CN117587055A - 一种提高微生物核苷产量的方法 - Google Patents
一种提高微生物核苷产量的方法 Download PDFInfo
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- CN117587055A CN117587055A CN202210971519.9A CN202210971519A CN117587055A CN 117587055 A CN117587055 A CN 117587055A CN 202210971519 A CN202210971519 A CN 202210971519A CN 117587055 A CN117587055 A CN 117587055A
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- glyceraldehyde
- phosphate dehydrogenase
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Abstract
本发明涉及基因工程技术领域，尤其涉及一种提高微生物核苷产量的方法。所述方法包括：提高所述微生物中3‑磷酸甘油醛脱氢酶的表达水平。本发明研究发现，提高微生物中3‑磷酸甘油醛脱氢酶的表达水平可以提高微生物生产核苷(例如鸟苷、腺苷和肌苷)的能力。本发明提供的方法可以适用于多种微生物核苷产量的提高，例如解淀粉芽孢杆菌、枯草芽孢杆菌、短小芽孢杆菌和大肠杆菌等，在核苷生产领域有重要意义。
Description
技术领域
本发明涉及基因工程技术领域，尤其涉及一种提高微生物核苷产量的方法。
背景技术
核苷是一类糖苷的总称。核苷是核酸和核苷酸的组成成分。核苷是由D-核糖或D-Z-脱氧核糖与嘧啶碱或嘌呤碱缩合而成。核苷一般为无色结晶，不溶于普通有机溶剂，易溶于热水，熔点为160～240℃。由D-核糖生成的核苷称核糖核苷，参与RNA组成，由D-α-脱氧核糖生成的核苷称脱氧核糖核苷，参与DNA组成。D-核糖与腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶或尿嘧啶缩合生成相应的腺嘌呤核糖核苷、鸟嘌呤核糖核苷、胞嘧啶核糖核苷、胸腺嘧啶核糖核苷和尿嘧啶核糖核苷，它们分别简称为腺苷(A)、鸟苷(G)、胞苷(C)、胸苷(T)和尿苷(U)。
鸟嘌呤核苷(鸟苷)和次黄嘌呤核苷(肌苷)在食品和医药行业有着广泛的作用。在食品领域，鸟苷和肌苷分别是鸟苷酸二钠和肌苷酸二钠的重要前体，而鸟苷酸二钠与肌苷酸二钠组合使用作为食品增鲜剂，广泛应用于鸡精、酱油等调味品中。在医药领域，鸟苷和肌苷可以作为多种抗病毒药物的医药中间体，如无环鸟苷、三氮唑核苷、三磷酸鸟苷钠等都需要鸟苷作为合成原料。肌苷是肌苷酸的重要前体，而肌苷酸可以作为合成腺苷酸(AMP)和鸟苷酸(GMP)的前体，适用于各种原因引起的白细胞减少症、血小板减少症、各种心脏疾患、急性及慢性肝炎、肝硬化等病症，此外还可治疗中心视网膜炎、视神经萎缩等。
腺苷即腺嘌呤核苷，化学名为6-氨基-9-β-D-呋喃核糖基-9-氢嘌呤，它是腺嘌呤核苷酸脱磷酸后的产物，属于重要的核苷酸衍生物。腺苷是一种遍布人体细胞的内源性核苷，可直接进入心肌经磷酸化生成腺苷酸，参与心肌能量代谢，同时还参与扩张冠脉血管，增加血流量。腺苷对心血管系统和肌体的许多其它系统及组织均有生理作用。腺苷除了可以用作治疗心脏的特效药物之外，还是用于合成三磷酸腺苷(ATP)、腺嘌呤、腺苷酸、阿糖腺苷的重要中间体，广泛应用于医药等行业。
目前，微生物发酵是生产核苷的主要方法，主要使用的微生物包括枯草芽孢杆菌、解淀粉芽孢杆菌或短小芽孢杆菌等。在生长菌株的选育与改造过程中，通过使用紫外诱变、硫酸二乙酯诱变育种，定向选育核苷高产菌株；或者根据细菌中核苷酸的代谢路径和调节机理，深入了解菌株遗传背景及菌株特性，通过代谢工程手段，有目的性地对菌株进行改造，以获得性状优良、能够高产核苷的生产菌株。但目前核苷菌种的发酵性能仍较差、核苷的转化率仍较低，不能满足大规模工业化生产的需求。
