CN114134045B - 一种同时高产维生素d3和二十碳五烯酸的微拟球藻工程株及其制备方法和应用 - Google Patents
一种同时高产维生素d3和二十碳五烯酸的微拟球藻工程株及其制备方法和应用 Download PDFInfo
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- CN114134045B CN114134045B CN202111463237.XA CN202111463237A CN114134045B CN 114134045 B CN114134045 B CN 114134045B CN 202111463237 A CN202111463237 A CN 202111463237A CN 114134045 B CN114134045 B CN 114134045B
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- nannochloropsis
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- dehydrocholesterol
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Classifications
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—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
- C12N1/12—Unicellular algae; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
- C12Y103/01021—7-Dehydrocholesterol reductase (1.3.1.21)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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Abstract
本发明涉及基因工程技术领域，特别涉及一种同时高产维生素D3和二十碳五烯酸的微拟球藻工程株及其制备方法和应用。该工程株为敲除DWARF5(DWF5)基因的微拟球藻。本发明利用CRISPR CAS9技术，在不引入任何外源分子标签的前体下，构建出DWF5基因的敲除株，得到胆固醇含量显著降低、7‑去氢胆固醇(7‑dehydrocholesterol)含量显著提高的工程株，其遗传性状稳定。7‑去氢胆固醇是维生素D3的前体，藻细胞经日光或紫外线照射，超过80％的7‑去氢胆固醇转化为维生素D3。维生素D3具促进骨骼生长、预防骨质疏松症、调节免疫的功能，并对白血病细胞，肿瘤细胞等的生长分化有调节作用。
Description
技术领域
本发明涉及基因工程技术领域，特别涉及一种同时高产维生素D3和二十碳五烯酸的微拟球藻工程株及其制备方法和应用。
背景技术
微拟球藻(Nannocloropsis oceanica)是一种光合自养的圆球状的单细胞生物，在缺氮胁迫条件下能产生大量的脂质积累，累积量高达其生物量干重的60％，故常用作生物柴油的潜在替代品。除此之外，它也能用作一系列高附加值产品的开发，如食品、饲料添加剂、色素、化妆品、药品等。而其代谢产物如叶绿素、甾醇、类胡萝卜素和一些植物激素，决定了对生长或产生有价值的化学物质至关重要的细胞特性。微拟球藻富含油脂，且有特别高含量的PUFA(polyunsaturated fatty acid，多不饱和脂肪酸)，如EPA(eicosapentaenoic acid，二十碳五烯酸)，它在功能食品、功能饵料等方面具有巨大的经济价值及广阔的应用前景。
胆固醇是构成人体组织细胞所不可或缺的重要组成物质，它不仅参与细胞膜的形成，而且是合成维生素D、胆汁酸及甾体激素的重要原料。但当人体血清胆固醇含量过高时，易导致高胆固醇血症的发生，对人体产生不利的影响。现代研究发现，动脉粥样硬化性、心脑血管疾病及冠心病等的发生与高胆固醇血症有密切的联系。因此，降低血清中过多的胆固醇含量对人身体健康是有利的。微拟球藻因其富含EPA，被广泛用于功能食品和功能饵料开发，但却因含有高的胆固醇而饱受诟病。
维生素D3(VD3)，又名胆骨化醇(colecalciferol)，具有促进骨骼生长、预防骨质疏松症、调节免疫的功能，并对白血病细胞，肿瘤细胞等的生长分化有调节作用。传统的合成方法是将VD3分子从中间三烯和侧链上切割成三个合成器，再通过缩合或偶联将三个部分拼接在一起。全合成过程非常复杂，收率很低。半合成主要是选择合适的天然甾体为原料，然后对A、B环和侧链进行改性，最后通过光化开环反应构建VD3。在实际生产中比较困难。
微拟球藻富含甾醇(>10mg/g DCW)，其中胆固醇占～70％，其合成的前体是7-脱氢胆固醇(Lu et al.,2014Regulation of the cholesterol biosynthetic pathway andits integration with fatty acid biosynthesis in the oleaginousmicroalgaNannochloropsis oceanica)。7-脱氢胆固醇经日光或紫外线(290～300nm)照射10min即可形成VD3。目前，还未见生产VD3的微藻工程株。
