CN102812124A - 用于产生脂质的经遗传改造的生物 - Google Patents
用于产生脂质的经遗传改造的生物 Download PDFInfo
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- CN102812124A CN102812124A CN201080038563XA CN201080038563A CN102812124A CN 102812124 A CN102812124 A CN 102812124A CN 201080038563X A CN201080038563X A CN 201080038563XA CN 201080038563 A CN201080038563 A CN 201080038563A CN 102812124 A CN102812124 A CN 102812124A
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- sterol
- yeast
- saccharomyces
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Abstract
本发明提供分离的经遗传改造的非哺乳动物生物,其中与相应的野生型生物相比,酰基辅酶A:固醇酰基转移酶/固醇O-酰基转移酶(EC2.3.1.26)和/或二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)和/或卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)和/或酰基辅酶A-蜡醇酰基转移酶(EC2.3.1.75)的活性被降低或消除;还提供所述生物的使用方法,用于制备所述生物的穿梭载体和产生所述生物的方法。
Description
发明领域
本发明涉及经遗传改造的非哺乳动物生物、尤其是微生物,其中某些酶活性被降低和/或增加,所述生物可用于脂质、尤其是中性脂质的工业化生物合成。本发明还涉及所述生物的用途,用于制备所述生物的穿梭载体和产生所述生物的方法。
发明背景和现有技术
脂质是疏水性或两亲性的小分子,其完全或部分通过硫酯如脂肪酸或聚酮化合物的碳负离子缩合和/或通过于异戊二烯单元如异戊烯醇或固醇的碳负离子缩合而产生。这一类包括经济上高度重要的若干物质。三酰甘油-脂质类包括例如油、脂或蜡类,其用于各种各样的用途,例如作为食物配料或用于烹饪,制造肥皂、皮肤产品、香水和其它个人护理及化妆品,制造油漆和其它木器处理品,在电子工业中作为生物可降解的绝缘体,制造生物可降解的水力流体,作为润滑剂,或者甚至作为可用于替代常规柴油的生物柴油的基料。类异戊二烯-脂质鲨烯例如可用作疫苗佐剂或其它药物、营养物、化妆品以及非处方药的辅料。鲨烯也可用作合成萜的结构单元。此外,鲨烯在工业上可用作生物可降解的润滑剂。其它经济上重要的脂质是诸如麦角固醇、酵母固醇、麦角甾二烯醇(episterol)、7-脱氢胆固醇或羊毛固醇等固醇,其可用作制备如皂苷、类固醇激素、维生素和药物的化合物的关键原料。
脂质、尤其是中性脂质,通常在细胞中是贮存于称为脂质粒(lipidparticle)的特定胞内细胞器中。这些颗粒的特征在于由高疏水性单层组成的简单结构,其中仅包含少量包埋的蛋白质。脂质贮存于脂质粒中,直到水解使其组分重返代谢和/或分解代谢途径中。这种脂质储备形成过程在自然界中广泛采用,所有种类的真核细胞都含有胞内脂质粒,其也可称为脂质体(lipid body)、脂滴(lipid droplet)、油脂体(oil body)或油脂小体(oleosome)。在酿酒酵母(Saccharomyces cerevisiae)中,脂质滴积聚了至多70%的细胞总脂含量。
因为大部分所述脂质源自如植物、动物或微生物等天然来源,所以进行尝试以增加活细胞中的脂质量,即在脂质粒中积聚更多脂质。为了这一目的,已知改造生物的代谢途径。文献EP-O 486 290A描述了麦角固醇代谢基因在酵母中的过量表达,导致细胞中麦角固醇含量增加。文献WO03/064652A公开了基于羊毛固醇-C14-脱甲基酶和HMG-辅酶A-还原酶活性的增加而制备酵母固醇的方法。在文献WO2004/083407A中描述了转基因生物,其具有降低的Δ22-去饱和酶活性和增加的HMG-辅酶A还原酶、羊毛固醇C14-脱甲基酶、鲨烯环氧酶和鲨烯合成酶活性。这些生物可用于产生固醇脂质麦角甾-5,7-二烯醇。因此,通过生物改造脂质产生生物,可显著提高所产生脂质的产率。
然而,所得脂质化合物通常不纯和混杂,需要进行分离和/或纯化,尤其是当所述脂质要用作进一步化学合成或改性的原料时。另外,任何纯化步骤,尤其是在工业化规模上进行时,都是昂贵而费力的并可能污染环境。
发明的技术问题.
因此,本发明的技术问题包括提供允许通过生物以更纯形式制备脂质的方式和方法,以避免费力而昂贵的纯化步骤。
发明概述和优选的实施方案
为了解决这个技术问题,本发明教导权利要求1的生物。在权利要求中依照权利要求1详细说明了优选的实施方案。其中术语“遗传改造”不仅包括其基因组经基因工程的现有技术方法改造的生物,而且还包括按照所需遗传改造而从突变(常规诱变)中选择出来的生物。
本发明是基于这一发现:能够对生物进行基因工程或生化改造,使得特定(中性)脂质能以更纯形式在脂质粒中积聚。本发明的生物经改造,改造方式使其消除或降低非必需的固醇基酰基酯(steryl acylester)和/或三酰甘油和/或蜡酯(wax ester)的合成,这取决于希望由生物所产生的更高纯度的脂质。除了以更纯形式积聚中性脂质之外,对潜在污染的固醇基酰基酯和/或三酰甘油和/或蜡酯的降低或消除也令人惊奇地导致所需脂质的水平和产率的增加。不受该理论的束缚,这可能是因为脂质粒中的贮存空间增加和/或因为所需脂质合成所用的物质的利用度增加。
术语“中性脂质”是指缺乏带电荷基团并因此而不能大量整合到双层膜上的脂质。该术语包括三酰甘油(TAG)、固醇基酰基酯(SAE)和蜡酯(WE)。鲨烯也属于“中性脂质”类。通常,脂质粒的内容物是不同中性脂质的混合物,其妨碍重组生物中所需特定中性脂质的有效产生。本发明通过改造产生生物,使得在所述生物所述脂肪粒中天然存在的至少一种类型的中性脂质不再由此合成,从而解决了这个问题。结果,所述脂质粒含有的其余中性脂质的组成就变得更纯,并因此对用于商业目的的制备而言更具吸引力。
通常,可使用权利要求1中的任一活性或不同(2、3或4种)活性的组合,取决于欲以更高纯度合成的中性脂质,或继而取决于在生物合成中作为不想要的污染物而欲除去的中性脂质。因此,本发明存在着各种实施方案,而在以下仅会更详细地描述某些具体的实施方案。
因此,在第一实施方案中,本发明的生物经改造,使其不再合成固醇基酰基酯(SAE)。固醇基酰基酯是具有长链脂肪酸的固醇酯。通常,SAE的合成是在细胞中通过涉及酰基辅酶A:固醇酰基转移酶/固醇O-酰基转移酶(EC2.3.1.26)的酶促机制来完成。所催化的反应是具有长链脂肪酸的固醇的酯化反应。因此,在一个优选的实施方案中,与相应的未经改造的生物、优选相应的野生型生物相比,本发明的生物经改造,使得酰基辅酶A:固醇酰基转移酶/固醇O-酰基转移酶(EC2.3.1.26)的活性被降低。
在酵母、尤其是酿酒酵母中,固醇酯化而得到固醇基酰基酯是由固醇酰基转移酶的两种同工酶(即Are1p和Are2p)来完成,所述酶对不同的固醇中间体具有不同的特异性亲和力。Are1p尤其是导致麦角固醇前体(例如羊毛固醇、酵母固醇、麦角甾-5,7-二烯醇)和酵母外源物质(例如7-脱氢胆固醇)的酯化。Are2p优先导致麦角固醇(在酵母中是麦角固醇生物合成途径的终产物)的酯化。如果所述生物是酵母生物、尤其是酵母属(Saccharomyces)和优选酿酒酵母的话,与相应的未经改造的酵母生物、优选相应的野生型生物相比,可降低或消除Are1p或Are2p的活性或Are1p和Are2p这两者的活性。如果同时消除这两种酶活性,在细胞中就不会发生固醇的酯化。如果在生物中相应地保持或调整产生某种固醇的酶活性,则仅消除一种活性并保持另一种的活性,在细胞中就可能根据需要得到某一类型固醇基酰基酯。
与其中所述生物的特征是不再合成固醇基酰基酯的本发明第一方面相关,本发明的生物原则上可以是任何可能的生物,植物生物、真菌生物或细菌生物。然而,优选的生物是源自在其脂质粒中天然就不贮存蜡酯、而仅贮存其它脂质(例如三酰甘油和固醇基酰基酯)的生物。因此,通过消除固醇基酰基酯合成的生物合成途径,使得所述生物在其脂质粒中基本上仅积聚三酰甘油,因而允许在所述生物中由脂质粒以相当纯形式产生三酰甘油。在其脂质粒中天然就不积聚蜡酯的生物的实例尤其是大多数动物、真菌细胞和大多数植物细胞,藻类和浮游生物细胞除外。在本发明第一方面相关的一个特别优选的实施方案中,所述生物是真菌生物,优选选自以下各属的真菌生物:耶氏酵母属(Yarrowia)、红酵母属(Rhodotorula)、油脂酵母属(Lipomyces)、假丝酵母属(Candida)、红冬孢酵母属(Rhodosporidium)、被孢霉属(Mortierella)、毛霉属(Mucor)、酵母属(Saccharomyces)、毕赤酵母属(Pichia)、克鲁维酵母属(Kluyveromyces)、曲霉属(Aspergillus)、青霉属(Penicillium)和网柄菌属(Dictyostelium)。特别优选的是以下真菌生物种:解脂耶氏酵母(Yarrowia lipolytics)、粘红酵母(Rhodotorula glutinis)、斯达油脂酵母(Lipomyces starkeyi)、弯假丝酵母(Candida curvata)、圆红冬孢酵母(Rhodosporidium tortuloides)、深黄被孢霉(Mortierellaisabellina)、爪哇毛霉(Mucor javonicus)、酿酒酵母(Saccharomycescerevisiae)、巴斯德毕赤酵母(Pichia pastoris)、克鲁维酵母(Kluyveromyces spec)、曲霉(Aspergillus spec)、青霉(Penicillium spec)或网柄菌(Dictyostelium spec)。在本发明第一方面的一个特别优选的实施方案中,所述生物是酿酒酵母。
在第二实施方案中,本发明的生物经改造,使其不再合成固醇基酰基酯(SAE)和蜡酯(WE)。
通常,WE的合成在细胞中通过涉及酰基辅酶A-蜡醇酰基转移酶(EC2.3.1.75)的酶促机制来完成。因此,在本发明第二方面的一个优选的实施方案中,与相应的未经改造的生物、优选相应的野生型生物相比,本发明的生物经改造,使得酰基辅酶A:固醇酰基转移酶/固醇O-酰基转移酶(EC2.3.1.26)和酰基辅酶A-蜡醇酰基转移酶(EC2.3.1.75)的活性被降低。
