WO2015062190A1 - 一种高山被孢霉重组基因表达系统及其构建方法和应用 - Google Patents

一种高山被孢霉重组基因表达系统及其构建方法和应用 Download PDF

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WO2015062190A1
WO2015062190A1 PCT/CN2014/072839 CN2014072839W WO2015062190A1 WO 2015062190 A1 WO2015062190 A1 WO 2015062190A1 CN 2014072839 W CN2014072839 W CN 2014072839W WO 2015062190 A1 WO2015062190 A1 WO 2015062190A1
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ura5s
pbig2
plasmid
gene
strain
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陈永泉
陈卫
郝光飞
陈海琴
赵建新
顾震南
张灏
郝丹辉
赵山山
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江南大学
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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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01038—Malate dehydrogenase (oxaloacetate-decarboxylating) (1.1.1.38)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/0104—Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) (1.1.1.40)

Definitions

  • the invention relates to a mountain spore recombination gene expression system and a construction method and application thereof.
  • the invention claims priority from the Chinese invention patent application date of October 30, 2013 and application number CN 201310524221.4.
  • the invention relates to a recombinant gene expression system of Mortierella alpina, a construction method and application thereof, and belongs to the technical field of bioengineering.
  • Mortierella alpina is an important oil-producing fungus with high arachidonic acid (AA) content, safe, and reasonable composition of polyunsaturated fatty acids (PUFAs). It has been used in industrial production of AA.
  • AA arachidonic acid
  • PUFAs polyunsaturated fatty acids
  • filamentous fungi The gene operating system of filamentous fungi has been lagging behind other species and has not been well established, mainly due to the fact that filamentous fungi are difficult to be transformed.
  • fungi with the characteristics of Mortierella alpina are most difficult to be transformed: multinuclear, non-separating, low in sporulation and insensitive to antibiotics. Therefore, there has been no report on the genetic transformation of such important industrial production microorganisms in China.
  • the choice of transformation method is also a key factor in determining whether filamentous fungi can be transformed.
  • the transformation and methods of filamentous fungi mainly consist of the following: protoplast transformation, electroporation transformation, gene gun transformation and Agrobacterium-mediated transformation.
  • protoplast transformation and electroporation transformation require degradation of the cell wall of the receptor to prepare protoplasts, which is difficult to culture, low in regeneration frequency and long in experimental period.
  • the gene gun transformation has the advantage of being convenient, it requires a large receptor base and the conversion cost is too high.
  • Agrobacterium transformation technology as one of the earliest applied to plants Conventional transformation techniques have been reported to have the ability to transform fungi as early as 20 years ago. To date, Agrobacterium transformation technology has been successfully applied to more than one hundred and twenty species of fungi.
  • Agrobacterium transformation has several outstanding advantages:
  • the receptor cells are extensive, can be spores or hyphae, and do not require the preparation of protoplasts; the transformation efficiency is high, the success rate is high, and the carrier can accommodate large fragments.
  • Heterologous DNA essentially a single copy is randomly inserted into the host chromosome; the homologous recombination efficiency can be improved. Therefore, the transformation method of Agrobacterium tumefaciens provides an important means of operation for the construction of the M. alpina expression system.
  • Malic enzyme (EC 1.1.1.40), which catalyzes the reaction of malic acid to pyruvate in cells, is an important source of NADPH production in living organisms. As early as the 1990s, malic enzyme was presumed to be an important factor in the fatty acid synthesis pathway of oily filamentous fungi. In another filamentous fungus belonging to the genus Mycobacterium, the activity of malic enzyme is inhibited by sesame powder, a chemical inhibitor that specifically inhibits the activity of the mucoidase of Mucor In the case, the total fat content in the cells was significantly affected.
  • malic acid was inferred to be an important rate-limiting step in the fatty acid synthesis of oil-producing fungi. Subsequently, in a systematic study of a series of NADPH-producing enzyme activities during the fermentation of Mortierella alpina, malic enzymes were also speculated to be closely related to intracellular fatty acid synthesis in Mortierella alpina. However, this theory has not been validated and applied in M. alpina due to the lack of an effective recombinant gene expression system.
  • the Mortierella alpina ATCC 32222 uracil auxotrophic strain disclosed in the patent application No. 201310347934.8 is used as a transforming strain, and a novel high expression recombinant malic enzyme is constructed by further genetic recombination method. Genetic expression system. The entire disclosure of the entire disclosure of the entire disclosure is hereby incorporated by reference.
  • the technical solution disclosed in Chinese Patent Application No. 201310347934.8 includes a strain of Mortierella alpina uracil auxotrophy, which is constructed by inactivating the picture 5 gene encoding the orotate phosphoribosyltransferase OPRTase in the Mortierella alpina ATCC 32222 genome. of.
  • the inactivation of the M. alpina uracil auxotrophic strain, the training 5 gene is achieved by deleting the 213 bp-230 bp sequence of pp bp of the 654 bp wra5 gene.
  • Chinese Patent Application No. 201310347934.8 also discloses a method for preparing the above-mentioned strain of Mortierella alpina uracil auxotrophy, which is caused by homologous recombination to delete the 213bp-230bp total 18bp sequence in the Mortierella alpina gene, thereby inactivating the wra5 gene.
  • the homology arms used were 1393 bp upstream of the wra5 gene at -1380 to +212 and a 1362 bp fragment downstream from +231 to +1592.
  • the specific steps were as follows: First, the wra5 knockout gene fragment was obtained, and the knockout plasmid pBIG4KOura5 was further constructed, and then Agrobacterium tumefaciens was transformed with the recombinant plasmid pBIG4KOura5, and finally transformed with the transformed plasmid Agrobacterium tumefaciens containing the plasmid pBIG4KOura5, and the transformed M. alpina was screened and identified to obtain a uracil auxotrophic strain.
  • the method used in the method is the Agrobacterium tumefaciens C58CL
  • the Agrobacterium tumefaciens starting vector used for gene knockout is: pBIG2RHPH2.
  • the knock-out gene fragment and the plasmid pBIG4 were digested with the restriction enzymes EcoR I and Kpn I, and the knock-out gene fragment was inserted into the plasmid pBIG4 by ligation to obtain pBIG4KOura5.
  • the knockout gene fragment in step 3 is obtained by the following steps, first designing the following primers according to the NCBI database
  • primers PI, P2 and primers P3 and P4 were used to amplify the upstream and downstream fragments, respectively, and then the upstream and downstream fragments were used as templates.
  • PI and P4 were added to the reaction system for fusion PCR reaction to obtain KO.
  • the ura5 knockout gene fragment was added to the reaction system for fusion PCR reaction to obtain KO.
  • the following primers are designed based on the sequence information of the plasmid pBluescript ll SK + : MCS upstream:
  • the MCS gene fragment of plasmid pBluescript ll SK+ in step 1) was then obtained by PCR.
  • the Agrobacterium tumefaciens-mediated gene knockout method is to transform Mortierella alpina by using Agrobacterium tumefaciens, specifically: taking 10 (L. agrobacterium tumefaciens mixed with ⁇ M. alpina spore liquid, uniform It was applied to a solid paper cultured on a cellophane IM, subjected to transformation culture, and then screened to obtain a strain of Mortierella alpina auxotrophy.
  • the specific steps of Agrobacterium tumefaciens transformation of Mortierella alpina are as follows:
  • Agrobacterium tumefaciens C58C1 containing plasmid pBIG4KOura5 stored at -80 °C was streaked onto a YEP solid medium plate containing 100 g/mL rifampicin and 100 g/mL kanamycin; inverted at 30 °C Incubate in the dark for 48 hours;
  • the cells were collected by centrifugation at 4000 g for 5 minutes, the supernatant was decanted, the cells were resuspended in 5 mL of IM medium, centrifuged at 4000 g for 5 minutes, the supernatant was decanted, and the cells were resuspended in 2 mL of IM medium;
  • the cellophane was transferred to a GY plate containing 100 g/mL spectinomycin, 100 g/mL cefotaxime, 0.05 g/L uracil, and cultured at 25-30 ° C until a large amount of spores were produced.
  • Agrobacterium tumefaciens C58Cl-pBIG4KOura5 obtained from Chinese patent application 201310347934.8 was deposited at the General Microbiology Center of China Microbial Culture Collection Management Committee on June 17, 2013. Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Academy of Sciences, Zip Code 100101, with accession number CGMCC No. 7730.
  • the technical problem to be solved by the present invention is to provide a recombinant gene expression system of Mortierella alpina.
  • the recombinant gene expression system of Mortierella alpina was transformed by Mortierella alpina ATCC 32222 uracil nutritional deficiency strain type by Agrobacterium tumefaciens transformation (ATMT).
  • the DNA sequence of the selection marker wra_ gene, malic enzyme 1 gene malEl and malic enzyme 2 gene malE2 of the recombinant gene expression system was derived from the Mortierella alpina ATCC 32222 genome group (DDBJ/EMBL/GenBank accession ADAG00000000, first version ADAG01000000).
  • the invention also provides a method for constructing a recombinant gene expression system of Mortierella alpina,
  • the ⁇ expression unit was digested with restriction enzymes EcoR I and Xbal, and inserted into the multiple cloning site (MCS) of EcoR I and Xbal alcohol-cut pET28a (+) to obtain plasmid pET28a-HPHs.
  • MCS multiple cloning site
  • OPRTase whey ribose transferase
  • Plasmid pET28a-ura5s was constructed by digesting the plasmid pET28a-HPHs with the replacement gene.
  • the ura5s expression unit was obtained by digesting the plasmid pET28a-ura5s with the restriction enzymes EcoR I and Xbal.
