CN114561333B - 一种转化支链氨基酸的工程菌及在制备治疗枫糖尿症产品中的应用 - Google Patents
一种转化支链氨基酸的工程菌及在制备治疗枫糖尿症产品中的应用 Download PDFInfo
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Abstract
本发明涉及一种转化支链氨基酸的工程菌及其在制备治疗枫糖尿症产品中的应用。本发明构建了包含基因LeuDH和基因MorA的载体，通过同源重组方式将其转入乳杆菌。经验证上述重组菌株能够有效表达亮氨酸脱氢酶及α‑羟基酸脱氢酶，并实现对支链氨基酸的降解作用，有望作为工程菌应用于枫糖尿症治疗药物的制备，对于枫糖尿症的临床治疗具有重要意义。
Description
技术领域
本发明属于生物医药技术领域，涉及一种将支链氨基酸转化为α-羟基酸的工程菌、所述工程菌的菌剂、药物组合物及其作为支链氨基酸降解剂的应用，具体涉及其在制备治疗枫糖尿症产品中的应用。
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。
枫糖尿症(MSUD)又称支链酮酸尿症，收录于2018年颁布的《第一批罕见病目录》。枫糖尿症是一种常染色体隐性遗传病，由于支链酮酸脱氢酶复合体的先天性缺陷，致使支链氨基酸(BCAA)分解代谢受阻。患者尿液和血液中积蓄支链氨基酸及其相应酮酸。临床表现主要侵犯神经系统，多表现进行性脑损害症状，小儿智能低下。
支链氨基酸是人体必需氨基酸，包括亮氨酸、异亮氨酸和缬氨酸，MSUD患者需要从食物中获取适量的支链氨基酸。针对急性代谢危象患者采用血液透析、全静脉营养、胰岛素、维生素B1等治疗方法，而MSUD患者的日常治疗方案为低蛋白质饮食，限制患者的支链氨基酸摄入量。但是长期的低蛋白质饮食治疗会影响MSUD患者的生长发育和心理健康，患者生活质量差。
苯丙酮尿症和MSUD都属于氨基酸代谢障碍疾病，苯丙酮尿症患者的苯丙氨酸羟化酶缺陷导致苯丙氨酸代谢障碍。目前利用大肠杆菌工程益生菌治疗苯丙酮尿症已经进入临床实验，通过构建可以降解苯丙氨酸的大肠杆菌工程益生菌，在患者肠道能降解食物中苯丙氨酸，减少患者的苯丙氨酸摄取量。但目前还没有可以治疗MSUD工程益生菌的研究报道。
发明内容
基于上述技术背景，本发明目的在于提供一种用于治疗枫糖尿症的重组益生菌工程菌，通过重组菌株表达亮氨酸脱氢酶基因及α-羟基酸脱氢酶基因。经验证，上述重组后的菌株能够定植于胃肠部位，降解和转化食物蛋白质来源的支链氨基酸，并降低机体内支链氨基酸及其酮酸的蓄积，有望应用于枫糖尿症的临床治疗。
因此，本发明提供以下技术方案：
本发明第一方面，提供一种转化支链氨基酸的工程菌，所述工程菌的出发菌株为乳杆菌，与野生株相比，被修饰为具有增强的亮氨酸脱氢酶和α-羟基酸脱氢酶基因表达。
上述工程菌可以表达亮氨酸脱氢酶、α-羟基酸脱氢酶两种酶。其中，亮氨酸脱氢酶将支链氨基酸降解为α-酮酸，α-羟基酸脱氢酶将α-酮酸继续转化为α-羟基酸，而α-羟基酸是一种营养物质，对人体无害。野生型微生物无这两个酶，只能通过转氨基途径将支链氨酸转化为α-酮酸，被人体吸收后无法继续转化，会蓄积在病人体内并致病。因此，本发明设计通过上述两种酶的合作关系，将机体原本无法代谢的支链氨基酸转化为营养物质，从而实现治疗效果。
考虑到上述工程菌应用于MSUD的治疗，首先采用益生菌作为出发菌株从而保证工程菌的安全性。乳杆菌属于乳酸菌，是一类重要的益生菌。大量研究表明，乳杆菌能调节胃肠道正常菌群、维持微生态平衡，抑制肠道内腐败菌和致病菌生长、提高机体免疫力等。利用乳杆菌作为出发菌株构建工程菌有望获得更好的生物相容性。
进一步的，所述出发菌株为包括但不限于嗜酸乳杆菌、干酪乳杆菌、卷曲乳杆菌、发酵乳杆菌、格氏乳杆菌、瑞士乳杆菌、约氏乳杆菌、副干酪乳杆菌、植物乳杆菌、唾液乳杆菌，德氏乳杆菌、鼠李糖乳杆菌、罗伊氏乳杆菌、发酵乳杆菌或瑞士乳杆菌中的一种。