发明内容
有鉴于此，本发明提供了一种同时高产维生素D3和二十碳五烯酸的微拟球藻工程株及其制备方法和应用。本发明获得一株低胆固醇、高7-脱氢胆固醇工程微拟球藻细胞，其胆固醇含量下降～90％，7-脱氢胆固醇含量实现了从检测不到到占总甾醇超过60％的含量，且性状可稳定遗传。经2小时光照，80％以上的7-脱氢胆固醇转变为VD3。同时，微拟球藻富含EPA，因此，可用于产业化生产高多不饱和脂肪酸、高VD3、低胆固醇的功能食品、保健品和海洋药物，为绿色健康产业服务。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种微拟球藻工程株，其为敲除DWF5基因的微拟球藻。
其中，DWF5基因全长为1338bp，核苷酸序列如SEQ ID NO.1所示。
本发明还提供了该微拟球藻工程株的制备方法，采用CRISPR/Cas9技术将微拟球藻中的DWF5基因敲除。
本发明还提供了该微拟球藻工程株的培养方法，将微拟球藻工程株接种于平板培养基中进行培养；平板培养基配方为：
优选地，平板培养基配方为：
作为优选，营养液母液的配方为：
硝酸钠 30～50g
一水合磷酸二氢钠 1～5g
水 补足至200mL。
优选地，营养液母液的配方为：
硝酸钠 40g
一水合磷酸二氢钠 2.66g
水 补足至200mL。
作为优选，维生素母液的配方为：
优选地，维生素母液的配方为：
作为优选，微量元素溶液的配方为：
优选地，微量元素溶液的配方为：
作为优选，抗生素溶液的配方为：
氨苄青霉素钠 0.5～2mg
头孢氨噻肟 0.5～2mg
水 补足至10mL。
优选地，抗生素溶液的配方为：
氨苄青霉素钠 1mg
头孢氨噻肟 1mg
水 补足至10mL。
本发明还提供了一种生产VD3的方法，对上述微拟球藻工程株进行高光诱导培养。
作为优选，高光诱导培养的条件为：将经过验证后的转化子接种至PBR中培养至对数生长期，即OD750＝2.0-3.5，置于光强为200μmol·photons·m-2·s-1，25℃，诱导2h测定7-脱氢胆固醇向VD3的转化率。
本发明还提供了该微拟球藻工程株在制备低胆固醇、高VD3、高二十碳五烯酸的功能食品和/或保健品中的应用。
本发明具有的技术效果如下：
传统VD3化学合成方法工艺复杂、操作难度高。目前还未见针对DWF5基因在微拟球藻中的研究，遗传工程策略往往引入外源抗性标签，会引发公众对转基因食品的顾虑。本发明利用CRISPR CAS9技术，在不引入任何外源分子标签的前体下，构建出微拟球藻DWF5基因的敲除株，得到胆固醇含量显著降低(从近70％下降至3.0％以下)、7-脱氢胆固醇(从检测不到到占总甾醇超过60％的含量；以提高最明显的dwf5-3为例)的工程株，经简单的光照处理后80％以上的7-脱氢胆固醇被转化为VD3。这一工程株遗传性状稳定，若将此工程株进行产业化生产，有助于填补低胆固醇、高VD3微藻这一领域上的空白，为将微拟球藻用于开发高VD3含量的健康食品的生产应用提供理论依据。将此工程株应用于开发成功能食品、保健品、特殊医学配方食品或海洋药物等，可以满足人民对于健康的需求。
具体实施方式
本发明公开了一种同时高产维生素D3和二十碳五烯酸微拟球藻工程株及其制备方法和应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明中所用试剂或仪器均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
1、平板培养基配制如下：
将10g琼脂粉溶入1L配制的人工海水中，配制成1％的琼脂液固体培养基中，放入灭菌锅中121℃，灭菌20min。灭菌完成后趁热在超净工作台上用移液器吸取5mL营养液母液、500μL微量元素溶液、1mL抗生素溶液以及3μL 100mg/mL的潮霉素B加入琼脂液中，摇匀，倒平板。(注：营养液母液及微量元素溶液均需要提前灭菌，121℃，20min；维生素母液和抗生素溶液均需要预先0.22μm膜过滤除菌)
表1营养液母液配方(200mL)
| 物料名称 | 分子式 | 级别 | 称量量(g) |
| 硝酸钠 | NaNO3 | AR | 40 |
| 一水合磷酸二氢钠 | NaH2PO4·H2O | AR | 2.66 |
表2维生素母液配方(100mL)
| 物料名称 | 分子式 | 级别 | 称量量(mg) |
| 维生素B12 | VitaminB12 | AR | 1 |
| 生物素 | Biotin | AR | 1 |
| 维生素B1 | VitaminB1 | AR | 20 |
表3微量元素溶液母液配方(200mL)
| 物料名称 | 分子式 | 级别 | 称量量(mg) |
| 乙二胺四乙酸二钠 | Na2EDTA | AR | 874 |
| 六水合氯化铁 | FeCl3.6H2O | AR | 730 |
| 五水硫酸铜 | CuSO4·5H2O | AR | 3.92 |
| 七水硫酸锌 | ZnSO4·7H2O | AR | 8.8 |
| 六水氯化钴 | CoCl2·6H2O | AR | 2.184 |
| 四水氯化锰 | MnCl2·4H2O | AR | 72 |
| 二水钼酸钠 | Na2MoO4·2H2O | AR | 2.52 |
表4抗生素溶液配方(10mL)
| 物料名称 | 分子式 | 级别 | 称量量(mg) |
