CN116926092A - 一种泛酸激酶基因RkPank及其应用 - Google Patents
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Abstract
本发明公开了一种泛酸激酶基因RkPank，其核苷酸序列如SEQ ID NO:1所示，该基因编码的氨基酸序列如SEQ ID NO:2所示；该基因是从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中分离得到，将该基因通过转化转入到红冬孢酵母YM25235中，实验结果显示RkPank基因的过表达会引起YM25235菌株胞内乙酰辅酶A水平提高，进一步导致类胡萝卜素合成水平的提高；本发明通过基因工程手段对微生物进行改造，来提高微生物体内乙酰辅酶A的水平，为类胡萝卜素和其他以乙酰辅酶A为前体的代谢产物的大规模商业化生产奠定基础。
Description
技术领域
本发明属于基因工程技术领域，涉及一种泛酸激酶基因RkPank及其应用。
背景技术
辅酶A（CoA）是参与酰基转移的大量酶的普遍共底物，是许多代谢反应中必不可少的辅因子，包括脂肪酸的合成和氧化、复杂的脂质合成，尤其是在PDH旁路中CoA乙酰化生成乙酰辅酶A。在大多数细菌和真核生物中，CoA是由泛酸通过泛酸激酶(PanK)、磷酸环腺苷酸-半胱氨酸连接酶(CoaB)、磷酸环腺苷酸半胱氨酸脱羧酶(CoaC)、泛氨酸-磷酸腺苷酰基转移酶(PPAT)和去磷辅酶a (dPCoA)激酶(DPCK)五步转化合成的。CoA生物合成的前体--泛酸（维生素B5）是一种普通的中间产物，可以由几种氨基酸从头形成，也可以使用特殊的渗透酶从细胞外摄取。
泛酸激酶催化泛酸磷酸化生成4’-磷酸泛酸，该反应是CoA生成途径的限速酶。泛酸激酶具有 3 种类型：Ⅰ型、Ⅱ型和Ⅲ型，其中，Ⅰ型以大肠埃希菌的泛酸激酶 (EcCoaA) 为代表；Ⅱ型以金葡菌的泛酸激酶（SaCoaA）为代表，主要存在于真核生物中，包括哺乳动物来源的4种亚型： PanK1α、PanK1β、PanK2 和 PanK3；Ⅲ型主要存在于病原微生物中，如幽门螺旋杆菌、铜绿假单胞菌、炭疽芽胞杆菌和结核分枝杆菌（Mtb）等。
乙酰辅酶A作为辅酶A巯基上最重要的硫酯衍生物，在糖和脂肪酸的氧化降解中起着核心作用，但对于合成代谢途径，如脂肪酸生物合成、类胡萝卜素生物合成、哺乳动物酮体的形成以及许多微生物C2底物的糖异生，也是不可或缺的。酵母产生乙酰辅酶A有三个主要途径:①PDH途径②PDH旁路③脂肪酸氧化。PDH旁路依靠乙酰辅酶A合成酶将辅酶A乙酰化生成乙酰辅酶A。
类胡萝卜素是一类橙黄色、橙红色或红色的多烯类化合物，其特有的抗氧化性可有效保护细胞中各种生物大分子不受自由基损伤，具有维持细胞正常生命代谢、防止机体衰老、提高机体免疫力等作用，被广泛应用于食品着色、营养添加、动物饲料等行业。市场上80% ~ 90%的类胡萝卜素通过传统化学合成法制备，但该方法由于自身的毒性问题使制备的类胡萝卜素在食品领域的应用受到严重限制。天然色素主要来源于植物和微生物，相较于植物源色素，微生物源色素主要采用发酵合成法制备，具有工艺绿色、稳定、发酵原料易得、发酵周期不受季节变化影响等优势。
发明内容
本发明提供了一种从红冬孢酵母（Rhodosporidium kratochvilovae）YM25235中分离得到泛酸激酶基因RkPank，该基因核苷酸序列如SEQ ID NO:1所示或与SEQ ID NO:1互补的核苷酸序列，该基因序列长为1686bp（碱基），该基因编码的氨基酸序列如SEQ ID NO:2所示的多肽。
本发明另一目的是将上述泛酸激酶基因RkPank应用在促进红冬孢酵母生产类胡萝卜素中。
