CN106488985B - 固醇代谢的抑制剂使微藻内甘油三酯累积的应用及其方法 - Google Patents
固醇代谢的抑制剂使微藻内甘油三酯累积的应用及其方法 Download PDFInfo
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Abstract
本发明涉及一种通过抑制固醇代谢使微藻内三酰基甘油累积的方法,所述抑制固醇代谢通过用固醇代谢的抑制剂孵育微藻来进行。本发明还涉及一种用于生产脂肪酸、生物燃料、药物组合物或化妆品组合物,和食品补充剂的方法,所述方法包括根据本发明的微藻内的三酰基甘油累积的步骤。最后,本发明关注于固醇代谢的抑制剂在微生物,并且优选微藻内使甘油三酯累积的应用。
Description
技术领域
本发明涉及一种通过抑制固醇代谢的方式使微藻内三酰基甘油累积的方法。本发明还涉及一种用于生产脂肪酸、生物燃料、药物组合物或化妆品组合物,和食品补充剂的方法,所述方法包括根据本发明的使微藻内三酰基甘油累积的步骤。最后,本发明关注于固醇代谢的抑制剂在微生物,并且优选微藻内使甘油三酯累积的应用。
背景技术
已知的是,用于营养目的的作物不能转变成用于生产油籽的作物(Durrett etal.,The Plant Journal(2008)54,593-607)。因此,努力在其它生物(如藻类)中进行油的生产(Chisti,Biotechnology Advances 25(2007)294-306;Chisti,Trends inBiotechnology,2008,Vol 26,No.3;Dismukes et al.,Current Opinion inBiotechnology 2008,19:235-240;Scott et al.,Current Opinion in Biotechnology2010,21:277-286;Singh et al.,Bioresource Technology 102(2011)26-34)。对藻类(下表1)中产生三酰基甘油(TAG,也被称为油)所进行的研究聚焦于细胞内液滴中TAG的增加。
表1:一些藻类中的油含量(来自Chisti,Biotechnology Advances 25(2007)294-306)
在EPOBIO报告(Micro-and macro-algae:utility for industrialapplication,September 2007,Editor:Dianna Bowles)中总结了微藻相对于陆地植物的优点。在陆地上可耕种的植物(作物)和在开放池或密闭反应器中生长的微藻均为生物燃料以及用于工业目的的TAG和脂肪酸的潜在来源(Dismukes et al.,Current Opinion inBiotechnology 2008,19:235-240)。然而,密集农业引起了密切的关注:作物应用暗示了作物由食物链到非食物链的转移。因此,需要努力开发新一代的基于光合微生物的生物燃料。
在用于生产TAG的植物中,微藻具有如下的主要优点:
-该生物资源不与用于动物或人类营养的农业资源竞争。
-能够在受控密闭的条件下,利用回收的人类其它活动产生的无机废弃物和有机废弃物,以环境友好的过程监控海藻的生长;并且微藻能够用于捕获工业副产气体(例如CO2)并将其转化成有价值的有机分子(Chisti,Biotechnology Advances 25(2007)294-306;Chisti,Trends in Biotechnology,2008,Vol 26,No.3;Chisti,Journal ofBiotechnology 167(2013)201-214)。
-海藻生物质产率高,当与陆地植物相比时,微藻显示出在成本节约下,极高的产率趋势(参见表2)。该产量是可变的并且取决于所采用的培养方法:其在开放池系统中是相对较低的;而在封闭光生物反应器(能够控制培养参数)中,该产量显著地提高。
-该生物资源并不依赖于地理位置或季节。
表2:主要作物(“C3”或“C4”型光合作用)和微藻(“EPOBIO计划”报告的摘录,纽约大学(09/2007),表4)的生物质产率的比较
该生物工业部分的经济可行性受到如下挑战:对总体生物质产量(即,每升产生的海藻有机物质的干重量)以及有价值分子的比例(即,每干重中TAG的足够高的比例用于工业提取和处理)的组合的当下制约(Chisti,Biotechnology Advances 25(2007)294-306;Chisti,Trends in Biotechnology,2008,Vol 26,No.3;Chisti,Journal ofBiotechnology 167(2013)201-214)。
具体地,微藻的油脂组成适用于生物柴油的生产(Dismukes et al.,CurrentOpinion in Biotechnology 2008,19:235-240;Scott et al.,Current Opinion inBiotechnology 2010,21:277-286)。由微藻生产生物柴油的基本原理是利用阳光将水和二氧化碳转化成生物质。随后通过施加外部刺激(如营养应力),和/或通过代谢的基因工程使该生物质特异性地定向用于产生生物燃料的油的合成(Dorval Courchesne et al.,Journal of Biotechnology 141(2009)31-41)。
