CN104312991B - CeDGAT1突变体及其应用 - Google Patents

CeDGAT1突变体及其应用 Download PDF

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CN104312991B
CN104312991B CN201410548711.2A CN201410548711A CN104312991B CN 104312991 B CN104312991 B CN 104312991B CN 201410548711 A CN201410548711 A CN 201410548711A CN 104312991 B CN104312991 B CN 104312991B
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胡赞民
郭雪洁
陈宇红
范成明
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Abstract

本发明提供了一种CeDGAT1突变体及其在提高细胞脂肪酸含量方面的用途，所述编码CeDGAT1突变体的基因的核酸序列如SEQ ID NO:1所示。利用本发明的编码CeDGAT1突变体的基因转化酵母细胞、植物细胞和微藻细胞能大幅提高细胞的总脂肪酸含量。

Description

CeDGAT1突变体及其应用
技术领域
本发明涉及一种二酰基甘油酰基转移酶突变体及其应用。具体地，涉及编码该突变体的基因序列的获得和酵母表达载体的构建，以及其大幅度提高酵母脂肪酸含量的用途。
背景技术
生物柴油生产的最大瓶颈是原料问题，因此寻找新的资源，开发潜在的生物柴油生产新原料成为生物柴油产业发展的重要途径。微藻是遍布全球水体的浮游植物，具有产能大、无污染、可再生、易培养、含有较多的脂类物质等优点，在能量转化和碳元素循环中具有重要的作用。有些微藻把光合作用产物转化为脂肪酸，进一步合成三酰甘油(TAG)，在细胞中以油滴的形式贮藏起来。微藻细胞中的三酰甘油可达到干重的20％-50％[1]，提取后通过转酯化后可转变为脂肪酸甲酯，即生物柴油[2]。大规模培养微藻是开发生物柴油，建立可再生能源基地的重要途径。微藻中的小球藻可以进行光自养培养、也可以进行无光照的异养培养，生长周期短，生长速度快，短时间可获得较高的生物产量和油脂产量，而且是少数几个可用于大规模培养的微藻。所以，小球藻是进行能源微藻研究的一个潜在的理想材料[3-4]。
三酰甘油(TAG)在大部分生物体中都是最主要的储藏脂类，包括脊椎动物、油料作物、真菌以及微藻。TAG不仅在生物体生长、发育和繁衍过程中扮演着重要的角色，而且作为一种可再生的生物能源产品，可用于生物柴油的生产，具有非常广泛的应用。在微藻中，TAG一般在逆境条件下积累于油体[5]。TAG的生物合成有依赖乙酰辅酶A和不依赖乙酰辅酶A的途径。依赖乙酰辅酶A的合成途径，被称作Kennedy途径，也是合成TAG的主要途径。该途径TAG组装是在内质网上进行的。首先，在质体中，乙酰-CoA在乙酰-CoA羧化酶(ACCase)的作用下形成丙二酸单酰-CoA；丙二酸单酰-CoA进行逐步的缩合反应，从二碳单酰基载体蛋白(ACP)开始，依次把二碳单位带入脂酰链，经过数次循环聚合以及去饱和反应后形成不同碳链长度的酰基-ACP，然后在酰基辅酶A合成酶(ACS)的作用下合成酰基辅酶A，并从质体转移到内质网或胞质中，成为真核生物内质网三酰甘油合成的底物。游离脂肪酸被酯化生成酯酰辅酶A(CoA)后，在膜结合的甘油-3-磷酸酰基转移酶(GPAT)，溶血磷脂酸酰基转移酶(LPAT)，二酰甘油酰基转移酶(DGAT)3种酰基转移酶以及磷脂酸磷酸水解酶(PAPase)作用下，依次把甘油骨架第一至三位经酰基化合成三酰甘油，该生物合成过程就是通常所说的起主导作用的Kennedy途径[6-7]。