  • the ura5s expression unit was replaced with the HPH expression unit in the plasmid pBIG2RHPH2, and the plasmid transformation plasmid pBIG2-ura5s was further constructed, and then the Agrobacterium tumefaciens was transformed with the recombinant plasmid pBIG2-ura5s, and finally the transformed Agrobacterium tumefaciens containing the plasmid pBIG2-ura5s was transformed.
  • the auxotrophic form of Mortierella alpina was transformed and the transformed Alternaria sp. was screened and identified to obtain a phenotype complementary strain, thereby realizing the genetic transformation of Mortierella alpina.
  • a malic enzyme 1 overexpression vector was constructed.
  • the malic enzyme 1 gene malEl was obtained from the M. alpina cDNA by PCR.
  • the malEl gene fragment and the plasmid pET28a-HPHs were digested with the restriction enzymes BspHI and BamHI, Ncol and BamHI, respectively, and the malEl gene fragment was inserted into the plasmid pET28a-HPHs between the Ncol and BamHI sites by ligation.
  • pET28a-malEl The plasmid pET28a-malEl was digested with restriction endonucleases Spel and Xbal to obtain a malEl expression unit.
  • the malEl expression unit was inserted into the Xbal-cut plasmid pBIG2-ura5s to obtain the plasmid pBIG2-ura5s-malEl. Then, the recombinant plasmid pBIG2-ura5s-malEl was used to transform Agrobacterium tumefaciens, and finally transformed with Agrobacterium tumefaciens C58C1 pBIG2-ura5s-malEl, a transformed BGA2-ura5s-malE 1 containing plasmid BIG2-ura5s-malE 1
  • the auxotrophic type was screened and identified by the transformed M. alpina, and the phenotype complementary strains of M. alpina MA-malEl- ⁇ , MA-malEl-2 and MA-a/-3 were obtained, thereby realizing the construction of malic acid. Enzyme The 1 gene is overexpressed in the homologous state of Mortierella alpina.
  • plasmid pBIG2-ura5s and the plasmid pET28a-HPHs a universal vector for the manipulation of the M. alpina gene was constructed. The specific steps are shown in Figure 2.
  • the non-coding intron DNA fragment IT was obtained from the M. alpina genome by PCR.
  • the IT gene fragment and plasmid pET28a-HPHs were digested with restriction endonucleases Ncol and Bamffl, respectively, and the IT fragment was substituted for the plasmid pET28a-HPHs by ligation to obtain plasmid pET28a-ITs.
  • the ITs expression unit was obtained by double digestion of the plasmid pET28a-ITs with the restriction enzymes Spel and Xbal.
  • the ITs expression unit was inserted into the Xbal-cut plasmid pBIG2-ura5s, and the M. alpina gene was used to operate the universal vector pBIG2-ura5s-ITs.
  • a malic enzyme 2 overexpression vector was constructed based on the general vector pBIG2-ura5s-ITs of the M. alpina gene.
  • the a/2 gene and pBIG2-ura5s-ITs were digested with Kpnl and Xmal, respectively, and ligated with ligase to obtain the a/ ⁇ expression plasmid pBIG2-ura5s-malE2.
  • the recombinant plasmid pBIG2-ura5s-malE2 was used to transform Agrobacterium tumefaciens, and finally the transformed plasmid containing the plasmid pBIG2-ura5s-malE2 was used to dry the Agrobacterium tumefaciens C58C1 pBIG2-ura5s-malE2.
  • the auxotrophic type was screened and identified by transformed Mortierella alpina, and the phenotype complementary strains MA- a/ ⁇ -l, MA-malE2-2 and MA-malE2-3 were obtained to realize the construction of malic enzyme 2 gene. Homologous overexpression of Mortierella alpina.
  • the present invention provides a M. alpina recombinant gene expression system constructed by transforming a Mortierella alpina ATCC 32222 uracil auxotrophic strain by an ATMT method.
  • the recombinant gene expression system was used to construct the overexpression strain of Malic Enzyme 1 (ME1) and Malic Enzyme 2 (ME2).
  • the plasmid D4 used therein (Mackenzie DA, Wongwathanarat P, Carter AT, et al. Isolation and use of a homologous liistone 114 promoter and a ribosomal DNA region in a transformation vector for the oil-making fungus Mortierella alpiiia [J], Applied And environmental microbiology, 2000, 66(1 1 ): 4655-4661 ), plasmid pBIG2RHPH2 and Agrobacterium tumefaciens C58C1 ( Tsuji G, Fujii S, Fujihara N, et al.
  • the M. alpina uracil auxotrophic strain provides a prerequisite for the genetic manipulation of PUFAs producing strains.
  • the method of the present invention finally obtains a phenotype complementary strain by genetic engineering method based on the existing uracil auxotrophic strain, and realizes the malic enzyme 1 gene and the malic enzyme 2 gene in Mortierella alpina. Homologous overexpression.
  • This complementary strain is of great significance for further study of the relationship between malic enzyme and fatty acid synthesis in Mortierella alpina cells, and can be used as a candidate strain for high-level production of fatty acids.
  • strain preservation information related to the present invention is as follows:
  • Agrobacterium tumefaciens C58C1 pBIG2-ura5s-malEl deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on September 24, 2013, Address 100101, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China The number is CGMCC No. 8250;
  • Agrobacterium tumefaciens C58C1 pBIG2-ura5s-malE2 deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on September 24, 2013, Address 100101, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China The number is CGMCC No. 8261;
  • Agrobacterium tumefaciens C58C1 pBIG2-ura5s-ITs deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on September 24, 2013, Address 100101, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China The number is CGMCC No. 8249.
  • Figure 1 is a schematic representation of the construction of plasmid pBIG2-ura5s-malEl for transformation of Mortierella alpina Figure.
  • Fig. 2 is a schematic view showing the construction of a plasmid pBIG2-ura5s-malE2 for transformation of Mortierella alpina.
  • Figure 3 is an electrophoresis diagram of the identified recombinant strain agarose gel.
  • Figure 4 is an electrophoresis map of the identified recombinant strain agarose gel.
  • Figure 5 is a graph showing the transcription levels, translation levels, enzyme activity assays, and fat group test results of the malEl overexpressing strain ME1.
  • Figure 6 is a graph showing the transcription level, enzyme activity assay and fatty acid test results of malE2 overexpressing strain ME2.
  • Example 1 Bioinformatics analysis of Mortierella alpina ATCC 32222 genome Protein coding sequence predicted by M. alpina ATCC32222 genomic information (DDBJ/EMBL/GenBank accession ADAG00000000, first version ADAGO 1000000) by BLAST against protein database NR (www .ncbi.nlm.nih.gov) , KOGs and COGs, KEGG, UniReflOO and Swiss-Prot, BRENDA search alignment. The protein structure database was aligned using InterProScan. It is predicted that the coding sequence of the 5 gene coding OPRTase is 654 bp in length. The predicted coding sequence of the malEl gene encoding ME1 was 1752 bp in length. The predicted a/2 gene coding sequence encoding ME2 was 1857 bp in length.
  • Total RNA was dissolved in enzyme-free water and stored at -80 °C.
  • URA5F ACATCATGACCATCAAGGAATACCAGCGCG
  • malElY CATGCGTCATGACTGTCAGCGAAAACACC
  • TACGCGGATCCTTAGAGGTGAGGGGCAAAGG ATCGGGGTACCATGTTGAGGAATCCTGCTCTCA malE2R TAATTCCCCCGGGTCAGGGGTGCGATTCCAG ITF:
  • PCR product was ligated to pEGM-T easy (Promega, Mandison, WI, USA) vector, identified by 3730 sequencing, and transformed into E. coli TOP10 and stored at -80 °C.
  • pEGM-T easy Promega, Mandison, WI, USA
  • the HPH expression unit was obtained by PCR.
  • HPH expression unit and plasmid ET28a were digested with restriction endonucleases EcoR I and Xba I, and the kit was recovered and ligated using T4 ligase.
  • the ligation system was ( ⁇ : HPH expression unit 2 L, vector l L, 10xT4 ligase buffer 1 ⁇ , ⁇ 4 ligase ⁇ , sterile water 5 L, overnight at 4 °C.
  • the ligation product was transformed into E. coli TOP10 competent state.
  • the conversion method is as follows:
  • the results showed that the connections were successful.
  • the plasmid pET28a-HPHs was obtained.
  • the ura5 gene fragment and the plasmid pET28a-HPHs were digested with the restriction enzymes BspHI and BamHI, Ncol and BamHI. After the kit was recovered, the T4 ligase was used for ligation, transforming the TOP10 competent state, picking up the positive transformant, and extracting the plasmid. , sequencing verification, the results show that the connection is successful.
  • the plasmid pET28a-ura5s was obtained.
  • the PCR reaction was carried out using the primers HPH and HPHR using the plasmid pET28a-ura5s as a template.
  • the ura5s expression unit was obtained.
  • the ura5 expression unit and plasmid pBIG2RHPH2 were digested with restriction endonucleases Spel and Xbal, Xbal, and the kit was recovered, ligated with T4 ligase, transformed into TOP10 competent state, picked positive transformants, extracted plasmid, and verified by sequencing. The results indicate that the connection was successful.
  • the plasmid pBIG2-ura5s was obtained.
  • the SOC resuscitation medium is composed of 20g/L Tryptone, 5g/L yeast powder, 0.5g/L NaCl, 2.5mM KCl, 10mM MgCl, 2. 20mM glucose; YEP solid medium is component 10g /L Tryptone, 10g/L yeast powder, 5g/L NaCl, 20g/L agar.
  • the malEl gene fragment and the plasmid pET28a-HPHs were digested with restriction endonucleases BspHI and BamHI, Ncol and BamHI. After the kit was recovered, the T4 ligase was used for ligation, and the TOP10 competent state was transformed, and the positive transformants were picked and extracted. The plasmid was verified by sequencing and the results showed that the connection was successful. The plasmid pET28a-malEl was obtained.
