CN116606868B - 一种乙酰CoA合成酶基因RkACS2及其应用 - Google Patents
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Abstract
本发明公开了一种乙酰CoA合成酶基因RKACS2，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RKACS2基因的过表达会引起YM25235菌株油脂合成水平的提高；本发明通过基因工程手段对微生物进行改造，来提高微生物体内油脂的产量，为油脂大规模商业化生产奠定基础。
Description
技术领域
本发明属于生物技术领域和遗传工程领域，涉及一种乙酰CoA合成酶基因RkACS2及其应用。
背景技术
油脂(lipids)也称为脂质或脂类，是生物体内一类重要的有机分子，它们所包括的范围广泛，化学结构多样，生理功能也各不相同。常见的功能性油脂包括许多不饱和脂肪酸，如亚油酸(LA)、α-亚麻酸(ALA)、γ-亚麻酸(GLA)、花生四烯酸(ARA)、二十碳五烯酸(EPA)以及二十二碳六烯酸(DHA)等。ALA、DHA、EPA三种脂肪酸被称为ω-3脂肪酸，有研究表明，ω-3脂肪酸具有抗炎症、抗血栓和动脉粥样斑块形成、降低血脂、舒张血管的功效，经常食用ω-3脂肪酸可以显著降低得心血管疾病的风险。而LA、ARA和GLA则被称为ω-6脂肪酸，LA是神经酰胺的基本成分，在皮肤的结构完整性和屏障功能中具有特定而独特的作用，皮肤细胞外基质形成角质层通透性屏障流动性取决于LA含量，LA也因此常被用于治疗炎症性皮肤病。另外，适当摄入ARA，可提高免疫力并且有利于预防心脑血管疾病的发生。GLA则具有抗炎特性和抑制血小板聚集作用，可以影响炎症过程以及血栓形成。许多功能性油脂是人体不可缺少的，但自身无法合成，需从外界摄取获得。随着社会的进步，人们健康意识的不断提高，功能性油脂需求量越来越大，但目前功能性油脂生产量不够，供应不足。功能性油脂主要来源于植物、鱼类以及产油微生物，但是其中植物生产油脂会受到地理、季节和气候等条件限制，且生产周期较长，鱼类生产的油脂则成本高、易受污染、品质不稳定，且植物和鱼类所产的油脂产量难以满足全球需求。
微生物油脂生产与植物和动物脂质生产相比有许多优势，如很少会受到地理、季节性和气候限制，生产成本较低，生产周期短，产量客观以及所产油脂品质稳定等。酵母和藻类是常见的两类用于生产油脂的微生物，但酵母相对于藻类更有优势，包括潜在的更快生长、更高的密度生长、对病毒感染的敏感性更低，以及使用低pH生长条件控制细菌污染的能力，因此用产油酵母发酵生产油脂越来越受到关注。目前已发现的油性酵母有160多种，它们分别属于耶氏酵母属、隐球菌属、粘红酵母属和红冬孢酵母属等酵母属，其中许多产油酵母通过培养条件优化和基因工程改造来提高油脂产量。我们从油脂代谢途径相关基因着手，利用基因工程技术有目的的改造菌株以优化现有菌株的油脂转化率，提高油脂产量。
在酵母中存在两种乙酰CoA合成酶(ACS)的同工酶，分别是ACS1和ACS2。其中ACS2存在一个大的氨基端域和一个小的羧基端域，且具有AMP结合家族重要保守残基序列，包括AMP结合域序列(ILYSSGTTGKPK)、参与腺苷酸和ATP结合反应的重要结构序列以及CoA结合位点序列，属于AMP结合家族，是一种新的ACS蛋白。ACS2基因为组成型表达基因，其在酵母的整个生长过程中均表达，其除了在乙酸盐代谢中发挥作用外还在葡萄糖代谢中起重要作用，但目前机制尚不清楚。
发明内容
本发明提供了一种乙酰CoA合成酶基因RkACS2，该基因是从红冬孢酵母(Rhodosporidium kratochvilovae)YM25235中分离得到；该基因核苷酸序列如SEQ ID NO:1所示或为与SEQ ID NO:1互补的核苷酸序列，该基因序列长为2094bp(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乙酰CoA合成酶基因RkACS2应用在促进红冬孢酵母(Rhodosporidium kratochvilovae)生产油脂中。
本发明中红冬孢酵母(Rhodosporidium kratochvilovae)YM25235分离自云南丽江程海湖，具有生产周期短、遗传稳定、生产安全等优点。
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，将载体pRH2034进行双酶切、回收，用一步克隆法将目的片段和载体连接，获得重组质粒pRHRkACS2，连接产物转入大肠杆菌中，通过PCR筛选出阳性单克隆，重组质粒pRHRkACS2用BamHⅠ、EcoRⅤ两个限制性内切酶进行酶切验证，验证阳性的克隆子培养后提取质粒，测序，获得片段大小为2094bp的乙酰CoA合成酶基因RkACS2；利用PEG介导的原生质体法将重组载体pRHRkACS2转化至红冬孢酵母YM25235中，筛选转化子，获得RkACS2的过表达菌株，过表达菌株经培养，提取油脂，并测定油脂含量。