二酰甘油酰基转移酶(DGAT)催化TAG合成途径即Kennedy途径的最后一步，也是该途径唯一的限速酶。DGAT被认为是在TAG合成过程中起主要作用的酶，普遍存在于所有已研究的真核生物中。目前为止，共发现3类DGAT基因家族，包括DGAT1、DGAT2和DGAT3基因家族[8-9]。Cases等[10]在小鼠中克隆了第一个DGAT1基因后，Hobbs等[11]在拟南芥中克隆了DGAT1基因，该基因在拟南芥中仅有一个拷贝。AtDGAT1基因在野生型拟南芥中过量表达研究发现，DGAT1转录水平和活性明显提高(10％-70％)，油脂积累增加，种子平均重量增加[12]。随后，油菜、蓖麻、烟草、大豆、玉米、橄榄、红花、向日葵、火焰卫矛和百脉根等植物中DGAT1基因也相继被克隆[13-16]。近年来，Lock[17]等通过将一个BnDGAT1反义基因转化到油菜DH12075中发现，DGAT1基因的表达量、总体DGAT蛋白的酶活性和种子的含油量都有明显的下降，同时，种子的产量以及萌发率也比转化前降低，而且种子发育严重畸形。这些结果表明，DGAT除了在总油脂形成中有重要作用以外，它还影响种子的正常发育，但是具体的调控机制目前还不清楚。目前，在一些微藻中也克隆到了DGAT1基因，Boyle等人[18]和Guiheneuf等人[5]分别在衣藻和三角褐指藻中克隆到DGAT1基因。莱茵衣藻和三角褐指藻中DGAT1基因的功能已被验证和具体阐述。DGAT2基因家族的成员在动物，植物和酵母中都存在，并且与DGAT1基因家族没有明显的同源性。目前对DGAT2的研究较少，仅在拟南芥(GenBank收录号NM 115011)、油菜(GenBank收录号AY 916129)、蓖麻少数植物中克隆出来。近两年在莱茵衣藻[19-20]和Ostreococcus tauri[21]中也有报道。Saha[8]等人从发育中的花生子叶细胞质中克隆得到了DGAT3基因，该基因与DGAT1和DGAT2基因家族的相似性不足10％，但是其编码的蛋白具有类似DGAT蛋白功能基序，而且与TAG合成密切相关。
近年来，对植物油脂的广泛需求极大的促进了DGAT相关基因的研究，尤其是在利用基因工程方法改良优质品质方面获得了较大的发展。2008年，Zheng等[22]通过对高油玉米DGAT1的研究发现，在DGAT1-2蛋白的F469位点处的一个苯丙氨酸的插入是提高油含量和油酸含量的重要决定因素，阐明了油含量以及组成差异的分子基础。2008年，Xu等人发现将旱金莲DGAT1蛋白SnRK1作用区S197位点处的Ser突变为Ala，可将DGAT1酶活性提高38％-80％，在拟南芥中表达该突变体可使种子含油量提高20％-50％[23]。
发明内容
本发明发现将来源于椭圆小球藻(Chlorella ellipsoidea)的CeDGAT1蛋白Y553位点处的Tyr突变为Ala后，把编码该CeDGAT1突变体的基因转入酿酒酵母INVSC1后，相比转入野生型CeDGAT1基因的酵母INVSC1，可显著提高其主要脂肪酸组分的含量，包括棕榈酸(C16:0)提高了17.24％，棕榈油酸(C16:1)提高2.85％，硬脂酸(C18:0)提高22.76％，油酸(C18:1)提高0.43％。酵母细胞总脂肪酸的含量也提高了约6.75％。相比转入空载的酿酒酵母INVSC1，可大幅度提高其主要脂肪酸组分的含量，包括棕榈酸(C16:0)提高了约282％，棕榈油酸(C16:1)提高204％，硬脂酸(C18:0)提高220％，油酸(C18:1)提高187％。酵母细胞总脂肪酸的含量也提高了约217％。总脂肪酸及各主要脂肪酸组分的含量这种幅度的提高还未见报道。前期工作表明野生型CeDGAT1就是能大幅度提高转基因酿酒酵母INVSC1脂肪酸含量的高活性DGAT。CeDGAT1突变体的活性比野生型CeDGAT1更高，具有重要的应用价值。