  • the PCR reaction was carried out using the primers HPH and HPHR using the plasmid pET28a-malEl as a template.
  • the malEl expression unit was obtained.
  • the malEl expression unit and the plasmid pBIG2-ura5s were digested with the restriction enzymes Spel and Xbal, Xbal, and the kit was recovered, ligated with T4 ligase, transformed into TOP10 competent state, picked positive transformants, extracted plasmid, and sequenced. Verify that the connection is successful.
  • the plasmid pBIG2-ura5s-malE 1 was obtained.
  • Example 6 Construction of the universal vector pBIG2-ura5s-ITs and the ME2 expression plasmid pBIG2-ura5s-malE2 of the M. alpina gene operation
  • the primers ITF and ITR were used to carry out a PCR reaction using the M. alpina genome as a template to obtain an intron DNA fragment IT.
  • the IT gene fragment and the plasmid pET28a-HPHs were digested with the restriction enzymes Ncol and BamHI respectively. After the kit was recovered, the IT fragment was replaced with the plasmid pET28a-HPHs by the ligation reaction to obtain the plasmid pET28a-ITs.
  • the plasmid pET28a-ITs was digested with the restriction enzymes Spel and Xbal dihydric alcohol, and the ITs expression unit was recovered by the kit.
  • the ITs expression unit was inserted into the Xbal alcohol-cut plasmid pBIG2-ura5s by ligase ligation, and the TOP10 competent state was transformed, the positive transformant was picked, the plasmid was extracted, and the result was confirmed by sequencing. Obtaining the M. alpina gene operation universal vector pBIG2-ura5s-ITs.
  • the malE2 gene and pBIG2-ura5s-ITs were digested with Kpnl and Xmal respectively, ligated with ligase, transformed into TOP10 competent state, positive transformants were picked, plasmids were extracted, and sequencing was confirmed. The results showed that the ligation was successful.
  • the malE2 expression plasmid pBIG2 -ura5 s-malE2 was obtained.
  • Example 7 Agrobacterium tumefaciens-mediated transformation of Mortierella alpina
  • the cells were collected by centrifugation at 4000 g for 5 minutes, and the supernatant was decanted. The cells were resuspended in 5 mL of IM medium, centrifuged at 4000 g for 5 minutes, and the supernatant was decanted. The cells were resuspended in 2 mL of IM medium.
  • the cellophane was transferred to a GY plate containing 100 g/mL spectinomycin, 100 g/mL cefotaxime. Incubate at 25-30 ° C until colonies with obvious growth are produced.
  • the composition of MM medium is 1.74g/L ⁇ 2 ⁇ 0 4 , 1.37g/L KH 2 P0 4 , 0.146g/L NaCl , 0.49g/L MgS0 4 -7H 2 , 0.078g/L CaCl 2 0.0025 g/L FeSO 4 -7H 2 O , 0.53 g/L (NH 4 ) 2 S0 4 , 7.8 g/L MES, 1.8 g/L glucose, 0.5% glycerol.
  • the IM medium was composed of 200 ⁇ M acetosyringone (AS) added to the MM medium.
  • the SC medium is a component 5g/L Yest Nitrogen Base w/o Amino Acids and Ammonium Sulfate, 1.7g/L (NH 4 ) 2 S0 4 , 20g/L glucose, 20mg/L adenine, 30mg/L Tyrosine Acid, lmg/L Methionine ⁇ ⁇ acid, 2mg/L Histidine group acid, 4mg/L Lysine lysine, 4mg/L Tryptophan chromic acid, 5mg/L Threonine sucrose, 6mg/L Isoleucine Amino acid, 6mg/L Leucine bright acid, 6mg/L Phenylalanine phenylalanine, 2mg/L Arginine arginine is a component.
  • Example 8 Screening and identification of recombinant strains
  • Colonies apparently grown in (4) were inoculated separately onto GY solid plates containing 1 mg/mL 5-FOA and 1 mg/mL 5-FOA. Incubate at 25 °C for 2-4 days.
  • TrpCRl CAAATGAACGTATCTTATCGAGATCC
  • TrpCR2 AGGCACTCTTTGCTGCTTGG
  • M is the marker.
  • A is the PCR product of the primers HisproFl and TrpCRl;
  • B is the PCR product of the primers HisproF2 and TrpCR2.
  • M is a wild-type control, MAU1 is a recipient strain control, and no PCR is used for PCR reaction with two pairs of primers.
  • pBIG2-ura5s and pBIG2-ura5s-malEl are positive controls for plasmids;
  • MAUC1, MAUC2, and MAUC3 are recombinant strains transformed with pBIG2-ura5s, and primers A and B can be used to amplify 818 bp and 861 bp, respectively.
  • MA-maH, MA-malEl-2, MA-malEl-3 is a recombinant strain transformed with pBIG2-ura5s-malEl, which can be expanded with primer pairs A and B, respectively.
  • pBIG2-ura5s-malE2 is a positive control using plasmid as a template
  • Mk-malE2- 1 , MA-malE2-2, MA-malE2-3 is a recombinant strain transformed with pBIG2-ura5s-malE2, using primer pair A
  • Two product bands were amplified by B and B: 818 bp, 2021 bp, and 861 bp, 2064 bp, which were consistent with the positive control using plasmid pBIG2-ura5s-malE2 as a template.
  • the cDNA of the recombinant strain was obtained by operating according to the description in Example 2 and Example 3.
  • the RT-qPCR reaction was carried out using an ABI-Prism 7900 sequence detection system (Applied Biosystems, CA) according to the instructions of SYBR Green PCR Master Mix (Applied Biosystems, CA).
  • the reaction system was: 10 ⁇ SYBR Green PCR Master Mix, 0.5 ⁇ for each of the two primers, 8 ⁇ without enzyme water, and 1 ⁇ template.
  • the PCR cycle was set to 50 ° C for 2 min, 95 ° C lO min, 40 cycles. 18S rRNA is used as an internal reference gene. All samples were tested in three replicates. The result is shown in Fig. 5A. M.
  • alpina is a wild-type control
  • MAUI, MAU2, and MAU3 are receptor strain controls
  • MAUC1, MAUC2, and MAUC3 are pBIG2-ura5s recombinant strains, and the expression of malEl is not affected by the ura5 selectable marker gene
  • MA-malEl-l, MA-malEl-2 and MA-malEl-3 were pBIG2-ura5s-malEl recombinant strains, and the expression levels of the mallEl gene were significantly higher than those of the control strains.
  • M is a wild-type control
  • MAU1 is a recipient strain control
  • MA-a/ ⁇ -l is a recipient strain control
  • MA-a/ ⁇ -2 is a recombinant strain transformed with pBIG2-ura5s-malE2
  • the a/2 gene expression levels shown were significantly higher than the control strains.
  • Example 10 Intracellular ME1 protein of recombinant strains Western Blot assay
  • Liquid nitrogen-milled cells extract total cellular protein.
  • the PVDF membrane is immersed in 5% skim milk powder and incubated on a horizontal shaker at room temperature for 30-40 min.
  • the PVDF membrane was immersed in TBST buffer for 10 min at room temperature on a horizontal shaker. repeat three times.
  • the primary antibody with ME1 specificity (prepared by Shanghai Bioengineering Co., Ltd. according to the ME1 protein sequence) was dissolved in TBST at a ratio of 1:3000 on a horizontal shaker to incubate the PVDF membrane for 1 h.
  • the PVDF membrane was immersed in TBST buffer for 10 min at room temperature on a horizontal shaker. repeat three times.
  • the PVDF film was developed by the ECL method. Exposure to film in a dark room.
  • M. alpina is a wild type control; MAUI, MAU2 and MAU3 are receptor strain controls; MAUC1, MAUC2, MAUC3 are pBIG2-ura5s recombinant strains; MA-malEl-1, MA-malEl-2 and MA-malEl-3 are pBIG2-ura5s-malEl recombinant strain.
  • MA-malEl-l, MA-malEl-2, and MA-malEl-3 were significantly higher than those of the recipient control strain and the pBIG2-ura5s recombinant strain.
  • Example 11 Intracellular ME activity assay of recombinant strains
  • Liquid nitrogen-milled cells extract total cellular protein.
  • M. alpina is a wild type control; MAUI, MAU2 and MAU3 are receptor strain controls; MAUC1, MAUC2, MAUC3 are pBIG2-ura5s recombinant strains; MA-malEl-1, MA-malEl-2 and MA-malEl-3 are pBIG2-ura5s-malE 1 recombinant strain.
  • M alpina is a wild type control; MAU1 is a receptor strain control; MA-malE24, MA-malE2-2, and MA-malE2-3 are recombinant strains transformed with pBIG2-ura5s-malE2.
  • ME activity was significantly increased in all over-expressed strains of oversera.
  • the Prototheca sinensis prototrophic strain and the recombinant strain were cultured in a fermentation medium at 28 ° C, 500 rpm in a 5 L fermentor for 144 hours.
  • the total fatty acid content of the recombinant strains MA-malEl-1, MA-malEl-2, and MA-malEl-3 was 30% higher than that of the control strain; There is also a certain increase.
  • the total fatty acid content of the recombinant strains MA-malE2-l, MA-malE2-2, MA-malE2-3 was not significantly increased, but the intracellular AA content was significantly increased.
