CN114891715B - 一种提高产油微生物合成油脂能力的方法 - Google Patents
一种提高产油微生物合成油脂能力的方法 Download PDFInfo
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- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N1/005—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor after treatment of microbial biomass not covered by C12N1/02 - C12N1/08
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Abstract
本发明提供一种提高产油微生物合成油脂能力的方法,属于生物工程技术领域。本发明通过将半导体纳米颗粒分散在产油微生物的表面,构建无机‑产油微生物杂合系统,使其在光照条件下,能够吸收光能,产生的电子进入产油微生物细胞质,提供外源电子促进还原力NADPH的形成,提高油脂的合成能力。这种方法简单,易操作,不需要通过基因工程操作即可实现产油微生物菌株胞内NADPH含量提升,进而提高产油微生物合成油脂的能力,是一种简单易实现的提高产油微生物合成油脂能力的方法。
Description
技术领域
本发明属于生物工程技术领域,涉及一种提高产油微生物合成油脂能力的方法。
背景技术
微生物油脂(Microbial Oils)又称为单细胞油脂(Single Cell Oils,SCO),是指由某些霉菌、酵母、细菌和微藻等在一定的培养条件下,利用碳源在菌体内合成并积累生成的油脂。利用微生物生产油脂具有很多优点:原料来源丰富、微生物易于培养、生产过程环境友好、产品生理功能突出、能连续大规模生产等。
微生物积累的油脂主要成分为甘油三酯,其脂肪酸组成大多与动植物来源的油脂相似,因此可以替代动植物油脂产生生物柴油,从而缓解能源危机(赵宗保.加快微生物油脂研究为生物柴油产业提供廉价原料.中国生物工程杂志,2005,25(2):8-11)。某些产油微生物还可以积累富含γ-亚麻酸(γ-Linolenic Acid,GLA)、花生四烯酸(ArachidonicAcid,ARA)、二十碳五烯酸(Eicosapentaenoic Acid,EPA)、二十二碳六烯酸(Docosahexaenoic Acid,DHA)等多不饱和脂肪酸的甘油三酯,这些多不饱和脂肪酸因具有特殊的生理功能而成为人类重要的营养元素(Ji XJ,Ledesma-Amaro R.Microbial LipidBiotechnology to Produce Polyunsaturated Fatty Acids.Trends inBiotechnology.2020;38(8):832-834)。因此,微生物油脂被誉为“生命绿洲中的新油田”和“人类营养素的新资源”。为此,微生物油脂作为可再生绿色能源和功能性营养化学品的主要原料,其开发与利用多年来一直收到人们的关注。而作为这类产品生产的重要环节——微生物合成油脂过程是一直是研究的焦点。
英国Hull大学Colin Ratledge教授对产油微生物(Oleaginous Microorganisms)进行了如下定义:在适宜条件下这些微生物能将碳水化合物等碳源转化为菌体内大量贮存的油脂,并获得油脂超过细胞总量的20%(Ratledge C,Wynn JP.The biochemistry andmolecular biology of lipid accumulation in oleaginous microorganisms.Advancesin Applied Microbiology,2002,51:1-52)。研究证实,产油微生物之所以具有产油脂的表型特征,是由于其在氮限制(C/N比较高)条件下具有独特的生化代谢途径导致的。在氮限制条件下,发生在线粒体的TCA循环中的柠檬酸穿梭到细胞质,在柠檬酸裂解酶(ACL)催化下发生裂解反应,生成乙酰辅酶A和草酰乙酸;随后,草酰乙酸由苹果酸脱氢酶(MDH)还原成苹果酸,再在苹果酸酶(ME)的作用下氧化脱羧得到丙酮酸,同时释放出还原型辅酶Ⅱ(NADPH)。