发明内容
为了解决现有技术存在的问题，本发明提供一种提高微生物核苷产量的方法。
第一方面，本发明提供一种提高微生物核苷产量的方法，包括：
提高所述微生物中3-磷酸甘油醛脱氢酶的表达水平。
进一步地，通过如下任意一种或多种方法提高所述微生物中3-磷酸甘油醛脱氢酶的表达水平：
i)氨基酸突变；ii)启动子强化；iii)RBS强化；iv)增加拷贝数。
进一步地，所述氨基酸突变为将第99位的赖氨酸突变为天冬酰胺；和/或，所述启动子强化为在起始密码子前插入强启动子P43。
进一步地，所述3-磷酸甘油醛脱氢酶的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
进一步地，所述3-磷酸甘油醛脱氢酶由如SEQ ID NO.3或SEQ ID NO.4所示的核苷酸序列编码得到。
进一步地，所述微生物为能够生产核苷的微生物；优选为解淀粉芽孢杆菌、枯草芽孢杆菌、短小芽孢杆菌或大肠杆菌中的一种或多种。
第二方面，本发明提供所述方法构建得到的重组微生物。
第三方面，本发明提供一种3-磷酸甘油醛脱氢酶突变体，所述3-磷酸甘油醛脱氢酶突变体包括如SEQ ID NO.5或SEQ ID NO.6所示的氨基酸序列。
进一步地，所述3-磷酸甘油醛脱氢酶突变体由包括如SEQ ID NO.7或SEQ ID NO.8所示的核苷酸序列编码得到。
本发明进一步提供所述的3-磷酸甘油醛脱氢酶突变体在提高微生物生产核苷或其衍生物的能力中的应用。
核苷衍生物包括次黄嘌呤、肌苷酸、鸟嘌呤、鸟苷酸、核黄素或二乙酰鸟苷酸等。
本发明具备如下有益效果：
本发明研究发现，在微生物中强化3-磷酸甘油醛脱氢酶的表达可以有效提高微生物中核苷的产量，例如提升鸟苷、腺苷和肌苷等核苷中某一种或多种或全部种类的核苷的产量。本发明提供的方法可以创造出高效生产核苷的微生物，在核苷生产领域具有重要意义。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例中涉及的引物名称及引物序列如表1(SEQ ID NO.9-40)所示：
表1本发明实施例中使用的引物名称及序列信息
实施例1解淀粉芽孢杆菌中gapBK99N点突变菌株构建
以DSM7菌株基因组为模板，使用gapB99-1f/gapB99-1r，gapB99-2f/gapB99-2r引物对，pfu高保真DNA聚合酶扩增获得gapB基因的上、下游同源臂。获得片段进行胶回收并融合，扩增获得gapBK99N全长片段，进行胶回收(对应的ORF框的核苷酸序列如SEQ ID No:7所示，氨基酸序列如SEQ ID No:5所示)。
将pKSU质粒(pKSU质粒由南开大学王淑芳教授惠赠，参见Amarkerless genereplacement method for B.amyloliquefaciens LL3 an d its use in genomereduction and improvement of poly-γ-glutamic acid production[J],AppliedMicrobiology and Biotechnology,2014,98(21):8963-8973.Zhang W,Gao W,Feng J,etal DOI:10.1007/s00253-014-5824-2)使用XbaI/PstI进行双酶切并进行胶回收。使用组装试剂盒将酶切后的线性化质粒及gapBK99N片段进行组装，并转化至TransT1感受态中，后期进行鉴定筛选获得重组质粒pKSU-gapBK99N。分别转化至模式菌株DSM7及实验室构建的两株鸟苷生产菌B.s833、B.a836中(B.s833、B.a836已在CN112574934A专利中公开)，用含2.5μg/mL氯霉素的LB平板在30℃下筛选转化子，将获得的转化子接到5ml LB液体培养基中，42℃200rpm培养12h并传一代，稀释涂布至含5μg/mL氯霉素的LB平板获得一次重组子；将一次重组子接到5ml LB液体培养基中，42℃200rpm培养12h并传一代，稀释涂布含0.8μM 5-FU的LB平板筛选二次重组子，筛选获得三株gapBK99N点突变菌株，分别命名为B.a8369、B.s8356、B.a8357。