  • the fermentation medium is composed of 50 g/L glucose, 2.0 g/L L-ammonium tartrate, 7.0 g/L KH2P04, 2.0 g/L Na2HP04, 1.5 g/L MgS04-7H20, 1.5 g/L Yeast extract , 0.1 g/L CaC12-2H20 , 8 mg/L FeC13-6H20 , 1 mg/L ZnS04-7H20 , 0.1 mg/L CuS04-5H20 , 0.1 mg/L Co ( N03 ) 2-6H20 , 0.1 mg/L MnS04 -5H20 is composed.

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Abstract

提供一种重组高山被孢霉（Mortierella alpina）菌株及其应用。所述菌株用含苹果酸酶基因的根癌土壤杆菌转化高山被孢霉ATCC 32222的尿嘧啶营养缺陷型菌株构建而成。该重组菌株用于生产脂肪酸。

Description

说 明 书
一种高山被孢審重組基因表达系统及其构建方法和应用 本发明要求申请日为 2013年 10月 30 日、 申请号为 CN 201310524221.4的中国发明专利申请优先权。
【技术领域】
本发明涉及一种高山被孢霉的重组基因表达系统及其构建方法 和应用， 属于生物工程技术领域。
【背景技术】
高山被孢霉是一种重要的产油丝状真菌， 它具有花生四烯酸 ( AA )含量高、 安全、 多不饱和脂肪酸（PUFAs )组成合理等特点， 已经应用于工业生产 AA。 目前对高山被孢霉高产菌株的研究主要集 中在菌种选育和发酵条件优化等方面。由于缺乏一种有效的高山被孢 霉的基因操作系统， 目前还不能对这种极具价值的丝状真菌进行遗传 改造。这就为高山被孢霉脂肪酸合成途径的基础理论研究和基因工程 生产菌株的构建构成了不可逾越的障碍。
丝状真菌的基因操作系统一直落后于其它物种而没有被很好地 建立起来， 主要归咎于丝状真菌难以被转化的特点。 尤其以具有高山 被孢霉这类特点的真菌最难以被转化： 多核， 无隔， 产孢能力低且对 抗生素不敏感。 因此， 国内一直未见这种重要的工业生产微生物遗传 改造的报道。 除了不同丝状真菌种类自身的特点及偏好性， 转化方法 的选择也是决定丝状真菌能否被转化的关键因素。 目前， 丝状真菌的 转化和方法主要由以下几种： 原生质体转化， 电穿孔转化， 基因枪转 化和农杆菌介导转化方法。 其中， 原生质体转化和电穿孔转化需要将 受体的细胞壁降解制备原生质体， 培养难度大，再生频率低且实验周 期长。基因枪转化虽然具有筒单便捷的优点，但是需要较大的受体基 数且转化成本过高。农杆菌的转化技术作为一种最早被应用于植物的 常规转化技术， 早在 20年前就已经被报道具有转化真菌的能力。 到 目前为止，农杆菌的转化技术已经成功的应用到超过一百二十多种真 菌。 和其它转化方法相比， 农杆菌转化方法具有几个突出优点： 受体 细胞广泛，可以是孢子或菌丝，且不需要制备原生质体；转化效率高， 成功率高， 载体可容纳大片段的异源 DNA; 基本上为单拷贝随机插 入宿主染色体中； 可以提高同源重组效率。 因此， 根癌土壤杆菌的转 化方法为高山被孢霉表达系统的构建提供了重要的操作手段。
苹果酸酶（ malic enzyme; EC 1.1.1.40 ), 催化细胞内苹果酸生成 丙酮酸的反应， 是生物体内一种重要的 NADPH产生来源。 早在上世 纪九十年代，苹果酸酶就被推测为产油丝状真菌脂肪酸合成途径中的 重要因素。 在另外一种同属于接合菌亚门的丝状真菌一卷枝毛霉中， 苹果酸酶的活性在被芝麻粉（一种特异性抑制卷枝毛霉苹果酸酶活的 化学抑制剂）抑制的情况下， 细胞内总脂肪含量受到了显著影响。在 英国赫尔大学 Colin教授等人的相关研究基础上， 苹果酸醇被推断为 产油真菌脂肪酸合成过程中的重要限速步骤。 随后， 在对高山被孢霉 发酵过程中一系列产生 NADPH的酶活的系统研究中，苹果酸酶也被 推测与高山被孢霉细胞内脂肪酸合成密切相关。但是， 由于缺乏一种 有效的重组基因表达系统，此种理论在高山被孢霉中一直没有得到验 证和应用。 本发明以申请号为 201310347934.8的专利申请中公开的 Mortierella alpina ATCC 32222 尿嘧啶营养缺陷型菌株作为转化菌 株，在其基础上通过进一步的基因重组方法构建了一种新的能够高表 达重组苹果酸酶的遗传表达系统。 申请号为 201310347934.8的专利 申请所公开的全部内容均引入本申请作为参考。
中国专利申请 201310347934.8公开的技术方案包括一种高山被 孢霉尿嘧啶营养缺陷型菌株， 该菌株是通过失活 Mortierella alpina ATCC 32222基因组中编码乳清酸磷酸核糖转移酶 OPRTase的画5 基因构建而成的。 所述高山被孢霉尿嘧啶营养缺陷型菌株，訓5基因的失活是通过 缺失 654bp的 wra5基因中的 213bp-230bp共 18bp的序列而实现的。
中国专利申请 201310347934.8还公开一种制备上述高山被孢霉 尿嘧啶营养缺陷型菌株的方法， 通过同源重组使高山被孢霉 基 因中的 213bp-230bp共 18bp序列缺失从而使 wra5基因失活， 所使用 的同源臂分别是 wra5基因上游 -1180至 +212的 1393bp和下游 +231 至 +1592的 1362bp的片段， 具体步骤为： 首先获得 wra5敲除基因片 段， 并进一步构建敲除质粒 pBIG4KOura5 , 然后用重组质粒 pBIG4KOura5 转化根癌土壤杆菌， 最后用经转化的含质粒 pBIG4KOura5的根癌土壤杆菌转化高山被孢霉并对转化后的高山被 孢霉进行筛选和鉴定， 获得尿嘧啶营养缺陷型菌株。
所述方法使用的才艮癌土壤干菌为： Agrobacterium tumefaciens C58CL
基因敲除使用的根癌土壤杆菌起始载体为： pBIG2RHPH2。
基因敲除载体构建的具体步骤如下：
1 )用 PCR的方法获得质粒 pBluescript II SK+的 MCS基因片段；
2 ) 用限制性内切酶 EcoR I和 Xba I对 MCS基因片段和质粒 pBIG2RHPH2进行酶切， 并通过连接反应将 MCS基因片段插入质粒 pBIG2RHPH2的 EcoR I和 Xba I位点之间得到质粒 pBIG4;
3 )用融合 PCR的方法获得并连接 wra5基因的上下游序列， 得 到敲除基因片段；
4 ) 用限制性内切酶 EcoR I和 Kpn I对敲除基因片段和质粒 pBIG4进行酶切， 并通过连接反应将敲除基因片段插入质粒 pBIG4 获得 pBIG4KOura5。
优选地， 步骤 3 ) 中的敲除基因片段是通过下述步骤获得的， 首先根据 NCBI数据库设计如下引物
PI : GACCGGAATTCCGACGCTGACATTACACATTTATCC P2:
TGACGGTGGTGCAGGCCAGAGGGCCAAAGATGATGTCGTG CTCAATG
P3:
GTCATT
P4： TGCGGGGTACCCATGCGAATCACAGATATGG
然后以高山被孢霉 ATCC32222基因组为模板， 用引物 PI、 P2 和引物 P3、 P4分别扩增上下游片段， 再以上下游片段为模板， 在反 应体系中加入 PI、 P4进行融合 PCR反应， 获得 KO ura5敲除基因片 段。
优选地， 根据质粒 pBluescript ll SK+的序列信息设计如下引物： MCS上游：
TTTCGCTAGCACGACGTTGTAAAACGACGGCCAGT MCS下游：
AACAACAATTGGGGCTCCACCGCGGTGGCGGCCG
然后用 PCR的方法获得步骤 1 ) 中的质粒 pBluescript ll SK+ 的 MCS基因片段。
优选地所述的根癌土壤杆菌介导的基因敲除方法是采用根癌土 壤杆菌转化高山被孢霉， 具体为： 取 10( L根癌土壤杆菌与 ΙΟΟμ 高山被孢霉孢子液混合， 均匀涂布于铺有玻璃纸 IM固体培养基上， 进行转化培养， 然后筛选获得高山被孢霉尿嘧啶营养缺陷型菌株。
优选地， 根癌土壤杆菌转化高山被孢霉的具体步骤如下：
( 1 )取保存于 -80°C的含有质粒 pBIG4KOura5的根癌土壤杆菌 C58C1于含有 lOO g/mL利福平和 lOO g/mL卡那霉素的 YEP固体培 养基平板划线； 30°C倒置避光培养 48小时；
( 挑取单克隆接种至 20mL含有 lOO g/mL利福平和 lOO g/mL 卡那霉素的液体 YEP培养基中 30°C , 200rpm避光培养 24-48小时；
( 4000g离心 5分钟收集菌体， 倒掉上清， 加 5mL IM培养基重 悬菌体， 4000g离心 5分钟， 倒掉上清， 加 2mL IM培养基重悬菌体；
(4)用 IM培养基调整菌浓度至 OD600=0.9, 30 °C , 200rpm避光培 养至 OD600=1.5 ;
(5)收集高山被孢霉孢子， 用血球计数器计数， 调整孢子浓度到 106个每 10( L;
(6)取 10( L根癌土壤杆菌与 10( L孢子混合， 均匀涂布于铺有 玻璃纸 IM固体培养基上， 23 °C避光培养 48-96小时；
(7)将玻璃纸转移到含有 lOO g/mL壮观霉素， lOO g/mL头孢噻 肟， 0.05g/L尿嘧啶的 GY平板上， 25-30°C培养至产生大量孢子。