其中丙酮酸可透过线粒体膜返回线粒体,参与新一轮循环。而NADPH和上述步骤获得的乙酰辅酶A则作为辅助因子留在细胞质中,在脂肪酸合酶(FAS)的催化下进行链延伸参与脂肪酸的合成,脂肪酸碳链延伸到一定长度后积累到甘油三酯中,最终形成油脂(Ji XJ,Ren LJ,Nie ZK,Huang H,Ouyang PK.Fungal arachidonic acid-rich oil:research,development and industrialization.Critical Reviews in Biotechnology.2014;34(3):197-214)。在油脂合成过程中,脂肪酸的合成是关键步骤,该过程不仅需要连续供给乙酰辅酶A用于碳链延伸,还需要提供足够的NADPH。每一分子乙酰辅酶A单元在新生脂肪酸碳链上延伸时需消耗两当量的NADPH用于还原反应;因此,NADPH的供给成为制约微生物油脂合成效率的关键因素(Ji XJ,Huang H.Engineering Microbes to ProducePolyunsaturated Fatty Acids.Trends in Biotechnology.2019;37(4):344-346)。
能够将碳源转化为油脂是产油微生物的主要表型特征,而高效利用碳水化合物合成油脂是制约这一过程商业化的关键。以葡萄糖为例,以其为底物,产油微生物合成油脂的理论得率仅为0.32g油脂/g葡萄糖(Papanikolaou S,Aggelis G.Lipids of oleaginousyeasts.Part I:Biochemistry of single cell oil production.European Journal ofLipid Science and Technology.2011;113(8):1031-1051)。造成这一结果的直接原因在于产油微生物胞内的还原力NADPH供应不足,导致与充足的乙酰辅酶A前体来源无法匹配。在现有报道的研究中,为了提高产油微生物的油脂生产能力,提高底物转化率,一般通过基因工程强化能够合成NADPH的磷酸戊糖途径(Hao,G.et al.Metabolic engineering ofMortierella alpina for enhanced arachidonic acid production through theNADPH-supplying strategy.Appl.Environ.Microbiol.2016,82,3280–3288);或者通过引入外源基因将胞内充足的还原性辅酶I(NADH)转化为NADPH(Qiao K,Wasylenko TM,ZhouK,Xu P,Stephanopoulos G.Lipid production in Yarrowia lipolytica is maximizedby engineering cytosolic redox metabolism.Nature Biotechnology.2017;35(2):173-177)。上述方法,在一定程度上能够提高NADPH的供给从而促进产油微生物积累油脂,但这种方法并不适用于所有的产油微生物:一方面,对终端产品是面向人类食用的油脂而言,这种通过转基因获得的产品将会饱受大众的质疑与诟病;另一方面,大多数的产油微生物,属于基因背景不清晰的非模式菌株,基因工程改造工具不健全,这给通过类似的方法实现预期目标带来了难度。
因此,开发一种适用于所有产油微生物且不涉及转基因操作的方法,来有效提高产油微生物菌株胞内的NADPH的含量,以解决制约提高产油微生物合成油脂能力的问题是十分必要的。
发明内容
本发明的目的是提供一种不通过基因工程方法实现产油微生物菌株胞内NADPH含量提升,从而提高产油微生物合成油脂能力的方法。
为了实现上述目的,本发明是采用如下技术方案实现的:
本发明提供一种利用无机纳米材料来构建无机-产油微生物杂合系统,通过构建的杂合系统吸收光能给产油微生物提供外源电子以促进油脂合成。在该杂合系统中,无机纳米材料被组装在产油微生物的表面,通过施加光照,无机纳米材料产生的电子能够导入细胞质中并参与产油微生物的油脂合成代谢。该杂合系统对光的利用分为两个步骤,首先是无机纳米材料将从光源中所吸收的能量转化成电子,这些电子进入细胞质后能促进还原力NADPH的形成,从而促进了油脂的生物合成。如此,我们可以将无机纳米材料与产油微生物结合,显著提高产油微生物细胞内的NADPH水平,有益于以NADPH为重要合成因子的油脂的积累。
所选用的无机纳米材料为半导体纳米颗粒CdS(硫化镉)、InP(磷化铟)、ZnS(硫化锌)、CdSe(硒化镉)中的一种或几种。
在构建无机-产油微生物杂合系统时,通过纳米模块化的组装方式,将上述半导体纳米颗粒,组装在产油微生物表面。组装过程中,采用对微生物细胞具有高度生物相容性的天然多酚修饰半导体纳米颗粒,再按照一定比例与经过表面修饰的产油微生物进行掺杂,在此技术条件下,天然多酚修饰半导体纳米颗粒具有较低的细胞毒性,表面修饰保证了微生物细胞与细胞之间相互绝缘。
本发明中使用的产油微生物为经发酵培养后菌体油脂含量可超过细胞干重20%的细菌、酵母、真菌或微藻。它们包括但不限于:产油细菌,如Rhodococcus opacus、Nocardia sp.、Gordonia sp.,Acinetobacter sp.;产油酵母,如Yarrowia lipolytica、Rhodosporidium toruloides、Cutaneotrichosporon oleaginosus、Rhodotorulaglutinis、Lipomyces starkeyi、Trichosporon cutaneum;产油真菌Mortierella alpina、Mortierella isabellina、Mucor circinelloides、Cunninghamella elegans;产油微藻,如Schizochytrium limacinum、Crypthecodinium cohnii、Nannochloropsis oculata、Ulkenia sp.。
本发明中所述具有高度生物相容性的天然多酚为来源于植物的具有多元酚结构的次生代谢物,例如单宁酸、白藜芦醇、绿原酸、茶多酚。
所述半导体纳米颗粒分散在产油微生物表面。
所述半导体纳米材料颗粒的直径小于300nm。
产油微生物细胞表面修饰是通过以下步骤实现的:
将对数生长期的产油微生物细胞,离心洗去培养基后,用水重悬;将聚烯丙基胺溶液加入微生物细胞悬液中(微生物细胞悬液的吸光值OD600=2.0-4.0,获得细胞表面经过修饰,能够进行与多酚功能化的半导体纳米颗粒组装的产油微生物。
产油微生物细胞与聚烯丙基胺溶液的用量比例关系为1:2-1:3(v/v)。
所述天然多酚修饰半导体纳米颗粒是通过如下步骤实现的:
S1、将半导体纳米颗粒悬浮在装有MQ水的Eppendorf管中,将FeCl3·6H2O溶液和单宁酸溶液依次加入到半导体纳米颗粒悬浮液中,并加入Tris缓冲液(pH8.0,100mM),提高溶液pH至8.0;
S2、将上述获得的多酚功能化的半导体纳米颗粒先用MQ水洗涤3-4次,然后用70%乙醇10-15min洗涤孵育,最后用MQ水洗涤3-4次。在洗涤过程中,通过离心(8000rpm,5-10min)去除上清,随后用超声法分散悬浮液中的颗粒。
所述半导体纳米颗粒、FeCl3·6H2O、天然多酚三种用量关系范围为1:2:1(v/v)。
半导体纳米颗粒悬浮液浓度为0.5-5%,w/v;FeCl3·6H2O溶液浓度为2-6mg/mL,单宁酸溶液浓度为20-80mg/mL;
所述无机-产油微生物杂合系统的构建方法是通过如下步骤实现的:将产油微生物细胞和半导体纳米颗粒进行混合,使其进行自组装,震荡10-60秒,通过添加额外的金属离子Fe3+和等体积的PBS缓冲溶液实现颗粒在细胞上的稳定组装获得了无机-产油微生物杂合体。
产油微生物细胞和半导体纳米颗粒二者的用量关系范围0.5:1-1:1(V/V)。
具体是通过以下几个步骤实现的:
1)半导体纳米颗粒的制备;
2)天然多酚修饰半导体纳米颗粒;
3)产油微生物细胞表面修饰;
4)无机-产油微生物杂合体的构建。
半导体纳米颗粒的制备