本发明从红冬孢酵母(Rhodosporidium kratochvilovae)YM25235的总RNA基因中分离得到乙酰CoA合成酶基因RkACS2，该基因全长2094bp；将该基因转入到红冬孢酵母YM25235中，实验结果显示RkACS2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；RkACS2基因的过表达能够促进油脂的合成；本研究结果为揭示微生物提高油脂产量机制提供了参考，将有助于通过基因工程手段对其进行改造来提高油脂含量，对油脂的工业化生产提供良好的应用前景和经济效益，为大规模商业化生产油脂奠定基础。
附图说明
图1为本发明的红冬孢酵母YM25235的RkACS2基因PCR扩增图；其中1.DNA分子量标记DL5000；2.阴性对照；3.RkACS2的cDNA片段；
图2为重组质粒pRHRkACS2的质粒图谱；
图3为菌落PCR验证电泳图；1.DNA分子量标记DL5000；2.RkACS2的cDNA片段；3-7为1-5号的转化子；
图4为重组质粒pRHRkACS2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.RkACS2的cDNA片段；5.转化子基因组验证；
图5为过表达菌株YM25235/pRHRkACS2与对照菌株YM25235的油脂含量比较。
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：从红冬孢酵母(Rhodosporidium kratochvilovae)YM25235中分离乙酰CoA合成酶基因RkACS2及其过表达载体pRHRkACS2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 cDNA为模板进行聚合酶链式反应，根据转录组测序中发现的RkACS2序列，设计特异性引物RkACS2-F和RkACS2-R，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、组分和扩增条件如下：
RkACS2-F:(单下划线部分为BamHⅠ位点，双下划线部分为同源臂)；
RkACS2-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5min，再用95℃变性15s，59℃退火15s，72℃延伸40s，进行30个循环，最后72℃彻底延伸10min，反应完后取产物2μL，然后在浓度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2094bp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 IIOne Step Cloning Kit，南京诺唯赞生物科技有限公司)把扩增片段连接到载体pRH2034中，重组载体连接体系如下(20μL)：
连接体系配制完后，使用移液器轻轻吹打混匀，短暂离心将反应液收集至管底，然后37℃反应30min；降至4℃或立即置于冰上冷却；
取10μL获得的连接产物加到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的培养基悬浮菌体后涂布于含有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)中过夜培养后，将菌液送出进行测序(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2094bp，序列如SEQ ID NO:1所示的核苷酸序列，命名为RkACS2，与RkACS2基因的cDNA片段大小及序列一致，说明成功构建表达载体pRHRkACS2，重组载体pRHRkACS2的质粒图谱见图2。
实施例2：RkACS2基因与红冬孢酵母中油脂合成关系的分析
1、转化红冬孢酵母YM25235
挑取成功转入正确重组载体pRHRkACS2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，提取质粒(OMEGA Plasmid Mini Kit I，美国OMEGA公司)，并测量浓度，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30℃、200rpm振荡培养至菌液OD600为0.5，将培养物于4℃、4500rpm离心5min收集菌体；使用事先准备的柠檬酸缓冲液洗涤(30mM柠檬酸、83mM柠檬酸钠、600mM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℃、4000rpm离心5min收集菌体，置于冰上备用；配制裂解酶液(0.156g蜗牛酶、0.04g溶壁酶、0.04g溶壁酶，用ddH2O定容至5mL)，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10min收集菌体；用STC(1.2M山梨醇、10mMTris-HCl、100mMCaCl2)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2-5μg