具体的，所述出发菌株为嗜酸乳杆菌，嗜酸乳杆菌可定植于胃和小肠，而胃和小肠都是食物中蛋白质消化吸收的主要场所，嗜酸乳杆菌工程益生菌可以更有效地降解食物中支链氨基酸。本发明验证可行的一种实施方式中，所述出发菌株为嗜酸乳杆菌NCFM，是目前研究最为丰富的益生菌菌种，主要存在小肠中，能够释放乳酸、乙酸和一些对有害菌起作用的抗菌素，对致病微生物具有拮抗作用，是目前乳制品发酵、药品或益生菌制剂的常用菌株，应用于人体的安全性较高。
优选的，所述亮氨酸脱氢酶(LeuDH)来自于蜡样芽孢杆菌，亮氨酸脱氢酶可以将支链氨基酸降解为α-酮酸，是氨基酸转氨酶降解途径的有益补充，从而增加肠道内支链氨基酸的降解效果。具体的实例为蜡样芽孢杆菌(Bacillus cereus)DSM 626，经测定，上述菌株中分离得到的亮氨酸脱氢酶最适反应温度为37℃，与人体的温度接近，在30℃的环境中半衰期长达330h，在pH7.0～8.0的缓冲液中保存24h后仍保持原有酶活力的80％以上。上述特性决定了本发明使用的亮氨酸脱氢酶有望良好地适应人体肠道环境。进一步的，本发明对野生型蜡样芽孢杆菌(Bacillus cereus)DSM 626中的亮氨酸脱氢酶(LeuDH)进行了密码子优化，优化后的序列如SEQ ID NO.1所示。
优选的，所述α-羟基酸脱氢酶基因(MorA)来源于米曲霉，具体为米曲霉(Aspergillus Oryzae)RIB40。α-羟基酸脱氢酶可将上述α-酮酸继续转化为对人体有益的营养物质α-羟基酸。本发明同样针对野生型米曲霉(Aspergillus Oryzae)RIB40中的α-羟基酸脱氢酶基因(MorA)进行了密码子优化，优化后的序列如SEQ ID NO.2所示。
上述优选方案的一种实施方式中，所述工程菌中同时具有亮氨酸脱氢酶和α-羟基酸脱氢酶基因的表达，所述修饰包括引入外源活性基因，所述外源活性基因的拷贝数可依据重组菌株的表达效果进行调整。所述修饰的方式包括将目的基因连接至载体将其整合入宿主细胞基因组而增加，所述载体为包含目的基因核苷酸序列的构建体，并且能够在乳杆菌中进行表达；进一步的，所述载体为天然或重组质粒、黏粒、病毒、噬菌体中的一种。本发明验证可行的一种实施方式中，所述载体为重组质粒。
另外，所述载体插入染色体可以通过本领域中任何已知的方法执行，例如，同源重组。具体的实施方式中，生产脱氢酶的工程菌的构建方式如下：
(1)将基因LeuDH和基因MorA置于嗜酸乳杆菌启动子P32下游，采用化学合成方法合成启动子P32、基因LeuDH和基因MorA；
(2)将上述基因片段连接到pORI28载体；
(3)以嗜酸乳杆菌NCFM为出发菌株，通过同源重组方法将基因LeuDH和基因MorA整合到基因组上，获得基因LeuDH和基因MorA整合菌株。
上述基因LeuDH和基因MorA整合菌株，命名为工程益生菌MS312；该工程菌具有以下生理特点：革兰氏阳性杆菌，菌体末端呈圆形。固体平板上形成粗糙、凸起、边缘卷曲的菌落。代谢产生乳酸和乙酸。
所述工程菌的最适培养方式如下：MRS液体培养基，35～39℃，厌氧，150～250转/分钟振荡培养；进一步优选的培养方式为：37℃下厌氧，200转/分钟振荡培养。
本发明第二方面，提供一种菌剂，所述菌剂为第一方面所述生产亮氨酸脱氢酶及α-羟基酸脱氢酶工程菌的培养物或代谢物。
本发明第三方面，提供第一方面所述工程菌和/或第二方面所述菌剂作为支链氨基酸降解剂的应用。
经验证，上述工程菌在体外培养条件下能够稳定表达亮氨酸脱氢酶及α-羟基酸脱氢酶，并实现对支链氨基酸(亮氨酸、异亮氨酸、缬氨酸)的降解作用，可作为一种支链氨基酸降解剂加以应用。
一种具体的实施方式中，上述工程菌生产的亮氨酸脱氢酶及α-羟基酸脱氢酶可用于分解肠道中食物来源的支链氨基酸，从而改善MSUD患者尿液、血液中支链氨基酸及酮酸的积蓄情况，即用于枫糖尿症的治疗及改善。即所述作为支链氨基酸降解剂的应用，进一步的，为用于制备治疗枫糖尿症的产品，所述产品包括但不限于药物、保健食品；所述保健食品包括但不限于特殊医用食品、糖果、饮品等。
本发明第四方面，提供一种药物组合物，所述药物组合物中，第一方面所述工程菌或第二方面所述菌剂作为活性成分。