半导体纳米材料通过研磨获得纳米颗粒:取2.0-4.0g所述半导体纳米材料,研磨30-60min获得粉末;将获得的半导体材料粉末转移到5mL Eppendorf管中,用1.5mL MQ水悬浮研磨后的半导体材料粉末,并应用超声处理分散颗粒;获得的半导体材料颗粒在8000-10000rpm下离心5-10min。尺寸较大的颗粒留在底部,而尺寸较小的颗粒附着在管壁上;丢弃底部颗粒,将附着在管壁上的颗粒在MQ水中重悬;重复2-3次离心分离过程,获得直径小于300nm的半导体纳米颗粒。
天然多酚修饰半导体纳米颗粒
将半导体纳米颗粒(直径小于300nm)悬浮在装有2.0mL MQ水的5mL Eppendorf管中(控制其浓度在0.5-5%,w/v);将FeCl3·6H2O溶液(2-6mg/mL)和单宁酸溶液(20-80mg/mL)各0.5-0.8mL依次加入到半导体纳米颗粒悬浮液中。加入Tris缓冲液(pH8.0,100mM),提高溶液pH至8.0。将上述获得的多酚功能化的半导体纳米颗粒先用MQ水洗涤3-4次,然后用70%乙醇10-15min洗涤孵育,最后用MQ水洗涤3-4次。在洗涤过程中,通过离心(8000rpm,5-10min)去除上清,随后用超声法分散悬浮液中的颗粒。
产油微生物细胞表面修饰
取3-5mL对数生长期的产油微生物细胞,经离心后,用MQ水洗涤3-5次以去除培养基,用500μL MQ水重悬。在洗涤过程中,离心转速2000-4000rpm,时间2-4min。然后,将40-100μL聚烯丙基胺(PAH)溶液(3-8mg/mL)加入400-600μL微生物细胞悬液中(OD600=2.0-4.0);用MQ水洗涤细胞3-5次,以去除多余的多环芳烃分子,在洗涤过程中,将细胞在2000-4000rpm下离心2-5min。最后将微生物细胞重悬于500μLMQ水中,获得细胞表面经过修饰,能够进行与多酚功能化的半导体纳米颗粒组装的产油微生物。
无机-产油微生物杂合体的构建
为了构建无机-产油微生物杂合体,产油微生物细胞和半导体纳米颗粒的OD600分别为1.0-2.0和0.5-1.0,二者的OD600的比值控制在1.5-2.0。无机纳米颗粒和微生物细胞数量的比例因不同的纳米颗粒和微生物细胞类型的不同而不同。值得注意的是,虽然微生物细胞的OD600高于纳米颗粒,但由于大小的差异,纳米颗粒的实际数量远远高于微生物细胞。在组装过程中,混合悬浮液被涡旋震荡10-60秒,以促进纳米颗粒与微生物细胞之间的碰撞。然后,通过添加额外的金属离子Fe3+(最终浓度为0.03-0.05mg/mL)和等体积的PBS缓冲溶液(pH 8.0,10mM)实现颗粒在细胞上的稳定组装。用MQ水洗涤3-5次以去除游离的半导体纳米颗粒(2000rpm,2-5min离心)后,获得了无机-产油微生物杂合体。
光照来源为外源施加的LED光源,光照时期为无机-产油微生物杂合体接入发酵培养基时开始。
本发明的有益效果:
本发明构建的无机-产油微生物杂合系统,能够吸收光能给产油微生物提供外源电子促进油脂的合成。在该杂合系统中,半导体纳米颗粒分散在产油微生物的表面,通过施加光照后,半导体纳米颗粒产生的电子能够进入细胞质,促进还原力NADPH的形成,提高产油微生物的油脂合成能力。这种方法简单,易操作,不需要通过基因工程操作即可实现产油微生物菌株胞内NADPH含量提升,进而提高产油微生物合成油脂的能力,且对所有的产油微生物具有普适性,是一种简单易实现的提高微生物合成油脂能力的方法。
具体实施方式
下面的实施例对本发明作详细说明,但对本发明没有限制。
实施例1:无机-产油细菌杂合体的构建及其与出发菌株产油脂能力比较
1)产油菌种
采用的菌株为产油细菌-浑浊红球菌Rhodococcus opacus DSMZ 44193(购自德国微生物菌种保藏中心)
2)硫化镉CdS半导体纳米颗粒的制备
所采用的半导体纳米材料为硫化镉CdS(购自Sigma-Aldrich),通过人工研磨获得纳米颗粒:取3.0g上述CdS,研磨40min获得粉末;将获得的CdS粉末转移到5mL Eppendorf管中,用1.5mL MQ水悬浮研磨后的半导体材料粉末,并应用超声处理分散颗粒。获得的CdS颗粒在8000rpm下离心6min。尺寸较大的CdS颗粒留在底部,而尺寸较小的CdS颗粒附着在管壁上;丢弃底部CdS颗粒,将附着在管壁上的CdS颗粒在MQ水中重悬。为了获得直径小于300nm的CdS颗粒,重复2次离心分离过程。
