WO2020103853A1 - 一种用于提高卷枝毛霉产油量的二羧酸转运体 - Google Patents

一种用于提高卷枝毛霉产油量的二羧酸转运体

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WO2020103853A1
WO2020103853A1 PCT/CN2019/119644 CN2019119644W WO2020103853A1 WO 2020103853 A1 WO2020103853 A1 WO 2020103853A1 CN 2019119644 W CN2019119644 W CN 2019119644W WO 2020103853 A1 WO2020103853 A1 WO 2020103853A1
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mucor
dit
dicarboxylic acid
recombinant
gene
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PCT/CN2019/119644
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English (en)
French (fr)
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宋元达
杨俊换
杨武
李少琦
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山东理工大学
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Priority to US17/262,960 priority Critical patent/US11407793B2/en
Publication of WO2020103853A1 publication Critical patent/WO2020103853A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA

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  • the invention relates to the technical field of genetic engineering, in particular to a dicarboxylic acid transporter used for increasing the oil production of Mucor cristatum.
  • ⁇ -linolenic acid GLA
  • ALA ⁇ -linolenic acid
  • SDA shikonic acid
  • EPA Eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • ⁇ -linolenic acid is an essential fatty acid for the human body, and it is a precursor substance for the body to synthesize active polyunsaturated fatty acids, which needs to be obtained from the diet.
  • ⁇ -linolenic acid comes from plants, such as evening primrose, glass lettuce oil, and blackcurrant seed oil.
  • the plant has a short growth cycle and unstable output, which cannot meet market demand. Therefore, the search for new oil resources has become the focus of current research. Oil-producing microorganisms have attracted much attention due to their high oil content, short growth cycle, and broad spectrum of carbon source utilization.
  • the oil-producing microbial cell factory refers to the use of oil-producing microbes. During the fermentation process, an excessive amount of carbon sources, such as carbohydrates, hydrocarbons, etc. are added, so that the microbial intracellular fatty acids are stored in the body in a large amount of triglycerides, and then the The cells are transformed into factories.
  • Mucor curl is the first strain in the world that uses microorganisms to commercialize oil and fat production strains. Mucor cristatum was used as a model strain for the study of oil-producing microorganisms. However, a new strain WJ11 was isolated in this study. The lipid produced by it can account for 36% of the dry cell weight, and its genome sequencing has been completed The research on heritage background and oil production mechanism is more in-depth. At the same time, its genetic operation is simple and the genetic tools are relatively complete, which is more suitable for cell factory preparation. Mucor curl can produce a large amount of ⁇ -linolenic acid (GLA), which has important physiological functions for the human body, which is also its main commercial value.
  • GLA ⁇ -linolenic acid
  • the dicarboxylic acid transporter (2-oxoglutarate / malate transporter, dit) gene is one of the key factors for lipid synthesis.
  • the oleaginous fungi usually have sufficient carbon source and lack of other nutrients (such as nitrogen, phosphorus, sulfur, etc.).
  • the mitochondrial tricarboxylic acid cycle is blocked, resulting in a large accumulation of citric acid in the mitochondria. At this time, the citric acid is transported to The cytoplasm is cleaved by citrate lyase to produce fatty acid synthesis substrates acetyl-CoA and oxalate acetyl.
  • Acetyl-CoA is a precursor of fat synthesis in cells, which is then used to synthesize fatty acids through biochemical reactions, and fatty acids are stored in cells in the form of triglycerides to form microbial fats.
  • dicarboxylic acid transporters can transport dicarboxylic acids such as malic acid in the cytoplasm through the mitochondrial cell membrane into the mitochondria, thereby promoting the transfer of citric acid and promoting the synthesis of cellular oils and fats, so the dicarboxylic acid transport The body plays an important role in microbial oil synthesis and accumulation.
  • the object of the present invention is to provide a dicarboxylic acid transporter for increasing the oil production of Mucor cristatum to improve the oil production of Mucor cristatum.
  • the present invention provides a dicarboxylic acid transporter for improving the oil production of Mucor cristatum.
  • the amino acid sequence of the dicarboxylic acid transporter is shown in SEQ ID NO: 2.