上述目的通过以下技术方案实现，从红冬孢酵母YM25235中提取总RNA，然后反转录合成cDNA，以合成cDNA为模板，通过PCR扩增获得目的片段，将双酶切胶回收后的载体与目的片段连接，获得重组质粒pRHRkPank，连接产物转入大肠杆菌中，通过PCR筛选出阳性单克隆，验证阳性的克隆子培养后提取质粒，测序，获得片段大小为1686bp的泛酸激酶基因RkPank；利用PEG介导的原生质体法将重组载体pRHRkPank转化至红冬孢酵母YM25235中，筛选转化子，获得RkPank的过表达菌株，过表达菌株经培养，利用紫外-可见分光光度计测量总类胡萝卜素的含量。
本发明将泛酸激酶基因RkPank转入到具有生产周期短、遗传稳定、生产安全等优点的红冬孢酵母YM25235中，实验结果显示RkPank基因的过表达会引起细胞内这个基因的转录水平在一定程度上的提高，说明外源基因在菌体内发生转录；RkPank基因的过表达能提高胞内乙酰辅酶A水平进一步促进总类胡萝卜素的合成；本发明为揭示微生物提高类胡萝卜素产量机制提供了参考，为大规模商业化生产类胡萝卜素奠定基础。
附图说明
图1为本发明的红冬孢酵母YM25235的RkPank基因PCR扩增图；1.DNA分子量标记DL2000；2.阴性对照；3.RkPank的cDNA片段；
图2为重组质粒pRHRkPank的质粒图谱；
图3为菌落PCR验证电泳图；1.DNA分子量标记DL2000；2.阴性对照；3.RkPank的cDNA片段；4-8为转化子；
图4为重组质粒pRHRkPank转化YM25235菌株阳性克隆验证；图中1. DNA分子标量DL5000；2.阴性对照；3.野生型菌株特异性基因条带；4.RkPank的cDNA片段；5.转化子验证；
图5为过表达菌株YM25235/pRHRkPank与对照菌株YM25235的乙酰辅酶A含量比较；
图6为过表达菌株YM25235/pRHRkPank与对照菌株YM25235的总类胡萝卜素含量比较。
具体实施方式
下面结合附图和实施例对本发明作进一步详细说明，但本发明保护范围不局限于所述内容，实施例中使用的试剂和方法，如无特殊说明，均采用常规试剂，使用常规方法。
实施例1：泛酸激酶基因RKPank的分离及其过表达载体pRHRkPank的构建
采用生工生物工程（上海）股份有限公司的UNlQ-10柱式Trizol总RNA抽提试剂盒（产品编号: SK1321）提红冬孢酵母YM25235的总RNA，然后按照Vazyme公司试剂盒（产品编号：R212-02）HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper)进行反转录合成cDNA，取1μL为模板进行聚合酶链式反应，根据转录组测序中发现的RkPank序列，设计特异性引物RkPank-F和RkPank-R，以上述得到的cDNA模板在PCR仪（北京六一生物科技有限公司）上进行PCR扩增，反应所用引物、扩增条件如下：
RkPank-F: ，（单下划线部分为BamHⅠ位点，双下划线部分为同源臂）；
RkPank-R:，（单下划线部分为EcoRⅤ位点，双下划线部分为同源臂）；
PCR扩增体系如下（50μL）：
；
扩增条件：95℃预变性3min，再用95℃变性15s，66℃退火30s，72℃延伸2min，进行30个循环，最后72℃终延伸5min，反应完后取产物1μL，然后在浓度为1%的琼脂糖凝胶中进行电泳分析，结果如图1所示，扩增得到大小约1686bp的片段；将pRH2034经BamHⅠ、EcoRⅤ两个限制性内切酶进行双酶切；将以上两个片段用多功能DNA回收试剂盒（北京百泰克生物技术有限公司，产品编号：DP1502）回收，使用无缝克隆试剂盒（Vazyme ClonExpress II OneStep Cloning Kit，南京诺唯赞生物科技有限公司）把片段连接到载体pRH2034中，重组载体连接体系如下（20µL）：