就丰度而言,三类最重要的微藻为硅藻(硅藻纲,Bacillariophyceae),绿藻(绿藻纲,Chlorophyceae)以及金藻(金藻纲,Chrysophyceae)(EPOBIO定义)。硅藻为海洋、淡水和各种土壤生境中浮游植物多样性的重要门类。它们占高达25%的地球初级生产力。对该群组的真核生物的研究受益于对壳缝羽纹硅藻(pennate diatom)的模型,三角褐指藻(Phaeodactylum tricornutum)的研究。像其他微藻一样,硅藻被认为是用于替代化石燃料的烃类或来自石油化学的化学品的合理替代来源,基于如下的假定,其具有中性CO2平衡的优点:通过光合作用,CO2和水能够有效地转化成生物质并且能够控制碳代谢,从而它们使TAG累积极力丰富(energetically-rich)。已经对囊泡藻界(Chromalevolata)总门(包括三角褐指藻Phaeodactylum tricornutum)的不同浮游生物进行关注,关注于它们累积TAG的能力,有希望在工业化工艺中实现其初始产率、合适的稳定性以及物理性质。三角褐指藻Phaeodactylum tricornutum目前用于工业生产ω-3多元不饱和脂肪酸,但是当利用常规营养物不足的方法(诸如氮缺乏)来引起TAG累积时,该应用以及其他应用(诸如生物燃料)的工业实施仍然受限于生物质的生长迟缓和低产率(Chisti,Journal of Biotechnology167(2013)201-214)。这些方法具有一重要的缺陷:氮缺乏导致了对生长的限制。三角褐指藻Phaeodactylum tricornutum显示出用于工业实施的感兴趣的性质,如在没有硅的存在下的生长能力或可用于收获技术的细胞的沉降能力。可依赖于多种策略的组合对促进TAG累积进行尝试,这些策略包括:刺激脂肪酸和TAG的生物合成,阻断碳向替代代谢途径转移的路径以及最终阻止TAG的分解代谢。小分子能够在TAG代谢的三个方面中发挥作用。
通过抑制或阻断碳通量向替代代谢的代谢路径,能够促进微生物内油的累积。例如,众所周知的是,阻断碳水化合物以存储糖(诸如淀粉)形式的累积促进油的累积(Siautet al.,BMC Biotechnology 2011,11:7)。
然而,仍然需要一种替代方法,该替代方法在藻类生长于富含氮的介质中时,通过试图避免阻断碳水化合物储存代谢来引起油的累积。事实上,在CO2光合转化后产生的碳水化合物用作细胞内所有其他有机分子的碳源,从而阻断它们的储存对细胞生长具有非常严重的负面影响。也可阻断利用碳的其它代谢路径,并且能够对向TAG代谢的碳代谢进行重新导向。
为此目的,本发明人现已确定固醇的代谢为碳的替代下降(sink),在微藻内对其进行抑制引起油的累积。
发明内容
因此,本发明的第一主题是一种通过抑制固醇代谢,优选通过抑制固醇的合成引起微藻内TAG累积的方法,所述方法通过用固醇代谢的抑制剂孵育微藻克服了上文列出的缺点。
在本发明的框架内,术语“微藻”是指真核生物的微藻。
此外,在本发明的语境下,TAG通过脂肪酸与甘油骨架上1位、2位和3位上3个碳的酯化反应来得到。以下,通过3个脂肪酸(R1、R2、R3)与甘油骨架的酯化反应来合成TAG。
在本发明的语境下:
烷基选自(C1-C26)烷基,优选(C1-C18)烷基,并且更优选(C1-C6)烷基,诸如,甲基、乙基、正丙基、异丙基、正丁基、仲丁基、叔丁基和异丁基;
烯基选自具有至少一个碳碳双键且具有2至26个、优选2至18个,并且更优选1至6个碳原子的烃链。烯基的实例包括乙烯基、丙烯基、异丙烯基、2,4-戊二烯基;
炔基选自具有至少一个碳碳三键且具有2至26个、优选2至18个,并且更优选1至6个碳原子的烃链;
烷基烯基是指由如上所限定的烯基中,氢原子被烷基取代所衍生的任何基团;
炔基烯基是指由如上所限定的炔基中,氢原子被烷基取代所衍生的任何基团;
环烷基是指优选3至7个环碳的单价环烃基。所述环烷基可具有一个或多个双键,并且可任选地被取代。术语“环烷基”包括例如,环丙基、环己基、环己烯基等;
杂烷基是指如上所限定的烷基中,该烷基的任意碳中的一个或多个氢原子被选自由N、O、P、B、S、Si、Sb、Al、Sn、As、Se和Ge所组成的组中的杂原子所替代。碳原子和杂原子之间的键可为单键或双键。合适的杂烷基包括氰基、苯甲酰基、甲氧基、乙酰胺、硼酸盐/酯、砜、硫酸盐/酯、硫化环戊烷(thiane)、磷酸盐/酯、膦酸盐/酯等;
烷氧基选自(C1-C20)烷氧基,并且优选(C1-C4)烷氧基,诸如甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基和异丁氧基;
芳基是指自至少一个简单芳香环衍生的任何官能团或取代基;芳香环对应于具有离域π体系的任何平面环状化合物,其中,该环的每个原子包括p-轨道,所述p-轨道自身重叠。更具体地,术语“芳基”包括但并不限于,苯基、联苯基、1-萘基、2-萘基、蒽基、芘基以及它们的被取代的形式;
杂芳基是指自如上所限定的至少一个芳香环衍生的并且含有至少一个选自P、S、O和N的杂原子的任何官能团或取代基。术语“杂芳基”包括但不限于,呋喃、吡啶、吡咯、噻吩、咪唑、吡唑、噁唑、异噁唑、噻唑、异噻唑、四唑、吡唑、吡啶、吡嗪、嘧啶、哒嗪、苯并呋喃、异苯并呋喃、吲哚、异吲哚、苯并噻吩、苯并[c]噻吩、苯并咪唑、吲唑、苯并噁唑、苯并异噁唑、苯并噻唑、喹啉、异喹啉、喹喔啉、喹唑啉、噌啉、嘌呤和吖啶。本发明的芳基和杂芳基优选包括1至12个碳原子,并且更优选5或6个碳原子;
芳基烷基是指由如上所限定的烷基中,氢原子被芳基或杂芳基所取代而衍生的任何基团;
芳基烯基是指由如上所限定的烯基中,氢原子被芳基或杂芳基所取代而衍生的任何基团;