本发明第二方面相关的本发明的生物在脂质粒中主要积聚三酰甘油,因为固醇基酰基酯和蜡酯的合成被降低,优选被完全消除。因此所述生物适合制备、尤其是在工业化规模上制备更纯形式的三酰甘油。
本发明第二方面的生物优选源自天然产生蜡酯并在胞内脂质粒中贮存它们的生物。产生并贮存蜡酯的生物例如植物和昆虫(其中它们可用于提供疏水性的组织外被,以尽量降低表面脱水)、藻类、浮游生物和细菌。在本发明第二方面的一个优选的实施方案中,所述生物是细菌生物,优选以下各属的生物:分枝杆菌属(Mycobacterium)、链霉菌属(Streptomyces)、红球菌属(Rhodococcus)、诺卡氏菌属(Nocardia)、芽孢杆菌属(Bacillus)、棒状杆菌属(Corynebacterium)、埃希氏菌属(Escherichia)或乳酸杆菌属(Lactobacillus)。最优选的生物是大肠杆菌(Escherichia coli)。
此外,不仅可在本发明的生物中产生三酰甘油(其在所述生物中天然产生,或以其在所述生物中天然产生的数量来产生),而且还可对所述生物进行遗传改造,以增加一种或多种在所述生物中天然产生的特定三酰甘油的数量,或者可对所述生物进行遗传改造,以导致合成一种或多种在所述生物中并非天然产生的三酰甘油。
在一个特别优选的实施方案中,本发明的生物所积聚的三酰甘油是甘油和至少一种不饱和脂肪酸的酯,并且更优选所述不饱和脂肪酸是ω-3脂肪酸。原则上,所述ω-3脂肪酸可以是任何可能的ω-3脂肪酸,但优选是α-亚麻酸(ALA)、十八碳四烯酸、二十碳四烯酸、二十碳五烯酸(EPA)、二十二碳五烯酸(DPA)或二十二碳六烯酸(DHA)。
在另一个优选的实施方案中,三酰甘油中的不饱和脂肪酸是ω-6脂肪酸。原则上,所述ω-6脂肪酸可以是任何可能的ω-6脂肪酸,但优选是亚油酸、γ-亚麻酸、二十碳二烯酸、二同型-γ-亚麻酸、花生四烯酸、二十二碳二烯酸、二十二碳四烯酸、二十二碳五烯酸或十八碳三烯酸。
在另一个优选的实施方案中,三酰甘油中的不饱和脂肪酸是ω-9脂肪酸。原则上,所述ω-9脂肪酸可以是任何可能的ω-9脂肪酸,但优选是油酸、二十二碳一烯酸、二十碳三烯酸、二十二碳烯酸或神经酸。
可以进一步改进第一和第二实施方案,如下所述。三酰甘油通常是通过二酰基甘油与长链脂肪酸或磷脂的酯化而形成。与长链脂肪酸的酯化反应是由二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)催化的。与磷脂的酯化反应是由卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)催化的。在一个特别优选的实施方案中,本发明第一或第二方面的生物的特征是与相应的未经改造的生物、优选相应的野生型生物相比,二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)的活性和/或卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)的活性增加。上述两种酶中的一种或两种的活性增加导致所述生物的细胞中TAG合成的增加。
除此之外,也可对所述生物进行遗传改造,使得所述生物能够产生某些脂肪酸以掺入到TGA中或增加所述脂肪酸的产量,如果它们在所述生物中是天然产生的话,则以增加它们在所述生物中所产生的TGA中的含量。例如,如果要在所述生物所产生的TGA中增加ω-3脂肪酸(例如二十碳五烯酸(EPA)、二十二碳五烯酸(DPA)和/或二十碳六烯酸(DHA))的含量,则最好是增加以下酶的活性:Δ12脂肪酸去饱和酶(EC1.14.19)、Δ15-去饱和酶(EC1.14.19)、Δ6脂肪酸去饱和酶(EC1.14.19.3)、脂肪酸延长酶、Δ5-去饱和酶/Δ5-脂肪酸去饱和酶(EC1.14.19)和Δ4脂肪酸去饱和酶(EC1.14.19)。因此,在一个优选的实施方案中,所述生物的特征是与相应的未经改造的生物、优选相应的野生型生物相比,二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)和/或卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)的活性和选自以下至少一种酶的活性被增加:Δ12脂肪酸去饱和酶(EC1.14.19)、Δ15-去饱和酶(EC1.14.19)、Δ6脂肪酸去饱和酶(EC1.14.19.3)、脂肪酸延长酶、Δ5-去饱和酶/Δ5-脂肪酸去饱和酶(EC1.14.19)和Δ4脂肪酸去饱和酶(EC1.14.19)。例如,植物和某些真菌能够产生ω-3和ω-6脂肪酸,而例如酿酒酵母仅可合成ω-9脂肪酸。因此,如果本发明的生物是酿酒酵母,则需要向所述细胞中引入产生ω-3和/或ω-6脂肪酸所需的酶活性。将相应基因引入酵母细胞的方法以及相应的基因在文献中已有描述(参见例如WO2004/101575)。特定脂肪酸的异源生物(例如在酵母细胞中)的合成描述于例如Beaudoin等(Proc.Natl.Acad.Sci.USA 97(2000),6421-6426)和Veen等(Appl.Microbiol.Biotechnol.63(2004),635-646)。
本发明也涉及产生三酰甘油的方法,所述方法包括培养本发明第一或第二方面的生物。如上所述,这些生物在其脂质粒中能以基本纯的形式积聚大量三酰甘油。可按照本领域众所周知的方法从生物中分离出TAG。本领域已知如何从细胞中分离脂质粒和如何从其中所含中性脂质中分离它们。对于生物体酿酒酵母,所述方法的实例描述于所附实施例中。
本发明也涉及产生作为甘油和至少一种不饱和脂肪酸的酯的三酰甘油的方法,所述方法包括:
与相应的未经改造的生物、优选相应的野生型生物相比,在生物中
(i)通过抑制剂降低或消除酰基辅酶A:固醇酰基-转移酶/固醇O-酰基转移酶(EC2.3.1.26)和/或酰基辅酶A-蜡醇酰基转移酶(EC2.3.1.75)活性;和
(ii)增加二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)和/或卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)的活性。培养所述生物并分离TAG。
对于待产生的三酰甘油中所含有的不饱和脂肪酸而言,以上关于多不饱和脂肪酸的特性和待增加的酶活性所述的优选实施方案同样适用。在一个特别优选的实施方案中,多不饱和脂肪酸是ω-3脂肪酸,并且所增加的酶活性是与ω-3脂肪酸相关的上文进一步提及的至少一种酶活性。
在第三个实施方案中,本发明的生物经改造,使其不再合成三酰甘油(TAG)和固醇基酰基酯(SAE)。如上所述,通常TAG的合成通过二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)和卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)的酶活性实现。因此,在一个优选的实施方案中,本发明的生物经改造,使得与相应的未经改造的生物、优选相应的野生型生物相比,酰基辅酶A:固醇酰基转移酶/固醇O-酰基转移酶(EC2.3.1.26)或二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)和或卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)的活性降低。
在一个优选的实施方案中,本发明第三方面的生物是源自天然产生并贮存蜡酯的生物。所述生物已在上文描述。优选生物是如上已述的细菌生物,最优选是大肠杆菌。因为TAG和SAE的合成的消除,所述生物适合于在其脂质粒中主要积聚蜡酯。在一个特别优选的实施方案中,所述生物还具有的特征是与相应的未经改造的菌株、优选相应的野生型菌株相比,它表现出酰基辅酶A蜡醇酰基转移酶(EC2.3.1.75)活性增加。这将允许增加在生物中合成的蜡酯的含量。在生物细胞中增加所需酶活性的方式和方法是本领域技术人员已知的并进一步描述于下文。
在另一个优选的实施方案中,本发明第三方面的生物是源自天然就不产生并贮存蜡酯的生物。所述生物在本发明第一方面已有描述。优选所述生物是如上所述的真菌生物,更优选酵母属且最优选酿酒酵母。因为TAG和SAE的合成的消除,所述生物的脂质粒可用于积聚目标脂质,例如鲨烯,正如以下进一步所述。
在第四实施方案中,本发明的生物经改造,使其不再合成蜡酯(WE)、三酰甘油(TAG)和固醇基酰基酯(SAE)。在这一方面,所述生物在其脂质粒中基本上不贮存任何WE、TAG和SE。所述生物尤其可用于设计可涉及特别想要的脂质的脂质粒的生物。也就是说,例如,可将核酸分子引入所述生物,以允许合成另一类型的脂质,其则积聚在脂质粒中。由此,可提供在脂质粒中允许合成基本纯的所需脂质的生物。
在一个优选的实施方案中,本发明第四方面的生物经改造,使得与相应的未经改造的生物、优选相应的野生型生物相比,酰基辅酶A:固醇酰基转移酶/固醇O-酰基转移酶(EC2.3.1.26)、二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)、卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)和酰基辅酶A-蜡醇酰基转移酶(EC2.3.1.75)的活性降低。
在第五实施方案中,本发明的生物是这样的生物:所述生物在细胞中产生积聚在脂质粒中的中性脂质,并且经遗传改造,使其不再合成三酰甘油(TAG)并因此在所述脂质粒中不再含有在其脂质粒中天然存在的所述三酰甘油。如上所述,例如在酵母中,通过二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)和卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)的活性,完成TAG的广泛合成。因此,在一个优选的实施方案中,本发明第五方面的生物经遗传改造,使得与相应的未经改造的生物、优选相应的野生型生物相比,二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)和卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)的活性降低。
在本发明第五方面的一个优选的实施方案中,所述生物源自在其脂质粒中天然就不能产生并贮存蜡酯的生物。所述生物在上文已有描述。优选的生物是如上所述的真菌生物,更优选酵母属和最优选酿酒酵母。