中 国 专利 申请 201310347934.8 获得的根癌土壤杆菌 Agrobacterium tumefaciens C58Cl-pBIG4KOura5已于 2013年 06月 17 日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址北京 市朝阳区北辰西路 1号院 3号中国科学院微生物研究所，邮编 100101 , 保藏编号为 CGMCC No. 7730。
【发明内容】
本发明要解决的技术问题是提供一种高山被孢霉的重组基因表 达系统。所述高山被孢霉的重组基因表达系统通过根癌土壤杆菌转化 ( ATMT ) 的方法转化 Mortierella alpina ATCC 32222 尿嘧啶营养缺 陷菌株型来实现的。
所述重组基因表达系统的选择标记 wra_ 基因， 苹果酸酶 1基因 malEl和苹果酸酶 2基因 malE2的 DNA序列来源于 Mortierella alpina ATCC 32222 基 因 组 （ DDBJ/EMBL/GenBank accession ADAG00000000, first version ADAG01000000 ) 中。
本发明还提供了一种构建高山被孢霉重组基因表达系统的方法， 表达单元， 将 ΗΡΗ表达单元用限制性内切酶 EcoR I和 Xbal酶切， 插入到 EcoR I和 Xbal醇切过的 pET28a ( + ) 的多克隆位点 （ MCS ) 中， 得到质粒 pET28a-HPHs。 利用 PCR从高山被孢霉 cDNA中获得 (乳清酸麟酸核糖转移酶； OPRTase )基因， 并利用限制性内切 酶 BspHI和 BamHI酶切 ura5基因，将酶切过的 基因插入到 Ncol 和 BamHI酶切过的质粒 pET28a-HPHs中， 以替换的 基因， 构建 质粒 pET28a-ura5s。 用限制性内切酶 EcoR I和 Xbal酶切质粒 pET28a-ura5s得到 ura5s表达单元。 将 ura5s表达单元替换质粒 pBIG2RHPH2 中的 HPH表达单元， 进一步构建质粒转化质粒 pBIG2-ura5s, 然后用重组质粒 pBIG2-ura5s转化根癌土壤杆菌， 最后 用经转化的含质粒 pBIG2-ura5s的根癌土壤杆菌转化高山被孢霉尿嘧 啶营养缺陷型并对转化后的高山被孢霉进行筛选和鉴定，获得表型互 补菌株， 从而实现高山被孢霉的遗传转化。
进一步在质粒 pBIG2-ura5s的基础上， 构建苹果酸酶 1过表达载 体。用 PCR的方法从高山被孢霉 cDNA中获得苹果酸酶 1基因 malEl。 用限制性内切酶 BspHI和 BamHI, Ncol和 BamHI分别对 malEl基因 片段和质粒 pET28a-HPHs进行酶切， 并通过连接反应将 malEl基因 片段插入质粒 pET28a-HPHs的 Ncol和 BamHI位点之间得到质粒 pET28a-malEl。 用 限制性内切酶 Spel 和 Xbal 双酶切质粒 pET28a-malEl ,得到 malEl表达单元。将 malEl表达单元插入到 Xbal 酶切过的质粒 pBIG2-ura5s中，得到质粒 pBIG2-ura5s-malEl。然后用 重组质粒 pBIG2-ura5s-malEl转化根癌土壤杆菌， 最后用经转化的含 质粒 BIG2-ura5s-malE 1的才艮癌土壤干菌 Agrobacterium tumefaciens C58C1 pBIG2-ura5s-malEl转化高山被孢霉尿嘧啶营养缺陷型并对转 化后的高山被孢霉进行筛选和鉴定， 获得表型互补菌株高山被孢霉 MA-malEl-\ , MA-malEl-2和 MA- a/ -3 , 从而实现构建苹果酸酶 1基因在高山被孢霉的同源过量表达。
更进一步的在质粒 pBIG2-ura5s和质粒 pET28a-HPHs的基础上， 构建高山被孢霉基因操作通用载体。具体步骤如图 2 ,用 PCR的方法 从高山被孢霉基因组中获得非编码的内含子 DNA片段 IT。用限制性 内切酶 Ncol和 Bamffl分别对 IT基因片段和质粒 pET28a-HPHs进行 酶切，并通过连接反应将 IT片段取代质粒 pET28a-HPHs的 基因， 得到质粒 pET28a-ITs。 用限制性内切酶 Spel和 Xbal双酶切质粒 pET28a-ITs得到 ITs表达单元。将 ITs表达单元插入到 Xbal酶切过的 质粒 pBIG2-ura5s中，高山被孢霉基因操作通用载体 pBIG2-ura5s-ITs。
再进一步， 在高山被孢霉基因操作通用载体 pBIG2-ura5s-ITs的 基础上， 构建苹果酸酶 2过表达载体。 分别用 Kpnl和 Xmal双酶切 a/ 2基因和 pBIG2-ura5s-ITs, 用连接酶进行连接，得到 a/^表达 质粒 pBIG2-ura5s-malE2。然后用重组质粒 pBIG2-ura5s-malE2转化根 癌土壤杆菌， 最后用经转化的含质粒 pBIG2-ura5s-malE2的根癌土壤 干菌 Agrobacterium tumefaciens C58C1 pBIG2-ura5s-malE2转 ^匕高山 被孢霉尿嘧啶营养缺陷型并对转化后的高山被孢霉进行筛选和鉴定， 获得表型互补菌株 MA- a/^-l、 MA-malE2-2和 MA-malE2-3 , 从而 实现构建苹果酸酶 2基因在高山被孢霉的同源过量表达。
具体地， 本发明提供一种高山被孢霉重组基因表达系统， 该系统 是通过 ATMT的方法转化 Mortierella alpina ATCC 32222尿嘧啶营养 缺陷型菌株构建成的。并且利用这一重组基因表达系统构建了高山被 孢霉苹果酸酶 1 ( malic enzyme 1 ; ME1 )和苹果酸酶 2 ( malic enzyme 2; ME2 )过量表达菌株。
其中所使用的质粒 D4( Mackenzie D A， Wongwathanarat P, Carter A T, et al. Isolation and use of a homologous liistone 114 promoter and a ribosomal DNA region in a transformation vector for the oil- producing fungus Mortierella alpiiia[J], Applied and environmental microbiology, 2000, 66(1 1 ): 4655-4661 )， 质粒 pBIG2RHPH2 和 Agrobacterium tumefaciens C58C1 ( Tsuji G, Fujii S, Fujihara N, et al. Agrobacterium turne facien s-rnediated transformation for random insertional mutagenesis in Colletotrichum iageiiarimn[J], Journal of General Plant Pathology, 2003, 69(4): 230 239。 ) 均为公开获得。
高山被孢霉尿嘧啶营养缺陷型菌株为 PUFAs生产菌株的基因操 作提供了先决条件。本发明的方法在现有的尿嘧啶营养缺陷型菌株的 基础上， 通过基因工程方法最终获得了表型互补菌株， 实现了苹果酸 酶 1基因和苹果酸酶 2基因在高山被孢霉中的同源过量表达。该互补 菌株对于进一步研究苹果酸酶与高山被孢霉细胞内脂肪酸的合成的 相互关系具有重要意义， 可用作高水平生产脂肪酸的候选菌株。
本发明的涉及的菌种保藏信息如下：
Agrobacterium tumefaciens C58C1 pBIG2-ura5s-malEl , 于 2013 年 9月 24日保藏于中国微生物菌种保藏管理委员会 普通微生物中 心，地址 100101北京市朝阳区北辰西路 1号院 3号 中国科学院微生 物研究所， 保藏编号为 CGMCC No. 8250;
Agrobacterium tumefaciens C58C1 pBIG2-ura5s-malE2 , 于 2013 年 9月 24日保藏于中国微生物菌种保藏管理委员会 普通微生物中 心，地址 100101北京市朝阳区北辰西路 1号院 3号 中国科学院微生 物研究所， 保藏编号为 CGMCC No. 8261 ;
Agrobacterium tumefaciens C58C1 pBIG2-ura5s-ITs, 于 2013年 9 月 24日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地 址 100101北京市朝阳区北辰西路 1号院 3号 中国科学院微生物研究 所， 保藏编号为 CGMCC No. 8249。
【附图说明】
图 1为构建转化高山被孢霉所用质粒 pBIG2-ura5s-malEl的示意 图。
图 2为构建转化高山被孢霉所用质粒 pBIG2-ura5s-malE2的示意 图。
图 3为鉴定的重组菌株琼脂糖凝胶电泳图，
图 4为鉴定的重组菌株琼脂糖凝胶电泳图。
图 5为 malEl过表达菌株 MEl转录水平、 翻译水平、 酶活检测 及脂肪组检测结果图。
图 6为 malE2过表达菌株 ME2转录水平， 酶活检测及脂肪酸检 测结果图。
【具体实施方式】
以下通过实施例来进一步阐述本发明，下例实施例中未注明具体 条件的实验方法， 基本上都按照常见的分子克隆手册进行实验操作。