芳基炔基是指由如上所限定的炔基中,氢原子被芳基或杂芳基所取代而衍生的任何基团;
烷基芳基是指由如上所限定的烷基芳基中,氢原子被烷基所取代而衍生的任何基团。
根据本发明,卤原子选自溴、氯、氟和碘,并且优选溴、氯和氟。
根据本发明的固醇代谢抑制剂的酸加成盐可例如选自盐酸盐、氢溴酸盐、硫酸盐或硫酸氢盐、磷酸盐或磷酸氢盐、乙酸盐、苯甲酸盐、琥珀酸盐、富马酸盐、马来酸盐、乳酸盐、柠檬酸盐、酒石酸盐、葡糖酸盐、甲磺酸盐、苯磺酸盐和对甲苯磺酸盐。
根据优选的实施方式,在本发明的方法中,固醇代谢的抑制通过在含氮介质中,利用固醇代谢的抑制剂孵育微藻来实现。
上述固醇代谢的抑制剂的浓度可为1μM至1M,优选5μM至20μM。孵育步骤持续优选24小时至72小时。
本发明的微藻有利地选自硅藻门、囊泡藻界门(Chromalveolata phylum)以及原始色素体生物门(Archaeplastidae phylum)的微藻,并且更有利地选自硅藻门和囊泡藻界门的微藻。优选地,上述微藻选自硅藻微藻种的三角褐指藻Phaeodactylum tricornutum和假微型海链藻Thalassiosira pseudonana;囊泡藻界微藻种的微拟球藻Nannochloropsis,并且更优选海洋富油微拟球藻Nannochloropsis gaditana、海洋微拟球藻Nannochloropsis oceanica、盐生微拟球藻Nannochloropsis salina;以及原始色素体生物微藻种的衣藻Chlamydomonas,绿藻Ostreococcus,小球藻Chlorella。更优选地,上述微藻选自硅藻微藻种的三角褐指藻Phaeodactylum tricornutum和假微型海链藻Thalassiosira pseudonana,以及囊泡藻界微藻种的微拟球藻Nannochloropsis,并且更优选海洋富油微拟球藻Nannochloropsis gaditana、海洋微拟球藻Nannochloropsisoceanica、盐生微拟球藻Nannochloropsis salina。
固醇抑制剂由这样的分子组成:该分子在固醇的生物合成中对任何的蛋白活性,对从生物合成HMG-CoA还原酶开始,随后合成甲羟戊酸、环氧鲨烯以及衍生自环氧鲨烯的所有类固醇结构的路径(参见图1)具有影响。基于已证明利用对检测植物固醇是有效的胆固醇氧化酶/酯酶的处理(D.Kritchevsky and S.A.Tepper,Clinical Chemistry,Vol.25,No.8,1464-1465(1979)),通过诸如CellBioLabs商业化的比色法和荧光剂量法(总固醇检测分析试剂盒,比色法,参比STA-384或荧光方法,参照STA-390)能简单地实现抑制固醇代谢(优选使每细胞的总固醇含量降低20%)的化合物的鉴定;或者也可根据诸如在申请WO2010/046385和WO 97/03202中所示例出的那些方法。
根据本发明的实施方式,上述固醇代谢的抑制剂为式(I)的化合物或其盐:
其中:
R41和R42相同或不同,表示氢原子、烷基、烯基、炔基、或羟基、-COR4a或-COOR4a基团,其中,R4a表示氢原子,可选地被独立选自烷基或环烷基(优选C4至C6环烷基)的一个或多个基团取代的直链或支链的烷基,可选地被独立选自烷基或环烷基(优选C4至C6环烷基)的一个或多个基团取代的芳基,可选地被独立选自烷基或环烷基(优选C4至C6环烷基)的一个或多个基团取代的杂芳基;或者R41和R42一起形成通过双键连接的氧原子;
R43表示氢原子或烷基,优选C1至C3烷基;以及
R44、R45和R46,相同或不同,表示氢原子、烷基、烷氧基、羟基或通过双键连接的氧原子,所述烷基可选地被一个或多个卤原子取代,并且在所述烷基的链中可选地包括一个或多个亚砜官能团。
有利地,R41和R42,相同或不同,表示氢原子、腈基、羟基、C1-C2烷基或-COOR4a基团,其中,R4a为C1-C7且可选地被C4-C6环烷基取代;或者R41和R42一起形成通过双键连接的氧原子。
根据本发明的另一实施方式,上述固醇代谢的抑制剂为式(II)的化合物或其盐:
其中:
W3、X3、Y3和Z3表示碳原子、硫原子、氮原子或氧原子,并且优选地,W3、X3、Y3和Z3表示碳原子;
n3和n3'独立地为等于0或1的整数,并且优选地,n3和n3'等于1;
R31和R32相同或不同,表示氢原子,直链或支链的烷基、烯基、炔基、烷基烯基、炔基烯基、环烷基、烷基芳基、芳基烷基(优选苄基)、芳基烯基、芳基炔基、杂烷基、杂芳基;或者R31和R32一起形成包括5至6个碳原子的环烷基,所述环烷基的一个或两个碳原子能够被一个或两个杂原子,优选氮原子替代;或者R31或R32中的一个与R39一起形成5至6个碳原子的环烷基,所述环烷基的一个或两个碳原子能够被一个或两个杂原子,优选氧原子替代,所述R31或R32可选地被独立选自以下基团的一个或多个基团取代:直链或支链的烷基,环烷基、诸如炔基(所述炔基优选地为C5至C12的支链炔基),芳基烷基,芳基,杂芳基,羟基,卤素,硝基,-COR3a基团或-NR3aR3b基团,其中,R3a和R3b相同或不同,表示氢原子或直链或支链的烷基链;
R33表示氢原子;直链或支链的烷基链,诸如C1至C3烷基,或者腈基,并且,优选地R33表示氢原子;
R34、R35、R36、R37、R38和R39,相同或不同,表示,氢原子或卤原子,或者羟基,并且优选地,R34、R35、R36、R37、R38和R39为氢原子。