在第六实施方案中,本发明的生物是这样的生物:所述生物在细胞中产生在脂质粒中积聚的中性脂质,并且经遗传改造,使其不再合成蜡酯(WE)和三酰甘油(TAG)并因此在所述脂质粒中不再含有在其脂质粒中天然存在的三酰甘油和蜡酯。在一个优选的实施方案中,本发明第六方面的生物经遗传改造,使得与相应的未经改造的生物、优选相应的野生型生物相比,二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)、卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)和酰基辅酶A-蜡醇酰基转移酶(EC2.3.1.75)的活性降低。
在一个优选的实施方案中,本发明第五或第六方面的生物,即不再合成三酰甘油或者不再合成三酰甘油和蜡酯的生物,在其脂质粒中能够积聚固醇脂质的酰基酯。固醇脂质的酰基酯在脂质粒中的积聚可能是因为以下事实所致:所述生物天然合成所述酯;或可因为以下事实所致:已将导致生物合成固醇脂质酰基酯的相应酶的编码基因导入细胞。
固醇脂质,例如胆固醇及其衍生物,以及甘油磷脂和鞘磷脂,都是膜脂质的重要组分。也含同样的稠合4环核心结构的类固醇作为激素和信号转导分子而具有不同的生物学作用。C18类固醇包括***家族,而C19类固醇则包括雄激素例如睾酮和雄酮。C21亚类包括孕激素以及糖皮质激素和盐皮质激素。包含维生素D的不同形式的开环甾类化合物(secosteroid)的特征在于核心结构的B环的裂解。固醇的其它实例是胆汁酸及其缀合物,其在哺乳动物中是胆固醇的氧化衍生物并在肝脏中合成。
在一个优选的实施方案中,所述固醇脂质是固醇或类固醇。类固醇是特征在于具有通常以6-6-6-5方式排列的4个稠合环的碳骨架的类萜脂质。类固醇因与这些环连接的官能团和环的氧化状态不同而异。上百种不同的类固醇存在于植物、动物和真菌中。所有类固醇在细胞中由以下固醇产生:羊毛固醇(动物和真菌)或环阿屯醇(植物)。这两类固醇源自三萜鲨烯的环化。类固醇包括***、孕酮和睾酮。
固醇(或甾族醇(steroid alcohol))是在A环3位具有羟基的甾族亚类。它们是自乙酰辅酶A合成的两亲性脂质。整个分子相当扁平。A环上的羟基是极性的。脂族链的其余部分是非极性的。植物的固醇被称为植物固醇,而动物的固醇被称为动物固醇。最重要的动物固醇是胆固醇和某些类固醇激素;最重要的植物固醇是菜油固醇、谷固醇和豆固醇。固醇在真核生物的生理学上起重要作用。例如胆固醇构成细胞膜的组成部分,其中它的存在影响细胞膜的流动性并在发育信号转导中起到第二信使的作用。也已知植物固醇可封闭人类肠道中的胆固醇吸收部位,因而有助降低人体内的胆固醇。在人体内,固醇的作用是提供重要信号和代谢通信,例如昼夜节律、凝血。
在一个优选的实施方案中,所述固醇是胆固醇、胆固醇衍生物、7-脱氢-胆固醇、羊毛固醇、羊毛固醇衍生物、酵母固醇、酵母固醇衍生物、7-烯胆甾烷醇、7-烯胆甾烷醇衍生物、葫芦素、葫芦素衍生物、麦角甾二烯醇、麦角甾二烯醇衍生物、茶甾酮、茶甾酮衍生物、油菜素甾酮、油菜素甾酮衍生物、香蒲固醇、香蒲固醇衍生物、长春花甾酮、长春花甾酮衍生物、环桉烯醇、环桉烯醇衍生物、谷固醇、谷固醇衍生物、异岩藻固醇、异岩藻固醇衍生物、岩藻固醇、岩藻固醇衍生物、柳珊瑚固醇、柳珊瑚固醇衍生物、麦角固醇、麦角固醇衍生物、豆固醇或豆固醇衍生物。
具体地讲,在积聚7-脱氢-胆固醇的酰基酯的生物中,与相应的野生型生物相比,HMG-辅酶A-还原酶(EC1.1.1.34)、和/或Δ24-还原酶、和/或羊毛固醇C14-脱甲基酶/细胞色素P45051(EC1.14.13.70)、和/或鲨烯-环氧酶/鲨烯-单加氧酶(EC1.14.99.7)的活性可被增加。另外,与相应的野生型生物相比,SAM:C-24固醇甲基转移酶、和/或C-22固醇去饱和酶和/或C-5固醇去饱和酶的活性可被降低或消除。在积聚麦角固醇的酰基酯的生物中,与相应的野生型生物相比,HMG-辅酶A-还原酶(EC1.1.1.34)、和/或羊毛固醇C14-脱甲基酶/细胞色素P45051(EC1.14.13.70)和/或鲨烯-环氧酶/鲨烯-单加氧酶(EC1.14.99.7)的活性可被增加。在积聚麦角甾二烯醇的酰基酯的生物中,与相应的野生型生物相比,HMG-辅酶A-还原酶(EC1.1.1.34)、和/或羊毛固醇C14-脱甲基酶/细胞色素P450 51(EC 1.14.13.70)、和/或鲨烯-环氧酶/鲨烯-单加氧酶(EC1.14.99.7)的活性可被增加,和/或与相应的野生型生物相比,Δ22-去饱和酶/细胞色素P450 61(EC1.14.14.1)活性可被降低。
在另一个优选的实施方案中,所述类固醇是雄酮、雄酮衍生物、睾酮、睾酮衍生物、***、***衍生物、雄烯二酮、雄烯二酮衍生物、卡普睾酮、卡普睾酮衍生物、美雄醇、美雄醇衍生物、勃拉睾酮、勃拉睾酮衍生物、表雄酮、表雄酮衍生物、美雄诺龙(mestanolone)、美雄诺龙衍生物、二氢睾酮(stanolone)、二氢睾酮衍生物、司腾勃龙(stenbolone)、司腾勃龙衍生物、表睾酮、表睾酮衍生物、皮质醇、皮质醇衍生物、醛固酮、醛固酮衍生物、孕烯诺龙、孕烯诺龙衍生物、可的松、可的松衍生物、皮质酮、皮质酮衍生物、炔诺酮、炔诺酮衍生物、尿皮质醇或尿皮质醇衍生物。
在本发明第五和第六方面的一个优选的实施方案中,能积聚固醇脂质的酰基酯的生物是以下生物:天然具有导致相应固醇脂质合成的生物合成途径并且其中,更优选的是,与相应的未经改造的生物相比相应途径的酶活性被增加,以达到相应固醇脂质的更高积聚。
在一个优选的实施方案中,本发明第一至第六方面的生物在其脂质粒中能积聚异戊烯醇脂质。异戊烯醇脂质是由主要通过甲羟戊酸(MVA)途径而产生的5-碳前体异戊烯基二磷酸酯和二甲基烯丙基二磷酸酯合成而来。异戊烯醇脂质包括类异戊二烯、醌和氢醌、聚异戊烯醇、hopanoid和某些其它少量种类。简单的类异戊二烯(线状醇、二磷酸酯等)是通过依次加入C5单元而形成,并且按照这些萜单元数目而分类。含有大于40个碳的结构称为多萜。类胡萝卜素是重要的简单类异戊二烯,其功能是作为抗氧化剂和维生素A的前体。另一类生物学上重要的分子是例如醌和氢醌,其含有与非类异戊二烯起源的醌型核心相连接的类异戊二烯尾。维生素E和维生素K,以及泛醌,就是这一类的实例。细菌合成聚异戊烯醇(称为细菌异戊烯醇),其中与氧连接的末端类异戊二烯单元仍然是不饱和的,而在动物聚异戊烯醇(多萜醇类)中末端类异戊二烯则被还原。
在一个优选的实施方案中,积聚在脂质粒中的异戊烯醇脂质是类异戊二烯。所述类异戊二烯包括例如C5类异戊二烯、C10类异戊二烯(单萜)、C15类异戊二烯(倍半萜)、C20类异戊二烯(二萜)、C25类异戊二烯(二倍半萜)、C30类异戊二烯(三萜)、C40类异戊二烯(四萜)、多萜和类视黄醇。
在一个特别优选的实施方案中,所述类异戊二烯是三萜。三萜由六个异戊二烯单元组成并具有基本分子式C30H48-50。这一类包括例如3S-鲨烯-2,3-环氧化物、鲨烯、前鲨烯二磷酸酯、四膜虫醇(tetrahymanol)、α-香树素、β-香树素、羽扇豆醇、羽扇豆醇乙酸酯、蒲公英固醇、印楝素A1、新苦木素、苦木素和3-乙酰基-1-巴豆酰基印楝素。线状三萜鲨烯,鲨鱼鱼肝油的主要成分,源自两分子法尼基焦磷酸酯还原性偶联。鲨烯再经生物合成加工而产生羊毛固醇或环阿屯醇,这是所有类固醇的结构前体。
在一个特别优选的实施方案中,脂质粒中积聚的异戊烯醇脂质是鲨烯或鲨烯衍生物。鲨烯衍生物包括与主链碳原子连接的一个或多个、特别是1-10个或1-4个额外甲基或乙基,而非如鲨烯中与主链碳原子连接的氢原子。
在一个特别优选的实施方案中,本发明的生物、优选本发明第一、第三和第五方面的生物,是在其脂质粒中不产生和贮存蜡酯的生物,例如如上所述的真菌生物,和更优选酵母属的真菌生物,最优选酿酒酵母。按照本发明第一、第三和第五方面而经改造、使其不合成三酰甘油和/或固醇基酰基酯的所述生物,能够在其脂质粒中合成并贮存异戊烯醇脂质、尤其是鲨烯,因此允许在脂质粒中以基本纯或更纯形式产生这样的异戊烯醇脂质。因此,已经通过在酵母生物中消除TAG和/或SAE生物合成的合成途径,可大大提高所述生物产生的鲨烯的产量和纯度。
可通过以下方式达到对所述生物的鲨烯产生的另一改进:改造所述生物的代谢以使导致鲨烯合成的酶活性增加并使将鲨烯转化为其它化合物的途径的酶活性降低。
鲨烯生物合成途径的基因是已知的并将其克隆到例如酿酒酵母中。主要的瓶颈酶是HMG-辅酶A-还原酶(HMG1)(Basson等(MoI.Cell.Biol.8(1988),3793-3808)。因此,在一个优选的实施方案中,本发明第一、第三或第五方面的生物在其脂质粒中能够积聚鲨烯并且特征在于与相应的未经改造的生物、优选与相应的野生型生物相比HMG-辅酶A-还原酶(EC1.1.1.34)的活性增加。可通过本领域技术人员众所周知的并且也详述于下文的方式和方法,使HMG-辅酶A-还原酶活性增加。在一个优选的实施方案中,通过在所述生物中表达仅编码酶的催化区、而不编码膜结合结构域的HMG-辅酶A-还原酶基因,而达到使HMG-辅酶A-还原酶活性增加。这样的改变已经描述于EP-A 486 290。通过这样的改造,避免了麦角固醇生物合成途径的中间体对HMG-辅酶A-还原酶的反馈调节。在另一个优选的实施方案中,本发明生物的HMG-辅酶A-还原酶编码基因处于异源启动子控制之下,所述异源启动子即对HMG-辅酶A-还原酶基因而言是外源的启动子,尤其是该启动子是其活性不受麦角固醇生物合成途径的中间体调节的启动子。合适的启动子的实例是ADH1启动子,尤其是表现出接近组成型表达的“平均”ADH1启动子(Ruohonen等,Journal of Biotechnology 39(1995),193-203)。