本发明提供了一个编码CeDGAT1突变体的基因，其cDNA核苷酸序列如SEQ ID NO:1所示，该基因cDNA全长为2142bp。
本发明还提供了含本发明所述基因(CeDGAT1突变体基因)的酵母表达载体。
本发明在一个方面提供了包含本发明所述基因的酵母、植物或藻类，所述植物包括拟南芥、烟草、油菜、向日葵、大豆、蓖麻、棉花、橄榄、和红花等。优选地，所述藻类选自小球藻，更优选地选自椭圆小球藻(Chlorella ellipsoidea)。所述酵母优选地选自酿酒酵母，更具体地是酿酒酵母INVSC1。
本发明提供一个编码椭圆小球藻CeDGAT1突变体的基因，其可用于利用基因工程方法提高酵母、植物或藻类油脂等方面。
更具体地，本发明提供以下各项：
1.CeDGAT1蛋白突变体，其氨基酸序列如SEQ ID NO:2所示。
2.编码根据1所述的CeDGAT1蛋白突变体的基因。
3.根据2所述的基因，其核酸序列如SEQ ID NO:1所示。
4.载体，其包含根据2或3所述的基因，所述载体可以通过将根据2或3所述的基因转入到pYES2.0(可购自Invitrogen公司)，pBIN19、pBI121、pBI221(可购自Clontech)，pCambia 1300(可购自Cambia公司)或pGreen(可购自the John Innes Centre)中制备。
5.宿主细胞，其包含根据1所述的CeDGAT1蛋白突变体、根据2或3所述的基因或根据4所述的载体。
6.一种制备具有高棕榈酸、棕榈油酸、硬脂酸和/或油酸含量，或具有高总脂肪酸含量的酵母、植物或藻类的细胞的方法，所述方法包括将根据2或3所述的基因或根据4所述的载体转入所述酵母、植物或藻类的细胞中，所述酵母优选是酿酒酵母，更优选是尿嘧啶缺陷型酿酒酵母INVSC1；所述藻类优选是小球藻，更优选是椭圆小球藻(Chlorellaellipsoidea)；所述植物优选选自拟南芥、烟草、油菜、向日葵、大豆、番茄、蓖麻、棉花、芝麻或花生。
7.根据1所述的CeDGAT1蛋白突变体、根据2或3所述的基因或根据4所述的载体在制备具有高总脂肪酸含量的酵母、植物或藻类的细胞中的用途，所述酵母优选是酿酒酵母，更优选是尿嘧啶缺陷型酿酒酵母INVSC1；所述藻类优选是小球藻，更优选是椭圆小球藻(Chlorella ellipsoidea)；所述植物优选选自拟南芥、烟草、油菜、向日葵、大豆、番茄、蓖麻、棉花、芝麻或花生，所述具有高总脂肪酸含量的酵母、植物或藻类的细胞优选是具有高棕榈酸、棕榈油酸、硬脂酸和/或油酸含量的酵母、植物或藻类的细胞。
8.根据1所述的CeDGAT1蛋白突变体、根据2或3所述的基因或根据4所述的载体在提高酵母、植物或藻类的细胞中棕榈酸、棕榈油酸、硬脂酸和/或油酸含量，或总脂肪酸含量中的用途，所述酵母优选是酿酒酵母，更优选是尿嘧啶缺陷型酿酒酵母INVSC1；所述藻类优选是小球藻，更优选是椭圆小球藻(Chlorella ellipsoidea)；所述植物优选选自拟南芥、烟草、油菜、向日葵、大豆、番茄、蓖麻、棉花、芝麻或花生。
9.利用根据6所述的方法制备的具有高棕榈酸、棕榈油酸、硬脂酸和/或油酸含量，或具有高总脂肪酸含量的酵母、植物或藻类的细胞。
10.根据9所述的酵母、植物或藻类的细胞在生产生物柴油中的用途。
附图说明
图1.含本发明的椭圆小球藻CeDGAT1突变体基因的酵母表达载体pYES-CeDGAT1Y553A。
图2.GC-MS法测定的酵母各脂肪酸组分含量以及总脂肪酸含量(CK为转空载酵母，pYES-CeDGAT1为转入野生型CeDGAT1基因的酵母，pYES-CeDGAT1Y553A为转入本发明的CeDGAT1突变体基因的酵母)。