式(II)的虚线表示单键或双键。具体地,当W3-X3键中的W3或X3中之一或者Y3-Z3键中的Y3或Z3中之一为硫或氧时,所述W3-X3键或Y3-Z3键分别为单键。当W3和X3,或Y3和Z3为碳或氮时,所述W3-X3键或Y3-Z3键分别为双键。
根据优选的实施方式,R31和R32相同或不同,表示氢原子、直链或支链的烷基、烯基、炔基、炔基烯基、环烷基、烷基芳基、芳基烷基(并且优选苄基)、芳基烯基、芳基炔基、杂烷基、杂芳基;或者R31和R32一起形成包括5至6个碳原子的环烷基,所述环烷基的一个或两个碳原子可能被一个或两个杂原子(优选氮原子)替代;或者R31和R32中的一个与R39一起形成5至6个碳原子的环烷基,所述环烷基的一个或两个碳原子可能被一个或两个杂原子(优选氧原子)替代,所述R31或R32可选地被独立选自以下基团的一个或多个基团取代:直链或支链的烷基,环烷基、诸如炔基(所述炔基优选为C5至C12的支链炔基),芳基烷基,芳基,杂芳基,羟基,卤素,硝基,-COR3a基团或-NR3aR3b基团,其中,R3a和R3b相同或不同,表示氢原子或直链或支链的烷基链。
根据更优选的实施方式,R31和R32相同或不同,表示可选地被独立选自直链或支链的烷基的一个或多个基团取代的,C1-C6直链或支链的烷基链或芳基烷基。有利地,R31表示C1-C6直链或支链的烷基链,并且R32为被C1-C6直链或支链的烷基链取代的苄基,或被直链或支链的炔基取代的烯基,诸如,-(CH2)n3”-CH=CH-C≡C-C(CH3)3,其中,n3”为1至6。
根据本发明的另一实施方式,上述固醇代谢的抑制剂为式(III)的化合物或其盐:
其中:
n1的范围为1至12,并且优选n1=2,
R11表示氢原子;-COR1a基团,其中,R1a为选自直链或支链的烷基链,可选的取代的苄基,或可选的取代的芳基(诸如苯基),所述直链或支链的烷基链可选地被独立选自羟基、卤素、硝基的一个或多个基团取代,所述芳基最终被独立选自氢原子和甲基的一个或多个基团取代;
R12、R13和R14,相同或不同,表示氢原子;直链或支链的烷基链;羟基;-CH2OH基团;-COOR1b基团,其中,R1b表示氢原子,或直链或支链的烷基链;-OSiR1cR1dR1e基团,其中,R1c、R1d和R1e,相同或不同,表示氢原子,或直链或支链的烷基链;或者R12和R13一起稠合形成环外亚甲基(exo methylene group);
R15表示羟基;如上所限定的-OSiR1cR1dR1e基团;-COOR1f基团,其中R1f表示氢原子,或直链或支链的烷基链;-OCOR1g基团,其中R1g表示直链或支链的烷基链,优选C1-C3烷基链,并且更优选C1烷基链;或R15为与四氢吡喃酮环形成烯属不饱和的碳。
根据优选的实施方式,R11为-COR1a基团,其中,R1a为直链或支链的烷基链,并且优选R11为-COCH(CH3)C2H5基团或-COC(CH3)2C2H5基团。
有利地,R14表示C1-C6烷基链,优选-CH3基团;并且R12和R13表示氢原子。可替代地,R12和R14可表示C1-C6烷基链,优选-CH3基团,并且R13为氢原子。
根据另一优选的实施方式,n1=2,并且R15表示羟基。
根据本发明的优选实施方式,上述固醇代谢的抑制剂选自乙炔雌二醇、美伐他汀、辛伐他汀、布替萘芬、特比萘芬、雌素酮。最优选的式(I)的固醇代谢抑制剂为乙炔雌二醇和雌素酮。最优选的式(II)的固醇代谢抑制剂为布替萘芬。最优选的式(III)的固醇代谢抑制剂为美伐他汀和辛伐他汀。
根据本发明的固醇代谢的抑制剂均对“甲羟戊酸酯(mevalonate)”路径发挥作用,而不对固醇合成的“非甲羟戊酸酯”路径(参见图1)发挥作用。
更具体地,
式(I)的固醇代谢的抑制剂用作类固醇结构类似物,与参与固醇代谢的蛋白相互作用,包括雌素酮(Merola and Arnold,Science,Vol.144,301-302(1964))和乙炔雌二醇(Koopen et al.,Journal of Lipid Research,Vol.40,1999),
式(II)的固醇代谢的抑制剂用作鲨烯环氧酶抑制剂(Belter et al.,Biol.Chem.,Vol.392,1053-1075(2011)),以及
式(III)的固醇代谢的抑制剂用作HMG-CoA还原酶抑制剂(Liu et al.,Mol.Biol.Rep.(2010)37:1391-1395)。
本发明的另一主题为一种用于生产脂肪酸的方法,所述方法包括:根据本发明所限定的引起微藻内三酰基甘油累积的步骤;随后对在微藻内累积的三酰基甘油进行提取的步骤。
本发明还涉及一种用于生产生物燃料的方法,所述方法包括以下步骤:
(i)根据本发明所限定的引起微藻内三酰基甘油累积的步骤;随后
(ii)对步骤(i)中在微藻内累积的三酰基甘油进行提取的步骤;以及
(iii)对步骤(ii)中回收的三酰基甘油进行酯交换的步骤,例如如Zhang et al.,Bioresource Technology 147(2013)59-64中所述。
本发明还涉及一种生产药物组合物或化妆品组合物的方法,所述方法包括以下步骤:
(i’)根据本发明所限定的引起微藻内三酰基甘油累积的步骤;随后
(ii’)对步骤(i’)中在微藻内累积的三酰基甘油进行提取的步骤;以及
(iii’)将至少一种药学上可接受的赋形剂或化妆品上可接受的赋形剂加入到步骤(ii’)中回收的三酰基甘油的步骤。
本发明还涉及一种用于生产人类食品补充剂和动物饲料补充剂的方法,所述方法包括以下步骤:
(i”)根据本发明所限定的引起微藻内三酰基甘油累积的步骤;随后
(ii”)对步骤(i”)中在微藻内累积的三酰基甘油进行提取的步骤;以及