在某些生物、例如酵母中,可将鲨烯转化为其它化合物、尤其是麦角固醇和麦角固醇生物合成途径的中间体。导致麦角固醇((22E)-麦角甾-5,7,22-三烯-3-β-醇)形成的化学反应和途径发生在麦角、酵母和霉菌中。在这些生物中,由鲨烯单加氧酶/鲨烯环氧酶(EC1.14.99.7;也称为鲨烯环氧酶或ERG1)将鲨烯转化为(S)-2,3-环氧鲨烯,接着再通过2,3-氧化鲨烯-羊毛固醇环化(EC5.4.99.7;ERG7)将其转化为羊毛固醇。再由细胞色素P450羊毛固醇14a-脱甲基酶(EC1.14.13.70;ERG11),使用NADPH和O2,将羊毛固醇转化为4,4-二甲基-胆甾-8,14,24-三烯醇,再由C14固醇还原酶(EC1.3.1.70;ERG24),使用NADPH,将其转化为4,4-二甲基-8,24-胆甾二烯醇。再通过C-4固醇甲基氧化酶(EC1.14.13.72;ERG25)的作用,将该化合物进一步转化为4-甲基-8,24-胆甾二烯醇。该物质由C-3固醇脱氢酶(EC1.1.1.170;ERG26)转化为3-酮-4-甲基酵母固醇。随后通过3-酮固醇还原酶(EC1.1.1.270;ERG27)将3-酮-4-甲基酵母固醇转化为酵母固醇。酵母固醇本身再与S-腺苷-L-甲硫氨酸一起,通过SAM:C-24固醇甲基转移酶(EC2.1.1.41;ERG6)的作用而转化为粪固醇和S-腺苷-高半胱氨酸。粪固醇再通过C-8固醇异构酶(ERG2)的作用转化为麦角甾二烯醇,其再通过C-5固醇去饱和酶(EC1.14.21.6;ERG3),通过使用NADPH和O2,而转化为5,7,24(28)-麦角甾三烯醇。该化合物通过C-22固醇去饱和酶(EC1.14.14;ERG5)的作用进一步转化为5,7,22,24(28)-麦角甾四烯醇。5,7,22,24(28)-麦角甾四烯醇再通过C-24固醇还原酶(EC1.3.1.71;ERG4)的作用转化为麦角固醇。如果希望本发明的生物在脂质粒中积聚鲨烯和如果该生物来源于能天然合成麦角固醇或起始自鲨烯的麦角固醇途径的中间体的生物,则需要它降低所述生物的从鲨烯到麦角固醇的途径的一种或多种上述酶活性。原则上,上述麦角固醇途径的上述酶的任何一种、不止一种或所有都可被降低。在一个优选的实施方案中,鲨烯单加氧酶(EC1.14.99.7;也称为鲨烯环氧酶或ERG1)的活性降低,然而,并非完全消除,因为某些固醇合成是酵母细胞生存所必需的。也可降低以下涉及麦角固醇生物合成的一种或多种酶的活性:SAM:C-24固醇甲基转移酶(EC2.1.1.41)、C-22固醇去饱和酶(EC1.14.14)和C-5固醇去饱和酶(EC1.14.21.6)。所有这些都适用于本发明的上述任何实施方案。
作为本发明的组成部分,但作为独立特性,使用关于经遗传改造的生物的以下特征。
在本发明内,也已经发现基因SAK1和/或HAP4在酵母酿酒中的组成型过量表达导致脂质、尤其是鲨烯和固醇的产量的大量增加。基因SAK1和HAP4涉及呼吸-发酵变化分布(respiro-fermentative fluxdistribution)。
基因SAK1编码上游丝氨酸/苏氨酸激酶,其负责Snf1p/Snf4p复合物的磷酸化。磷酸化产生位于核内的活性Snflp/Snf4p复合物。Snflp/Snf4p复合物属于蛋白质丝氨酸/苏氨酸激酶并在“二次转换(diauxic shift)”期间在免除葡萄糖阻遏中起到主要作用。二次转换是指当培养基中的葡萄糖或其它发酵性碳源被消耗时,从发酵代谢向呼吸代谢的转换。Snf1/Snf4复合物的范围远达分别负责葡萄糖阻遏和解除阻遏的级联的层次(hierarchy)。在二次转换过程中,在酿酒酵母的6000个基因中约有1/4的表达水平显著改变。这些重大改变主要涉及从发酵模式向呼吸模式的转换的主要代谢。在发酵模式中,酵母细胞代谢可发酵碳源例如葡萄糖,产生乙醇和二氧化碳。在呼吸模式中,所产生的乙醇被呼吸而得到三磷酸腺苷(ATP),其在细胞中提供能量。在二次转换过程中,Snf1p/Snf4p复合物被Snf1-激酶Sak1p、Tos1p和Elm1p磷酸化并因此被激活。活化的复合物位于核内,其中它影响两种转录因子Mig1p和Cat8p。锌指蛋白Mig 1p是转录阻遏蛋白,其募集蛋白Tup1p和Cyc8p,与作为复合物的大量葡萄糖阻遏基因的某种共有序列结合,因此该共有序列的基因下游的转录被阻遏。Cat8p是转录激活蛋白并且当培养基中的葡萄糖被耗尽时诱导至少34个基因表达。这些基因是乙醛酸旁路的主要基因,所述旁路是C2化合物例如乙醇或乙酸代谢所必需的。除此之外,负责柠檬酸循环和乙醛酸旁路的中间体的胞内转运的基因也受到Cat8p的调节。活性Snf1p/Snf4p复合物使Mig 1p磷酸化,而该蛋白质失活并从核中移出并使Cat8p和Sip 1p(一种功能性同源物)磷酸化,其导致这两种蛋白质失活。活性Snflp/Snf4p复合物位于核内,因此显著促进了对葡萄糖阻遏的免除和从发酵向呼吸的转换,因为它影响阻遏级联的若干转录因子。因此,本发明的生物可经进一步开发,使其具有更多活性Snf1p/Snf4p复合物,甚至在葡萄糖存在时。这样的改造可通过蛋白Sak1p的转录失调(transcriptional deregulation)而达到,所述蛋白是磷酸化的主要激酶并因此激活Snf1p/Snf4p复合物。
Hap2/3/4/5蛋白复合物调节大量的葡萄糖阻遏基因。这些基因是呼吸链和柠檬酸循环的主要基因。作为激活物,它在二次转换期间诱导这些基因转录并强烈促进呼吸-发酵平衡转向呼吸方向的转移。基因HAP2、HAP3和HAP5是组成型表达的。天然HAP4仅在非发酵性碳源上生长时才表达。在培养HAP4-过量表达的突变型菌株期间,观察到该突变导致生长率、生物量和乙酸产量提高并使甘油和乙醇的形成降低。Hap2/3/4/5蛋白复合物通过增加呼吸量、线粒体生物发生和通过柠檬酸循环的碳通量而增殖呼吸***。
在本发明内的另一观察结果就是,在生物例如酵母、尤其是酿酒酵母中,基因FLD1的缺失导致脂质产量、尤其是鲨烯产量的意想不到的显著增加。基因FLD1编码涉及脂质滴形态、数目和大小的seipin蛋白。据报道FLD1的过量表达导致脂质粒融合,其导致在酵母细胞中形成明显增大、但数量更少的脂质粒。在野生型酵母菌株中,鲨烯是固醇生物合成途径的中间体并且不在细胞内积聚。通过在酵母中表达如EP-A 486 290所述的仅编码酶的催化部分、但不编码膜结合结构域的HMG-辅酶A-还原酶基因,而达到的HMG-辅酶A-还原酶的活性增加,导致鲨烯在细胞中积聚。这样的积聚是因为避免了麦角固醇生物合成途径的中间体对HMG-辅酶A-还原酶的反馈调节。在本发明框架内观察到在表现出HMG-辅酶A-还原酶活性增加的酵母菌株中,基因FLD1的缺失导致脂质产量、尤其是鲨烯产量的意想不到的显著增加。
对于SAK1、HAP4和FLD1的上述发现导致进一步改进和如上所述的本发明六方面中的任一方面的实施方案。然而,这些发现对于不包含上述改造、但具有合成相应未经改造生物通常能合成的所有脂质的能力的生物而言也具有独立价值。在这种情况下,未经改造的生物包括除了野生型之外的所述生物,其具有与相应的野生型生物相比增加的HMG-辅酶A-还原酶(EC1.1.1.34)活性。
因此,本发明也包括分离的经遗传改造的非哺乳动物生物,其中与相应的野生型生物相比,基因HAP4和/或SAK1是转录失调的和/或过量表达的和/或其中基因FLD1被阻遏或失活或缺失。具体地讲,可将基因HAP4和SAK1中的一个或这两者置于组成型活化启动子例如(任选)ADH 1启动子控制之下。在该变体的另一个实施方案中,与相应的野生型生物相比,基因REG1被阻遏或失活或缺失。除此之外,本发明的所有前述解释都以类似方式适用于该独立方面并且先前所公开的任何特征也都可结合到该独立方面。
本发明也涉及产生第二脂质的方法,其中使用和培养本发明的生物,和其中从所述生物中分离出第二脂质。例如,本发明产生异戊烯醇脂质的方法包括培养如上所述的本发明第三或第四方面的生物,所述生物在其脂质粒中能积聚异戊烯醇脂质。对于所积聚的异戊烯醇脂质,同样适用于以上对于本发明第三和第四方面的生物所述的优选实施方案。同样适用于相应生物中降低或增加的酶活性。作为另一实例,产生固醇基酰基酯的方法包括培养本发明第五或第六方面的生物。
与相应的未经改造的生物相比,如上所述的蛋白质或酶活性的降低优选至少50%,更优选至少75%,甚至更优选至少80%,特别优选至少90%和最优选100%。100%的降低是指无蛋白质或所述酶的酶活性在所述生物中存在。与相应的未经改造的生物相比,如上所述的酶或蛋白质活性增加优选至少10%,更优选至少50%,甚至更优选至少200%,特别优选至少1000%。术语“增加”也包括在未经改造的生物不含任何可检测的所述活性的情况下,存在(任何可检测的)酶活性或蛋白质。术语“活性增加”还包括在所有这些情况下的蛋白质量的增加,其中所述蛋白质并非酶(例如HAP4)。然后以上增加参数以类似方式用于数量。酶活性可通过以下方式测定:向预定量的浸提物中加入预定量的能催化所述浸提物反应而得到产物的酶,并任选额外加入所需反应组分,然后通过在预定的时间周期内测定所合成的产物量。测定上述酶活性的具体方法公开于例如文献WO 03/064650A1。所述文献中未描述的上述酶活性的测定以类似方式进行。
在细胞中降低给定酶活性的方法是例如降低或消除所述酶的编码基因的基因表达,例如,通过使用相应基因上游的弱启动子或者通过所述基因和/或相关启动子的完全或部分缺失,和/或在生物的细胞内加入酶的抑制剂以抑制所翻译的酶和/或将siRNA掺入所述细胞以降低活性转录物的量和/或使基因突变以产生活性更低的变体。
基因表达的降低是指与相应的未经改造的生物、优选相应的野生型生物中的所述核苷酸序列的基因表达水平相比,编码相应酶的核苷酸序列的基因表达水平被降低。同样适用于上文所述关于活性的水平降低。检测基因表达水平的方式和方法包括例如测定所合成的相应的mRNA或蛋白质的量或者测定相应蛋白质的酶活性。在一个优选的实施方案中,通过例如在RNA印迹中检测mRNA的量来测定基因表达水平。在另一个优选的实施方案中,通过例如在蛋白质印迹中检测所合成的相应蛋白质的量,或者通过测定相应的酶活性量来测定基因表达水平。也可通过使目标基因无功能而达到基因表达的降低。使基因无功能一个可能的方式就是基因中断。
增加酶活性的方法包括细胞转化,使转录失调,使酶的编码基因(异源或同源)处于组成型活性(同源或异源)启动子的控制之下,和/或使酶的编码(异源或同源)基因的拷贝数增加;和/或通过突变使活性增加。
在实施例中提供了使酶活性增加或降低的具体实例。然而,技术人员也可不用本发明的具体公开内容,而是利用本领域众所周知的其它方法。
本发明所用的酶的合适基因序列公开如下,但也可使用具有同样酶活性的其它基因序列。因此,具体的基因序列或所编码的蛋白质序列并非本发明的相关结构特征,而是相同EC(酶学委员会(EnzymeCommision))编号之下的分类。
酰基辅酶A:固醇酰基转移酶/固醇O-酰基转移酶(EC2.3.1.26)的基因序列包括:NC_001135.4,NC_001147.6,NM_005891,NM_144784,NM_153728。
二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)的基因序列包括:NC_001147.5,XM_002478787,NM_123089,XM_002378082,NM_032564,NM_001012345,NM_010046,XM_002146497。
卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)的基因序列包括:NC_001147.6,NM_008490,NM_001162568,NM_000229,NM_001005715,NM_017024,NM_001082190。
酰基辅酶A-蜡醇酰基转移酶(EC2.3.1.75)的基因序列包括:NM_123089,NM_177448.