实施例2实时定量荧光PCR验证解淀粉芽孢杆菌工程菌株中gapB表达水平
将工程菌B.a8357和对照菌株DSM7在LB培养基上培养至对数生长期，取1mL菌液用适量溶菌酶处理后提取总RNA进行逆转录，以cDNA为模板进行实时定量PCR反应。反应条件:95℃预变性10min；95℃15s，60℃1min，40个循环。反应结束后，以细菌16S rR NA为参比，根据2-ΔΔCT法计算相关基因的转录水平。结果显示工程菌B.a8357中的gapB表达水平较DSM7中提高了5倍。
实施例3解淀粉芽孢杆菌中gapB启动子强化菌株构建
以DSM7菌株基因组为模板，使用P43-gapB-1f/P43-gapB-1r，P43-gapB-2f/P43-gapB-2r引物对，pfu高保真DNA聚合酶扩增获得上下游同源臂。使用P43-F/P43-R，以PBE43质粒(PBE43质粒为全基因合成，参考文献Effects of overexpression of key enzymegenes on guanosine accumulation in Bacillus amyloliquefaciens)为模板，扩增获得P43启动子片段。以上述获得的三个片段为模板，P43-gapB-1f/P43-gapB2r为引物，将三片段进行融合PCR，获得全长片段P43-gapB，进行胶回收，按照实施例1中的构建方式构建获得质粒pKSU-P43-gapB，并将其转化至DSM7模式菌株及实验室构建的两株鸟苷生产菌株B.s833、B.a 836中，获得的菌株命名为B.a 8370、B.s 8358、B.a 8359。
实施例4解淀粉芽孢杆菌中gapB二拷贝菌株构建
以DSM7菌株基因组为模板，使用gapB2nd-1f/1r，gapB2nd-3f/3r引物对，pfu高保真DNA聚合酶扩增获得片段gapB2nd-F1，gapB2nd-F3；以B.a 8359菌株为模板，P43-gapB-2f/gapB2nd-2r引物对扩增片段gapB2nd-F2；以上述3个片段为模板，gapB2nd-1f/gapB2nd-3r为引物进行融合PCR获得P43-gapB2nd全长片段，进行胶回收，按照实施例1中的构建方式构建获得质粒pKSU-P43-gapB2nd，并将其分别转化至DSM7模式菌株及实验室构建的两株鸟苷生产菌株B.s833、B.a 836中，获得的菌株命名为B.a8372、B.s 8360、B.a 8361。
实施例5解淀粉芽孢杆菌中P43-gapBK99N菌株的构建
以实施例1中获得的点突变菌株B.a8357为模板，P43gapB99-1f/P43-R，P43-gapB-2f/gapB99-2r引物对进行扩增，获得两个片段；再将上述获得的片段进行融合，获得全长片段，进行胶回收，按照实施例1中的构建方式获得质粒pKSU-P43-gapBK99N，并将其分别转化至DSM7模式菌株及实验室构建的两株鸟苷生产菌株B.s833、B.a 836中，获得的菌株命名为B.a 8373、B.s 8362、B.a 8363。
实施例6解淀粉芽孢杆菌中P43-gapBK99N二拷贝菌株的构建
以DSM7菌株基因组为模板，使用引物P43-gapB992nd-1f/1r，P43-gappB992nd-3f/3r引物为模板，扩增获得P43-gapBK99N2nd-F1和P43-gapBK99N2nd-F3。以实施例5中获得的B.a8363菌株为模板，引物P43-F/P43-gapB992nd-2r引物扩增获得P43-gapBK99N2nd-F2片段，将上述3个片段进行融合获得P43-gapBK99N2nd全长片段，按照实施例1中方法，构建获得质粒pKSU-P43-gapBK99N2nd质粒，将其分别转化至DSM7模式菌株及实验室构建的两株鸟苷生产菌株B.s833、B.a 836中，获得的菌株命名为B.a 8374、B.s 8364、B.a 8365。
