部分为Spe I酶切位点)
gRNA-R：5’-AGCGGCCGCAAAAAAGCACCGAC-3’(下划线部分为Not I酶切位点)
PCR反应体系为：DNA模板(50ng)1μL、正反向引物(10μM)各1μL、dNTP 2μL、5×高保真酶反应缓冲液5μL、高保真DNA聚合酶0.5μL，补足ddH2O至25μL。PCR反应程序为： 95℃预变性5min，95℃30s，62-64℃30s，72℃2min，30个循环，最后72℃延伸10min。
利用电转化技术将构建的质粒pCRISPR-nadG(如图2所示)导入梭菌WK(菌株保藏号： GDMCC61493)中，使用2mm电击杯和3.5ms的电转时间，在电压1.8kV条件下完成整个电转化过程，将电击后的菌体置于10mL RCM(增强型梭菌培养基)中，于37℃恒温培养箱中静置6h后添加100μg/mL氨苄青霉素，再置于在150rpm条件下继续扩摇2h完成菌体复苏。随后将已复苏的菌体涂布于含有100μg/mL氨苄青霉素的RCM琼脂平板上，置于37℃厌氧培养工作站培养至阳性克隆子形成，挑取克隆子使用蛋白酶K预处理后进行菌液PCR反应以验证质粒是否成功导入梭菌，最终获取工程菌株WK-dCas9ΔnadG。
实施例2：qPCR定量分析工程菌中nadG基因的转录抑制效率
本发明选择前期经实验确定的用于对产溶剂梭菌Clostridium sp.WK表达量分析内参基因(recA)，并使用Primer Premier 5软件再设计用于检测nadG基因的扩增引物，如下：
qPCR-recA-F：5’-TCCTCCAGTTGTTGTTTCT-3’
qPCR-recA-R：5’-CTCAGTTGCGGCTTTAGT-3’
qPCR-nadG-F：5’-TCTAATATCCGTCAAACC-3’
qPCR-nadG-R：5’-AAAGATGCGATGTCAAT-3’
使用Omega公司的细菌基因组提取试剂盒提取梭菌WK的基因组DNA，通过引物PCR和TA克隆将目的基因nadG和内参基因recA连接到pMD19-T载体上，以对应的质粒DNA 建立相应标准曲线。通过提取RNA并反转录为cDNA采用qPCR对nadG基因的相对表达量水平进行分析(丁鹏et al.,2005)。结果显示，在以30g/L葡萄糖为底物进行发酵时，工程菌株WK-dCas9ΔnadG在发酵前48h内的nadG基因表达量水平均比野生菌WK低，其中基因的最大抑制效率在32h可以达到最大值为37％(如图3)，该结果证明基于CRISPR-dCas9系统靶向nadG基因的转录表达确实起到了对该基因明显抑制效果。
实施例3：工程菌株的胞内还原力水平分析及其ABE发酵效果评价
菌株胞内还原力水平的测定主要分析NAD(P)H、ATP的浓度和NADPH/NAD(P)+指标大小，本发明通过碧云天NAD(P)+/NAD(P)H检测试剂盒完成菌株胞内NAD(P)H、NAD(P)+的定量测定，而通过碧云天增强型ATP检测试剂盒完成对菌株胞内ATP含量的定量测定。
选取产溶剂菌株(含工程菌株与野生型菌株)发酵前48h的细胞，每12h取样一次，测定其胞内还原力水平变化情况。其中样品处理操作过程如下：将2mL菌液样品在4℃5000rpm条件下离心5min，弃去上清液，然后用PBS洗涤菌体1-2次，并置于超净工作台中完全吹干菌体，然后准确称取菌体重量，记录菌体干重。用移液器准确加入200μL冰浴预冷的裂解液(主要成分含50mM Tris-HCl(pH7.4)，150mM NaCl，0.1％SDS，1％Triton X-100，1％sodium deoxycholate，40μg/mL蛋白酶K)，并轻轻吹打以促进细胞的裂解。之后置于4℃5000 rpm离心5min，取上清液作为待测样品备用。
通过绘制NAD(P)H和ATP的标准曲线、样品还原力水平测定，以及计算NAD(P)H、NAD(P)+及NAD(P)H/NAD(P)+的数值进行分析。结果如图4和表1所示。结果显示，在产酸及产溶剂期(即发酵前48h内)，工程菌株每克干重细胞中的NAD(P)H及NAD(P)H/NAD(P)+浓度和ATP水平均明显高于野生型菌株。这说明通过抑制NADH-醌氧化还原酶亚基G基因 (nadG)的表达确实可以明显提高菌体胞内NADH的供给水平，增加胞内还原力水平。
在分别以30g/L葡萄糖为底物进行发酵过程中，工程菌株WK-dCas9ΔnadG在溶剂产量上均比野生型菌株有了明显的提高，其中从丁醇生成情况看，在96h丁醇产量才达到峰值时，野生型菌株和工程菌株的丁醇产量分别检测为7.15和7.93g/L，工程菌株的丁醇产量明显提高10.9％。同样的，工程菌株丙酮和乙醇的产量也分别提高了14.6％(3.22g/L)和27.3％(0.75 g/L)，由于胞内还原力水平的提升，菌株WK-dCas9ΔnadG的总ABE产量从野生菌的10.29g/L 提升到了11.90g/L，总体提升比例为15.6％(如图5)。
表1菌株改造前后胞内还原力水平测定对比分析
Figure BDA0003708181810000051
参考文献：
[1]Hsu,P.,Lander,E.S.,Zhang,F.,2014.Development and applications ofCRISPR-Cas9 for genome engineering.Cell,157(6),1262–1278.