3)硫化镉CdS半导体纳米颗粒功能化
这里所有溶液均为新鲜配制,并通过0.2μm孔径滤膜过滤后立即使用。制备工艺如下:首先将上一步骤获得的CdS半导体纳米颗粒悬浮在装有2.0mL MQ水的5mL Eppendorf管中(控制其浓度在2%,w/v;将FeCl3·6H2O溶液(5mg/mL)和单宁酸溶液(40mg/mL)各0.5mL依次加入到CdS半导体纳米颗粒悬浮液中。加入Tris缓冲液(pH8.0,100mM),提高溶液pH至8.0。将上述获得的多酚功能化的CdS半导体纳米颗粒先用MQ水洗涤3次,然后用70%乙醇10min洗涤孵育,最后用MQ水洗涤3次。在洗涤过程中,通过离心(8000rpm,6min)去除上清,随后用超声法分散悬浮液中的CdS半导体纳米颗粒。
4)浑浊红球菌细胞表面修饰
取3mL对数生长期的浑浊红球菌细胞,经离心后,用MQ水洗涤3次以去除培养基,用500μL MQ水重悬。在洗涤过程中,离心转速3000rpm,时间2min。然后,将50μL聚烯丙基胺(PAH)溶液(5mg/mL)加入450μL微生物细胞悬液中(OD600=3.0);用MQ水洗涤细胞3次,以去除多余的多环芳烃分子,在洗涤过程中,将细胞在3000rpm下离心3min。最后将微生物细胞重悬于500μL MQ水中,获得的浑浊红球菌细胞表面经过修饰,能够进行与多酚功能化的CdS半导体纳米颗粒组装。
5)硫化镉CdS-浑浊红球菌杂合体的构建
为了构建硫化镉CdS-浑浊红球菌杂合体,浑浊红球菌细胞和CdS半导体纳米颗粒的OD600分别为1.0和0.5。在组装过程中,混合悬浮液被涡旋震荡30秒,以促进CdS半导体纳米颗粒与浑浊红球菌细胞之间的碰撞。然后,通过添加额外的金属离子Fe3+(最终浓度为0.03mg/mL,和等体积的PBS缓冲溶液(pH 8.0,10mM)实现CdS半导体纳米颗粒在浑浊红球菌细胞上的稳定组装。用MQ水洗涤3次去除游离的CdS半导体纳米颗粒(2000rpm,3min离心)后,获得了硫化镉CdS-浑浊红球菌杂合体。
6)发酵培养
培养基:40g/L葡萄糖,3.3088g/L KH2PO4,7.9552/Lg K2HPO4,14.2g/L(NH4)2SO4,2g/L MgSO47H2O,2.86mg/L H3BO4,15mg/L CaCl2,1ml/L储液体(2g/L NaMoO42H2O,5g/LFeNaEDTA),1ml/L 微量元素(0.5g/L FeSO47H2O,0.4g/L ZnSO4H2O,0.02g/LMnSO4H2O,0.01g/L NiCl26H20,0.05g/L CuSO45H2O,0.01g/L MnCl2,0.05g/LCoCl26H2O)。
将上述构建好的硫化镉CdS-浑浊红球菌杂合体作为种子接种至150mL三角瓶的上述培养基中(接种量0.5%(v/v))(以游离的浑浊红球菌以相同接种量接种至另一个三角瓶中作为对照),接种完备,即开始施加LED光照(光照强度400lux),监测这一过程中浑浊红球菌产油脂情况,如表1所示,可以发现CdS-浑浊红球菌杂合体由于CdS的引入,增强了油脂合成能力。
表1
实施例2:无机-产油酵母杂合体的构建及其与出发菌株产油脂能力比较
1)产油菌种
采用的菌株为产油酵母-解脂耶氏酵母Yarrowia lipolytica ATCC 20362(购自美国菌种保藏中心)
2)磷化铟InP半导体纳米颗粒的制备
所采用的半导体纳米材料为磷化铟InP(购自Sigma-Aldrich),通过人工研磨获得纳米颗粒:取3.0g上述InP,研磨40min获得粉末;将获得的InP粉末转移到5mL Eppendorf管中,用1.5mL MQ水悬浮研磨后的半导体材料粉末,并应用超声处理分散颗粒。获得的InP颗粒在8000rpm下离心6min。尺寸较大的InP颗粒留在底部,而尺寸较小的InP颗粒附着在管壁上;丢弃底部InP颗粒,将附着在管壁上的InP颗粒在MQ水中重悬。为了获得直径小于300nm的InP颗粒,重复2次离心分离过程。
3)磷化铟InP半导体纳米颗粒功能化