具体实施方式
下面参考实施例和附图详细描述本发明。本领域的普通技术人员可以理解的是，下述实施例是举例说明的目的，其不应以任何方式解释为对本发明的限制。本发明的保护范围由后附的所限定。
实施例1.含有编码CeDGAT1突变体基因的酵母表达载体的获得
前期实验室已将来源于椭圆小球藻的野生型CeDGAT1基因构建到酵母表达载体pYES2.0(购自Invitrogen公司)上，并将其命名为pYES-CeDGAT1(具体构建过程请参见发明专利申请201310316565.6)。现提取pYES-CeDGAT1重组质粒，以其为模板进行定点突变PCR扩增。
上下游引物(见SEQ ID NO:3和SEQ ID NO:4)分别是：
5’-CGTTCCTACTTTGGCCGGCTGGCTCATCA-3’
5’-TGATGAGCCAGCCGGCCAAAGTAGGAACG-3’。
其中下划线为引入的突变位点。具体操作参照Site-directed Gene MutagenesisKit(购自碧云天)说明书进行。
基因定点突变反应体系如下：
按照上面的顺序依次加入各种试剂。适当混匀后，加入1μL Pfu DNA Polymerase混匀后按如下程序进行PCR反应：
PCR反应后，直接在PCR产物中加入1μL Dpn I，混匀后37℃孵育1小时。将Dpn I消化完毕的PCR产物直接用于转化大肠杆菌感受态细胞，涂布到含有适当抗生素的平板上，培养过夜。挑取单克隆测序，测序验证获得了含有编码CeDGAT1突变体基因的酵母表达载体pYES-CeDGAT1Y553A。其载体图见图1。
实施例2.酵母表达载体pYES-CeDGAT1Y553A转化酿酒酵母
接种尿嘧啶缺陷型酿酒酵母INVSC1(购自Invitrogen公司)于10mL YPD培养基中，30℃震荡培养过夜。次日将菌液接种到50mL YPD培养基中，稀释到OD600＝0.4，继续培养2-4h至OD600在0.5-0.6之间，5,000rpm冷冻离心1min,用40mL 1×TE(10mM Tris，pH7.5，1mMEDTA)悬浮沉淀，5,000rpm冷冻离心，用2mL 1×LiAc(10mM乙酸锂，pH7.5)/0.5×TE悬浮沉淀，室温放置10min。将100μL酵母悬浮液与1ug酵母表达载体pYES-CeDGAT1Y553A和100ug变性鲑鱼精DNA混匀，然后加入700μL 1×LiAc/40％PEG-3350/1×TE，混匀。30℃培养30min，加入88μL DMSO，混匀，42℃热激7min。10,000rpm离心10s，去上清，用1mL 1×TE悬浮沉淀，10,000rpm离心10s，去除上清。用50-100μL 1×TE悬浮沉淀，涂布于SC-U基本培养基上，30℃培养2天。
2天后，从培养基平板上挑取菌落，参考博迈德酵母高纯度质粒小量快速提取试剂盒说明书，进行质粒提取，然后将提取的质粒作为模板进行PCR验证。最后对验证为阳性的酵母质粒和相应的菌落进行保存。
实施例3.CeDGAT1突变体在酿酒酵母中的诱导表达
将转化有pYES-CeDGAT1Y553A的酿酒酵母INVSC1的单菌落接种于5mL SC-U培养基中，200rpm，30℃培养过夜。取过夜培养的菌液转接到50mL含1％棉子糖和2％酵母表达诱导物D-半乳糖的SC-U培养液中，使其OD600约为0.1，加入NP-40(终浓度为1％，有利于酵母细胞悬浮)，200rpm，20℃，培养72h诱导表达。将转化有pYES2.0空载和pYES-CeDGAT1的酵母转化子设为对照，对作为对照的酵母转化子进行同样操作。
实施例4.酵母脂肪酸的提取与检测
1.酵母脂肪酸的提取