| 氨苄青霉素钠 | C16H18N3NaO4S | AR | 1 |
| 头孢氨噻肟 | C16H16N5O7S2Na | AR | 1 |
2、利用CRISPR CAS9技术，构建出微拟球藻DWARF5基因的敲除株。
需敲除的微拟球藻DWARF5基因序列如SEQ ID NO.1所示。
电穿孔转化参照Wang等(Wang et al.,Genome editing ofmodel oleaginousmicroalgaeNannochloropsis spp.by CRISPR/Cas9)。基因敲除参照Lu等(Lu et al.,Roleof an ancient light-harvesting protein of PSI in light absorption andphotoprotection)。转化后的藻液涂于含300μg/L潮霉素的f/2平板中，挑选至液体培养基(步骤1中配制的平板培养基不加琼脂即为液体培养基)中进行培养，平板培养温度为25℃，光照强度为50μmol·photons·m-2·s-1。培养21天。
3、单克隆培养
待平板上长出肉眼可见的较大的单克隆时，挑取若干单克隆至含f/2液体培养基的50mL三角瓶中。置于培养箱中25℃，50μmol·photons·m-2·s-1下培养10-15天。
4、PCR验证
待生长至肉眼可见绿色时取藻液进行藻液PCR验证，用XD-001F与XD-001R引物对进行扩增，PCR产物大小约为973bp。
XD-001F：ATGTGGCTCAACAATAATGG
XD-001R：TAGCACCGGCCAGGAGGAGGAGG
并将PCR产物送测序，用基因组序列进行blastn比对，确认是否在PAM区发生移码突变，使得该基因失效。
选取其中的三株转化子，分别命名为dwf5-1、dwf5-2、dwf5-3。
5、甾体谱测定
将经过验证后的转化子与野生型均接种至PBR中培养至对数生长期，即OD750＝2.0-3.5，离心收集藻液，将样品冷冻真空干燥后，用于后续GC-MS的分析。选取对数期dwf5-3，过暗培养，收集部分藻体；余下部分置于光强为200μmol·photons·m-2·s-1，25℃，光照处理2h。离心收集藻液，将样品冷冻真空干燥后，用于后续GC-MS的测定，分析7-脱氢胆固醇向VD3的转化率(注：无论野生型还是突变体，均选用了三个生物学重复中差异最大的一个用作展示说明)。
GC-MS分析技术参数：流量1mL/min，起始温度170℃，持续1min，然后以20℃/min上升到280℃，停留至少15min，离子源温度为150℃，进样量1μL，柱箱温度为170℃。
表5微拟球藻在高光胁迫处理下的GC/MS甾醇数据分析
为了评价不同处理的效果，每个样品在上述条件下建立三个生物重复。采用单因素方差分析评价各处理的差异，然后进行p值检验。数据以平均值±标准差(n≥3)表示。p值＜0.05时，差异被认为是显著的。
由表可见，与野生型相比，突变体的胆固醇相对含量显著降低、7-去氢胆固醇的含量显著增加。
以dwf5-3为例，经光照处理2小时后，80％以上的7-脱氢胆固醇被转化为VD3。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 海南大学
<120> 一种同时高产维生素D3和二十碳五烯酸的微拟球藻工程株及其制备方法和应用
<130> MP21025081
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1338
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgtggctca acaataatgg caagtcctcg ggccttttgc ccggccgcga gagtgtgggg 60
ccccttgcgc tcatgtgcat cacgcccgtc ttcatcttca ttctttggta caccatgcag 120
cacttggggg gcgacttcgg caaactcatc gataacttcc atacgaatgg atggggttac 180
ctcaaggtga tcgttccgac gccctttgac cccaccgctt ggaaggtgat tctctcctac 240
atggctgtgg agctggcctt catgaggctc ctcccaggca agaccttcaa ggcgaccgtg 300
acaccggcag gaaacgtacc cgtgtataag gctaacggta tgcaagcctt ctttgcctcc 360
ctcttcctct tcttcctcct gcagcaatac ggccctgcgt acggcctgca cgtctcctgg 420
gtctaccacc acatgggcga gcttctttcg gccatgaacg tcttttctct tgccttctgc 480
tttttcctcc tggtcaaggg cctcaccttt ccaagttcct ctgattcagg ctcctcgggc 540
aactggatca ttgacttcta ctggggcacg gagctgtacc cgcgcgtact tggcttcgac 600
atgaagatgt tcaccaactg ccggtttggg atgatgttct gggcgttggg cattctctgc 660