本发明第五方面，提供一种枫糖尿症治疗药物，所述药物中，包括第一方面所述工程菌、第二方面所述菌剂和/或第三方面所述药物组合物。
优选的，所述治疗药物中，还包括药学上述所必需的辅料。
优选的，所述治疗药物为固体制剂或液体制剂，所述固体制剂为包括但不限于片剂、丸剂、胶囊剂、粉剂、微胶囊制剂中的一种；所述液体制剂为溶液剂、混悬剂或乳剂；进一步优选的，所述治疗药物为固体制剂，为粉剂或片剂。
以上一个或多个技术方案的有益效果是：
(1)本发明首次采用乳杆菌构建用于治疗枫糖尿症的工程益生菌，利用嗜酸乳杆菌主要定植于蛋白质消化、氨基酸吸收场所胃和小肠的特征，有效降解和转化食物中支链氨基酸，降低患者的支链氨基酸摄取量。
(2)本发明提供的工程益生菌，有助于治疗枫糖尿症患者，提高患者生活质量，具有重要的临床治疗价值和社会效益。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为实施例2中所述工程益生菌MS312对MSUD小鼠血液中支链氨基酸浓度的影响。
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。
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。以下实施例中采用的出发菌株嗜酸乳杆菌NCFM为市售途径购买获得。
实施例1
一、工程益生菌MS312构建
以嗜酸乳杆菌NCFM出发菌株，在基因组上整合亮氨酸脱氢酶基因LeuDH和α-羟基酸脱氢酶基因MorA，包括如下步骤：
本实施例中全基因合成及测序由上海生工生物工程有限公司完成。分子生物学实验包括感受态细胞制备、电转化等参照《分子克隆实验指南》(第三版)进行。
设计含启动子P32(SEQ ID NO.3)、亮氨酸脱氢酶基因LeuDH(SEQ ID NO.1)和α-羟基酸脱氢酶基因MorA(SEQ ID NO.2)的基因片段MSUD1，用全基因合成的方法获得DNA片段并构建到克隆载体pUC57，获得pUC57-MSUD1载体。
以嗜酸乳杆菌NCFM基因组为模板，L-For和L-Rev为引物，PCR扩增获得上游同源臂L-arm。以NCFM基因组为模板，R-For和R-Rev为引物，PCR扩增获得下游同源臂R-arm。以pUC57-MSUD1载体为模板，MSUD1-For和MSUD1-Rev为引物，PCR扩增获得MSUD1片段。L-arm、R-arm和MSUD1片段利用重组酶方法克隆到pORI28载体，获得pORI28-MSUD1载体。
将pORI28-MSUD1载体和pTRK669辅助质粒通过电击转化一起转入NCFM菌株，并用Val-For和Val-Rev引物进行PCR筛选，获得LeuDH和MorA基因整合到基因组上的重组菌株。然后通过42℃和在无抗生素培养基上连续传代消除质粒，最终获得工程菌MS312。
表1引物列表
二、工程益生菌MS312降解支链氨基酸活性
将NCFM出发菌株和MS312工程菌分别接种于MRS肉汤培养基中，37℃、250rpm培养至OD600＝1.0，离心收集菌体，并用PBS缓冲液洗涤2次。最后用含0.5％葡萄糖、40mg/L亮氨酸、异亮氨酸或缬氨酸的PBS缓冲液悬浮稀释至OD600＝1.0，37℃、250rpm培养，分别在0小时、2小时、4小时和8小时取样，离心收集上清样品，检测亮氨酸、异亮氨酸或缬氨酸的浓度。每个处理做3个重复，结果取平均值，检测结果见表2。统计分析显示，MS312工程菌处理样品中支链氨基酸浓度下降速度显著高于NCFM对照处理，表明MS312工程菌具有较好的降解支链氨基酸能力。
表2益生菌处理条件下支链氨基酸的平均浓度(mg/L)
实施例2工程益生菌MS312对枫糖尿症小鼠的影响