；
37℃反应30min；降至4℃或立即置于冰上冷却；
取10µL上的上述接产物加到100µL DH5α感受态细胞中，轻轻混匀，冰浴30min，42℃热激90s后立即放置在冰上冷却90s，向连接体系中加入900µL LB液体培养基，于37℃、100rpm振荡培养1h，在5000rpm下离心10min后弃900µL上清，剩余约100µL左右培养基悬浮菌体并涂布含有100µg/mL壮观霉素(Spe+)的LB固体平板，于37℃倒置培养过夜，随机挑取5个平板上生长的白色菌落并编号为1-5号，通过菌落PCR验证阳性克隆，结果见图3，从图中可以看出挑取的五株单克隆菌株通过菌落PCR均扩增出与目的片段大小相同的特异性条带，说明挑取的五株DH5α菌株中均成功转入重组质粒；将验证阳性的克隆子接入LB液体培养基（含100µg/mL壮观霉素）中过夜培养，OMEGA Plasmid Mini Kit I（美国OMEGA公司）提取质粒，测序（昆明擎科生物科技有限公司）；测序结果显示，所扩增得到的片段大小为1686bp，序列如SEQ ID NO:1所示的核苷酸序列，命名为RkPank，与RkPank基因的cDNA片段大小及序列一致，说明成功构建表达载体pRHRkPank，重组载体pRHRkPank的质粒图谱见图2。
实施例2：RkPank基因在促进红冬孢酵母生产类胡萝卜素中的应用
1、转化红冬孢酵母YM25235
挑取成功转入正确重组载体pRHRkPank的DH5α菌株单克隆，接入LB液体培养基（含100µg/mL壮观霉素）中过夜培养，OMEGA Plasmid Mini Kit I（美国OMEGA公司）提取质粒，并测量浓度为300μg/ml，置于-20℃储存备用；挑取红冬孢酵母YM25235单菌落接种于5mL的YPD液体培养基中，于30℃、200rpm振荡培养过夜；将过夜培养的菌液按1%的接种量转接至50mL的YPD液体培养基中，在30℃、200rpm下振荡培养至菌液OD600为0.5，将培养物于4℃、4500rpm离心6 min收集菌体；使用事先准备的柠檬酸缓冲液洗涤（30mmol/L柠檬酸、83mmol/L柠檬酸钠、600mmol/L甘露醇、NaOH调pH值到5.4）菌体两次，并用1mL柠檬酸缓冲液悬浮菌体，于4℃、4000 rpm离心5min收集菌体，置于冰上备用；配制裂解酶液（0.075g蜗牛酶、0.03g广东微生物溶壁酶，0.03gSIGMA溶壁酶用ddH2O定容至4mL），用0.22μm无菌滤膜过滤酶液，置于50mL无菌离心管中备用；取4mL酶液与菌液混合后置于30℃、90rpm振荡培养酶解2.5h，将培养物于4℃、1300rpm离心11min收集菌体；用STC（1.2mol/L山梨醇、10mmol/LTris-HCl、100mmol/L CaCl2）于冰上洗涤收集的菌体两次，制成酵母感受态细胞；将酵母感受态细胞按每管100μL分装到5mL无菌离心管中备用；向100μL感受态细胞中加入10μL（3μg）pRHRkPank重组质粒并轻轻混匀，冰上孵育10min，加入200μL预冷的PTC（50%PEG、10mmol/LTris-HCl、100mmol/L CaCl2），冰浴10min，再加入800μL预冷的PTC，并轻轻混匀，冰浴10min，4℃、1500rpm离心10min收集菌体；加入1.6mL 0.4M蔗糖YPD液体培养基悬浮，30℃、90rpm振荡培养12h复苏菌体；将复苏的菌体于1300rpm离心10min收集菌体，弃上清剩余100μL培养基悬浮菌体，最后涂布于含130μg/mL潮霉素B（HygB+）的YPD固体培养基上，于30℃倒置培养2d；将涂布后得到的转化子编号，并转接到含150μg/mL潮霉素（Hyg