(iii”)将至少一种食品添加剂加入到步骤(ii”)中回收的三酰基甘油中的步骤。
本发明的方法在引起微藻内三酰基甘油累积的步骤之后,可包括一个或多个提取步骤。上述提取步骤可利用溶剂或其它本领域技术人员熟知的其它提取方法来进行。
本发明的另一主题涉及固醇代谢的抑制剂在微生物,尤其是微藻,更优选硅藻门的微藻,并且进一步优选硅藻微藻种的三角褐指藻Phaeodactylum tricornutum中使甘油三酯累积的应用。
有利地,本发明涉及选自式(I)、式(II)和式(III)的化合物,诸如乙炔雌二醇、雌素酮、布替萘芬、美伐他汀、辛伐他汀、特比萘芬的固醇代谢的抑制剂在微生物,尤其是微藻内,更优选硅藻门的微藻,并且进一步优选硅藻微藻种的三角褐指藻Phaeodactylumtricornutum内使甘油三酯累积的应用。本发明还涉及选自式(I)、式(II)和式(III)的化合物,诸如乙炔雌二醇、雌素酮、布替萘芬、美伐他汀、辛伐他汀、特比萘芬的固醇代谢的抑制剂的组合以及它们与已知能增强微藻中TAG累积的其它方法(尤其是营养不足,优选氮不足)的组合的应用。
附图说明
除了上述设定外,本发明还包括在以下说明书的剩余部分以及所附附图中进行的其他设定,其中:
图1示出了经由甲羟戊酸的固醇生物合成路径的示意图,并且示出了美伐他汀和辛伐他汀(Liu et al.,Mol.Biol.Rep.(2010)37:1391-1395)、雌素酮(Merola andArnold,Science,Vol.144,301-302(1964)、布替萘芬和特比萘芬(Belter et al.,Biol.Chem.,Vol.392,1053-1075(2011))的作用;
图2示出了在富氮N(+)介质或氮缺乏N(-)介质中培养72h后,经共聚焦显微镜直观化的褐指藻细胞的图片,在左侧(A和C)细胞以相衬(“相”)的方式示出;在右侧(B和D),示出在488nm处激发尼罗红且在519nm处发光后的细胞;
图3a至图3h示出了乙炔雌二醇、美伐他汀、辛伐他汀和雌素酮的剂量响应;以及
图4示出了通过尼罗红染色估算TAG含量,以及利用马拉瑟细胞(Malassez cell)以等份(aliquote fraction)的方式计数来估算细胞,乙炔雌二醇、美伐他汀和辛伐他汀的剂量响应;
图5示出了布替萘芬对分别在ESAW 1N1P介质和ESAW 10N10P介质中生长的海洋富油微拟球藻Nannochloropsis gaditana中尼罗红累积的影响。通过计算每百万细胞中的相对荧光单位来对尼罗红荧光性进行标准化;
图6示出了乙炔雌二酮对分别在ESAW 1N1P介质和ESAW 10N10P介质中生长的海洋富油微拟球藻Nannochloropsis gaditana中尼罗红累积的影响。通过计算每百万细胞中的相对荧光单位来对尼罗红荧光性进行标准化。
具体实施方式
实施例
1)材料和方法
1)1-三角褐指藻Phaeodactylum tricornutum菌株和生长条件
在各实验中使用三角褐指藻Phaeodactylum tricornutum(Pt1)Bohlin菌株8.6CCMP2561(海洋生物的菌种保藏,现被称为NCMA:海洋藻类和微生物群国家中心)。
使Pt1在20℃下,250mL烧瓶中,于根据加拿大微生物培养中心(Canadian Centerfor the Culture of Microorganisms)的规定制备的“富集的人工海水”(enrichedartificial seawater,ESAW)介质中生长。
为了制备ESAW介质,制备四种单独的溶液:两种盐溶液(溶液1和溶液2),一种营养溶液以及一种维生素溶液。将盐加入到蒸馏的去离子水(DDW)中。当溶液1和溶液2中的盐完全溶解后,将溶液1和溶液2混合在一起。用DDW来稀释总体积。
表3:ESAW盐溶液1和ESAW盐溶液2的组成
表4:营养物富集原液
*甘油磷酸酯二钠可用Na2HPO4的等摩尔原液替换。
**Na2EDTA.2H2O在痕量金属Fe(NH4)2(SO4)2.6H2O和FeCl3.6H2O之前加入。
***Fe(NH4)2(SO4)2.6H2O可用FeCl3的等摩尔原液替换。
利用2g的Na2CO3将溶液5调节至pH=6。
可对溶液4进行加热以使铁溶解。
表5:维生素原液
维生素原液 | 原液溶度(g.L<sup>-1</sup>) | 终浓度(mM) |
硫胺素 | 0.1 | 2.97×10<sup>-1</sup> |
维生素B12 | 0.002 | 1.47×10<sup>-3</sup> |
生物素 | 0.001 | 4.09×10<sup>-3</sup> |
为了制备ESAW介质,利用玻璃纤维预过滤器,使溶液过滤通过0.45μm膜过滤器。在第一使用前用10%HCl的对烧瓶进行酸洗,并且用蒸馏水进行冲洗。将1mL的营养物富集原液1、营养物富集原液2、营养物富集原液4、营养物富集原液5、营养物富集原液6和营养物富集原液7,2mL的营养物富集原液3,以及2mL的维生素原液(表4和表5)加入至1L的经过滤的盐溶液中。为了降低高压灭菌过程中的沉淀,加入1.44mL的1N HCl以及0.12g的碳酸氢钠。随后通过高压灭菌对所得到的ESAW介质进行杀菌。
使细胞在12:12光(450μEinstein-1sec-1)/黑暗周期(Einstein被限定为1摩尔(6.022×1023)光子的能量)下生长。利用1/5的前培养物在新鲜介质上进行接种,来每周对细胞进行继代培养。富氮N(+)介质不含氮源。氮缺乏N(-)介质含有0.05g/L NaNO3。为了监控细胞生长,使用含有组蛋白H4蛋白的基因修饰的菌株,其中,所述组蛋白H4蛋白融合有黄色荧光蛋白(Siaut et al.,Gene 406(2007)23-35)。