实施例7枯草芽孢杆菌中gapBK99N点突变菌株的构建
为确定gapB发生K99N点突变是否能提高腺苷产量，以实验室构建的腺苷生产菌株B.subtilis A5(菌株B.subtilis A5参见CN110257315B)菌株基因组为模板，使用A5-gapB99-1f/1r，A5-gapB99-2f/2r引物对扩增左右同源臂，并融合获得A5-gapBK99N全长片段(对应的ORF框的序列信息见SEQ ID No:8，氨基酸序列信息见SEQ ID No:6)。按照实施例1中的构建方式获得重组质粒pKSU-A5-gapBK99N。转化至B.subtilis A5菌株中，筛选获得gapBK99N菌株，命名为B.subtilis A0006。同时转化至野生菌168中，筛选获得菌株命名为B.subtilis A0017。
实施例8实时定量荧光PCR验证枯草芽孢杆菌工程菌株中gapB表达水平
按照实施例2中方式，检测了工程菌B.subtilis A0006及野生型菌株B.subtilis168中gapB基因表达水平，结果显示B.subtilis A0006中gapB表达水平较B.subtilis 168提高了9倍。
实施例9枯草芽孢杆菌中gapB启动子强化菌株的构建
以B.subtilis A5菌株基因组为模板，A5-gapB99-1f/A5-P43-gapB-1r，A5-P43-gapB-2f/A5-gapB99-2r引物扩增获得A5-P43-gapB-F1和A5-P43-gapB-F3片段，使用实施例2中获得的P43启动子片段，将上述3个片段进行融合，获得P43-gapB全长片段。按照实施例1中构建方法获得pKSU-A5-P43-gapB，将其转化至B.subtilis A5菌株中，筛选获得的的菌株命名为B.subtilis A0007。同时转化至野生菌168中，筛选获得菌株命名为B.subtilisA0018。
实施例10枯草芽孢杆菌中P43-gapB二拷贝菌株的构建
以B.subtilis A5菌株基因组为模板，A5-P43-gapB2nd-1f/A5-P43-gapB2nd-1r，A5-P43-gapB2nd-3f/A5-P43-gapB2nd-3r引物对扩增获得A5-P43-gapB2nd-F1，A5-P43-gapB2nd-F3片段。以B.subtilis A0007菌株为模板，使用P43-F/A5-P43-gapB2nd-2r引物对扩增获得A5-P43-gapB2nd-F2片段，将上述片段进行融合获得A5-P43-gapB2nd全长片段，按照实施例1中方法构建获得pKSU-A5-P43-gapB2nd质粒，转化至B.subtilis A5菌株中，筛选获得的菌株命名为B.subtilis A0008。
实施例11枯草芽孢杆菌中P43-gapBK99N菌株的构建
以pKSU-A5-P43-gapB质粒为模板，A5-gapB99-1f/A5-gapB99-1r，A5-gapB99-2f/A5-gapB99-2r引物对扩增获得A5-P43-gapBK99N-F1，A5-P43-gapBK99N-F2片段。将上述获得的F1和F2片段进行融合获得A5-P43-gapBK99N全长片段，按照实施例1中方法构建获得pKSU-A5-P43-gapBK99N质粒，转化至B.subtilis A5菌株中，筛选获得的菌株命名为B.subtilisA0009。同时转化至野生菌168中，筛选获得菌株命名为B.subtilis A0019。
实施例12枯草芽孢杆菌中P43-gapBK99N二拷贝菌株的构建