B+）YPD固体培培养基上，于30℃倒置培养2d；所得转化子接入5mL YPD培养基，于30℃、200rpm振荡培养120h，以YM25235野生菌株作为对照，观察颜色，筛选出颜色较YM25235不同的转化子；挑取筛选出的转化子，然后按照上海生工生物工程股份有限公司DNA 提取试剂盒说明书中步骤提取酵母转化子的基因组 DNA，后进行PCR验证，结果如图5所示，从图中可以看出以酵母转化子的基因组为模板通过PCR可扩增出与RkPank的cDNA片段大小相同的条带，重组转化子的基因验证正确，说明RkPankcDNA片段已成功连入酵母转化子的基因组中；
2、RkPank基因过表达的红冬孢酵母YM25235中乙酰辅酶A含量及类胡萝卜素检测
将RkPank过表达菌株采用 YPD液体培养基在28℃培养至120h，以YM25235作为对照，使用乙酰辅酶A测定试剂盒（Solarbio）测定乙酰辅酶A含量：将试剂二和试剂三加入到试剂四中混匀制成工作液分装保存于-20℃；离心收集菌体并用去离子水清洗，称取0.1g湿菌体加入1mL试剂一并研磨5min，然后8000g、4℃下离心10min，取上清，置冰上待测；分光光度计预热30min，用蒸馏水于340nm调零，取920μL工作液和100μL样本至1mL石英比色皿，混匀立即记录340nm处20s的吸光值记为A1和80s的吸光值A2，计算ΔA=A2-A1。
乙酰辅酶A含量（nmol/g鲜重）=(1640×ΔA＋0.012)÷W， W为样本质量（g）；
乙酰辅酶A含量如图5所示，野生型菌株乙酰辅酶A含量为1095.18±15nmol/g，RkPank过表达菌株乙酰辅酶A含量为1505.67nmol/g；过表达菌株较野性型菌株乙酰辅酶A含量提高了37.48％；结果表明泛酸激酶基因RkPank的过表达能引起红冬孢酵母YM25235菌株中乙酰辅酶A含量的提高；
将RkPank过表达菌株采用 YPD液体培养基在28℃下培养168h，以野生型红冬孢酵母YM25235菌株为对照，4500r离心5min收集菌体，55℃烘箱过夜，称取0.4g干菌体加入石英砂研磨10min，再加入10mL丙酮甲醇混合液（4:1）提取类胡萝卜素，4500r、4℃离心5min后利用紫外-可见分光光度计在450nm下测定吸光度并计算总类胡萝卜素的含量（mg/g干菌体），结果如图6所示；由图可知，过表达菌株YM25235/pRHRkPank的总类胡萝卜素合成量较野生型红冬孢酵母YM25235菌株明显提高，野生型红冬孢酵母YM25235菌株的类胡萝卜素合成量为5.53±0.06mg/g，而过表达菌株YM25235/pRHRkPank类胡萝卜素合成量为8.255.±0.06mg/g，即过表达菌株YM25235/pRHRkPank类胡萝卜素合成量是对照菌提升49.28％；结果显示泛酸激酶基因RkPank的过表达能引起红冬孢酵母YM25235菌株中总类胡萝卜素含量的增加，RkPank基因能够促进总类胡萝卜素的合成。
Claims (2)
1.一种泛酸激酶基因RkPank，其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的泛酸激酶基因RkPank在促进红冬孢酵母生产类胡萝卜素中的应用。
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| CN116286900B (zh) * | 2022-10-28 | 2024-04-26 | 昆明理工大学 | 一种乙酸渗透酶A基因RkAcpa及其应用 |
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