1)2-油滴的尼罗红染色的原理
如Ren等人所述(Biotechnology for Biofuels 2013,6:143),通过尼罗红(SigmaAldrich)荧光染色(激发波长485nm;发光波长525nm)能够监控油滴(oil droplet)的累积。对细胞进行稀释,并将其调节至与尼罗红荧光性线性相关的细胞浓度。将尼罗红溶液(40μL的2.5μg.mL-1原液浓度,在100%DMSO中)加入到160μL细胞悬浮液中。通过用尼罗红荧光强度除以细胞数来确定荧光比度。随后,利用Zeiss AxioScope.A1显微镜(FITC滤光器;激发波长488nm;发光波长519nm)使尼罗红染色过的油滴直观化。
油脂滴能够被直观化。在图2中,在富氮N(+)介质或氮缺乏N(-)介质中培养72h后,通过共聚焦显微镜使指藻Phaeodactylum细胞直观化。在左侧,细胞以相衬(“相”)的方式示出。在右侧,示出在488nm处尼罗红激发且在519nm处发光后的细胞。油脂滴可被清楚地直观化。
该原理是简单地通过利用分光荧光计测量三角褐指藻Phaeodactylumtricornutum中油的存在(在这些条件下,对细胞内其它荧光素,诸如在530nm处激发且在580nm处发光的叶绿素的检测降低)。
1)3-用于油水平检测的替代方法
可替代地,利用溶剂或其它提取方法对油进行提取,通过薄层色谱法对油进行分离和纯化,并且使其进行甲醇化以产生脂肪酸甲酯,通过与电离火焰检测器或质谱仪联用的气相色谱法对其进行量化。
从200mg的冷冻干燥的三角褐指藻Phaeodactylum tricornutum细胞中提取TAG以防止油脂降解。简单地说,在收获之后,将细胞立即冷冻在液氮中。使经冷冻干燥的细胞团重新悬浮在4mL的煮沸的乙醇中5分钟,随后在室温下加入2mL的甲醇和8mL的氯仿。随后,用氩气饱和该混合物,并且在室温下搅拌1h。在通过玻璃丝过滤后,用3mL的氯仿/甲醇(2:1,v/v)冲洗细胞剩余物。为了促进两相的形成,随后将5mL的1%NaCl加入到滤液中。在氩气下对氯仿相进行干燥,随后将油脂提取物重新溶解在纯氯仿中。为了分离TAG,利用己烷/乙醚/乙酸(70:30:1,v/v),油脂在硅胶薄层色谱(TLC)板(Merck)上移动。随后,在8-苯胺基-1-萘磺酸以2%粉碎在甲醇中后,利用UV灯使油脂直观化。随后,将它们从TLC板上刮落以进行进一步的分析。为了TAG的酰基剖析和量化,在100℃下,1小时内,利用3mL的2.5%的H2SO4甲醇溶液来使脂肪酸甲基化(涵盖21:0的标准量)。通过加入3mL的水和3mL的己烷来使反应停止。在BPX70(SEG)柱上,利用气液色谱法(Perkin Elmer)对己烷相进行分析。通过比较甲基化的脂肪酸的保留时间和标准物的保留时间来鉴定该甲基化的脂肪酸,并且利用用于校准的21:0,通过表面峰值方法来量化该甲基化的脂肪酸。上述提取和量化至少进行3次。
1)4-细胞标准化的原理
利用荧光标记物(reporter),如Siaut等人(Gene 406(2007)23-35)所述的含有与黄色荧光蛋白(YFP)融合的组氨酸H4蛋白的基因构造的菌株,能够估算样品中的细胞数(Siaut et al.,Gene 406(2007)23-35的图5A)。
为了细胞计数,我们可使用该被称为“ptYFP”的菌株,并且测量在515nm处激发后,YFP在530nm处发射的荧光;或者使用任何的菌株,并且通过用Malassez网格(grid)(供应商:Mareinfeld)计数来估算细胞数。
1)5-利用固醇代谢的抑制剂孵育三角褐指藻Phaeodactylum tricornutum,并且检测由该处理引起的油累积
在第一天,我们通过在每个孔上加入经紫外光照射消毒的4mm玻璃珠,来制备48孔板。
我们制备处于指数生长期的微藻的新鲜悬浮液。对于基于YFP荧光使细胞标准化,使在N(+)ESAW介质中培养的含有YFP标记物(ptYFP)的三角褐指藻Phaeodactylumtricornutum的细胞以3,500rpm离心5分钟。对于基于利用Malassez网格的计数使细胞标准化,使在N(+)ESAW介质中培养的三角褐指藻Phaeodactylum tricornutum的Pt1细胞以3,500rpm离心5分钟。弃去上清,并且使团块(pellet)悬浮在N(-)ESAW中。随后,使微藻以3,500rpm离心5分钟。弃去上清,并且使团块悬浮在N(-)ESAW中。随后,将细胞在N(-)ESAW中稀释至1×106个细胞/mL。随后,将样品分为两批。使一批补充有1μL/mL的46.7g/L NaNO3原液以得到细胞在N(+)ESAW介质中的悬浮液。另一批保持没有NaNO3以得到N(-)ESAW培养物,来作为用于高油脂累积的对照。在48孔透明板中,布置有1×106细胞/mL的450μL/孔。
对于剂量响应分析,随后利用50μL的下列物质,通过0μM、1μM、10μM或100μM的固醇代谢的抑制剂使48孔板的每个孔进行恰当的孵育:50μL的固醇代谢的抑制剂的10倍浓缩液(5%DMSO:95%N(+)ESAW);或50μL的富氮介质,没有任何的固醇代谢的抑制剂(5%DMSO:95%N(+)ESAW);或者氮缺乏介质,没有任何的固醇代谢的抑制剂(5%DMSO:95%N(-)ESAW),作为阳性对照。
对于单剂量效果的分析,利用0以及选定浓度的固醇代谢的抑制剂(50μM)进行孵育。