  • the present invention also provides a gene dit encoding the dicarboxylic acid transporter according to the above scheme, and the nucleotide sequence of the gene dit is shown in SEQ ID NO: 1.
  • the present invention also provides a recombinant vector containing the gene dit described in the above scheme.
  • the recombinant vector is capable of expressing Mucor cristatum dicarboxylic acid transporter, and the vector is a Mucor cristatum expression vector.
  • the recombinant vector uses pMAT1552 as the original vector.
  • the present invention also provides a recombinant bacterium comprising the recombinant vector described in the above scheme.
  • the recombinant bacteria are capable of expressing Mucor cristatum dicarboxylic acid transporter.
  • the recombinant bacteria use Mucor cristatum as the host bacteria.
  • the Mucor cristatum comprises Mu402 deficient strain Mucor cristatum.
  • the present invention also provides the application of the dicarboxylic acid transporter or the gene dit or the recombinant vector or the recombinant bacteria in the above solution to increase the oil production of Mucor cristatum.
  • the technical solution of the present invention is: extracting the mRNA of Mucor circinelloides WJ11 strain and reversing the cDNA, designing specific primers to PCR amplify the dicarboxylic acid transporter (dit) gene and connecting the gene to the integrated plasmid pMAT1552 , the recombinant plasmid was then electroporated into strain Mucor circinelloides deficient Mu402 protoplast, positive clones for fermentation, fermentation conditions were: the medium Kendrick, 28 °C, 700rpm, the intake air amount 1v / vmin - 1 , pH 6.0. During the fermentation process, samples are collected according to the law of oil accumulation to determine the content and composition of the oil.
  • the present invention provides a dicarboxylic acid transporter for increasing the oil production of Mucor cristatum, a recombinant strain Mc-Dit constructed by using a gene encoding the dicarboxylic acid transporter, and a control Compared with the bacteria Mc1552, the production of intracellular fat increased by 33.76%, and the content of intracellular fat could reach 17.67% of the total fatty acids.
  • the present invention utilizes Mucor cristatum as a model strain of a cell factory to study microbial lipid production, and uses homologous recombination genetic engineering methods to improve the oil production of Mucor cristatum, and provides guidance for vigorously promoting the industrial application of Mucor cristatum, and The use of microbial oils and fats to provide polyunsaturated fatty acids with high nutritional value meets the growing demands of people for good health and high-quality life.
  • FIG. 1 is a PCR verification diagram of a recombinant strain of Mucor cristatum, where M represents the molecular weight of a standard nucleic acid; 0 represents a control strain Mc1552; 1 to 3 represent a recombinant strain of Mucor cristatum Mc-Dit;
  • Fig. 2 is a graph showing the determination of the expression level of the dit gene mRNA of the recombinant strain of Mucor cristatum.
  • the dicarboxylic acid transporter (dit) gene (000239.15, 2129bp) (the nucleotide sequence of which is shown in SEQ ID NO: 1) was found, and the information analysis was carried out through the gene sequence.
  • the coding region of the sequence is a 1701bp base sequence, which can encode 566 amino acids (the amino acid sequence of which is SEQ ID NO: 2), and the expected molecular weight is 60.62kDa, PI 6.56.
  • the protein encoded by this sequence and the dicarboxylic acid transporter from Mucor ambiguus (dit) gene (NCBI gene ID: GAN03794.1) and Choanephora cucurbitarum's malate transporter YflS gene (NCBI gene ID: OBZ90888.1) are 67% and 75% homologous, respectively, therefore, the gene was initially determined
  • the Mucor circinelloides WJ11 strain was inoculated into a 100-mL Kendrick medium (glucose 30g / L, MgSO 4 ⁇ 7H 2 O 1.5g / L, ammonium tartrate 3.3g / L, KH 2 PO 4 7.0g / L , Na 2 HPO 4 2.0g / L, yeast extract 1.5g / L, CaCl 2 0.076g / L, FeCl 3 ⁇ 6H 2 O 8mg / L, ZnSO 4 ⁇ 7H 2 O 1mg / L, CuSO 4 ⁇ 5H 2 O 0.1mg / L, Co (NO 3 ) 2 ⁇ 6H 2 O 0.1mg / L, MnSO 4 ⁇ 5H 2 O 0.1mg / L) in 500mL baffled Erlenmeyer flask, 28 °C, 150rpm, 24h , Collect the bacterial cells by suction filtration to extract DNA.