取诱导培养好的酵母液，4,000rpm离心5min，室温收集菌体；经去离子水反复悬浮、室温离心洗涤三次，50℃烘干；取100mg酵母干粉充分研磨，加入3mL 7.5％KOH-CH3OH，加15-20μL的d17:0(购自sigma公司，浓度30mg/mL)，70℃水浴3-5h；加入2mL HCl酸化至其pH值达2.0；加入2mL 14％BF3-CH3OH(购自Aldtich公司)溶液，70℃水浴1.5h；加入1mL0.9％NaCl溶液，4mL正己烷抽提一次，N2吹干；300μL乙酸乙酯溶解。该实验每次每个样品平行做两份，共重复三次。
2.终产物GC-MS检测分析实验
所用GC/MS仪为TurboMass(PerkinElmer公司)；GC条件：色谱柱：BPX-70，30m×0.25mm×0.25um。柱温：120℃，气化室温度230℃。取1μL终产物上样，分流比10:1。
3.GC-MS结果分析
研究表明，把编码CeDGAT1突变体的基因转入酿酒酵母INVSC1后，相比转野生型CeDGAT1基因的酵母INVSC1，可显著提高其主要脂肪酸组分的含量，包括棕榈酸(C16:0)提高了17.24％，棕榈油酸(C16:1)提高2.85％，硬脂酸(C18:0)提高22.76％，油酸(C18:1)提高0.43％。酵母细胞总脂肪酸的含量也提高了约6.75％。相比转pYES2.0空载酿酒酵母INVSC1，可大幅度提高其主要脂肪酸组分的含量，包括棕榈酸(C16:0)提高了约282％，棕榈油酸(C16:1)提高204％，硬脂酸(C18:0)提高220％，油酸(C18:1)提高187％。酵母细胞总脂肪酸的含量也提高了约217％。酵母各脂肪酸组分及含量GC-MS测定结果见图2(注：**表示：P<0.01)。
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Claims (14)

1.CeDGAT1蛋白突变体，其氨基酸序列如SEQ ID NO:2所示。
2.编码根据权利要求1所述的CeDGAT1蛋白突变体的基因。
3.根据权利要求2所述的基因，其核酸序列如SEQ ID NO:1所示。
4.载体，其包含根据权利要求2或3所述的基因。
5.宿主细胞，其包含根据权利要求1所述的CeDGAT1蛋白突变体、根据权利要求2或3所述的基因或根据权利要求4所述的载体，其中所述宿主细胞是非植物细胞。
6.一种制备具有高棕榈酸、棕榈油酸、硬脂酸和/或油酸含量，或具有高总脂肪酸含量的酵母的细胞的方法，所述方法包括将根据权利要求2或3所述的基因或根据权利要求4所述的载体转入所述酵母的细胞中，所述酵母是酿酒酵母。
7.根据权利要求6所述的方法，其中所述酵母是尿嘧啶缺陷型酿酒酵母INVSC1。
8.根据权利要求1所述的CeDGAT1蛋白突变体、根据权利要求2或3所述的基因或根据权利要求4所述的载体在制备具有高总脂肪酸含量的酵母的细胞中的用途，所述酵母是酿酒酵母。
9.根据权利要求8所述的用途，其中所述酵母是尿嘧啶缺陷型酿酒酵母INVSC1。
10.根据权利要求8所述的用途，其中所述具有高总脂肪酸含量的酵母的细胞是具有高棕榈酸、棕榈油酸、硬脂酸和/或油酸含量的酵母的细胞。
11.根据权利要求1所述的CeDGAT1蛋白突变体、根据权利要求2或3所述的基因或根据权利要求4所述的载体在提高酵母的细胞中棕榈酸、棕榈油酸、硬脂酸和/或油酸含量，或总脂肪酸含量中的用途，所述酵母是酿酒酵母。
12.根据权利要求11所述的用途，其中所述酵母是尿嘧啶缺陷型酿酒酵母INVSC1。
13.利用根据权利要求6-7中任一项所述的方法制备的具有高棕榈酸、棕榈油酸、硬脂酸和/或油酸含量，或具有高总脂肪酸含量的酵母的细胞。
14.根据权利要求13所述的酵母的细胞在生产生物柴油中的用途。
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