在所有的情况下,利用封口膜对板的边缘进行密封。在20℃、100rpm,12h/12h光/暗下,通过顶部照明在孵育器中使板孵育48小时。
在孵育48小时之后,在以下激发/发光波长处测量荧光:530/580nm(用于在尼罗红加入之前,估算基线荧光)和515/530nm(用于估算YFP荧光)。在该第一次测量之后,加入40μL的尼罗红(2.5μg/mL原液浓度,在100%DMSO中)。将这些板混合,并且在室温下在免受光下孵育20分钟。随后利用分光荧光计测量尼罗红荧光(激发530nm/发光580nm)。
1)6-利用固醇代谢的抑制剂孵育海洋富油微拟球藻Nannochloropsis gaditana,并且检测由该处理引起的油累积
在两个不同条件的海洋富油微拟球藻Nannochloropsis gaditana中进行该实验:细胞在含47mg.L-1NaNO3和3mg.L-1NaH2PO4的ESAW(介质1N1P)中孵育,或细胞在含470mg/LNaNO3和30mg.L-1NaH2PO4的ESAW(介质10N10P)中孵育。
通过以3500rpm离心10分钟来收集处于指数生长期的细胞。弃去上清,并且使细胞重新悬浮在相同体积的ESAW介质(1N1P或10N10P)中。再一次,使培养物以3500rpm离心10分钟,并且弃去上清。使团块重新悬浮在ESAW(1N1P或10N10P)中以得到2×106个细胞/mL的浓度。利用Malassez计数池对细胞进行计数,在计数前,停留10分钟以用于细胞沉降。
将20毫升的在ESAW(10N10P)和ESAW(1N1P)中的2×106个细胞/mL的海洋富油微拟球藻Nannochloropsis gaditana分配到无菌玻璃锥形瓶中。制备在DMSO中的固醇代谢的抑制剂的原液。将固醇代谢的抑制剂以终浓度为10μM、30μM或100μM加入到20mL的海洋富油微拟球藻Nannochloropsis gaditana样品中。样品中DMSO的最大终浓度为1%(v/v)。将所有的培养物在100rpm、12h/12h光/暗周期、50μE.m-2.s-1、20℃下孵育7天。
每一天,从每个烧瓶中取出一等份,以进行尼罗红染色和细胞计数。将160μL/每样品加入到黑色96孔板中,并且使其沉降10分钟。为了检测任何的背景噪声,在530nm激发处和580nm发光处分别测量荧光。将40μL的DMSO中的2.5μg.mL-1尼罗红加入到各个孔中,并且彻底混合。在孵育20分钟后,在530nm激发处和580nm发光处分别测量尼罗红荧光。
通过计算每百万细胞的相对荧光单位来使尼罗红荧光标准化。结果被表示为在完全介质(ESAW(10N10P)或ESAW(1N1P))中培养的海洋富油微拟球藻Nannochloropsisgaditana的尼罗红荧光的百分比。
1)7-固醇代谢的抑制剂
从Prestwick实验室中获知固醇代谢的抑制剂引起三角褐指藻Phaeodactylumtricornutum的细胞内油脂滴累积的能力,随后,从Sigma-Aldrich.购买该固醇代谢的抑制剂。
表6:选自Prestwick实验室的固醇代谢的抑制剂
2)结果
在10μM的美伐他汀、布替萘芬、辛伐他汀、雌素酮、乙炔雌二醇和特比萘芬的存在下,孵育三角褐指藻Phaeodactylum tricornutum 48h。
在所有的情况下,基于尼罗红染色,每个细胞中存在的油增加至少1.5倍。
图3a至图3h示出了通过用尼罗红染色对TAG含量进行估算,以及通过监控表达的组蛋白H4标记物基因的YFP荧光对细胞进行估算,对于乙炔雌二醇、美伐他汀和雌素酮的剂量响应。在左侧,图3a至图3h示出了以未处理细胞的百分率表示的尼罗红水平以及以未处理细胞的百分率表示的通过YFP荧光估算的细胞数。右侧示出了每个细胞的油含量的增加,以用未处理细胞的百分率表示的尼罗红荧光/YFP荧光的比表示。与未处理的细胞相比时,每个细胞油含量随着药物浓度和固醇代谢抑制的水平而增加,并且范围在120%至400%。
图4示出了通过用尼罗红染色对TAG含量进行估算,以及通过利用Malassez细胞以等份计算来对细胞进行估算,对于乙炔雌二醇(A)、美伐他汀(B)和辛伐他汀(C)的剂量响应。在图4的直方图中,白色柱表示以未处理细胞的百分率表示的估算的细胞数,并且黑色柱表示以未处理细胞的百分率表示的每106个细胞的尼罗红。当与未处理的细胞相比时,每个细胞的油含量以及因此固醇代谢抑制的水平由于在50μM抑制剂下孵育而增加,并且范围在120%至350%。
在布替萘芬和乙炔雌二醇的存在下,孵育海洋富油微拟球藻Nannochloropsisgaditana。
图5和图6示出了布替萘芬和乙炔雌二醇对分别在ESAW 1N1P介质和ESAW 10N10P介质中生长的海洋富油微拟球藻Nannochloropsis gaditana中尼罗红累积的影响。通过计算每百万细胞中相对荧光单位来对尼罗红荧光性进行标准化。
在不同介质中以及能够观察的时段(至少7天)中,布替萘芬和乙炔雌二醇均能够引起海洋富油微拟球藻Nannochloropsis gaditana中油的累积。
Claims (15)
1.一种通过抑制固醇代谢引起微藻内三酰基甘油累积的方法,所述抑制固醇代谢通过用固醇代谢的抑制剂孵育微藻来进行;
所述固醇代谢的抑制剂为下式之一的化合物或其盐:
其中:
R41和R42,相同或不同,表示氢原子、烷基、烯基、炔基,或羟基、-COR4a或-COOR4a基团,其中,R4a表示氢原子,未取代的或被独立选自烷基或环烷基的一个或多个基团取代的直链或支链的烷基,未取代的或被独立选自烷基或环烷基的一个或多个基团取代的芳基,未取代的或被独立选自烷基或环烷基的一个或多个基团取代的杂芳基;或者R41和R42一起形成通过双键连接的氧原子;
