CN116515917A

CN116515917A - C derived from Mortierella alpina 16/18 Elongase and use thereof

Info

Publication number: CN116515917A
Application number: CN202310607236.0A
Authority: CN
Inventors: 陈海琴; 王瑜鑫; 常璐璐; 杨波; 陈卫
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2023-05-26
Filing date: 2023-05-26
Publication date: 2023-08-01

Abstract

The invention discloses a C derived from Mortierella alpina _16/18 Elongase and application thereof belong to the technical field of bioengineering. The invention provides a gene MaELO4 with a coding nucleotide sequence shown as SEQ ID NO.1, and the gene is inserted into saccharomyces cerevisiae, so that the screened MaELO4 has higher activity of prolonging C16 fatty acid into C18 fatty acid and has the function of isomerizing trans-LA into cis-LA. The invention provides the gene codingC _16/18 The use of elongases in the conversion of LC-PUFAs yields Saccharomyces cerevisiae with high yields of C18 fatty acids, particularly cis-oleic acid.

Description

C derived from Mortierella alpina 16/18 Elongase and use thereof

Technical Field

The invention relates to a C _16/18 Fatty acid elongase and application thereof in lipid synthesis belong to the technical fields of genetic engineering and microbial engineering.

Background

Microbial oils are a class of lipids produced by microorganisms that are similar in composition to traditional vegetable and animal oils. The microbial oil is a novel biomass resource with wide application prospect, and has many advantages such as strong reproducibility, short production period, low production cost and the like. Microbial oils and fats can overcome the problem of insufficient sources of traditional long chain polyunsaturated fatty acids (Long Chain Polyunsaturated fatty acids, LC-PUFAs) and are therefore considered viable alternatives to vegetable oils and fish oils. At present, the research on microbial oil is mainly focused on producing high-added-value functional long-chain polyunsaturated fatty acid, special industrial oil and the like, has industrial development and production conditions, has promotion effect on sustainable development of socioeconomic performance, and is applied to commercial production by partial strains at present. With the rapid development of microbiology and molecular biology, microbial grease has become a research hotspot in the current LC-PUFAs field, and the purpose of producing the required LC-PUFAs in a sustainable manner is achieved by modifying and optimizing the fatty acid synthesis path through genetic engineering technology.

The microbial synthesis of PUFAs comprises the steps of catalyzing by fatty acid synthase (Fatty acid synthase, FAS) to obtain palmitic acid C16:0, and adding two carbon units to the carboxyl terminal of a fatty chain by taking palmitic acid as an initiator to form long-chain fatty acid. The fatty acid elongation process involves a four-step reaction in which malonyl-CoA is first condensed with long chain acyl-CoA under the catalysis of 3-ketoacyl-CoA synthase to produce β -ketoacyl-CoA, wherein the acyl moiety is elongated by two carbon atoms. The beta-ketoacyl-CoA formed is then reduced to beta-hydroxyacyl-CoA by the beta-ketoacyl-CoA reductase, dehydrated to alkenyl-CoA by the dehydratase, and finally reduced by the alkenyl reductase to yield elongated acyl-CoA. Extension in mitochondria is independent of processes other than fatty acid synthesis, is addition and reduction of acetyl units, and is the reverse reaction of fatty acid degradation processes. It has been confirmed that the first step of the elongation processIs the rate limiting step in fatty acid elongation, and the condensed enzyme is called "elongase". C (C) _16/18 Elongase is the first enzymatic reaction in the synthesis of long chain polyunsaturated fatty acids and plays an important role in determining the total carbon content into the fatty acid biosynthetic pathway, whose enzymatic activity will limit the yield of longer chain fatty acids.