[2]Wang,Y.,Zhang,Z.T.,Seo,S.O.,Lynn,P.,Lu,T.,Jin,Y.S.,Blaschek,H.P.,2016.Gene transcription repression in Clostridium beijerinckii using CRISPR-dCas9.Biotechnol.Bioeng.,113(12),2739-2743.
[3]Xu,T.,Li,Y.,Shi,Z.,Hemme,C.L.,Li,Y.,Zhu,Y.,Van Nostrand,J.D.,He,Z.,Zhou,J.,2015.Efficient genome editing in Clostridium cellulolyticum viaCRISPR-Cas9 nickase.Appl Environ Microbiol.,81(13), 4423-4431.
[4]丁鹏,王家宁,黄永章,2005.利用TA克隆载体构建pET15b-SOD1重组质粒.郧阳医学院学报,24(2),65-68.
序列表
<110> 广州乾相生物科技有限公司
<120> 转录抑制质粒pCRISPR-nadG及其在提高产溶剂梭菌胞内还原力中的应用
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atggtcattg atggcaatag gatagaattt gataaagaaa agaatatctt agatttagtt 60
agaaaagcag gaattgattt acctactctt tgttactata cagatttatc agtttatggg 120
gcatgcagaa tgtgtgtagt ggaggatgaa agaggtagca ttttaacatc ttgctctaca 180
ccaccaaaag atgcgatgtc aattagaact aatacaccaa agcttcaaaa gtatcgtaag 240
gtaattttag aattgttgct tgcaacacat tgtagagatt gcactatatg tgaaaagaat 300
ggaaagtgta agttacaaaa attagcatca cgttttggtt tgacggatat tagatttaaa 360
agcattcagg gaaaaaaagc tttagatacg tcatctaaag caatcattag agatcctaat 420
aaatgcattt tatgtggaga ctgtgttagg atgtgtagtg aaatacaaag tgtaggagcg 480
attgatttcg tcaatagggg ttcaaatatg gtagtaagtc cagcgtttgg gaaaagcctt 540
gcagagacag attgtgtgaa ttgtgggcaa tgtgcaactg tatgtccgac aggagcaata 600
gttgttaaga gtgatattaa aaatgtttgg aaatcaatat ataaccctaa gcagagggtt 660
gtagcacaag ttgctcctgc agtgcgagtc gctttaggtg aagaattcgg catagaatct 720
ggtgaaaatg ttatggggaa aattgttgca gccatgagaa aattaggctt tgaaaatatt 780
tatgatactt ctctaagtgc tgatttaaca gtaattgaag aatcaaaaga attcttaaag 840
aagttagagt ctgatgataa taaatttcca ctatttactt cttgttgccc agcatgggta 900
agatatgtag aaaataaata tcctgagcta ttgccgtatg tatctagttg taaatcacca 960
atggagatgt ttggggctgt tgttaaggcg tatttcaaag aaaaagattc tttagagaat 1020
agagaaacaa tatctgtagc agttatgccg tgcacagcta aaaaagcaga ggcggctaga 1080
gaagaattta taagggataa tattccggat gttgattatg taattactac agcggaacta 1140
tgtgcaatga taaaagaaat tggaatacag tttgatgaaa ttgaggcaga agcatcagat 1200
ataccactat ctctttactc aggcgcagga gtaatatttg gtgttacggg aggagtaacg 1260
gaagctgtta tccgtgaagt tgttaaagat aaatcttcaa gagtattaaa ggacatagaa 1320
tttataggtg ttcgaggtat gaaaggagta aaaacatgtg aacttcaagt taaagatgaa 1380
tccattagaa tcggtattgt tagtggactt agaaatgcag aagatttaat tggaaaaatt 1440
aagagtgggg aagaacattt tgactttata gaagttatgg catgtcctgg aggatgtata 1500
gctggtgcgg ggcaaccttt tggattaatg gaagagaaaa atgaaagggc aaagggacta 1560
tataagatag ataaggttac gcaaattaaa agaagcgaag aaaatctagt ggttaaatct 1620
ttatatgaag gattgctaaa gaataggacg aaagaattac tacatgttca ttatgataaa 1680
agtgaacatt aa 1692
<210> 2
<211> 563
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Asp Glu Arg Gly Ser Ile Leu Thr Ser Cys Ser Thr Pro Pro Lys Asp
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Ala Met Ser Ile Arg Thr Asn Thr Pro Lys Leu Gln Lys Tyr Arg Lys