HMG-CoOA-还原酶的基因序列包括:NC_001145,NM_106299,NC_003421.2,NC_009784.1,NC_003028.3,NC_007308.3,和图5的序列(截短的,tHMG1)。
C-24固醇甲基转移酶的基因序列包括:NC_001145,NC_000911.1,NC_003423.3,XM_505173,XM_716615。
C-22固醇去饱和酶的基因序列包括:NC_003424.3,NC_009046.1,NC_001145.2,XM_500188,XM_711840。
C-5固醇去饱和酶的基因序列包括:NC_001144,S46162,NG_009446,NM_053642,N M_001035356,XM_503090,XM_708519。
HAP4v的基因序列包括:NC_001143.7,XM_448596,XM_001645329。
SAK1的基因序列包括:NC_001137.2,XM_502591,XM_448319,XM_453478,NM_208704。
REG1的基因序列包括:NC_001136.8,XM_500990,XM_448729,XM_455276。
FLD1的基因序列包括:NC_001144.4,NM_210286,XM_001647166,XM_449778。
7-脱氢胆固醇还原酶的基因序列包括:NM_103926,NM_001360,NM_007856,NM_203904,NM_001014927,NM_201330,NM_022389,NM_001131727,NM_001087087,XM_001497598,XM_001174160,XM_001099101,BM490402,CA753545。
24-脱氢胆固醇还原酶的基因序列包括:NM_014762,NM_001016800,NM_001094456,NM_001008645,NM_001103276,NM_001080148,NM_053272,NM_00103128,XM_001488247,AB125202,XM_001153751。
羊毛固醇固醇14-脱甲基酶的基因序列包括:NC_001140.5,XM_500518,EF059165,XM_445876,XM_454109。
鲨烯单加氧酶的基因序列包括:NC_001139.8,M64994,XM_503994,XM_706801,XM_455763。
合适的启动子(异源表达的相应的不同酶的启动子可以相同或不同)的基因序列包括:NC_001142,NC_001139,NC_001147,NC_001139,NC_001148,NC_001135,NC 001136。
通常本发明的生物(无论是上述哪一方面)可以是任何可能的非人类生物,优选非哺乳动物生物。合适生物的实例在权利要求中提供。
图1显示来自所构建的酿酒酵母突变株的总脂质提取物的薄层色谱。
图2图示了在酿酒酵母中鲨烯的生物合成。
图3图示了在酿酒酵母中三酰甘油的生物合成。
图4图示了在酿酒酵母中固醇基酰基酯的生物合成。
图5图示了截短的HMG辅酶A-还原酶,tHMG1的序列。
以下实施例仅用于说明本发明。
以下材料与方法用于实施例。
1.限制性裂解
质粒(1-10μg)的限制性裂解在30μl的批次(batch)中进行。为此,将DNA溶于24μl H2O,并与3μl相应的缓冲液、1μl RSA(牛血清白蛋白)和2μl酶混合。根据DNA的量,酶浓度为1单位/μl或5单位/μl。在某些情况下,再将1μl RNase加入到该批次中,以降解tRNA。将该限制性批次在37℃孵育2小时。用微型凝胶控制限制酶解。
2.凝胶电泳
在微型凝胶或宽-微型凝胶仪器中进行凝胶电泳。微型凝胶(约20ml,8包)和宽-微型凝胶(50ml,15或30包)是由含1%琼脂糖的TAE组成。1*TAE用作流动缓冲液。将样品(10μl)与3μl终止液(stoppersolution)混合并上样。用HindIII切割的I-DNA用作标准(条带在:23.1kb;9.4kb;6.6kb;4.4kb;2.3kb;2.0kb;0.6kb)。为了分离,在80V电压下进行45-60分钟。然后,将凝胶在溴化乙锭溶液中染色并在紫外灯下用视频文件***INTAS或用橙色滤色片摄影来记录。
3.凝胶洗脱
经凝胶洗脱分离所需片段。将限制性制备物用于几包微型凝胶并分离。仅[λ]-HindIII和“牺牲痕迹(sacrifice trace)”在溴化乙锭溶液中着色,在紫外灯下观察,并标记所需片段。结果,DNA受到保护避免了溴化乙锭和紫外灯对剩余包的破坏。通过将染色和未染色胶块进行比对,根据标记,可切下未染色胶块的所需片段。将带有待分离片段的琼脂糖块加入到透析管中,用少量TAE缓冲液封闭,避免气泡,并放入BioRad-微型凝胶仪器中。流动缓冲液是由1*TAE组成,电压为100V,进行40分钟。然后,改变流动极性达2分钟,以松开粘附在透析管上的DNA。将含有DNA片段的透析管缓冲液移入反应容器中,进行乙醇沉淀。为此,将部分(1/10)体积的3M乙酸钠、tRNA(1μl/50μl溶液)和2.5倍体积的冰冷的96%乙醇加入到DNA溶液中。将该批次在-20℃孵育30分钟,然后在12,000rpm于4℃离心30分钟。DNA沉淀经干燥后溶于10-50μl H2O(根据DNA的量)。
4.Klenow处理
通过Klenow处理补平DNA片段的突出端,使得形成“平端”。每1μg DNA中,将以下批次移液在一起:在这种情况下,DNA应当来源于乙醇沉淀,以防来自抑制Klenow-聚合酶的污染物。在37℃孵育30分钟,然后在70℃再孵育5分钟,终止反应。通过乙醇沉淀,从该批次得到DNA并将其溶于10μl H2O。
5.连接
将待连接的DNA片段合并。13.1μl的终体积含有约0.5μg DNA,载体-***片段比例为1∶5。将样品在70℃孵育45秒,冷却至室温(约3分钟),再在冰上孵育10分钟。然后加入连接缓冲液:2.6μl 500mmolTrisHCl,pH 7.5和1.3μl 100mmol MgCl2,并将其在冰上再孵育10分钟。加入1μl 500mmol DTT和1μl 10mmol ATP之后,在冰上加入1μl连接酶(1单位/μl)再过10分钟。整个处理都应在尽可能少的摇动下进行,以防止邻接的DNA末端再分离。连接在14℃进行过夜。
6.大肠杆菌转化
用连接制备物DNA转化感受态(Component)大肠杆菌NM522细胞。作为阳性对照,供应含50ng pScL3质粒的批次,而作为空白对照,供应不含DNA的批次。对于每次转化制备,将100μl 8%PEG溶液、10μl DNA和200μl感受态细胞(大肠杆菌NM522)移液至桌面离心管。将各批放在冰上过30分钟并间歇性振摇。然后,进行热冲击:42℃1分钟。对于再生,将1ml LB培养基加入到细胞中并在振荡器上在37℃孵育90分钟。将100μl的各未稀释批次、1∶10稀释液和1∶100稀释液涂布于LB+氨苄青霉素平板上并在37℃孵育过夜。
7.从大肠杆菌分离质粒(微量制备)
在37℃和120rpm,将大肠杆菌菌落在桌面离心管中的1.5mlLB+氨苄青霉素培养基中培养过夜。第二天,将细胞在5000rpm于4℃离心5分钟,将沉淀溶于50μl TE-缓冲液。将每批与100μl 0.2NNaOH、1%SDS溶液混合,混合并放在冰上过5分钟(细胞裂解)。然后,加入400μl乙酸钠/NaCl溶液(230μl H2O、130μl 3M乙酸钠和40μl 5M NaCl),将该批混合并在冰上再放置15分钟(蛋白沉淀)。在11,000rpm离心15分钟后,将含有质粒-DNA的上清液移入Eppendorf管。如果上清液并未完全澄清,就再离心一次。将上清液与360μl冰冷的异丙醇混合并在-20℃孵育30分钟(DNA沉淀)。将DNA离心(15分钟,12,000rpm,4℃),弃去上清液,沉淀在100μl冰冷的96%乙醇中洗涤,在-20℃孵育15分钟,再次离心(15分钟,12,000rpm,4℃)。沉淀经快速真空干燥并溶于100μl H2O。通过限制性分析表征质粒-DNA。为此,对10μl各批次进行限制酶切并通过在宽-微型凝胶中进行凝胶电泳而分离(参见上文)。
8.在大肠杆菌上分离质粒(大量制备)
为了分离更大量质粒-DNA,进行大量制备方法。两管(plunger)100ml LB+氨苄青霉素培养基中接种菌落或100μl冷冻培养物,其携带欲分离的质粒,将其在37℃和120rpm孵育过夜。第二天将培养物(200ml)移入GSA烧杯并在4000rpm(2600*g)离心10分钟。将细胞沉淀重悬于6ml TE-缓冲液中。为了消化细胞壁,加入1.2ml溶菌酶溶液(20mg/ml TE-缓冲液),并将其在室温下孵育10分钟。然后,用12ml 0.2NNaOH、1%SDS溶液进行细胞裂解,并在室温下再孵育5分钟。通过加入9ml冷的3M乙酸钠溶液(pH 4.8)沉淀蛋白质并在冰上孵育15分钟。离心(GSA:13,000rpm(27,500*g),20分钟,4℃)之后,将含有DNA的上清液移入新的GSA烧杯,再用15ml冰冷的异丙醇沉淀DNA并在-20℃孵育30分钟。将DNA沉淀在5ml冰冷的乙醇中洗涤并风干(约30-60分钟)。然后,将其重悬于1ml H2O。通过限制性分析检查质粒。通过将稀释液上样到微型凝胶上而测定浓度。为了降低盐含量,进行30-60分钟微透析(孔径0.025μm)。
9.酵母转化
对于酵母转化,制备酿酒酵母AH22菌株的预培养物。一管20mlYE培养基中接种100μl冷冻培养物并在28℃和120rpm孵育过夜。在一管100ml YE培养基中接种10μl、20μl或50μl预培养物的相同条件下进行主要培养。
9.1产生感受态细胞
第二天,用Thoma chamber对各管计数,用容纳3-5*107细胞/ml的管继续该程序。通过离心(GSA:5000rpm(4000*g),10分钟)收获细胞。将细胞沉淀物重悬于10ml TE-缓冲液中并分装两个桌面离心管(各5ml)。在6000rpm将细胞离心3分钟并洗涤2次,每次各用5mlTE-缓冲液。然后,将细胞沉淀物按109细胞溶于330μl乙酸锂缓冲液,移至无菌50ml锥形瓶并在28℃振摇1小时。结果,细胞处于转化的感受态。
9.2转化