  • Kendrick medium glucos
  • the dicarboxylic acid transporter (dit) gene (scaffold00239.15, 2129bp) was found (its nucleotide sequence is shown in SEQ ID NO: 1), and the specific primer Mudit was designed according to the gene sequence -F and Mudit-R, PCR was carried out using Mucor cristatum cDNA as template, Mudit-F: 5'–ACTTTTATATACAAAATAACTAAATCTCGAGATGCCAAAAGAGCCGTCTAT–3 '(as shown in SEQ ID NO: 3), Mudit-R: 5'–ACTAGTCGCAATTGCCGCGGCTCGAGTCAACACCAGCCCAAAAGTT– '(As shown in SEQ ID NO: 4).
  • the PCR reaction was tested in accordance with the PrimeSTAR HS DNA Polymerase (Takara) specification.
  • the reaction conditions were 95 ° C denaturation for 3 min and then the cycle was started, then 95 ° C denaturation for 30 sec, 55 ° C annealing for 30 sec, 72 ° C extension for 1 min, a total of 30 cycles, and then 72 Extend at °C for 10 minutes, and lower the temperature to 4 °C for 5 minutes.
  • Amplified to obtain 2129bp PCR fragment the recovered fragment was ligated with XholI endonuclease-treated vector pMAT1552 by one-step cloning technology, the ligation product was mixed with E.
  • YPG medium yeast extract 3g / L, peptone 10g / L, glucose 20g / L, leucine 20 ⁇ g / mL, uracil 200 ⁇ g / mL, pH 4.5
  • YPG medium yeast extract 3g / L, peptone 10g / L, glucose 20g / L, leucine 20 ⁇ g / mL, uracil 200 ⁇ g / mL, pH 4.5
  • the remaining cells cultured in the shake flask after coating were separated by vacuum filtration with a Büchner funnel, and the genomic DNA of Mucor cristatum was extracted (refer to the instructions of the plant quick DNA extraction kit), using this as a template with 1552-F and 1552 -R is the primer (the pair of primers is 600bp upstream and downstream of the insertion site of the target gene in the plasmid), and PCR verification is performed.
  • the internal reference gene is actin, and its amplification primer sequence is:
  • the amplification conditions were: preheating at 95 ° C for 10 min, then 95 ° C for 30 s, 59 ° C for 10 s, and 72 ° C for 30 s (45 cycles).
  • the dit gene expression results are shown in Figure 2. In Mc-Dit, the dit gene was successfully expressed, and the gene expression level decreased after 24 hours, but the gene expression level was still at a relatively high level compared with the control.
  • Example 7 Determination of fatty acid composition and content of Mc-Dit recombinant strain of Mucor cristatum
  • Kendrick medium was used to cultivate the recombinant strain Mc-Dit of K. brevis in a 2L fermentor.
  • the fermentation conditions were 28 ° C, 700 rpm, air intake 1v / v min -1 and pH maintained at 6.0.
  • Mucor twig collect the whole fermentation broth sample, vacuum filter with a Buchner funnel, separate the fermentation broth and the bacterial cells, collect the fermentation broth and store at -20 °C for use, wash the bacterial cells three times with distilled water, and then Freeze dry for use.
  • Gas chromatographic analysis was used to analyze fatty acid methyl esters using commercial fatty acid methyl ester standards (37 fatty acid methyl ester mixed standards) as standard samples.
  • Gas chromatography is GC-6890N from Agilent, USA. Measurement conditions: Gas chromatography conditions: splitless injection, chromatographic column is DM-FFAP (30m ⁇ 0.32mm, 0.22 ⁇ m), hydrogen ion flame detector, carrier gas is nitrogen, gas Both the chamber temperature and the detector temperature are 250 ° C, and the injection volume is 1 ⁇ L.