以pKSU-A5-P43-gapB2nd质粒为模板，A5-P43-gapB2nd-1f/A5-gapB99-1r，A5-gapB99-2f/A5-P43-gapB2nd-3r引物对扩增获得A5-P43-gapB K99N2nd-F1，A5-P43-gapBK99N2nd-F2片段。将上述获得的F1和F2片段进行融合获得A5-P43-gapB K99N2nd全长片段，按照实施例1中方法构建获得pKSU-A5-P43-gapB K99N2nd质粒，转化至B.subtilis A5菌株中，筛选获得的菌株命名为B.subtilis A0010。同时转化至野生菌168中，筛选获得菌株命名为B.subtilis A0020。
实施例13突变菌株产核苷性能验证
1、将甘油中保存的菌种37℃过夜培养划出单克隆。
2、挑单菌落接种至30mL种子培养基(g/L：葡萄糖20，酵母粉5，玉米浆干粉5，磷酸二氢钾3，硫酸镁0.5，硫酸亚铁0.02，硫酸锰0.01，pH7.0～7.2，121℃下灭菌20min。110rpm37℃培养7～8h)。
3、按10％v/v接种量转接至30ml发酵培养基(g/L：葡萄糖120，酵母粉3.5，磷酸二氢钾3，硫酸铵25，硫酸锰0.01，硫酸镁5，谷氨酸钠10，玉米浆干粉15，碳酸钙25，pH7.0～7.2，121℃下灭菌20min)中，摇床转速130rpm，35℃培养70h(B.s8356、B.a8357、B.s8358、B.a8359、B.s8360、B.a8361、B.s8362、B.a8363、B.s8364、B.a8365、B.a8369、B.a8370、B.a8372、B.a8373、B.a8374菌株发酵72h；B.subtilis A0006-0009，B.subtilis A0017-0020菌株发酵48h)。
4、使用液相色谱仪对发酵液中产苷进行检测(表2)
表2突变菌株摇瓶发酵产鸟苷、肌苷及腺苷评估结果(三次重复均值)
注：表中数值为0的，意为检测值<0.05，可以忽略不计。
从上表可以看出，将gapB的99位K突变为N，将其引进不同出发菌株中，其对应的核苷产量均有不同程度的提升。说明该位点对提高菌株的核苷生产能力是有效的。同时将gapB野生型蛋白或突变后的蛋白在不同出发菌株中进行启动子强化表达或二拷贝强化表达，菌株的核苷生产能力也有不同程度提高。说明gapB的强化表达或包含K99N突变的蛋白强化表达对提高菌株生产核苷均有正效果。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (10)
1.一种提高微生物核苷产量的方法，其特征在于，包括：
提高所述微生物中3-磷酸甘油醛脱氢酶的表达水平。
2.根据权利要求1所述的方法，其特征在于，通过如下任意一种或多种方法提高所述微生物中3-磷酸甘油醛脱氢酶的表达水平：
i)氨基酸突变；ii)启动子强化；iii)RBS强化；iv)增加拷贝数。
3.根据权利要求2所述的方法，其特征在于，所述氨基酸突变为将第99位的赖氨酸突变为天冬酰胺；和/或，所述启动子强化为在起始密码子前插入强启动子P43。
4.根据权利要求1-3任一项所述的方法，其特征在于，所述3-磷酸甘油醛脱氢酶的氨基酸序列如SEQ ID NO.1所示。
5.根据权利要求4所述的方法，其特征在于，所述3-磷酸甘油醛脱氢酶由如SEQ IDNO.2所示的核苷酸序列编码得到。
6.根据权利要求1-5任一项所述的方法，其特征在于，所述微生物为能够生产核苷的微生物；优选为解淀粉芽孢杆菌、枯草芽孢杆菌、短小芽孢杆菌或大肠杆菌中的一种或多种。
7.权利要求1-6任一项所述的方法构建得到的重组微生物。
8.一种3-磷酸甘油醛脱氢酶突变体，其特征在于，所述3-磷酸甘油醛脱氢酶突变体包括如SEQ ID NO.3或SEQ ID NO.4所示的氨基酸序列。
9.根据权利要求8所述的3-磷酸甘油醛脱氢酶突变体，其特征在于，所述3-磷酸甘油醛脱氢酶突变体由包括如SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列编码得到。
10.权利要求8或权利要求9所述的3-磷酸甘油醛脱氢酶突变体在提高微生物生产核苷或其衍生物的能力中的应用。
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