实施例 1 : 高山被孢霉 ATCC 32222基因组生物信息学分析 根据高山被孢霉 ATCC32222基因组信息（ DDBJ/EMBL/GenBank accession ADAG00000000, first version ADAGO 1000000 )预测的蛋白 质编码序列经 BLAST对蛋白数据库 NR (www.ncbi.nlm.nih.gov) , KOGs 和 COGs , KEGG , UniReflOO和 Swiss-Prot, BRENDA搜索 比对。 使用 InterProScan对蛋白质结构数据库进行比对。 预测得到编 码 OPRTase的画5基因 coding序列全长 654bp。 预测得到的编码 ME1的 malEl基因 coding序列全长 1752bp。 预测得到的编码 ME2 的 a/ 2基因 coding序列全长 1857bp。
实施例 2: 高山被孢霉总 RNA提取
(1)取出适量在液氮中冻存的菌体于无菌无醇研钵中充分研磨。
(2)加入 TRIzol (Invitrogen, Carlsbad, CA, USA) 试剂 1 mL继续研 磨后室温放置至溶解。
( 吸取 1 mL( 中液体于无酶离心管中，加入 20(^L三氯曱烷混均。 (4)12000 ΐη, 4°C , 离心 15 min吸上清于新的无酶离心管中。 (5)加入等体积异丙醇,静置 15 min, 12000 rpm, 4°C ,离心 15 min。
(6)无醇枪头吸取去出异丙醇尽量吸干。
(7)沉淀用 70%乙醇洗一次， 12000 rpm, 4°C , 离心 15 min。
(8)无酶水溶解总 RNA, -80°C储存。
( 浓度测定：取 2 μ 总 RNA于离心管中 Nanodrop2000测定浓度。 (10)跑胶:取 1 总 RNA跑 1.2% 琼脂糖电泳检测总 RNA完整性。 实施例 3： 获得層5基因， malEl基因和 malE2基因以及 IT片 段
(1)取 0.5-^g 总 RNA为模板， 根据 PrimeScript RT reagent kit (TaKaRa, Otsu, Shiga, Japan) 试剂盒说明进行操作， 获得高山被孢霉 cDNA。
(2)根据基因组生物信息学分析结果， 针对预测的 Mra_ 基因， malEl基因和 malE2以及 IT基因编码序列设计引物 （酶切位点用下 划线表示）：
URA5F: ACATCATGACCATCAAGGAATACCAGCGCG
URA5R: TCGGGATCCCTAAACACCGTACTTCTCC
malElY: CATGCGTCATGACTGTCAGCGAAAACACC malEIR TACGCGGATCCTTAGAGGTGAGGGGCAAAGG malE2¥： ATCGGGGTACCATGTTGAGGAATCCTGCTCTCA malE2R TAATTCCCCCGGGTCAGGGGTGCGATTCCAG ITF:
GCATGCCATGGAGAAGCTTGGTACCGCTAGCTCCCAAGCG AATTTGTCATCTCG ITR:
CGCGGATCCGAGCTCCCCGGGGGACTCGAGAGCATACGGA AGTCCATCAGTTACG
(3)以 cDNA为模板， 用以上两对引物进行 PCR反应， 得到 基因和 a/ 基因。
(4)将得到的 PCR产物连接到 pEGM-T easy (Promega, Mandison, WI, USA) 载体上，经 3730测序鉴定后，转化大肠杆菌 TOP10中 -80°C 保存。
实施例 4: 选择标记质粒 pBIG2-ura5s的构建
根据质粒 pD4的序列信息设计引物：
HPHF:
GAGACGAATTCGCCCGTACGGCCGACTAGTTTTAGTTGATG TGAG
HPHR:
TGGG
用 PCR的方法获得 HPH表达单元。
用限制性内切酶 EcoR I和 Xba I对 HPH表达单元和质粒 ET28a 进行双酶切，试剂盒回收后，使用 T4连接酶连接。连接体系为（ ΙΟμυ: HPH表达单元 2 L, 载体 l L, 10xT4连接酶 buffer 1 μΐ, Τ4连接酶 ΙμΙ, 无菌水 5 L, 4 °C过夜连接。
连接产物转化大肠杆菌 TOP10感受态。 转化方法如下：
(1)无菌状态下取 10( L感受态细胞， 加入 2 L连接产物， 混匀。
(2)将 (1)中感受态移入电转杯中， 避免气泡。
(3)将电转杯放入 Bio-Rad电转仪， 调到预设程序档位， 电转。
(4)电转后的感受态移至含有 900μΙ SOC复苏培养基的离心管中， 37 °C , lOOrpm 1小时。
(5)取 20(^L涂布 lOO g/mL卡那霉素抗性 YEP固体培养基平板。 倒置 37°C培养过夜。
挑取阳性转化子， 提取质粒， 测序验证， 结果表明连接成功。获 得质粒 pET28a-HPHs。 用限制性内切酶 BspHI和 BamHI , Ncol和 BamHI对 ura5基因 片段和质粒 pET28a-HPHs进行酶切， 试剂盒回收后， 使用 T4连接酶 连接， 转化 TOP10感受态， 挑取阳性转化子， 提取质粒， 测序验证， 结果表明连接成功。 获得质粒 pET28a-ura5s。
利用引物 HPHF和 HPHR,以质粒 pET28a-ura5s为模板进行 PCR 反应。 得到 ura5s表达单元。
用限制性内切酶 Spel和 Xbal , Xbal对 ura5表达单元和质粒 pBIG2RHPH2进行酶切， 试剂盒回收后， 使用 T4连接酶连接， 转化 TOP10感受态， 挑取阳性转化子， 提取质粒， 测序验证， 结果表明连 接成功。 获得质粒 pBIG2-ura5s。
其中， SOC复苏培养基是以组分 20g/L Tryptone, 5g/L酵母粉， 0.5g/L NaCl, 2.5mM KCl, 10mM MgCl, 2. 20mM 葡萄糖构成的； YEP 固体培养基是以组分 10g/L Tryptone, 10g/L 酵母粉， 5g/L NaCl， 20g/L琼脂构成的。
实施例 5: ME1表达质粒 pBIG2-ura5s-malEl的构建
用限制性内切酶 BspHI和 BamHI , Ncol和 BamHI对 malEl基因 片段和质粒 pET28a-HPHs进行双酶切， 试剂盒回收后， 使用 T4连接 酶连接， 转化 TOP10感受态， 挑取阳性转化子， 提取质粒， 测序验 证， 结果表明连接成功。 获得质粒 pET28a-malEl。
利用引物 HPHF和 HPHR,以质粒 pET28a-malEl为模板进行 PCR 反应。 得到 malEl表达单元。
用限制性内切酶 Spel和 Xbal , Xbal对 malEl表达单元和质粒 pBIG2-ura5s进行酶切， 试剂盒回收后， 使用 T4连接酶连接， 转化 TOP10感受态， 挑取阳性转化子， 提取质粒， 测序验证， 结果表明连 接成功。 获得质粒 pBIG2-ura5s-malE 1。
实施例 6 : 高山被孢霉基因操作通用载体 pBIG2-ura5s-ITs以及 ME2表达质粒 pBIG2-ura5s-malE2的构建 利用引物 ITF和 ITR以高山被孢霉基因组为模板进行 PCR反应， 获得内含子 DNA片段 IT。
用限制性内切酶 Ncol和 BamHI分别对 IT基因片段和质粒 pET28a-HPHs进行酶切， 试剂盒回收后， 通过连接反应将 IT片段取 代质粒 pET28a-HPHs的 基因， 得到质粒 pET28a-ITs。
用限制性内切酶 Spel和 Xbal双醇切质粒 pET28a-ITs, 试剂盒回 收得到 ITs表达单元。 通过连接酶连接， 将 ITs表达单元插入到 Xbal 醇切过的质粒 pBIG2-ura5s中，转化 TOP10感受态，挑取阳性转化子， 提取质粒， 测序验证， 结果表明连接成功。 获得高山被孢霉基因操作 通用载体 pBIG2-ura5s-ITs。
分别用 Kpnl和 Xmal双酶切 malE2基因和 pBIG2-ura5s-ITs , 用 连接酶进行连接， 转化 TOP10感受态， 挑取阳性转化子， 提取质粒， 测序验证， 结果表明连接成功。 得到 malE2 表达质粒 pBIG2 -ura5 s-malE2。
实施例 7: 根癌土壤杆菌介导转化高山被孢霉
在已有的国内外文献有关根癌土壤杆菌转化方法报道的基础上， 做了适当的优化调整， 具体成功实施例如下：
(1)取保存于 -80°C的含有质粒 pBIG2-ura5s或 pBIG2-ura5s-malEl 的根癌土壤杆菌 C58C1于含有 lOO g/mL利福平和 lOO g/mL卡那霉 素的 YEP固体培养基平板划线。 30 °C倒置避光培养 48小时。
( 挑取单克隆接种至 20mL含有 lOO g/mL利福平和 lOO g/mL 卡那霉素的液体 YEP培养基中 30°C , 200rpm避光培养 24-48小时。
( 4000g离心 5分钟收集菌体， 倒掉上清。 加 5mL IM培养基重 悬菌体， 4000g离心 5分钟， 倒掉上清。 加 2mL IM培养基重悬菌体。
(4)用 IM培养基调整菌浓度至 OD600=1.0。 30 °C , 200rpm避光培 养至 OD600=1.5。
(5)收集高山被孢霉尿嘧啶营养缺陷型菌株 （ 申请号为 201310347934.8的专利申请中公开的 Mortierella alpina ATCC 32222 尿嘧啶营养缺陷型菌株）孢子， 用血球计数器计数， 调整孢子浓度到 每 ΙΟΟμΙ Ο7个。
(6)取 10( L根癌土壤杆菌与 10( L孢子混合， 均匀涂布于铺有 玻璃纸 IM固体培养基上。 23 °C避光培养 48-96小时。
(7)将玻璃纸转移到含有 lOO g/mL壮观霉素， lOO g/mL头孢噻 肟的 GY平板上。 25-30°C培养至有明显生长的菌落产生。