R43表示氢原子或烷基;以及
R44、R45和R46,相同或不同,表示氢原子,烷基、在链中含有一个或多个亚砜官能团的烷基、烷氧基、羟基或通过双键连接的氧原子,所述烷基是未取代的或者被一个或多个卤原子取代;
其中:
W3、X3、Y3和Z3表示碳原子、硫原子、氮原子或氧原子;
n3和n3'独立地为等于0或1的整数;
R31和R32,相同或不同,表示氢原子、直链或支链的烷基、烯基、炔基、烷基烯基、炔基烯基、环烷基、烷基芳基、芳基烷基、芳基烯基、芳基炔基、杂烷基、杂芳基;或者R31和R32一起形成包括5至6个碳原子的环烷基或环烷基中的一个或两个碳原子被一个或两个杂原子替代的包括5至6个碳原子的环烷基;或者R31和R32之一与R39一起形成包括5至6个碳原子的环烷基或环烷基的一个或两个碳原子被一个或两个杂原子替代的包括5至6个碳原子的环烷基,所述R31或R32是未取代的或者被独立选自以下基团的一个或多个基团取代:直链或支链的烷基,环烷基,炔基,芳基烷基,芳基,杂芳基,羟基,卤素,硝基,-COR3a或-NR3aR3b,其中,R3a和R3b相同或不同,表示氢原子,或直链或支链的烷基链;
R33表示氢原子、直链或支链的烷基链,或者腈基;
R34、R35、R36、R37、R38和R39,相同或不同,表示氢原子,或者卤原子,或者羟基;
其中:
n1为1至12,
R11表示氢原子;-COR1a基团,其中,R1a为选自直链或支链的烷基链,取代的或未取代的苄基,或取代的或未取代的芳基,所述直链或支链的烷基链是未取代的或者被独立选自羟基、卤素、硝基的一个或多个基团取代,所述芳基最终被独立选自卤原子和甲基的一个或多个基团取代;
R12、R13和R14,相同或不同,表示氢原子;直链或支链的烷基链;羟基;-CH2OH基团;-COOR1b基团,其中,R1b表示氢原子,或直链或支链的烷基链;-OSiR1cR1dR1e基团,其中,R1c、R1d和R1e,相同或不同,表示氢原子,或直链或支链的烷基链;或者R12和R13一起形成环外亚甲基;
R15表示羟基;-OSiR1cR1dR1e基团;-COOR1f基团,其中R1f表示氢原子,或直链或支链的烷基链;-OCOR1g基团,其中R1g表示直链或支链的烷基链;或R15为与四氢吡喃酮环形成烯属不饱和的碳。
2.根据权利要求1所述的方法,其中,所述孵育的步骤在含氮介质中进行。
3.根据权利要求1或2所述的方法,其中,所述微藻选自硅藻门的微藻、囊泡藻界门的微藻以及原始色素体生物门的微藻。
4.根据权利要求3所述的方法,其中,所述微藻选自:硅藻微藻种的三角褐指藻和假微型海链藻;囊泡藻界微藻种的微拟球藻;以及原始色素体生物微藻种的衣藻、绿藻、小球藻。
5.根据权利要求4所述的方法,其中,所述微藻选自海洋富油微拟球藻、海洋微拟球藻、盐生微拟球藻。
6.根据权利要求1所述的方法,其中,n3和n3'等于1。
7.根据权利要求1所述的方法,其中,R11表示-COR1a基团,其中,R1a为选自直链或支链的烷基链,取代或未取代的苄基,或取代或未取代的苯基,所述直链或支链的烷基链是未取代的或者被独立选自羟基、卤素、硝基的一个或多个基团取代,所述取代的苯基被独立选自卤原子和甲基的一个或多个基团取代。
9.根据权利要求1所述的方法,其中,所述固醇代谢的抑制剂选自乙炔雌二醇、雌素酮、布替萘芬、美伐他汀、辛伐他汀、特比萘芬。
10.一种用于生产脂肪酸的方法,所述方法包括:根据权利要求1所述的引起微藻内三酰基甘油累积的步骤。
11.一种用于生产生物燃料的方法,所述方法包括以下步骤:
(i)根据权利要求1所述的引起微藻内三酰基甘油累积的步骤;随后
(ii)对步骤(i)中在微藻内累积的三酰基甘油进行提取的步骤;以及
(iii)对步骤(ii)中回收的三酰基甘油进行酯交换的步骤。
12.一种用于生产药物组合物或化妆品组合物的方法,所述方法包括以下步骤:
(i’)根据权利要求1所述的引起微藻内三酰基甘油累积的步骤;随后
(ii’)对在步骤(i’)中在微藻内累积的三酰基甘油进行提取的步骤;以及
(iii’)将至少一种药学上可接受的赋形剂或化妆品上可接受的赋形剂加入到步骤(ii’)中回收的三酰基甘油中的步骤。
13.一种用于生产食品补充剂的方法,所述方法包括以下步骤:
(i”)根据权利要求1所述的引起微藻内三酰基甘油累积的步骤;随后
(ii”)对在步骤(i”)中在微藻内累积的三酰基甘油进行提取的步骤;以及
(iii”)将至少一种食品添加剂加入到步骤(ii”)中回收的三酰基甘油中的步骤。
14.固醇代谢的抑制剂在微藻内使甘油三酯累积的应用,所述固醇代谢的抑制剂选自如权利要求1中所限定的式(I)、式(II)和式(III)的化合物。
15.根据权利要求14所述的应用,其中,所述固醇代谢的抑制剂选自乙炔雌二醇、雌素酮、布替萘芬、美伐他汀、辛伐他汀、特比萘芬或它们的组合。
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modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae;Yu et al;《microbial cell factories biomed central,London,NL》;20111102;第10卷(第1期);全文 * |
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