tacgcgcagg cccaggtcga ggcagacggg ttcctctcca acgccatgct ggtgagcgtg 720
accctgcaac tcgtgtacat caccaagttc tttcactggg agacgggcta cctctgctcc 780
atggacatcc agcacgaccg ggccggctac tacatttgtt ggggctgcct ggtctgggtc 840
ccctccgtct atacctctcc gtcctacttt ctggtcaacc acgccgccca ggatatctcc 900
agcctcacgg ccgtcctcct cctcctggcc ggtgctatct gtgtcgccat caactactgg 960
gccgaccgcc agcgccaggt cttccgcgcc actgacggca agtgtaccat ctggggaaag 1020
ccgcctgtat tcatcacggc ctcctacacc accgaagcgg gccagaagcg ctcctccctt 1080
ctgctggcct cgggctggtg gggcgtggcg cggcactttc attatgtgcc cgagatcttg 1140
ggcgcgttcc tctggagctg cccggcgggc tttcatggct ttcgctattt cttggcttat 1200
ttctatgtta tttttctgac cccgttactc tttgaccgcg ccttccgtga cgacgcgcgg 1260
tgccgtgaca aatatgggaa gcactgggag aagtattgcg cgctcgtgcc ctacaagatt 1320
atccccggtg tcctttaa 1338
Claims (9)
1.一种微拟球藻工程株，其特征在于，其为敲除DWF5基因的微拟球藻；
所述DWF5基因的核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述微拟球藻工程株的制备方法，其特征在于，采用CRISPR/Cas9技术将微拟球藻中的DWF5基因敲除。
3.权利要求1所述微拟球藻工程株的培养方法，其特征在于，将所述微拟球藻工程株接种于平板培养基中进行培养；每1L所述平板培养基的组成如下：
4.根据权利要求3所述的培养方法，其特征在于，所述营养液母液的组成为：
硝酸钠30～50g
一水合磷酸二氢钠1～5g
水补足至200mL。
5.根据权利要求3所述的培养方法，其特征在于，所述维生素母液的组成为：
6.根据权利要求3所述的培养方法，其特征在于，所述微量元素溶液的组成为：
7.一种生产维生素D3的方法，其特征在于，对权利要求1所述微拟球藻工程株进行高光或紫外诱导培养。
8.根据权利要求7所述的方法，其特征在于，所述高光诱导培养的条件为：200μmol·photons·m-2·s-1，25℃，2h光照；所述紫外诱导条件的波谱范围为290～300nm。
9.权利要求1所述微拟球藻工程株在制备低胆固醇、高维生素D3的功能食品和/或保健品中的应用。
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| CN104946535A (zh) * | 2014-03-26 | 2015-09-30 | 中国科学院青岛生物能源与过程研究所 | 一种可以调控微藻生长和其它功能的生长调节剂及其鉴定方法和应用 |
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| CN104946535A (zh) * | 2014-03-26 | 2015-09-30 | 中国科学院青岛生物能源与过程研究所 | 一种可以调控微藻生长和其它功能的生长调节剂及其鉴定方法和应用 |
Non-Patent Citations (3)
| Title |
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| Clade-Specific Sterol Metabolites in Dinoflagellate Endosymbionts Are Associated with Coral Bleaching in Response to Environmental Cues;Yandu Lu 等;mSystems;第5卷(第5期);第1-16页 * |
| Diatoms synthesize sterols by inclusion of animal and fungal genes in the plant pathway;Carmela Gallo 等;Scientific ReportS;第10卷;第1-13页 * |
| Regulation of the cholesterol biosynthetic pathway and its integration with fatty acid biosynthesis in the oleaginous microalga Nannochloropsis oceanica;Yandu Lu 等;Biotechnology for Biofuels;第7卷;第1-15页 * |
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