  • Heating program Initial temperature 80 °C, first rise to 200 °C with 8 °C / min heating rate, then increase to 205 °C with 1 °C / min heating rate, and finally increase to 240 °C with 4 °C / min heating rate, keep 5min.
  • pentadecanoic acid C15: 0
  • record the size of each fatty acid composition peak area and calculate the total fatty acid content.
  • the results are shown in Table 1.
  • the over-expression strain Mc-Dit's intracellular oil fatty acid composition did not change much, but the over-expression strain Mc-Dit strain's total oil fatty acid content increased by 33.76%, and the intracellular oil content can reach up to 17.67% of the total fatty acid .
  • the protein encoded by the 000239.15 gene in Mucor twitch WJ11 is a dicarboxylic acid transporter, and the protein is successfully expressed in the recombinant strain Mc-Dit, and the protein is involved in the oil synthesis process of Mucor twig.
  • the transporter can effectively increase the intracellular oil production of this strain.

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Abstract

提供了一种用于提高卷枝毛霉产油量的二羧酸转运体及其编码基因dit基因，该dit基因是从高产油卷枝毛霉WJ11中克隆获得的，将该dit基因转化到卷枝毛霉缺陷型菌株Mu402中，通过同源重组将该dit基因整合到卷枝毛霉的基因组上，得到重组菌株Mc-Dit，该Mc-Dit菌株油脂总脂肪酸含量提高了33.76％，胞内油脂含量最多可以达到总脂肪酸的17.67％。

Description

一种用于提高卷枝毛霉产油量的二羧酸转运体
本申请要求于2018年11月23日提交中国专利局、申请号为CN201811402942.7、发明名称为“一种用于提高卷枝毛霉产油量的二羧酸转运体”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及基因工程技术领域，尤其涉及一种用于提高卷枝毛霉产油量的二羧酸转运体。
背景技术
现在，人民生活水平日益提高，越来越多的人重视身体健康与生活质量。膳食油脂的营养成分与慢性病的发生密切相关，油脂中重要的活性多不饱和脂肪酸，如γ-亚麻酸(GLA)、α-亚麻酸(ALA)、紫草酸(十八碳四烯酸、SDA)、二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)，可以调节体内脂质代谢，预防多种慢性疾病的发生。γ-亚麻酸为人体必需脂肪酸，是人体合成活性多不饱和脂肪酸的前体物质，需要从膳食中获得。目前工业化的γ-亚麻酸大多来源于植物，如月见草、玻璃苣油和黑加仑籽油。然而植物因受产地、气候的影响，生长周期短、产量不稳定，不能满足市场需求。因此，寻找新型油脂资源成为当前研究的热点。产油微生物由于其油脂含量高，生长周期短，碳源利用广谱等特点备受关注，通过微生物产生的油脂以及其衍生品来代替部分植物资源，可能在未来生物产油产业中发挥重要作用。产油微生物细胞工厂是指利用产油微生物，发酵过程中通过添加过量的碳源，如碳水化合物、碳氢化合物等，使得微生物胞内脂肪酸以甘油三酯的方式大量储存在自身体内，进而将细胞转变成工厂。