对于每次转化制备,将15μl鲱鱼精DNA(10mg/ml)、10μl待转化的DNA(约0.5μg)和330μl感受态细胞移液到桌面离心管中并在28℃孵育30分钟(勿振摇!)。然后,加入700μl 50%PEG 6000,在28℃将其再孵育1小时,勿振摇。然后在42℃热击5分钟。将100μl上清液接种到选择培养基(YNB,Difco),以选择亮氨酸原养型。在G418抗性选择的情况下,在热攻击之后进行细胞再生(参见9.3再生阶段)。
9.3再生阶段
因为选择标记是G418抗性,所以细胞需要时间来表达抗性基因。将转化制备物与4ml YE培养基混合并在28℃在振荡器(120rpm)上孵育过夜。第二天,将细胞离心(6,000rpm,3分钟),溶于1ml YE培养基,并将100μl或200μl涂布于YE+G418平板上。将各板在28℃孵育数天。
10.PCR的反应条件
聚合酶链式反应的反应条件必须针对单个情况来优化并且对任何批次不一定有效。因此,所用DNA的量、盐浓度和解链温度都可以改变。对于我们的问题表述,已经证明将以下物质合并在Eppendorf管中是有利的,其适用于热循环器:将5μl超级缓冲液、8μl dNTP(各0.625μM)、5′-引物、3′-引物和0.2μg基质DNA(溶于足量水,得到总体积50μl,用于PCR制备)加入到2μl(-0.1U)Super Taq聚合酶中。将该批简单离心并用一滴油覆盖。对于扩增,选择介于37至40循环之间。
11.从酿酒酵母中分离脂质粒
酵母细胞在50ml WMVIII基本培养基上在28℃培养72小时,同时以250rpm往复振摇。按照Leber等(Leber R,Zinser E,Zellnig G,Paltauf F,Daum G.Characterization of lipid particles of the yeast,Saccharomyces cerevisiae.Yeast.1994年11月;10(11):1421-1428),通过离心收获细胞并分离和纯化脂质粒。对于固醇分析,将脂质粒在30%甲醇KOH中在室温下皂化16小时,用于通过GC对总固醇进行定量测定,或者将固醇直接用氯仿/甲醇(4∶1)萃取并通过TLC分析以区分游离的和酯化的固醇或通过GC对游离固醇进行定量测定。
12.鲨烯和固醇分析
为了定量测定细胞总脂质和脂质粒的总脂质,将样品皂化,然后进行GC分析。将125OD600的细胞在100℃在0.5N HCl中处理20分钟并让其冷却至室温。然后加入3g KOH和12.5ml甲醇和连苯三酚(2g/l)。为了皂化,将混合物在70℃水浴中孵育2小时。将水解酯提取在正己烷中。将非皂化部分重悬于2ml正己烷。通过GC定量测定鲨烯和固醇,用鲨烯和胆固醇作为内标。在Hewlett-Packard 5890气相色谱的毛细管柱上分离鲨烯和固醇(25m×0.25mm×0.25μm[膜厚度];Chrompack CPSil5),程序调至150-250℃。温度开始是150℃持续2min;然后以15℃/分钟的速度增加到终温度250℃,在此温度持续20min。线速度为30cm/s,氦气用作载气,并以***方式进行注射。注射体积为1μl。计算各峰面积并关联至1克细胞干重。一式两份地测定每个样品。使用麦角固醇和鲨烯的标准品用于鉴定。
提取中性脂质并通过Sorger和Daum(J.Bacteriol.184(2002),519-524)的方法而定量测定。具体地讲,为了定量测定中性脂质,将提取物上样到硅胶60板并通过使用石油醚-***-乙酸(25∶25∶1,体积比)的溶剂***到距离的前三分之一进行色谱展层。然后简单干燥各板并用石油醚-***(49∶1,体积比)的溶剂***进一步展层到板顶部。通过在TLC小室中用碘蒸气对薄层板染色,观察中性脂质。通过光密度扫描进行定量测定。
对于鲨烯和固醇分析,酿酒酵母菌株的标准培养方法是:
预培养:在100ml摇瓶中的20ml WMVIII培养基中接种20μl相应的甘油贮液并在30℃和150rpm培养48小时。主要培养:在250ml具有挡板的摇瓶中的50ml WMVIII培养基中接种1%预培养物并在30℃和150rpm培养72小时。
实施例1
酿酒酵母AH22ura3中的基因ARE1和ARE2的缺失
载体pUG6(U,Heck S,Fiedler T,Beinhauer JD和Hegemann JH(1996)。重复用于芽殖酵母的一种新的有效的基因破坏盒(Nucleic Acids Res 24 2519-24)用于基因ARE1和ARE2的缺失。
通过同样方法相继完成了这两个基因的缺失。首先缺失ARE1,然后再缺失ARE2。质粒制备之后,通过PCR扩增pUG6片段,得到由loxP-kanMX-loxP组成的工具。构建了引物,以使ARE1 respec.ARE2编码序列的5′和3′序列融合到pUG6载体的loxP区。
所得PCR产物由KanR基因、loxP位点和ARE1 respec.ARE2同源区组成,用于酿酒酵母AH22ura3的整合转化。酵母中的同源重组导致靶序列的缺失。
针对G418的抗性已用于选择阳性克隆。在该酵母菌株中,ARE1respec.ARE2编码区已经缺失。为了制备更多基因缺失的菌株,已从菌株中除去G418抗性。为了该目的,所述菌株已经转化了pSH47(Guldner等,1996)。载体携带cre-重组酶以摆脱loxP位点侧接的KanR基因。
为了处理pSH47,在5-FOA(5-氟乳清酸)(1g/L)琼脂板上对菌株进行反向选择。所得菌株同时携带基因ARE1和ARE2的缺失。
实施例2
酿酒酵母AH22ura3中基因DGA1和LRO1的缺失
载体pUG6(Guldner等,1996)用于基因DGA1和LRO1的缺失。
通过同样方法相继完成了这两个基因的缺失。首先缺失DGA1,然后再缺失LRO1。质粒制备之后,通过PCR扩增pUG6片段,得到由loxP-kanMX-loxP组成的工具。构建了引物,以使DGA1respec.LRO1编码序列的5′和3′序列融合到pUG6载体的loxP区。
所得PCR产物由KanR基因、loxP位点和DGA1respec.LRO1同源区组成,用于酿酒酵母AH22ura3的整合转化。酵母中的同源重组导致靶序列的缺失。
针对G418的抗性已用于选择阳性克隆。在该酵母菌株中,DGA1respec.LRO1编码区已经缺失。为了制备更多基因缺失的菌株,已从菌株中除去G418抗性。为了该目的,所述菌株已经转化了pSH47(Guldner等,1996)。载体携带cre-重组酶以摆脱loxP位点侧接的KanR基因。
为了处理pSH47,在5-FOA(5-氟乳清酸)(1g/L)琼脂板上对菌株进行反向选择。
所得菌株同时携带基因DGA1和LRO1的缺失。
实施例3
酿酒酵母AH22ura3are1are2中基因DGA1和LRO1的缺失
载体pUG6(GuI dner等,1996)用于基因DGA1和LRO1的缺失。
通过同样方法相继完成了这两个基因的缺失。首先缺失DGA1,然后再缺失LRO1。质粒制备之后,通过PCR扩增pUG6片段,得到由loxP-kanMX-loxP组成的工具。构建了引物,以使DGA1respec.LRO1编码序列的5′和3′序列融合到pUG6载体的loxP区。
所得PCR产物由KanR基因、loxP位点和DGA1respec.LRO1同源区组成,用于酿酒酵母AH22ura3的整合转化。酵母中的同源重组导致靶序列的缺失。
针对G418的抗性已用于选择阳性克隆。在该酵母菌株中,DGA1respec.LRO1编码区已经缺失。为了制备更多基因缺失的菌株,已从菌株中除去G418抗性。为了该目的,所述菌株已经转化了pSH47(Guldner等,1996)。载体携带cre-重组酶以摆脱loxP位点侧接的KanR基因。
为了处理pSH47,在FOA(5-氟乳清酸)(1g/L)琼脂板上对菌株进行反向选择。
所得菌株同时携带基因ARE1、ARE2、DGA1和LRO1的四重缺失。
实施例4
t-HMGI在实施例1-3所得的酵母菌株和作为参考菌株的AH22ura3中的表达,使用附加型质粒
使用标准方法,通过PCR,自酿酒酵母S288C基因组DNA(Mortimer和Johnston(Genetics 113(1986),35-43))扩增tHMG的DNA序列(Basson等(MoI.Cell.Biol.8(1988),3793-3808))。在此情况下所用的引物是DNA寡聚体tHMG-5′和tHMG-3′。在Klenow处理之后,将所得DNA-片段引入克隆载体pUC19(Yanisch-Perron等(1985):Improved M13 phage cloning vectors and host strains:nucleotidesequences of the M13mp18 and pUC19 vectors.载于:Gene.Bd.33,S.103-119),得到载体pUC19-tHMG。在质粒分离和用内切核酸酶EcoRI和BamHI对pUC 19-tHMG进行限制酶切之后,将所得片段引入同样经EcoRI和BamHI处理的酵母表达载体pPT2b(Lang和Looman(Appl.Microbiol.Biotechnol.44(1995),147-156))。所产生的质粒pPT2b-tHMG含有截短的ADH1-启动子(Bennetzen和Hall(Yeast 7(1982),475-477))和TRP 1-终止子(Tschumper G,Carbon J.Sequence ofa yeast DNA fragment containing a chromosomal replicator and the TRP1gene.Gene.1980Jul;10(2):157-166),发现tHMG-DNA片段就在这两者之间。通过内切核酸酶EcoRV和NruI,从载体pPT2b-tHMG中分离DNA部分,所述DNA部分含有所谓的中等长度的ADH 1-启动子、tHMG基因和TRP1-终止子。将该DNA部分引入到经内切核酸酶SphI和DNA聚合酶处理过的酵母载体YEp13(Fischhoff等(Gene 27(1984),239-251))。
用YEpH2转化酿酒酵母AH22URA3are1are2、
AH22URA3dga1lro1、AH22URA3are1are2dga1lro1和AH22URA3。使用YEp13作为参考质粒。
实施例5