这里所有溶液均为新鲜配制,并通过0.2μm孔径滤膜过滤后立即使用。制备工艺如下:首先将上一步骤获得的InP半导体纳米颗粒悬浮在装有2.0mL MQ水的5mL Eppendorf管中(控制其浓度在0.5-5%,w/v);将FeCl3·6H2O溶液(5mg/mL)和单宁酸溶液(40mg/mL)各0.5mL依次加入到InP半导体纳米颗粒悬浮液中。加入Tris缓冲液(pH8.0,100mM),提高溶液pH。将上述获得的多酚功能化的InP半导体纳米颗粒先用MQ水洗涤3次,然后用70%乙醇10min洗涤孵育,最后用MQ水洗涤3次。在洗涤过程中,通过离心(8000rpm,6min)去除上清,随后用超声法分散悬浮液中的InP半导体纳米颗粒。
4)解脂耶氏酵母细胞表面修饰
取3mL对数生长期的解脂耶氏酵母细胞,经离心后,用MQ水洗涤3次以去除培养基,用500μL MQ水重悬。在洗涤过程中,离心转速3000rpm,时间2min。然后,将50μL聚烯丙基胺(PAH)溶液(5mg/mL)加入450μL微生物细胞悬液中(OD600=3.0);用MQ水洗涤细胞3次,以去除多余的多环芳烃分子,在洗涤过程中,将细胞在3000rpm下离心3min。最后将微生物细胞重悬于500μL MQ水中,获得的解脂耶氏酵母细胞表面经过修饰,能够进行与多酚功能化的InP半导体纳米颗粒组装。
5)磷化铟InP-解脂耶氏酵母杂合体的构建
为了构建磷化铟InP-解脂耶氏酵母杂合体,解脂耶氏酵母细胞和InP半导体纳米颗粒的OD600分别为1.0和0.5。在组装过程中,混合悬浮液被涡旋震荡30秒,以促进InP半导体纳米颗粒与解脂耶氏酵母细胞之间的碰撞。然后,通过添加额外的金属离子Fe3+(最终浓度为0.03mg/mL)和等体积的PBS缓冲溶液(pH 8.0,10mM)实现InP半导体纳米颗粒在解脂耶氏酵母细胞上的稳定组装。用MQ水洗涤3次去除游离的InP半导体纳米颗粒(2000rpm,3min离心)后,获得了磷化铟InP-解脂耶氏酵母杂合体。
6)发酵培养培养基:20g/L蛋白胨,10g/L酵母膏,6g/L KH2PO4,2g/L K2HPO4,1.5g/L MgSO4.7H2O,50g/L葡萄糖。
将上述构建好的磷化铟InP-解脂耶氏酵母杂合体作为种子接种至150mL三角瓶的上述培养基中(接种量0.5%(v/v))(以游离的解脂耶氏酵母以相同接种量接种至另一个三角瓶中作为对照),接种完备,即开始施加LED光照(光照强度400lux),监测这一过程中解脂耶氏酵母产油脂情况,如下表2所示,可以发现磷化铟InP-解脂耶氏酵母杂合体由于InP的引入,增强了油脂合成能力。
表2
实施例3:无机-产油真菌杂合体的构建及其与出发菌株产油脂能力比较
1)产油菌种
采用的菌株为产油真菌-高山被孢霉Mortierella alpina ATCC 32222(购自美国菌种保藏中心)
2)硫化锌ZnS半导体纳米颗粒的制备
所采用的半导体纳米材料为硫化锌ZnS(购自Sigma-Aldrich),通过人工研磨获得纳米颗粒:取3.0g上述ZnS,研磨40min获得粉末;将获得的ZnS粉末转移到5mL Eppendorf管中,用1.5mL MQ水悬浮研磨后的半导体材料粉末,并应用超声处理分散颗粒。获得的ZnS颗粒在8000rpm下离心6min。尺寸较大的ZnS颗粒留在底部,而尺寸较小的ZnS颗粒附着在管壁上;丢弃底部ZnS颗粒,将附着在管壁上的ZnS颗粒在MQ水中重悬。为了获得直径小于300nm的ZnS颗粒,重复2次离心分离过程。
3)硫化锌ZnS半导体纳米颗粒功能化
这里所有溶液均为新鲜配制,并通过0.2μm孔径滤膜过滤后立即使用。制备工艺如下:首先将上一步骤获得的ZnS半导体纳米颗粒悬浮在装有2.0mL MQ水的5mL Eppendorf管中(控制其浓度在0.5-5%,w/v);将FeCl3·6H2O溶液(5mg/mL)和单宁酸溶液(40mg/mL)各0.5mL依次加入到ZnS半导体纳米颗粒悬浮液中。加入Tris缓冲液(pH8.0,100mM),提高溶液pH。将上述获得的多酚功能化的ZnS半导体纳米颗粒先用MQ水洗涤3次,然后用70%乙醇10min洗涤孵育,最后用MQ水洗涤3次。在洗涤过程中,通过离心(8000rpm,6min)去除上清,随后用超声法分散悬浮液中的ZnS半导体纳米颗粒。