枫糖尿症小鼠模型购自生物技术公司，基因型为C57BL/6J-Bckdhaem1。在正常喂养条件下，该模型小鼠具备枫糖尿症的典型症状。实验采用为6周龄C57BL/6J-Bckdhaem1小鼠，利用无支链氨基酸饲料喂养，每天定时灌胃100μL含亮氨酸、异亮氨酸和缬氨酸的纯化水作为支链氨基酸补给。动物实验开始后，将小鼠分成3组，每天定时灌胃2次，分别灌胃200μL生理盐水、NCFM益生菌或MS312工程益生菌，每次益生菌服用量约1*1010cfu/只小鼠。益生菌喂养14天后采血和采尿取样，检验血液中支链氨基酸浓度(图1)。结果显示，口服MS312工程益生菌后，C57BL/6J-Bckdhaem1小鼠血液中支链氨基酸浓度相比于口服生理盐水和NCFM益生菌都明显降低，说明MS312工程益生菌能够降解和转化食物中支链氨酸，减少小鼠的支链氨基酸摄取量。
综上所述，本发明所构建的嗜酸乳杆菌工程益生菌MS312能够降解和转化食物中支链氨基酸，显著降低枫糖尿症基因小鼠血液中支链氨基酸浓度，有望应用于治疗枫糖尿症。
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
<110> 山东第一医科大学（山东省医学科学院）
<120> 一种转化支链氨基酸的工程菌及在制备治疗枫糖尿症产品中的应用
<130> 2010
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Claims (11)
1.一种转化支链氨基酸的工程菌，其特征在于，所述工程菌的出发菌株为嗜酸乳杆菌NCFM，与野生株相比，被修饰为具有增强的亮氨酸脱氢酶和α-羟基酸脱氢酶基因表达；
所述亮氨酸脱氢酶来源于蜡样芽孢杆菌（Bacillus cereus）DSM 626；
所述亮氨酸脱氢酶进行了密码子优化，优化后的序列如SEQ ID NO.1所示；
所述α-羟基酸脱氢酶基因来源于米曲霉；
为米曲霉（Aspergillus Oryzae）RIB40；
所述α-羟基酸脱氢酶基因进行了密码子优化，优化后的序列如SEQ ID NO.2所示。
2.如权利要求1所述转化支链氨基酸的工程菌，其特征在于，所述工程菌中同时具有亮氨酸脱氢酶和α-羟基酸脱氢酶基因的表达。
3.如权利要求2所述转化支链氨基酸的工程菌，其特征在于，所述转化支链氨基酸的工程菌的构建方式如下：
（1）将基因LeuDH和基因MorA置于嗜酸乳杆菌启动子P32下游，采用化学合成方法合成启动子P32、基因LeuDH和基因MorA；
（2）将上述基因片段连接到pORI28载体；
（3）以嗜酸乳杆菌NCFM为出发菌株，通过同源重组方法将基因LeuDH和基因MorA整合到基因组上，获得基因LeuDH和基因MorA整合菌株。
4.一种菌剂，其特征在于，所述菌剂为权利要求1-3任一项述转化支链氨基酸的工程菌的培养物或代谢物。
5.权利要求1-3任一项所述工程菌和/或权利要求4所述菌剂作为支链氨基酸降解剂的应用。
6.如权利要求5所述的应用，其特征在于，所述作为支链氨基酸降解剂的应用为用于制备抗枫糖尿症的产品。
7.如权利要求6所述的应用，其特征在于，所述产品包括药物、保健食品；所述保健食品包括特殊医用食品、糖果或饮品。
8.一种药物组合物，其特征在于，所述药物组合物中，权利要求1-3任一项所述工程菌或权利要求4所述菌剂作为活性成分。
9.一种枫糖尿症治疗药物，其特征在于，所述药物中，包括权利要求1-3任一项所述工程菌、权利要求4所述菌剂和/或权利要求8所述药物组合物。
10.如权利要求9所述枫糖尿症治疗药物，其特征在于，所述治疗药物中，还包括药学上所必需的辅料；
或，所述治疗药物为固体制剂或液体制剂，所述固体制剂为片剂、丸剂、胶囊剂、粉剂、微胶囊制剂中的一种；所述液体制剂为溶液剂、混悬剂或乳剂。
11.如权利要求10所述枫糖尿症治疗药物，其特征在于，所述治疗药物为固体制剂，为粉剂或片剂。
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| CN114008209A (zh) * | 2019-04-12 | 2022-02-01 | 马萨诸塞大学 | Aav介导的枫糖尿症(msud)基因疗法 |
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