(8)及时挑取明显生长的菌落， 转移至含有 lOO g/mL壮观霉素， lOO g/mL头孢噻肟的 SC平板上， 待鉴定。
其中， MM培养基成分是以组分 1.74g/L Κ2ΗΡ04 , 1.37g/L KH2P04 , 0.146g/L NaCl , 0.49g/L MgS04-7H2 , 0.078g/L CaCl2 , 0.0025g/L FeSO4-7H2O , 0.53g/L (NH4)2S04 , 7.8g/L MES , 1.8g/L葡萄 糖， 0.5% 甘油构成的。 IM培养基是在 MM培养基的基础上添加 200μΜ乙酰丁香酮 （AS ) 构成的。 SC培养基是以组分 5g/L Yest Nitrogen Base w/o Amino Acids and Ammonium Sulfate , 1.7g/L (NH4)2S04, 20g/L 葡萄糖， 20mg/L腺嘌呤， 30mg/L Tyrosine络氨酸， lmg/L Methionine曱疏氛酸, 2mg/L Histidine 组氛酸, 4mg/L Lysine 赖氛酸, 4mg/L Tryptophan 色氛酸, 5mg/L Threonine 苏氛酸, 6mg/L Isoleucine 异亮氛酸, 6mg/L Leucine 亮氛酸, 6mg/L Phenylalanine 苯 丙氨酸， 2mg/L Arginine精氨酸为组分构成的。
实施例 8: 重组菌株的筛选和鉴定
(1)将挑取在 SC平板上的菌落于 25-30°C培养 3-5天，至明显生长。
(2)挑取菌落边缘的新生菌丝，接种于新鲜的含有 lOO g/mL壮观 霉素， lOO g/mL头孢噻肟的 SC平板上直至产孢子。
(3)用 3mL生理盐水沖刷共培养的平 JDi表面， 收集液体于一个无 菌 1.5mL离心管中。 过 25μηι滤膜。
(4)取 200μΙ^涂布于新鲜的含有 lOO g/mL壮观霉素， lOO g/mL 头孢噻肟的 SC平板上直至产孢子。 共传代 3次。
(5)将 (4)中明显生长的菌落分别接种到含有 lmg/mL 5-FOA和不 含有 lmg/mL 5-FOA的 GY固体平板上。 25 °C培养 2-4天。
(6)观察高山被孢霉在两种平板上的生长情况。 挑出不在 lmg/mL 5-FOA平板上生长的菌落， 接种于 GY斜面上。
(7)提取具有尿嘧啶营养缺陷型表型高山被孢霉基因组。设计两对 与启动子和终止子特异性结合的引物进行 PCR验证：
HisproF 1： CACACACAAACCTCTCTCCCACT
TrpCRl : CAAATGAACGTATCTTATCGAGATCC
HisproF2: GTGTTCACTCGCATCCCGC
TrpCR2: AGGCACTCTTTGCTGCTTGG
进行 PCR反应， 鉴定为重组菌株（图 3 , 4 )。 M为 marker。 A 为引物 HisproFl和 TrpCRl的 PCR产物； B为引物 HisproF2和 TrpCR2 的 PCR产物。 M 为野生型对照， MAU1为受体菌株对照， 用 两对引物进行 PCR反应均无产物。 图 3 中 pBIG2-ura5s 和 pBIG2-ura5s-malEl是质粒为模板的阳性对照； MAUC1 , MAUC2, MAUC3为 pBIG2-ura5s转化的重组菌株，用引物对 A和 B分别可扩 增出 818bp和 861bp的条带， 与以质粒 pBIG2-ura5s为模板的阳性对 照 一 致 ； MA-maH , MA-malEl-2 , MA-malEl-3 为 pBIG2-ura5s-malEl 转化的重组菌株，用引物对 A和 B分别可扩增出 两条产物条带： 818bp、 1916bp和 861bp、 1959bp , 与以质粒 pBIG2-ura5s-malEl 为模板的 阳 性对照一致 。 图 4 中 ， pBIG2-ura5s-malE2是以质粒为模板的阳性对照； Mk-malE2- 1 , MA-malE2-2 , MA-malE2-3 是 pBIG2-ura5s-malE2 转化的重组菌株， 用引物对 A和 B分别可扩增出两条产物条带： 818bp、2021bp和 861bp、 2064bp, 与以质粒 pBIG2-ura5s-malE2为模板的阳性对照一致。
(8)重组菌株保藏于 GY斜面上。 实施例 9： 阳性转化子的 malEl基因和 a/ 2基因转录水平 RT-qPCR检测
根据预测的 mcdEl基因、 mcdE2基因序列和内参 18SrDNA序列 设计引物：
malEl TV: GGCTGTTGCCGAAGGGACT
malEl T : GGCAAAGGTGGTGCTGATTTC
malE2RTF CCTTGCAGGACCGTAACGAGA
malE2RTR CCTGGAGCGACGATAAATGGA
18SRTF: CGTACTACCGATTGAATGGCTTAG
18SRTR: CCTACGGAAACCTTGTTACGACT
根据实施例 2和实施例 3中的描述进行操作获得重组菌株的 cDNA。 使用 ABI-Prism 7900 sequence detection system (Applied Biosystems, CA) 按照 SYBR Green PCR Master Mix (Applied Biosystems, CA) 的说明进行 RT-qPCR反应。 反应体系为： 10 μΐ SYBR Green PCR Master Mix, 两种引物各 0.5 μΐ, 8 μΐ无酶水， 1 μΐ模 板。 PCR循环设置为 50°C 2 min, 95°C lO min, 40个循环。 18S rRNA 作为内参基因。 所有样品测三个重复。 结果如图 5A所示。 M. alpina 为野生型对照； MAUI , MAU2 , MAU3为受体菌株对照； MAUC1 , MAUC2, MAUC3为 pBIG2-ura5s重组菌株， 所示 malEl表达量未受 ura5选择标记基因影响； MA-malEl-l , MA-malEl-2 , MA-malEl-3 为 pBIG2-ura5s-malEl重组菌株， 所示 malEl基因表达量均明显高于对 照菌株。 如图 6A所示， M 为野生型对照； MAU1为受体菌株 对照； MA- a/^-l , MA- a/^-2 , MA-malE2-3为 pBIG2-ura5s-malE2 转化的重组菌株， 所示 a/ 2基因表达量均明显高于对照菌株。
实施例 10: 重组菌株细胞内 ME1蛋白 Western Blot检测
(1)液氮研磨菌体提取细胞总蛋白。
(2)测定蛋白浓度后， 点样量 10 每泳道于 Bio-Rad电泳仪中跑 SDS-PAGE电泳， 至 Marker完全分离。
(3)于 Bio-Rad电泳仪中将蛋白凝胶中的蛋白转移到 PVDF膜上。 转膜条件 50V, 3h。
(4)转膜完成后， 将 PVDF膜浸泡在 5%脱脂奶粉中于水平摇床上 室温孵育 30-40min。
(5)将 PVDF膜浸泡在 TBST緩沖液中于水平摇床上室温孵育 10min。 重复三次。
(6)将具有 ME1特异性的一抗 （由上海生物工程有限公司根据 ME1蛋白质序列制备） 以 1 :3000的比例溶于 TBST中于水平摇床上 孵育 PVDF膜 lh。
(7)将 PVDF膜浸泡在 TBST緩沖液中于水平摇床上室温孵育 10min。 重复三次。
(8)将羊抗兔二抗以 1 :5000用 5%脱脂乳稀释孵育 PVDF膜 lh。
(9)将 PVDF膜浸泡在 TBST緩沖液中于水平摇床上室温孵育 10min。 重复三次。
(10)将 PVDF膜用 ECL法显影。 在暗室中用胶片曝光。
结果如图 5B所示。其中 M. alpina为野生型对照； MAUI , MAU2 , MAU3为受体菌株对照； MAUC1 , MAUC2, MAUC3为 pBIG2-ura5s 重 组 菌 株 ； MA-malEl-l , MA-malEl-2 , MA-malEl-3 为 pBIG2-ura5s-malEl重组菌株。 由图中结果可以看出， 重组菌株 MA-malEl-l , MA-malEl-2 , MA-malEl-3中的 MEl蛋白的水平明显 高于受体对照菌株和 pBIG2-ura5s重组菌株。
实施例 11 : 重组菌株细胞内 ME活性测定
(1)液氮研磨菌体提取细胞总蛋白。
(2)配制活性测定体系： 80 mM KH2P04/KOH H 7.5 , 0.6 mM NADP+, 3 mM MgCb, 粗蛋白液（蛋白约 30 g )。
(3) 30°C保温 2 min, 待数值基本稳定后， 加入苹果酸（ pH 6.8 ), 终浓度 25 mM。
(4)于 340 nm处测定 3 min,根据单位时间内吸光值的变化计算酶 活。
测定结果如图 5C。其中 M. alpina为野生型对照； MAUI , MAU2 , MAU3为受体菌株对照； MAUC1 , MAUC2, MAUC3为 pBIG2-ura5s 重 组 菌 株 ； MA-malEl-l , MA-malEl-2 , MA-malEl-3 为 pBIG2-ura5s-malE 1重组菌株。 如图 6B M alpina为野生型对照； MAU1为受体菌株对照； MA-malE24 , MA-malE2-2 , MA-malE2-3 为 pBIG2-ura5s-malE2 转化的重组菌株。 如图 5C、 6B所示， 所有苹 果酸酶过量表达菌株中 ME活性均有显著提高。
实施例 12: 高山被孢霉脂肪组提取与检测
(1)将高山被孢霉原养型菌株与重组菌株于发酵培养基中， 28 °C , 500rpm在 5L发酵罐中培养 144小时。