卷枝毛霉是全球首次利用微生物商业化培养生产油脂的菌株。卷枝毛霉被用来作为产油微生物研究的模式菌种，然而，本研究中分离获得新菌株WJ11，其所产生的脂质可以占到细胞干重的36％，且其基因组测序已完成，遗产背景与产油机制研究较为深入，同时其基因操作简单，遗传工具较为齐全，比较适于进行细胞工厂制备。卷枝毛霉能够大量产生对人体具有重要生理功能的γ-亚麻酸(GLA)，这也是其主要的商业价值。
二羧酸转运体(2-oxoglutarate/malate transporter，dit)基因是脂质合成的关键因素之一。产油真菌通常在碳源充足，其它营养成分(如氮、磷、硫等)缺乏的条件下，线粒体中三羧酸循环受阻，导致柠檬酸大量积累在线粒体中，此时柠檬酸被转运到细胞质中由柠檬酸裂解酶裂解产生脂肪酸合成底物乙酰辅酶A和草酸乙酰。乙酰辅酶A为细胞中油脂合成前体物质，进而通过生化反应被用于合成脂肪酸，而脂肪酸则以甘油三酯的形式储存在细胞内，从而形成微生物油脂。据报道，二羧酸转运体能够将细胞质内的苹果酸等二羧酸透过线粒体细胞膜转运到线粒体内，进而促进柠檬酸的转出，为细胞油脂合成起到促进作用，因此二羧酸转运体对于微生物油脂合成与积累具有重要作用。
发明内容
本发明的目的在于提供一种用于提高卷枝毛霉产油量的二羧酸转运体，以提高卷枝毛霉产油量。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种用于提高卷枝毛霉产油量的二羧酸转运体，所述二羧酸转运体的氨基酸序列如SEQ ID NO:2所示。
本发明还提供了一种编码上述方案所述二羧酸转运体的基因dit，所述基因dit的核苷酸序列如SEQID NO:1所示。
本发明还提供了一种包含上述方案所述基因dit的重组载体。
优选的，所述重组载体能够表达卷枝毛霉二羧酸转运体，所述载体为卷枝毛霉表达载体。
优选的，所述重组载体以pMAT1552作为原始载体。
本发明还提供了一种包含上述方案所述重组载体的重组菌。
优选的，所述重组菌能够表达卷枝毛霉二羧酸转运体。
优选的，所述重组菌以卷枝毛霉作为宿主菌。
优选的，所述卷枝毛霉菌包括卷枝毛霉缺陷型菌株Mu402。
本发明还提供了上述方案所述二羧酸转运体或者所述基因dit或者所述重组载体或者所述重组菌在提高卷枝毛霉产油量中的应用。
本发明的技术方案是：提取卷枝毛霉(Mucor circinelloides)WJ11菌株的mRNA反转出cDNA，设计特异引物PCR扩增二羧酸转运体(dit) 基因将该基因连接至整合型质粒pMAT1552上，然后将该重组质粒电转化到卷枝毛霉缺陷型菌株Mu402的原生质体中，挑选阳性克隆进行发酵培养，发酵条件为：采用Kendrick培养基，28℃，700rpm，进气量1v/vmin -1，pH 6.0。发酵过程中，根据油脂积累规律采集样品，进行油脂含量与组成的测定。
本发明的有益效果：本发明提供了一种用于提高卷枝毛霉产油量的二羧酸转运体，利用编码该二羧酸转运体的基因构建得到的重组菌株Mc-Dit，与对照菌Mc1552相比，生成的胞内油脂产量提高了33.76％，胞内油脂含量可以达到总脂肪酸的17.67％。本发明利用卷枝毛霉作为研究微生物产脂的细胞工厂的模式菌株，利用同源重组的基因工程方法来提高卷枝毛霉油脂产量，为大力推广卷枝毛霉的工业化应用提供指导，并且能够用微生物油脂提供高营养价值的多不饱和脂肪酸，符合人民对身体健康和高质量生活的日益增长的要求。
附图说明
图1是卷枝毛霉重组菌株的PCR验证图，其中M代表标准核酸分子量；0代表对照菌株Mc1552；1～3代表卷枝毛霉重组菌株Mc-Dit；
图2是卷枝毛霉重组菌株的dit基因mRNA表达水平测定图。
具体实施方式
下面结合实施例对本发明进一步说明。
实施例1：二羧酸转运体(dit)基因信息学分析