pRHRkACS2重组质粒并轻轻混匀(通常加入片段体积不应超过10μL)，冰上孵育10min，加入200μL预冷的PTC(50％PEG、10mMTris-HCl、100mMCaCl2)，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1.6mL0.4M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130ug/mL潮霉素B(HygB+)的0.4M蔗糖YPD固体培养基上，于30℃倒置培养2-3天；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；将转化子分别接入5mL YPD培养基，于30℃、200rpm振荡培养96h，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4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RkACS2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RkACS2 cDNA片段已成功连入酵母转化子的基因组中。
2、RkACS2基因过表达的红冬孢酵母YM25235中油脂含量分析
将含pRHRkACS2的过表达菌株在28℃条件下培养120h，提油脂，以野生型红冬孢酵母YM25235菌株为对照，测定油脂的含量(％DCW)，含量如图5所示；由图可知，过表达菌株YM25235/pRHRkACS2的油脂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菌株的油脂合成量5.87±0.18g/g，而过表达菌株YM25235/pRHRkACS2油脂合成量为7.85±0.21g/g，即过表达菌株YM25235/pRHRkACS2油脂合成量是对照菌的1.34倍；结果显示乙酰CoA合成酶基因RkACS2的过表达能引起红冬孢酵母YM25235菌株中油脂含量的增加，RkACS2基因能够促进油脂的合成。
Claims (2)
1.一种乙酰CoA合成酶基因RkACS2，其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的乙酰CoA合成酶基因RkACS2在促进红冬孢酵母(Rhodosporidium kratochvilovae)生产油脂中的应用。
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| Engineering Rhodosporidium toruloides with a membrane transporter facilitates production and separation of carotenoids and lipids in a bi-phasic culture;Jaslyn J.L Lee等;Appl Microbiol Biotechnol;20151103;第1-9页 * |
| Firrincieli,A.等.Rhodotorula graminis WP1 uncharacterized protein (RHOBADRAFT_35572), partial mRNA.Genbank Database.2020,Accession No：XM_018413285.1. * |
| Firrincieli,A.等.uncharacterized protein RHOBADRAFT_35572 [Rhodotorula graminis WP1].Genbank Database.2020,Accession No：XP_018271860.1. * |
| 乙酰辅酶A合成酶的酶学性质及其在甲羟戊酸合成中的应用;胡红;张汝兵;秦杰明;咸漠;;应用与环境生物学报;20160625(第03期);第357-362页 * |
| 红冬孢酵母Δ~(12)-脂肪酸脱氢酶基因的克隆和表达;罗敏周;杨晓霞;季秀玲;林连兵;魏云林;张琦;;云南大学学报(自然科学版);20160710(第04期);第669-675页 * |
| 葡萄糖饥饿胁迫下乙酰 CoA合 成酶基因与红冬孢酵母类胡萝卜素含 量变化的关系研究;杨晓霞;中国知网;20231231;第1-90页 * |
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