使用染色体整合质粒YDpUHK3,在实施例1-3所得的酵母菌株和作为参考菌株的AH22ura3中染色体整合和过量表达t-HMG1
载体YEpH2用内切核酸酶EcoRV和NruI处理。由此产生具有以下区域的DNA-片段:来自四环素抗性基因的转录活化区(Sidhu和Bollon(10(1990)157-166))、中等长度ADH 1-启动子、tHMG和TRP1-终止子(表达盒)。将该DNA-片段引入经StuI处理的载体YDpU(Berben等1991 Berben G.,Dumont J.,Gilliquet V,BoIIe P-A.und Hilger F.(1991):″The YDp plasmids:a uniform set of vectors bearing versatilegene disruption cassettes for″Saccharomyces cerevisiae″.″Yeast 7,475-477)。由此产生的载体YDpUH2/12用内切核酸酶SmaI处理并与编码卡那霉素抗性的DNA序列连接(Webster,T.D.,Dickson,R.C.(1983)Direct selection of Saccharomyces cerevisiae resistant to theantiobiotic G418 following transformation with a DNA vector carryingthe kanamycin-resistancegene of Tn903.Gene 26:243-252)。所产生的构建体(YDpUHK3)用EcoRV处理。用该构建体转化酿酒酵母AH22菌株。用线状载体转化酵母,正如在本实施例中一样,导致整个质粒在URA3基因座的染色体整合。为了从所整合载体中消除并非表达盒部分的区域(大肠杆菌复制起点、大肠杆菌的氨苄青霉素抗性基因、TEF-启动子和卡那霉素抗性基因),通过FOA选择,让已转化的酵母接受选择压力(Boeke等(Methods in Enzymology 154(1987),164-175)),以促进尿嘧啶营养缺陷型酵母生长。在选择中所述尿嘧啶营养缺陷型菌株携带所述的AH22tH3ura8并具有tHMG1-表达盒作为在URA3-基因中的染色体整合。
已经评价了表1所示的酵母菌株的鲨烯产率/含量。其中,已将所述菌株在WMVIII培养基中在30℃和150rpm振摇中培养72小时。在0.5M煮沸的HCl中破坏细胞之后,脂质用20ml正己烷萃取2次并通过GC/MS分析/定量测定(细节请参见第12项)。得到以下数据(表1)。
表1
实施例6
酿酒酵母AH22tH3ura8are1are2中基因FLD1的缺失
载体pUG6(Guldner等,1996)已用于缺失基因FLDI。
质粒制备之后,通过PCR扩增pUG6片段,得到由loxP-kanMX-loxP组成的工具。构建了引物,以使FLD1编码序列的5′和3′序列融合到pUG6载体的loxP区。
所得PCR产物由KanR基因、loxP位点和FLD1同源区组成用于酿酒酵母AH22tH3ura8are1are2的整合转化。酵母中的同源重组导致靶序列的缺失。
针对G418的抗性已用于选择阳性克隆。在该酵母菌株中,FLD1编码区已经缺失。为了制备更多基因缺失的菌株,从已菌株中除去G418抗性。为了该目的,所述菌株已经转化了pSH47(Guldner等,1996)。载体携带cre-重组酶以摆脱loxP位点侧接的KanR基因。
为了处理pSH47,在FOA(5-氟乳清酸)(1g/L)琼脂板上对菌株进行反向选择。
所得菌株同时携带基因ARE1、ARE2和FLD1的三重缺失并命名为AH22tH3ura8are1are2fld1。
已经评价了表2所示的酵母菌株的鲨烯产率/含量。为此,已将所述菌株在WMVIII培养基中在30℃和150rpm振摇中培养72小时。在0.5M煮沸的HCl中破坏细胞之后,脂质用20ml正己烷萃取2次并通过GC/MS分析/定量测定(细节请参见第12项)。得到以下数据(表2)。
表2
实施例7
基因SAK1和HAP4在实施例1-5所得的酵母菌株中的附加型过量表达
为了进行基因SAK1和HAP4的附加型过量表达,将这些基因分别克隆到表达载体pFlat1和pFlat3中。为此,使用在5’端引入NotI限制位点和在3’端引入XhoI限制位点的引物,由酿酒酵母S288c菌株的染色体DNA扩增这两个基因。所得PCR片段,以及载体pFlat1和pFlat3都用限制性内切核酸酶XhoI和NotI切割。将经限制酶切的PCR片段和线状载体连接起来,得到载体pFlat1-SAK1和pFlat3-HAP4。这些载体分别携带基因SAK1和HAP4拷贝,其邻接组成型形式的ADH1启动子和TRP1终止子,以提供SAK1和HAP4的强组成型表达。
为了构建质粒pFlat3,用SphI切割质粒YEp24,并***来自质粒pPT2B的含有ADH1启动子和TRP1终止子(其间间隔了质粒pUC19的多克隆位点)的900bp的SphI片段。通过***含有NotI和XhoI限制位点的多接头,而延伸所述多克隆位点。携带用于选择的URA3基因的所得质粒pFlat1通过NcoI限制酶切而线状化,用Klenow聚合酶补平,并整合含酵母LEU2基因的YDpL的末端补平的BamHI片段。所得载体就是pFlat3。
将质粒pFlat1-SAK1和pFlat3-HAP4以及作为对照的空质粒pFlat1和pFlat 3转化到实施例1-6所得的酵母菌株中。
已经评价了表3所示的酵母菌株的鲨烯产率/含量。为此,已将所述菌株在WMVIII培养基中在30℃和150rpm振摇中培养72小时。在0.5M煮沸的HCl中破坏细胞之后,脂质用20ml正己烷萃取2次并通过GC/MS分析/定量测定(细节请参见第12项)。得到以下数据(表3)。
表3
实施例8
通过薄层色谱评价所构建菌株的中性脂质组成。按照第12项进行总脂质提取和薄层色谱。图1显示野生型菌株AH22ura3、HMG-辅酶A还原酶失调的菌株AH22tH3ura8、以及两个双重缺失菌株AH22tH3ura8Δare1Aare2和AH22tH3ura8Δdga1Δlro1的总脂质/中性脂质的组成,所述野生型菌株具有失调的HMG-辅酶A还原酶AH22tH3ura8。图1表明野生型菌株(AH22ura3,第1和2道)产生极少量鲨烯,相比之下,在第3至8道的表达失调的HMG-辅酶A还原酶的菌株产生大量鲨烯。负责形成固醇酯类(are1,are2)和三酰甘油(dga1,lro1)的酶的编码基因的缺失,导致相应菌株中完全缺乏这些成分(在第5至8道的黑框所示)。通过鲨烯、胆固醇油酸酯、三油酸酯、油酸酯和麦角固醇的标准品鉴别脂质成分(未显示)。
Claims (12)
1.一种分离的经遗传改造的非哺乳动物生物,其中与相应的野生型生物相比,酰基辅酶A:固醇酰基转移酶/固醇O-酰基转移酶(EC2.3.1.26)和/或二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)和/或卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)和/或酰基辅酶A:长链醇O-酰基转移酶(EC2.3.1.75)活性降低或消除,并且其中与相应的野生型生物相比,HMG-辅酶A-还原酶(EC1.1.1.34)的活性增加。
2.权利要求1的生物,
其中不再合成在相应的野生型生物的脂质粒中积聚的至少第一脂质、优选三酰甘油和/或固醇基酰基酯和/或蜡酯,和
其中在经遗传改造的生物的脂质粒中积聚不同于第一脂质的第二脂质,
其中第二脂质优选地是中性脂质,更优选选自异戊烯醇脂质,尤其是类异戊二烯,甚至更优选是三萜,和最优选选自鲨烯、鲨烯衍生物、以及固醇脂质优选固醇或类固醇的酰基酯,其中鲨烯衍生物包括与主链碳原子连接的一个或多个、特别是1-10个或1-4个额外甲基或乙基,而非如鲨烯中与主链碳原子连接的氢原子。
3.权利要求1或2的生物,其中与相应的野生型生物相比,选自以下的一种或两种或所有酶的活性被降低或消除:SAM:C-24固醇甲基转移酶(EC2.1.1.41)、C-22固醇去饱和酶(EC1.14.14.-)和C-5固醇去饱和酶(EC1.14.21.6)。
4.权利要求1-3中任一项的生物,其中与相应的野生型生物相比,选自以下的一种或两种或所有酶的活性被增加:鲨烯单加氧酶(EC1.14.99.7)、固醇14-脱甲基酶(EC1.14.13.70)和7-脱氢胆固醇还原酶(EC1.3.1.21)。
5.权利要求1-4中任一项的生物,其中除了权利要求1的改变的活性之外,HAP4p和/或SAK1p的活性和/或数量增加和/或其中REG1p和/或FLD1p的活性和/或数量降低或消除。
6.权利要求1-5中任一项的生物,
其中所述固醇是胆固醇、胆固醇衍生物、7-脱氢-胆固醇、羊毛固醇、羊毛固醇衍生物、酵母固醇、酵母固醇衍生物、7-烯胆甾烷醇、7-烯胆甾烷醇衍生物、葫芦素、葫芦素衍生物、麦角甾二烯醇、麦角甾二烯醇衍生物、茶甾酮、茶甾酮衍生物、油菜素甾酮、油菜素甾酮衍生物、香蒲固醇、香蒲固醇衍生物、长春花甾酮、长春花甾酮衍生物、环桉烯醇、环桉烯醇衍生物、谷固醇、谷固醇衍生物、异岩藻固醇、异岩藻固醇衍生物、岩藻固醇、岩藻固醇衍生物、柳珊瑚固醇、柳珊瑚固醇衍生物、麦角固醇、麦角固醇衍生物、豆固醇或豆固醇衍生物,或
其中所述类固醇是雄酮、雄酮衍生物、睾酮、睾酮衍生物、***、***衍生物、雄烯二酮、雄烯二酮衍生物、卡普睾酮、卡普睾酮衍生物、美雄醇、美雄醇衍生物、勃拉睾酮、勃拉睾酮衍生物、表雄酮、表雄酮衍生物、美雄诺龙、美雄诺龙衍生物、二氢睾酮、二氢睾酮衍生物、司腾勃龙、司腾勃龙衍生物、表睾酮、表睾酮衍生物、皮质醇、皮质醇衍生物、醛固酮、醛固酮衍生物、孕烯诺龙、孕烯诺龙衍生物、可的松、可的松衍生物、皮质酮、皮质酮衍生物、炔诺酮、炔诺酮衍生物、尿皮质醇或尿皮质醇衍生物。
7.权利要求1-6中任一项的生物,
其中所述生物是原核生物,优选细菌,更优选选自分枝杆菌属(Mycobacterium)、链霉菌属(Streptomyces)、红球菌属(Rhodococcus)、诺卡氏菌属(Nocardia)、芽孢杆菌属(Bacillus)、大肠杆菌(Escherichiacoli)、棒状杆菌属(Corynebacterium)、醋酸杆菌属(Acetobacter)、不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、乳酸杆菌(Lactobacillus spec)或链霉菌(Streptomyces spec),