根据已测序的WJ11的基因组信息，查找到二羧酸转运体(dit)基因(000239.15，2129bp)(其核苷酸序列如SEQ ID NO:1所示)，通过基因序列进行信息学分析，该序列编码区为1701bp碱基序列，可编码566个氨基酸(其氨基酸序列如SEQ ID NO:2)，预计分子量为60.62kDa，PI 6.56，该序列编码的蛋白与来自Mucor ambiguus的二羧酸转运体(dit)基因(NCBI基因ID：GAN03794.1)和Choanephora cucurbitarum的苹果酸转运体YflS基因(NCBI基因ID：OBZ90888.1)的同源性分别为67％和75％，因此，初步确定该基因可以编码卷枝毛霉WJ11的二羧酸转运体。
实施例2：重组质粒构建
将卷枝毛霉(Mucor circinelloides)WJ11菌株接种在含100mL  Kendrick培养基(葡萄糖30g/L，MgSO 4·7H 2O 1.5g/L，酒石酸铵3.3g/L，KH 2PO 47.0g/L，Na 2HPO 42.0g/L，酵母提取物1.5g/L，CaCl 20.076g/L，FeCl 3·6H 2O 8mg/L，ZnSO 4·7H 2O 1mg/L，CuSO 4·5H 2O 0.1mg/L，Co(NO 3) 2·6H 2O 0.1mg/L，MnSO 4·5H 2O 0.1mg/L)的500mL带挡板的锥形瓶中，28℃，150rpm，培养24h，抽滤收集菌体，提取DNA。根据已测序的WJ11的基因组信息，查找到二羧酸转运体(dit)基因(scaffold00239.15,2129bp)(其核苷酸序列如SEQ ID NO:1所示)，根据基因序列设计特异引物Mudit-F和Mudit-R，以卷枝毛霉cDNA为模板进行PCR，Mudit-F：5’–ACTTTTATATACAAAATAACTAAATCTCGAGATGCCAAAAGAGCCGTCTAT–3’(如SEQ ID NO:3所示)，Mudit-R：5’–ACTAGTCGCAATTGCCGCGGCTCGAGTCAACACCAGCCCAAAAGTT–3’(如SEQ ID NO:4所示)。
PCR反应参照PrimeSTAR HS DNA Polymerase(Takara)说明书进行试验，反应条件为95℃变性3min后开始循环，然后95℃变性30sec，55℃退火30sec，72℃延伸1min，共30个循环后，再于72℃延伸10min，降温至4℃保持5min。扩增得到2129bp的PCR片段，回收片段与经过XholI内切酶处理的载体pMAT1552通过一步法克隆技术进行连接，连接产物与大肠杆菌Top10感受态细胞混匀后进行热击转化，转化产物加入1mL LB液体培养基(蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L)37℃孵育1h后涂布于含100mg/L氨苄青霉素的LB平板(蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L，琼脂1.5％)。经37℃培养过夜，挑选菌落，接入LB液体培养基，8～10h后提取质粒进行序列测定，将序列正确的质粒命名为pMAT1552-Dit。
实施例3：卷枝毛霉原生质体制备
将卷枝毛霉Mu402菌株孢子接种到YPG培养基(酵母提取物3g/L，蛋白胨10g/L，葡萄糖20g/L，亮氨酸20μg/mL，尿嘧啶200μg/mL，pH 4.5)的平板中，28℃，培养1天。取单克隆菌丝点植于YPG培养基的平板，28℃培养3～4天孢子即可长好。取孢子生长良好的平板，每个平板加入5～6mL的YPG培养基，用灭菌的涂布棒刮取孢子，将孢子悬浮液收集于 灭菌的50mL离心管中，用血球计数板计算浓度并用pH 4.5的YPG调整孢子浓度为1×10 7个/mL。取12.5mL上述孢子悬液于灭菌的250mL锥形瓶中，置于4℃冰箱过夜使孢子充分吸水膨胀。将锥形瓶置于30℃，250rpm的摇床培养至孢子萌发。1100rpm离心后用5mL pH 6.5的PS缓冲液[18.22g山梨醇与20mL PBS缓冲液(NaCl 137mM，KCl 2.7mM，Na 2HPO 410mM，KH 2PO 42mM)]洗两次，将培养基洗去。用5ml PS缓冲液重悬，并加入终浓度为4mg/mL的裂解酶和0.06U/mL的壳聚糖酶，置于30℃，60rpm的摇床孵育90min以除去细胞壁。100×g离心后用0.5M 4℃预冷的山梨醇洗两次，加入800μL 0.5M的山梨醇轻轻吹吸重悬沉淀，得到原生质体，分装100μL/管以备使用。
