CN116103174A - 一种毕赤酵母突变菌株及其在溶菌酶生产中的应用 - Google Patents
一种毕赤酵母突变菌株及其在溶菌酶生产中的应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域，具体涉及一种毕赤酵母突变菌株及其在溶菌酶生产中的应用。所述突变菌毕赤酵母RONG2‑5（Pichia pastoris RONG2‑5），是通过紫外诱变的方法筛选获得的，溶菌酶产量得到显著提高，已于2022年6月27日保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2022976。所述突变菌可广泛应用于溶菌酶的生产，有利于降低该酶的生产成本，促进其在饲料中的广泛应用。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种毕赤酵母突变菌株及其在溶菌酶生产中的应用。
背景技术
溶菌酶(Lysozyme)是一种能水解致病菌中黏多糖的水解酶，它是一种碱性酶，又称为胞壁质酶(muramidase)或N-乙酰胞壁质聚糖水解酶(N-acetylmuramideglycanohydrlase)，主要通过破坏细胞壁中的N-乙酰胞壁酸和N-乙酰氨基葡糖之间的β-1,4糖苷键，使细胞壁不溶性黏多糖分解成可溶性糖肽，导致细胞壁破裂内容物逸出而使细菌溶解。溶菌酶还可与带负电荷的病毒蛋白直接结合，与DNA、RNA、脱辅基蛋白形成复盐，使病毒失活。因此，溶菌酶具有抗菌、消炎、抗病毒等重要作用。
1992年FAO/WTO的食品添加剂协会公布:溶菌酶应用于食品工业中是安全的，我国卫生部2010年第23号公告批准溶菌酶等物质为食品添加剂。溶菌酶的化学本质为蛋白质，在人及动物的胃肠内可以被消化、吸收，因此不会在体内残留且安全性很高，在食品、药品和饲料等行业具有广泛的应用前景。它在食品工业中可当作天然防腐剂，可延长肉制品、水产品、乳制品、果蔬等的储存时间；在药品领域具有抗病毒、抗感染、调节肠道菌群、止血及凝血等作用；在动物饲料工业上能提高动物的生产性能、调整动物肠道菌群、减少并替代抗生素的使用，是新型的环保饲料添加剂，同时也可以作为饲料防腐剂和杀菌剂，具有安全无毒等优点。
溶菌酶在自然界中来源广泛，根据来源不同可分为动物溶菌酶、植物溶菌酶、微生物溶菌酶，其中动物溶菌酶又分为鸡型溶菌酶(c型溶菌酶)、鹅型(g型)溶菌酶、无脊椎型溶菌酶三种。目前溶菌酶主要从鸡蛋清和蛋壳膜中提取。但这种 直接提取的生产方式，其规模小、成本高，不利于大规模化生产与应用。同时蛋清溶菌酶热稳定性差和对变性剂的难受性较差，其作为饲料添加剂时，饲料造粒过程中有高温处理步骤，由于蛋清溶菌酶不耐受高温，活力受损，严重限制了其应用。特别是近年来，随着饲料用的抗生素的管理不断加强，溶菌酶的市场需求正快速扩大，利用基因工程技术构建溶菌酶酵母表达系统，可对溶菌酶进行异源表达和微生物发酵生产，已成为溶菌酶生产技术研究的热点。
发明内容
本发明的目的是提供了一种毕赤酵母突变菌株及其在溶菌酶生产中的应用。所述突变菌株是通过紫外诱变的方法筛选获得的，溶菌酶产量得到显著提高，有利于降低该酶的生产成本，促进其广泛应用。
本发明一方面涉及一种重组质粒，其携带有溶菌酶基因。
所述溶菌酶基因的核苷酸序列为SEQ ID NO：1，编码的氨基酸序列为SEQ ID NO：2。
本发明一方面涉及一种毕赤酵母工程菌，其携带有上述重组质粒。
本发明还涉及一种毕赤酵母突变菌株，是以上述毕赤酵母工程菌为出发菌，通过紫外诱变的方法获得的。
所述突变菌株为毕赤酵母RONG2-5（
Pichia pastoris RONG2-5），已于2022年6月27日保藏于中国武汉 武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M2022976。
本发明还涉及一种发酵生产溶菌酶的方法，是以上述毕赤酵母突变菌为发酵菌株。
有益效果