(2)收集菌体， 冷冻干燥。
(3)取 1 OOmg干重菌丝， 加入 2mL 4mol/L盐酸。
(4) 80 °C水浴 0.5小时， -80°C 15分钟。 重复一次。 80°C水浴 0.5 小时。
(5)冷却至室温， 加入 ImL曱醇， 混匀。
(6)加入 ImL氯仿， 震荡 10分钟。 6000g离心 3分钟。 收集氯仿。
(7)重复 (6)两次。
(8)合并氯仿， 加入 ImL饱和氯化钠， 混勾， 3000g离心 3分钟。 收集氯仿层于新瓶。 剩余液体加入 ImL氯仿， 3000g离心 3分钟。合 并氯仿（4mL )。
(9)氮吹干燥， 加入 ImL乙醚， 转移至洁净的已经称重的瓶中。氮 吹干燥， 称重得到总脂肪重量。
(10) GC分析脂肪组构成。 分析结果见图 5D、 6C , 灰色代表花生 四烯酸， 浅灰色代表其它 ω6多不饱和脂肪酸， 白色代表其它脂肪酸。 其中 M alpina为野生型对照； MAUI , MAU2 , MAU3为受体菌株对 照； MAUC1 , MAUC2, MAUC3 为 pBIG2-ura5s 重组菌株； MA-malEl-l , MA-malEl-2 , MA-malEl-3 为 pBIG2-ura5s-malEl重 组 菌 株 ； MA-malE2-l , MA-malE2-2 , MA-malE2-3 为 pBIG2-ura5s-malE2重组菌株。 由图 5D所示结果可以看出，重组菌株 MA-malEl-l , MA-malEl-2 , MA-malEl-3中总脂肪酸含量相对于对 照菌株均有 30%的提高； 同时细胞内 AA的含量也有一定增加。 由图 6C所示结果可以看出， 重组菌株 MA-malE2-l , MA-malE2-2 , MA-malE2-3中总脂肪酸含量没有明显提高， 但是细胞内 AA含量显 著增加。
其中， 发酵培养基是以组分 50 g/L葡萄糖， 2.0 g/L L-酒石酸铵, 7.0 g/L KH2P04, 2.0 g/L Na2HP04, 1.5 g/L MgS04-7H20 , 1.5 g/L Yeast extract , 0.1 g/L CaC12-2H20 , 8 mg/L FeC13-6H20 , 1 mg/L ZnS04-7H20 , 0.1 mg/L CuS04-5H20 , 0.1 mg/L Co ( N03 ) 2-6H20 , 0.1 mg/L MnS04-5H20构成的。 虽然本发明专利已以较佳实施例公开如上，但其并非用以限定本 发明。 任何熟悉此技术的人， 在不脱离本发明的精神和范围内， 都可 做各种改动与修饰。因此本发明的保护范围应该以权利要求书所界定 的为准。

Claims

权 利 要 求 书
1. 一种过量表达苹果酸酶基因的同源重组高山被孢霉菌株， 其特征在于， 该菌株是用含苹果酸酶基因的根癌土壤杆菌转化高山被孢霉尿嘧啶营 养缺陷型菌株构建而成的。
2. 根据权利要求 1所述的一种过量表达苹果酸酶基因的同源重组高山被孢 霉菌株， 其特征在于， 该菌株是用包含质粒 pBIG2-ura5s-malEl或质粒 PBIG2-ura5s-malE2的根癌土壤杆菌转化高山被孢霉尿嘧啶营养缺陷型 菌株构建而成的。
3. 根据权利要求 1或 2所述的一种过量表达苹果酸酶基因的同源重组高山 被孢霉菌株， 其特征在于， 该菌株是用重组质粒 pBIG2-ura5s-malEl 或 PBIG2-ura5s-malE2转化根癌土壤杆菌后， 进一步用经转化的含质粒 pBIG2-ura5s-malEl或 pBIG2-ura5s-malE2的根癌土壤杆菌转化高山被孢 霉尿嘧啶营养缺陷型菌株构建而成的， 其中的高山被孢霉尿嘧啶营养缺 陷型菌株是 ura5基因中的 213bp-230bp之间共 18bp的序列缺失的 Mortierella alpina ATCC 32222菌株。
4. 一种构建权利要求 1或 2所述的同源重组高山被孢霉菌株的方法， 其具 体步骤如下：
a) 提取 Mortierella alpina ATCC 32222菌株的 R A， 通过反转录获取 cDNA，利用 PCR扩增分别获取 ura5基因、 内含子 DNA片段 IT、苹 果酸酶 1基因 malEl和苹果酸酶 2基因 malE2;
b) 分别构建重组质粒 pBIG2-ura5s-malEl 和 pBIG2-ura5s-malE2 ; c) 分别用构建获得的重组质粒 pBIG2-ura5s-malEl或
pBIG2-ura5s-malE2转化根癌土壤杆菌；
d) 分别用经过转化的含质粒 pBIG2-ura5s-malEl或 pBIG2-ura5s-malE2 的根癌土壤杆菌转化高山被孢霉尿嘧啶营养缺陷型菌株； e) 筛选鉴定转化菌株， 获得过量表达苹果酸酶 1或 2基因的同源重组 高山被孢霉菌株。
5. 一种根据权利要求 4所述的构建同源重组高山被孢霉菌株的方法， 其特 征在于步骤 c) 中所使用的根癌土壤杆菌为： Agrobacterium tumefaciens C58Cl o
6. 一种根据权利要求 5所述的构建同源重组高山被孢霉菌株的方法， 其特 征在于步骤 d)中所使用的高山被孢霉尿嘧啶营养缺陷型菌株是 ura5基 因中的 213bp-230bp之间共 18bp的序列缺失的 Mortierella alpina ATCC 32222菌株。
7. 一种根据权利要求 6所述的构建同源重组高山被孢霉菌株的方法， 其特 征在于构建 pBIG2-ura5s-malEl时， 先利用 ura5基因构建转化质粒 pBIG2-ura5s, 再利用转化质粒 pBIG2-ura5s和苹果酸酶 1基因 malEl进 一步构建重组质粒 pBIG2-ura5s-malEl ; 构建 pBIG2-ura5s-malE2时，先 利用转化质粒 pBIG2-ura5s和 IT基因片段构建转化质粒高山被孢霉基因 操作通用载体 pBIG2-ura5s-ITs， 再利用转化质粒 pBIG2-ura5s-ITs和苹 果酸酶 2基因 mcdE2进一步构建重组质粒 pBIG2-ura5s-malE2。
8. 一种根据权利要求 6所述的构建同源重组高山被孢霉菌株的方法， 其特 征在于构建 pBIG2-ura5s-malEl时步骤 b ) 包括如下步骤： 用 PCR的方 法从 pD4质粒上获得 HPH表达单元，将 HPH表达单元用限制性内切酶 EcoR I和 Xbal酶切， 插入到 EcoR I和 Xbal酶切过的 pET28a ( + )的多 克隆位点（MCS )中，得到质粒 pET28a-HPHs;利用限制性内切酶 BspHI 禾口 BamHI酶切 ura5基因， 将酶切过的 ura5基因插入到 Ncol和 BamHI 酶切过的质粒 pET28a-HPHs中， 以替换 hpt基因， 构建质粒
pET28a-ura5s; 用限制性内切酶 EcoR I和 Xbal酶切质粒 pET28a-ura5s 得到 ura5s表达单元；将 ura5s表达单元替换质粒 pBIG2RHPH2中的 HPH 表达单元，构建转化质粒 pBIG2-ura5s;用限制性内切酶 BspHI和 BamHI , Ncol和 BamHI分别对 malEl基因片段和质粒 pET28a-HPHs进行酶切， 并通过连接反应将 malEl基因片段插入质粒 pET28a-HPHs的 Ncol禾口 BamHI位点之间得到质粒 pET28a-malEl；用限制性内切酶 Spel和 Xbal 双酶切质粒 pET28a-malEl， 得到 malEl表达单元； 将 malEl表达单元 插入到 Xbal酶切过的质粒 pBIG2-ura5s中， 得到质粒
pBIG2-ura5s-malEl ; 构建 pBIG2-ura5s-malE2时步骤 b )包括如下步骤： 用 PCR方法从高山被孢霉基因组中获得非编码的内含子 DNA片段 IT, 用限制性内切酶 Ncol和 BamHI分别对 IT基因片段和质粒 pET28a-HPHs 进行酶切， 并通过连接反应将 IT片段取代质粒 pET28a-HPHs的 hpt基 因， 得到质粒 pET28a-Its， 用限制性内切酶 Spel和 Xbal双酶切质粒 pET28a-ITs得到 ITs表达单元， 将 ITs表达单元插入到 Xbal酶切过的质 粒 pBIG2-ura5s中， 高山被孢霉基因操作通用载体 pBIG2-ura5s-Its， 分 别用 Kpnl和 Xmal双酶切 malE2基因和 pBIG2-ura5s-ITs， 用连接酶进 行连接， 得到 malE2表达质粒 pBIG2-ura5s-malE2。
9. 一种根据权利要求 5所述的构建同源重组高山被孢霉菌株的方法， 其特 征在于步骤 a)扩增 Ura5基因 IT， 以及苹果酸酶 1基因 malEl和苹果酸 酶 2基因 malE2的引物序列如下：
URA5F : ACATCATGACCATCAAGGAATACCAGCGCG
URA5R: TCGGGATCCCTAAACACCGTACTTCTCC
malElF: CATGCGTCATGACTGTCAGCGAAAACACC
malEIR: TACGCGGATCCTTAGAGGTGAGGGGCAAAGG
malE2F： ATCGGGGTACC ATGTTGAGGAATCCTGCTCTCA
malE2R: TAATTCCCCCGGGTCAGGGGTGCGATTCCAG
ITF:
ATCTCG
ITR:
CAGTTACG。
10. 权利要求 1或 2所述的菌株用于生产制备脂肪酸的用途。
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