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Val Ile Leu Glu Leu Leu Leu Ala Thr His Cys Arg Asp Cys Thr Ile
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Cys Glu Lys Asn Gly Lys Cys Lys Leu Gln Lys Leu Ala Ser Arg Phe
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Gly Leu Thr Asp Ile Arg Phe Lys Ser Ile Gln Gly Lys Lys Ala Leu
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Asp Thr Ser Ser Lys Ala Ile Ile Arg Asp Pro Asn Lys Cys Ile Leu
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Ile Asp Phe Val Asn Arg Gly Ser Asn Met Val Val Ser Pro Ala Phe
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Gly Lys Ser Leu Ala Glu Thr Asp Cys Val Asn Cys Gly Gln Cys Ala
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Ala Pro Ala Val Arg Val Ala Leu Gly Glu Glu Phe Gly Ile Glu Ser
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Gly Glu Asn Val Met Gly Lys Ile Val Ala Ala Met Arg Lys Leu Gly
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Phe Glu Asn Ile Tyr Asp Thr Ser Leu Ser Ala Asp Leu Thr Val Ile
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Glu Glu Ser Lys Glu Phe Leu Lys Lys Leu Glu Ser Asp Asp Asn Lys
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Phe Pro Leu Phe Thr Ser Cys Cys Pro Ala Trp Val Arg Tyr Val Glu
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Asn Lys Tyr Pro Glu Leu Leu Pro Tyr Val Ser Ser Cys Lys Ser Pro
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Met Glu Met Phe Gly Ala Val Val Lys Ala Tyr Phe Lys Glu Lys Asp
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Ser Leu Glu Asn Arg Glu Thr Ile Ser Val Ala Val Met Pro Cys Thr
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Ala Lys Lys Ala Glu Ala Ala Arg Glu Glu Phe Ile Arg Asp Asn Ile
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Pro Asp Val Asp Tyr Val Ile Thr Thr Ala Glu Leu Cys Ala Met Ile
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Ile Pro Leu Ser Leu Tyr Ser Gly Ala Gly Val Ile Phe Gly Val Thr
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Gly Gly Val Thr Glu Ala Val Ile Arg Glu Val Val Lys Asp Lys Ser
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Ile Lys Arg Ser Glu Glu Asn Leu Val Val Lys Ser Leu Tyr Glu Gly
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Leu Leu Lys Asn Arg Thr Lys Glu Leu Leu His Val His Tyr Asp Lys
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Ser Glu His

Claims (6)

1.一种转录抑制质粒pCRISPR-nadG，为将特异性靶向nadG基因的gRNA连接到携带dCas9表达元件的质粒骨架载体而成，其中所述gRNA的序列为5’-GATGTTTGGGGCTGTTGTTA-3’。
2.权利要求1所述的转录抑制质粒pCRISPR-nadG，其特征在于所述质粒骨架载体为pdCASclos。
3.权利要求1-2中任一项所述的转录抑制质粒pCRISPR-nadG在提高产溶剂梭菌胞内还原力中的应用。
4.一种工程菌WK-dCas9ΔnadG，其特征在于：将权利要求1或2所述的转录抑制质粒pCRISPR-nadG转化到保藏编号为GDMCC61493的产溶剂梭菌（Clostridium sp. ）WK中而成。
5.权利要求4所述的工程菌WK-dCas9ΔnadG在产溶剂中的应用。
6.根据权利要求5所述的应用，其特征在于所述溶剂为丁醇、丙酮或乙醇。
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