4)高山被孢霉细胞表面修饰
取3mL对数生长期的高山被孢霉细胞,经离心后,用MQ水洗涤3次以去除培养基,用500μL MQ水重悬。在洗涤过程中,离心转速3000rpm,时间2min。然后,将50μL聚烯丙基胺(PAH)溶液(5mg/mL)加入450μL微生物细胞悬液中(OD600=3.0);用MQ水洗涤细胞3次,以去除多余的多环芳烃分子,在洗涤过程中,将细胞在3000rpm下离心3min。最后将微生物细胞重悬于500μL MQ水中,获得的高山被孢霉细胞表面经过修饰,能够进行与多酚功能化的ZnS半导体纳米颗粒组装。
5)硫化锌ZnS-高山被孢霉杂合体的构建
为了构建硫化锌ZnS-高山被孢霉杂合体,高山被孢霉细胞和ZnS半导体纳米颗粒的OD600分别为1.0和0.5。在组装过程中,混合悬浮液被涡旋震荡30秒,以促进ZnS半导体纳米颗粒与高山被孢霉细胞之间的碰撞。然后,通过添加额外的金属离子Fe3+(最终浓度为0.03mg/mL)和等体积的PBS缓冲溶液(pH 8.0,10mM)实现ZnS半导体纳米颗粒在高山被孢霉细胞上的稳定组装。用MQ水洗涤3次去除游离的ZnS半导体纳米颗粒(2000rpm,3min离心)后,获得了硫化锌ZnS-高山被孢霉杂合体。
6)发酵培养培养基:80g/L葡萄糖,10g/L酵母膏,4g/L KH2PO4,2g/L K2HPO4,3g/LNaNO3,0.6g/L MgSO4.7H2O
将上述构建好的硫化锌ZnS-高山被孢霉杂合体作为种子接种至150mL三角瓶的上述培养基中(接种量2%(v/v))(以游离的高山被孢霉以相同接种量接种至另一个三角瓶中作为对照),接种完备,即开始施加施加LED光照(光照强度400lux),监测这一过程中高山被孢霉产油脂情况,如下表3所示,可以发现ZnS-高山被孢霉杂合体由于ZnS的引入,增强了油脂合成能力。
表3
实施例4:无机-产油微藻杂合体的构建及其与出发菌株产油脂能力比较
1)产油菌种
采用的菌株为产油微藻-裂殖壶菌Schizochytrium limacinum SR21(购自日本大阪发酵研究所)
2)硒化镉CdSe半导体纳米颗粒的制备
所采用的半导体纳米材料为硒化镉CdSe(购自Sigma-Aldrich),通过人工研磨获得纳米颗粒:取3.0g上述CdSe,研磨40min获得粉末;将获得的CdSe粉末转移到5mLEppendorf管中,用1.5mL MQ水悬浮研磨后的半导体材料粉末,并应用超声处理分散颗粒。获得的CdSe颗粒在8000rpm下离心6min。尺寸较大的CdSe颗粒留在底部,而尺寸较小的CdSe颗粒附着在管壁上;丢弃底部CdSe颗粒,将附着在管壁上的CdSe颗粒在MQ水中重悬。为了获得直径小于300nm的CdSe颗粒,重复2次离心分离过程。
3)硒化镉CdSe半导体纳米颗粒功能化
这里所有溶液均为新鲜配制,并通过0.2μm孔径滤膜过滤后立即使用。制备工艺如下:首先将上一步骤获得的CdSe半导体纳米颗粒悬浮在装有2.0mL MQ水的5mL Eppendorf管中(控制其浓度在0.5-5%,w/v);将FeCl3·6H2O溶液(5mg/mL)和单宁酸溶液(40mg/mL)各0.5mL依次加入到CdSe半导体纳米颗粒悬浮液中。加入Tris缓冲液(pH8.0,100mM),提高溶液pH。将上述获得的多酚功能化的CdSe半导体纳米颗粒先用MQ水洗涤3次,然后用70%乙醇10min洗涤孵育,最后用MQ水洗涤3次。在洗涤过程中,通过离心(8000rpm,6min)去除上清,随后用超声法分散悬浮液中的CdSe半导体纳米颗粒。
4)裂殖壶菌细胞表面修饰
取3mL对数生长期的裂殖壶菌细胞,经离心后,用MQ水洗涤3次以去除培养基,用500μL MQ水重悬。在洗涤过程中,离心转速3000rpm,时间2min。然后,将50μL聚烯丙基胺(PAH)溶液(5mg/mL)加入450μL微生物细胞悬液中(OD600=3.0);用MQ水洗涤细胞3次,以去除多余的多环芳烃分子,在洗涤过程中,将细胞在3000rpm下离心3min。最后将微生物细胞重悬于500μL MQ水中,获得的裂殖壶菌细胞表面经过修饰,能够进行与多酚功能化的CdSe半导体纳米颗粒组装。