本发明首先构建得到重组表达溶菌酶基因的工程菌毕赤酵母RONG2，其摇瓶发酵上清液中溶菌酶酶活最高达到6544U/ml；然后以毕赤酵母RONG2为出发菌，通过紫外诱变筛选获得突变菌株毕赤酵母RONG2-5，能大幅度提高溶菌酶的表达量，其摇瓶发酵上清液中溶菌酶酶活高达10221U/ml，比出发菌提高了56.2%，取得了意料不到的技术效果。
所述突变菌株可作为溶菌酶的发酵生产菌株，有利于降低该酶的生产成本，促进其在饲料领域中的广泛应用。
具体实施方式
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法，例如MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001)和CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是，本领域的技术人员可以在本发明所记载的技术方案的基础上，采用本领域其它常规的方法、实验方案和试剂，而不限于本发明具体实施例的限定。
下面结合具体实施方式，对本发明做进一步阐述。
实施例1 溶菌酶基因的克隆
申请人以来源于Gallus gallus的溶菌酶基因（GenBank号为NP_990612.2）序列为基础，分析该酶的氨基酸序列，去除其自身的信号肽，再根据毕赤酵母的密码子偏好性，对其进行密码子优化，由华大基因公司进行全基因合成。将该溶菌酶基因命名为RONG2，其核苷酸序列为SEQ ID NO：1，编码的氨基酸序列为SEQ ID NO：2。
采用PCR反应克隆溶菌酶RONG2的基因片段，引物序列和反应条件如下：
引物1(F)：gcgcgaattcaaggtcttcggccgatgcgagctgg（下划线为
EcoR I的酶切位点）；
引物1(R)：taaagcggccgcttagaggcggcagcctcgaatccaa（下划线为
Not I的酶切位点）。
PCR条件为：94℃变性5min；然后94℃变性30s，56℃复性30s，72℃延伸40s，35个循环后，72℃保温10min。RONG2基因全长390bp。
实施例2毕赤酵母工程菌的构建
1、重组质粒的构建
将克隆得到的溶菌酶RONG2基因，用限制性内切酶
EcoR I和
Not I进行双酶切，100μl酶切体系如下：溶菌酶基因RONG2的PCR产物40 μl、10×H buffer 10 μl、10×BSA 10 μl、
EcoR I 5 μl、
Not I 5 μl、ddH2O 30 μl。37℃酶切4 h后，琼脂糖凝胶电泳回收。
将表达载体pPIC9K先用限制性内切酶
EcoR I进行单酶切，100 μl酶切体系如下：表达载体pPIC9K 20 μl、10×H buffer 10 μl、
EcoR I 5 μl、ddH2O 65 μl。37℃酶切4 h后，琼脂糖凝胶电泳回收。将回收片段再用限制性内切酶
Not I进行单酶切，100 μl酶切体系如下：pPIC9K回收片段 20 μl、10×H buffer 10 μl、10×BSA 10 μl、10×Triton 10 μl、
Not I 5 μl、ddH2O 45 μl。37℃酶切4 h后，琼脂糖凝胶电泳回收。
将经
EcoR I和
Not I双酶切的RONG2基因片段与表达载体pPIC9K连接，构建表达载体pPIC9K-RONG2。
连接体系如下：表达载体pPIC9K双酶切产物5 μl、RONG2基因双酶切产物3 μl、10×T4 ligase buffer 1 μl、T4 ligase 1 μl。22 ℃连接过夜，转化到大肠杆菌DH5α，挑取转化子测序验证。测序验证正确的转化子转接到LB+Amp液体培养基（0.5%酵母提取物，1%蛋白胨，1%NaCl，100 μg/mL氨苄青霉素，pH7.0）中，37℃过夜培养，提质粒，即为重组质粒pPIC9K-RONG2。
2、转化与筛选
将重组质粒pPIC9K-RONG2用
Sal I进行线性化，线性化产物用柱纯化试剂盒纯化后，通过电穿孔法转化毕赤酵母GS115，涂布MD平板（1.34% YNB，4×10-5%生物素，1%甘油、2%琼脂糖）。在MD平板上生长出的菌落即为毕赤酵母工程菌株，然后涂布含不同浓度遗传霉素G418的YPD平板（1%酵母提取物，2%蛋白胨，2%葡萄糖）上筛选多拷贝的转化子。