实施例4：重组菌株Mc-Dit构建
取100μL上述制备好的原生质体与1μg质粒pMAT1552-Dit或pMAT1552混匀电击转化，电击结束后立即加入1mL预冷的YPGS(0.5mol/L山梨醇，酵母提取物3g/L，蛋白胨10g/L，葡萄糖20g/L)，26℃，100rpm孵育1h，100×g离心除去YPGS，以YNBS[山梨醇91.1g/L，谷氨酸1.5g/L，(NH 4) 2SO 41.5g/L，酵母基本氮源0.5g/L，葡萄糖10g/L，调pH 4.5，灭菌后加入硫胺素和烟酸至终浓度为1μg/mL]重悬后均匀涂布于MMC选择培养基上[酪蛋白氨基酸10g/L，酵母基本氮源0.5g/L，葡萄糖20g/L，琼脂15g/L调pH 3.2，灭菌后加入硫胺素和烟酸至终浓度为1μg/mL]，28℃避光培养3～4天。随机挑取8个选择性平板上长出的单菌落菌丝于新的MMC平板，28℃培养2～3天收集孢子，将大约200～300个孢子分别接种于MMC和含尿嘧啶的MMC平板中，28℃培养2～3天计数，重复上述筛选步骤直到两个平板中孢子生长数量基本相同则说明得到稳定遗传的转化子。稳定遗传的转化子菌丝在YPG培养基平板，30℃培养5～7天后收取孢子，调整孢子浓度为1×10 7个/mL，-80℃保存于30％甘油管中。最终获得了卷枝毛霉重组菌株Mc-Dit和对照菌株Mc1552。将涂布后摇瓶培养的剩余菌体用布氏漏斗真空抽滤分离得到，提取卷枝毛霉基因组DNA(参照植物快捷DNA提取试剂盒说明书进行)，以此为模板以1552-F和1552-R为引物(该对引物为质粒中插入目的基因位点上下游600bp位置)，进行PCR验证。
1552-F:5’–CCTCGGCGTCATGATGTTTTTGTGTACCT–3’(如SEQ ID NO:5所示),
1552-R：5’–GGGATGTCTGCTGCTACCATGTCTCAT–3’(如SEQ ID NO:6所示)。
反应体系及扩增条件，95℃预变性3min，95℃变性30sec，60℃退火30sec，72℃延伸2min，循环数30，72℃补偿延伸10min。PCR验证结果如图1，卷枝毛霉重组菌株Mc-Dit得到的片段为2301bp，而对照菌株Mc1552在相应位置片段为600bp，说明质粒已成功转化进入卷枝毛霉中。
实施例6：二羧酸转运体基因表达水平测定
参照Trizol使用说明书提取3、24、48、27h发酵样品的mRNA,并用ReverTraAce qPCRRT Kit(Roche)反转为cDNA,利用RT-qPCR方法测定二羧酸转运体的表达水平，利用2 -ΔΔCt方法处理数据，测定过程采用的试剂盒为SYBR Green Realtime PCRMasterMix(Roche)，扩增引物序列为：
WJ11-dit-F 5’–CCATAAAGTGTCTTTGGCTATTACGCACC–3’(如SEQ ID NO:7所示)，
WJ11-dit-R 5’–ACCAAGAGCTCCAAAATAAGCGAGC–3’(如SEQ ID NO:8所示)。
内参基因为actin，其扩增引物序列为：
actin-F 5’–GATGAAGCCCAATCCAAGA–3’(如SEQ ID NO:9所示)，
actin-R 5’–TTCTCACGGTTGGACTTGG–3’(如SEQ ID NO:10所示)。
扩增条件为：95℃预热10min，之后95℃30s，59℃10s，72℃30s(45循环)。dit基因表达结果如图2所示。在Mc-Dit中，dit基因成功表达，24h后基因表达量降低，但是与对照相比基因表达水平仍处于较高水平。
实施例7：卷枝毛霉重组菌株Mc-Dit脂肪酸组成及含量测定
待测样品制备：在2L发酵罐中采用Kendrick培养基培养卷枝毛霉重组菌株Mc-Dit。发酵条件为28℃，700rpm，进气量1v/v min -1，pH维持6.0。根据卷枝毛霉产油规律，收集全发酵液样品，用布氏漏斗真空抽滤，分离发酵液和菌体，收集发酵液于-20℃保存待用，用蒸馏水洗涤菌体3遍，然后冷冻干燥备用。