或者其中所述生物是真核生物,优选真菌、植物、藻类或昆虫细胞,更优选选自解脂耶氏酵母(Yarrowia lipolytics)、粘红酵母(Rhodotorula glutinis)、斯达油脂酵母(Lipomyces starkeyi)、弯假丝酵母(Candida curvata)、圆红冬孢酵母(Rhodosporidium tortuloides)、深黄被孢霉(Mortierella isabellina)、爪哇毛霉(Mucor javonicus)、酵母属(Saccharomyces)、巴斯德毕赤酵母(Pichia pastoris)、克鲁维酵母(Kluyveromyces spec)、根霉属(Rhizopus)、镰孢霉属(Fusarium)、梭孢霉属(Fusidium)、赤霉属(Gibberella)、被孢霉属(Mortierella)、木霉属(Trichoderma)、曲霉(Aspergillus spec)、青霉(Penicillium spec)或网柄菌(Dictyostelium spec),尤其是酵母属(Saccharomyces)、酿酒酵母(Saccharomyces cerevisiae)、德尔布酵母(Saccharomyces delbruckii)、意大利酵母(Saccharomyces italicus)、椭圆酵母(Saccharomycesellipsoideus)、发酵性酵母(Saccharomyces fermentati)、克鲁维酵母(Saccharomyces kluyveri)、克鲁斯酵母(Saccharomyces krusei)、乳酸酵母(Saccharomyces lactis)、马克斯酵母(Saccharomyces marxianus)、小椭圆酵母(Saccharomyces microellipsoides)、孟他努酵母(Saccharomycesmontanus)、Saccharomyces norbensis、产油酵母(Saccharomycesoleaceus)、奇异酵母(Saccharomyces paradoxus)、巴斯德酵母(Saccharomyces pastorianus)、有孢酵母(Saccharomyces pretoriensis)、罗斯酵母(Saccharomyces rosei)、鲁氏酵母(Saccharomyces rouxii)、葡萄汁酵母(Saccharomyces uvarum)、路德类酵母(Saccharomycodesludwigii),克鲁维酵母属(Kluyveromyces)的酵母例如乳酸克鲁维酵母(K.lactis)、马克斯克鲁维酵母马克斯变种(K.marxianus var.marxianus)、耐热克鲁维酵母(K.thermotolerans),假丝酵母属(Candida)例如产朊假丝酵母(Candida utilis)、热带假丝酵母(Candida tropicalis)、白色假丝酵母(Candida albicans)、解脂假丝酵母(Candida lipolytica)和皱状假丝酵母(Candida versatilis),毕赤酵母属(Pichia)例如树干毕赤酵母(Pichiastipidis)、巴斯德毕赤酵母(Piachia pastoris)和嗜山梨醇毕赤酵母(Pichiasorbitophila)、隐球菌属(Cryptococcus)、德巴利酵母属(Debaromyces)、汉逊酵母属(Hansenula)、Saccharomycecopsis、类酵母属(Saccharomycodes)、裂殖酵母属(Schizosaccharomyces)、威克酵母属(Wickerhamia)、德巴利酵母属(Debayomyces)、有孢汉生酵母属(Hanseniaspora)、克勒克酵母属(Kloeckera)、接合酵母属(Zygosaccharomyces)、Ogataea、Kuraish ia、Komagataella、海洋酵母属(Metschn ikowia)、Williopsis、Nakazawaea、隐球菌属Cryptococcus、有孢圆酵母属(Torulaspora)、布勒掷孢酵母属(Bullera)、红酵母属(Rhodotorula)、Willopsis和掷孢酵母属(Sporobolomyces),或选自花生、油菜、云苔、向日葵、afflor、罂粟、芥菜、***、Rizinus、橄榄、苋菜、墨西哥鼠尾草籽、芝麻、金盏草、punica、月见草、毛蕊花、蓟、野玫瑰、榛子、杏仁、澳洲坚果、鳄梨、杨梅属植物、南瓜、亚麻、大豆、开心果、紫草、椰子、核桃、玉米、小麦、黑麦、燕麦、黑小麦、水稻、大麦、棉花、树薯、胡椒、万寿菊、茄科、土豆、棕榈、烟草、茄子、蚕豆、豌豆、苜蓿、咖啡、可可、茶、柳、牧草、physcomitrellaoder ceratodon,或者选自Cryptista、绿胞藻纲(Chloromonadophyceae)、黄藻纲(Xanthophyceae)、隐甲藻(Crypthecodinium)、金藻门(Chrysophyta)、硅藻门(Bacillariophyta)、褐藻门(Phaeophyta)、红藻门(Rhodophyta)、绿藻门(Chlorophyta)、定鞭藻门(Haptophvta)、Cryptista、Euqlenozoa、Dinozoa、Chlorarachniophvta,或者选自草地贪夜蛾(Spodoptera frugiperda)、粉纹夜蛾(Trichoplusia ni)、甘蓝夜蛾(Mamestra brassicae)、果蝇属(Drosophila)。
8.权利要求1-7中任一项的生物用于产生脂质、优选第二脂质的应用,其包括培养所述生物并从所述生物中分离脂质。
9.按照权利要求8而获得的第二脂质在制备辅料制剂中的用途,所述辅料制剂用于疫苗、作为生物可降解润滑剂、作为包含不同于第二脂质的化妆用或药用活性物质的化妆品和/或药物中的辅料,其中将第二脂质与化妆用或药用活性物质混合并制备制剂用于给药,优选局部或口服给药。
10.核酸构建体,尤其是质粒或整合表达盒,其包含编码具有以下一种或多种活性的蛋白质的一种核酸或多种相同或不同的核酸:酰基辅酶A:固醇酰基转移酶/固醇O-酰基转移酶(EC2.3.1.26)和/或二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)和/或卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)和/或鲨烯单加氧酶(EC1.14.99.7)和/或固醇14-脱甲基酶(EC1.14.13.70)和/或7-脱氢胆固醇还原酶(EC1.3.1.21)和/或HAP4p和/或SAK1p,
其中所述核酸处于至少一个优选组成型活性启动子的控制之下。
11.权利要求10的核酸构建体,其中还包含编码具有HMG-辅酶A-还原酶(EC1.1.1.34)活性的蛋白质的核酸,其中所述蛋白质处于优选组成型活性启动子的控制之下。
12.权利要求10或11的核酸构建体在制备权利要求1-7中任一项的生物中的用途,其中转化起源生物,其中所述起源生物是野生型生物或这样的起源生物:其中与相应的野生型生物相比,选自以下的一种或两种或所有酶的活性降低或消除:SAM:C-24固醇甲基转移酶(EC2.1.1.41)、C-22固醇去饱和酶和/或C-5固醇去饱和酶(EC1.14.21.6)和/或酰基辅酶A:固醇酰基转移酶/固醇O-酰基转移酶(EC2.3.1.26)和/或二酰基甘油酰基转移酶/二酰基甘油O-酰基转移酶(EC2.3.1.20)和/或卵磷脂胆固醇酰基转移酶/磷脂:二酰基甘油酰基转移酶(EC2.3.1.158)和/或酰基辅酶A:长链醇O-酰基转移酶(EC2.3.1.75)和/或REG1p和/或FLD1p。
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RU2617963C2 (ru) | 2017-04-28 |
AU2010288968B2 (en) | 2016-05-12 |
BR112012004289A2 (pt) | 2015-09-01 |
CA2773030A1 (en) | 2011-03-03 |
EP2292741A1 (en) | 2011-03-09 |
AU2010288968A1 (en) | 2012-02-09 |
US9598710B2 (en) | 2017-03-21 |
WO2011023298A1 (en) | 2011-03-03 |
KR20120069693A (ko) | 2012-06-28 |
JP2013502903A (ja) | 2013-01-31 |
EP2470647A1 (en) | 2012-07-04 |
EP2586859A1 (en) | 2013-05-01 |
EP2470647B1 (en) | 2017-04-19 |
JP5954544B2 (ja) | 2016-07-20 |
SG178517A1 (en) | 2012-04-27 |
US8841114B2 (en) | 2014-09-23 |
ES2632541T3 (es) | 2017-09-14 |
CN102812124B (zh) | 2016-05-18 |
EP2586859B1 (en) | 2018-04-18 |
RU2012111430A (ru) | 2013-10-20 |
US20150024009A1 (en) | 2015-01-22 |
IL217583A (en) | 2017-02-28 |
MX2012002409A (es) | 2012-07-03 |
BR112012004289A8 (pt) | 2018-02-14 |
US20120156249A1 (en) | 2012-06-21 |
ES2673548T3 (es) | 2018-06-22 |
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