5)硒化镉CdSe-裂殖壶菌杂合体的构建
为了构建硒化镉CdSe-裂殖壶菌杂合体,裂殖壶菌细胞和CdSe半导体纳米颗粒的OD600分别为1.0和0.5。在组装过程中,混合悬浮液被涡旋震荡30秒,以促进CdSe半导体纳米颗粒与裂殖壶菌细胞之间的碰撞。然后,通过添加额外的金属离子Fe3+(最终浓度为0.03mg/mL)和等体积的PBS缓冲溶液(pH 8.0,10mM)实现CdSe半导体纳米颗粒在裂殖壶菌细胞上的稳定组装。用MQ水洗涤3次去除游离的CdSe半导体纳米颗粒(2000rpm,3min离心)后,获得了硒化镉CdSe-裂殖壶菌杂合体。
6)发酵培养培养基:80g/L葡萄糖,0.4g/L酵母膏,2mL/L微量元素(Na2EDTA 6g/L,FeSO4 0.29g/L,MnCl2.4H2O 0.86g/L,ZnSO4 0.8g/L,CoCl2.6H2O 0.01g/L,Na2MoO4.2H2O0.01g/L,NiSO4.6H2O 0.06g/L,CuSO4.5H2O 0.6g/L),2mL/L维生素混合液(硫胺素50mg/L,生物素1mg/L,维生素B12 10mg/L)
将上述构建好的硒化镉CdSe-裂殖壶菌杂合体作为种子接种至150mL三角瓶的上述培养基中(接种量0.5%(v/v))(以游离的裂殖壶菌以相同接种量接种至另一个三角瓶中作为对照),接种完备,即开始施加LED光照(光照强度400lux),监测这一过程中裂殖壶菌产油脂情况,如下表4所示,可以发现CdSe-裂殖壶菌杂合体由于CdSe的引入,增强了油脂合成能力。
表4
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Claims (5)
1.一种提高产油微生物合成油脂能力的方法,其特征在于,采用对微生物细胞具有高度生物相容性的天然多酚修饰半导体纳米颗粒,再按比例与经过表面修饰的产油微生物进行掺杂,构建无机-产油微生物杂合系统;将构建的无机-产油微生物杂合系统接入发酵培养基,外源施加LED光源,提高产油微生物合成油脂能力;
所述天然多酚为单宁酸;
所述半导体纳米颗粒为硫化镉、磷化铟、硫化锌、硒化镉中的一种或几种;
所述天然多酚修饰半导体纳米颗粒是通过如下步骤实现的:
S1、将半导体纳米颗粒悬浮在装有MQ水的Eppendorf管中,将FeCl3∙6H2O溶液和天然多酚溶液各0.5-0.8mL依次加入到半导体纳米颗粒悬浮液中,并加入Tris缓冲液提高溶液pH至8.0;
S2、将上述获得的多酚功能化的半导体纳米颗粒先用MQ水洗涤3-4次,然后用70%乙醇10-15min洗涤孵育,最后用MQ水洗涤3-4次,在洗涤过程中,通过离心去除上清,随后用超声法分散悬浮液中的颗粒,离心条件为8000 rpm,5-10min;
产油微生物细胞表面修饰是通过以下步骤实现的:将对数生长期的产油微生物细胞,离心洗去培养基后,用水重悬;将聚烯丙基胺溶液加入微生物细胞悬液中,获得细胞表面经过修饰,能够进行与多酚功能化的半导体纳米颗粒组装的产油微生物;
所述无机-产油微生物杂合系统的构建方法是通过如下步骤实现的:将产油微生物细胞和半导体纳米颗粒进行混合,使其进行自组装,震荡10-60秒,通过添加额外的金属离子Fe3+和等体积的PBS缓冲溶液实现颗粒在细胞上的稳定组装获得了无机-产油微生物杂合体。
2.根据权利要求1所述的提高产油微生物合成油脂能力的方法,其特征在于,所述半导体纳米颗粒分散在产油微生物表面。
3.根据权利要求1所述的提高产油微生物合成油脂能力的方法,其特征在于,聚烯丙基胺溶液的浓度为3-8 mg/mL,重悬后的对数生长期产油微生物细胞的OD600值为2.0-4.0,二者的用量比例关系为2:1-3:1(V/V)。
4.根据权利要求1所述的提高产油微生物合成油脂能力的方法,其特征在于,所述FeCl3∙6H2O浓度为2-6 mg/mL、天然多酚溶液浓度为20-80 mg/mL。
5.根据权利要求1所述的提高产油微生物合成油脂能力的方法,其特征在于,产油微生物细胞和半导体纳米颗粒二者的初始OD600的比值控制在1.5-2.0。
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