采用酸处理与反复冻融结合的破壁方式，用有机溶剂提取重组菌 Mc-Dit干菌体中油脂，参照方法(Folch J,Lees M,Sloane-Stanley G,et al.A simple method forthe isolation and purification of total lipids from animal tissues.BiolChem,1957,226,497-509)，并作适当修改，具体方法如下：
①将冷冻干燥后的菌体研磨后，称取20mg干重菌体于5mL的玻璃瓶中，加入2mL 4M盐酸；
②80℃水浴1h，-80℃15min，重复一次；
③恢复至室温后，加入1mL甲醇和1mL氯仿，并用微量进样器加入100μL浓度为2.02μg/μL内标C15:0；
④置于混匀仪中旋转萃取0.5h，3000rpm离心3min，收集氯仿层于新的5mL的玻璃瓶中；
⑤向原玻璃瓶中再次加入1mL氯仿，重复④的过程并合并氯仿层；
⑥氮气吹干；
⑦加入1mL 10％的盐酸甲醇溶液，60℃水浴3h，期间每隔半小时震荡30sec；
⑧冷却至室温后加入2mL正己烷和1mL饱和NaCl溶液，漩涡震荡混匀，4000rpm离心3min，吸取正己烷层1mL，转移至气相瓶得到脂肪酸甲酯溶液。
采用气相色谱分析，以商业化的脂肪酸甲酯标准品(37种脂肪酸甲酯混标)为标样来对脂肪酸甲酯进行分析。气相色谱为美国安捷伦的GC-6890N，测量条件：气相色谱条件：不分流进样，色谱柱是DM-FFAP(30m×0.32mm，0.22μm)，氢离子火焰检测器，载气为氮气，气化室温度和检测器温度均为250℃，进样量1μL。升温程序：初温80℃，先以8℃/min的升温速率升至200℃，再以1℃/min升温速率升至205℃，最后以4℃/min的升温速率升至240℃，保持5min。以十五烷酸(C15：0)作为参照，记录各个脂肪酸组成峰面积的大小，计算总脂肪酸的含量。结果如表1，过表达菌株Mc-Dit胞内油脂脂肪酸组成变化不大，但在过表达菌株Mc-Dit菌株油脂总脂肪酸含量提高了33.76％，胞内油脂含量最多可以达到总脂肪酸的17.67％。
表1发酵培养对照型和dit过表达型菌株油脂含量
Figure PCTCN2019119644-appb-000001
Figure PCTCN2019119644-appb-000002
由此可确定卷枝毛霉WJ11中000239.15基因所编码的蛋白质为二羧酸转运体，该蛋白质在重组菌株Mc-Dit成功表达，并且，该蛋白质参与卷枝毛霉油脂合成过程，过表达该转运体可以有效提高该菌株胞内油脂的产量。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。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，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种用于提高卷枝毛霉产油量的二羧酸转运体，其特征在于，所述二羧酸转运体的氨基酸序列如SEQ ID NO:2所示。
  2. 一种编码权利要求1所述二羧酸转运体的基因dit，所述基因dit的核苷酸序列如SEQ ID NO:1所示。
  3. 一种包含权利要求2所述基因dit的重组载体。
  4. 根据权利要求3所述的重组载体，其特征在于，所述重组载体能够表达卷枝毛霉二羧酸转运体，所述载体为卷枝毛霉表达载体。
  5. 根据权利要求3或4所述的重组载体，其特征在于，所述重组载体以pMAT1552作为原始载体。
  6. 一种包含权利要求3所述重组载体的重组菌。
  7. 根据权利要求6所述的重组菌，其特征在于，所述重组菌能够表达卷枝毛霉二羧酸转运体。
  8. 根据权利要求6或7所述的重组菌，其特征在于，所述重组菌以卷枝毛霉菌作为宿主菌。
  9. 根据权利要求8所述的重组菌，其特征在于，所述卷枝毛霉菌包括卷枝毛霉缺陷型菌株Mu402。
  10. 权利要求1所述二羧酸转运体或者权利要求2所述基因dit或者权利要求3～5任意一项所述重组载体或者权利要求6～9任意一项所述重组菌在提高卷枝毛霉产油量中的应用。
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