3、摇瓶发酵验证
挑取单个多拷贝转化子分别接入BMGY培养基（2%蛋白胨，1%酵母提取物，100 mM磷酸钾缓冲液(pH6.0)，1.34% YNB，4×10-5%生物素，1%甘油）中，30℃、220rpm振荡培养24h后，再转入BMMY培养基（2%蛋白胨，1%酵母提取物，100 mM磷酸钾缓冲液(pH6.0)，1.34% YNB，4×10-5%生物素，0.5%甲醇）中，30℃、220rpm振荡培养，每24h添加0.5%的甲醇。诱导表达4d后，离心去除菌体，参照GB/T25879-2010所述方法分别检测发酵上清液中的溶菌酶酶活力。
结果显示，本发明构建得到的重组表达溶菌酶RONG2的毕赤酵母工程菌在摇瓶条件下，其发酵酶活最高达到6544U/ml。将该转化子命名为毕赤酵母RONG2（
Pichia
pastorisRONG2）。
实施例3 溶菌酶高产菌株的紫外诱变筛选
紫外诱变导致的突变随机性很强，突变产生的效果也是随机的，难以预测。因此，为了获得有效的正突变，技术人员通常需要进行多轮紫外诱变，筛选的工作量较大，而且存在无法获得有效正突变的可能性。但因为紫外诱变所需设备简单、费用少，并且可在短时间内获得大量突变体，因此，它现在仍是一种常用的诱变选育方法。
申请人以毕赤酵母RONG2为出发菌株，通过紫外诱变方法对其进行遗传学改造，进一步提高其溶菌酶的产量。
将毕赤酵母RONG2接种于YPD平板，30℃培养2-3天，使用无菌水洗菌体，制成悬浮液，稀释至1×106个/mL，紫外灯（40W）照射2-10min，距离约22cm，致死率达到90%以上，涂布平板，30℃培养48h。
第一轮紫外诱变共获得了约240个突变菌单菌落。将各个单菌落分别接种于装有200ul BMGY液体培养基的96孔板，30℃、250rpm振荡培养1天后，离心去掉上层培养基，再加入200ul BMMY培养基，30℃、250rpm振荡培养2天，每天添加0.5%的甲醇。诱导表达2天后，离心去除菌体，得到含溶菌酶的上清液，参照GB/T25879-2010所述方法测定上清液中溶菌酶酶活力。
结果显示，第一轮紫外诱变筛选获得的突变菌中，没有一株突变菌发酵上清液中溶菌酶酶活高于出发菌。申请人又按照上述方法继续进行了37轮诱变筛选，最终获得1株溶菌酶产量显著高于出发菌的突变菌株，命名毕赤酵母RONG2-5（
Pichia pastoris RONG2-5）。
在摇瓶发酵条件下，突变菌毕赤酵母RONG2-5发酵上清液中溶菌酶酶活力高达10221U/ml，比出发菌提高了56.2%，取得了意料不到的技术效果。
申请人已于2022年6月27日将毕赤酵母RONG2-5（
Pichia pastoris RONG2-5）保藏于中国武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M2022976。
本发明提供的毕赤酵母突变菌可用于溶菌酶的发酵生产，有利于降低该酶的生产成本，促进其在饲料领域的广泛应用。
Claims (6)
1.一种重组质粒，其特征在于，所述的重组质粒携带有溶菌酶基因。
2.如权利要求1所述的重组质粒，其特征在于，所述溶菌酶基因的核苷酸序列为SEQ IDNO：1，编码的氨基酸序列为SEQ ID NO：2。
3.一种毕赤酵母工程菌，其特征在于，所述的毕赤酵母工程菌携带有权利要求1或2所述的重组质粒。
4.一种毕赤酵母突变菌，其特征在于，所述的突变菌是以权利要求3所述的毕赤酵母工程菌为出发菌，通过紫外诱变的方法获得的。
5.如权利要求4所述的毕赤酵母突变菌，其特征在于，所述突变菌为毕赤酵母RONG2-5（Pichia pastoris RONG2-5），已于2022年6月27日保藏于中国武汉 武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2022976。
6.一种生产溶菌酶的方法，其特征在于，所述方法是以权利要求4或5所述的毕赤酵母突变菌为发酵菌株。
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