C _16/C18 The identification of elongase is less, and after RNAi interference is carried out on CeELO2 in caenorhabditis elegans, the C16:0 fatty acid is obviously increased, and the CeELO2 has C _16/18 Prolonging enzyme activity. After the ELOVL6 of the mice is knocked out, the fatty acids C16:0 and C16:1 in the mice are obviously accumulated, and the ELOVL6 is proved to be involved in the prolongation of the fatty acids from C16 to C18. ELO2 from saccharomyces cerevisiae has also been shown to be involved in the prolongation of c16:0 fatty acids, but several prolongation enzymes have not been used. After Katsuya overexpresses mouse rELO2 in Saccharomyces cerevisiae, the conversion of C18/C16 fatty acids was increased from 55.7% to 83.5%, by a factor of 0.58. After the prolongation enzyme NsFAE in the marine microalgae is overexpressed in saccharomyces cerevisiae, the conversion rate of the C18/C16 fatty acid of the transformant is improved from 43.42% to 58.6%, and the conversion rate is improved by 0.35 times. Eiji Sakuradani identified C in Mortierella alpina in 2009 _16/18 The conversion rate of the C18/C16 fatty acid of the elongase gene MALDE 1 and the yeast transformant is improved from 40.6% to 63.1%, and the conversion rate is improved by 0.55 times. The elongase has been widely used in the enhancement of LC-PUFAs production from microbial sources, such as the high yield of eicosapentaenoic acid (Eicosapentaenoic Acid, EPA) using yarrowia lipolytica and marine microalgae, the high yield of oleic acid by yarrowia lipolytica, the enhancement of eicosahexaenoic acid (decosahexaenoic acid, DHA) content in schizochytrium, and the like. In summary, C in the genetic engineering of oleaginous microorganisms _16/18 The elongase has a wide range of applications, so for higher conversion C _16/18 Prolonged enzyme identification is necessary.

Mortierella alpina (Mortierella alpina) is an oleaginous filamentous fungus with strong lipid synthesis capacity that can de novo synthesize a variety of functional LC-PUFAs including arachidonic acid (ARA) and eicosapentaenoic acid (EPA). Mortierella alpina has been commercially produced to replace or supplement the traditional source of LC-PUFAs. Extension of Mortierella alpinaEnzymes have been reported to have three isolated and characterized functions, in 2000, jennifer M.Parker-Barnes isolated from Mortierella alpina into the first elongase GLELO gene and by heterologous expression and substrate addition in Saccharomyces cerevisiae, it was determined that GLELO-encoded proteins could catalyze GLA (18:3, n 6) to DGLA (20:3, n 6). In 2008, the elongase gene MAELO was isolated from M.alpina 1S-4 by Eiji Sakuradani, and the gene silencing strain obtained by RNA interference confirmed that the enzyme encoded by the MAELO gene was involved in biosynthesis of 20-, 22-and 24-carbon saturated fatty acids in M.alpina 1S-4 as compared with the wild strain. Mortierella alpina contains abundant long-chain polyunsaturated fatty acids, and the separated elongase and desaturase are used as an advantageous element in the transformation of oleaginous microorganisms. C (C) _16/18 Elongase acts as the first rate-limiting enzyme for fatty acid elongation, determining the content of LC-PUFAs, therefore higher active C _16/18 The identification of the prolongation enzyme has great scientific research value and application prospect.

Disclosure of Invention

[ technical problem ]

The invention aims to provide a C capable of improving the capability of extending microorganism C16 fatty acid into C18 fatty acid _16/18 Fatty acid elongase.

Technical scheme

Extracting RNA from Mortierella alpina, performing reverse transcription to obtain cDNA, amplifying a target gene fragment by a PCR technology, inserting the target gene fragment into a Saccharomyces cerevisiae plasmid pYES2-NT C to obtain an expression vector pYES2-MaELO4, selecting a positive transformant, and completing sequencing verification. Then transferring into INVSc1 in Saccharomyces cerevisiae by using lithium acetate method, and performing activity verification on the obtained recombinant strain by using the expression level of protein encoded by Westernblot candidate MaELO4 gene. The invention confirms that the screened MaELO sequence has C _16/18 Prolonging the enzyme activity and providing the application of the enzyme coded by the gene in the aspect of converting LC-PUFAs.

A first object of the present invention is to provide C _16/18 The use of elongases in catalyzing long chain polyunsaturated fatty acids is disclosed, said use being in catalyzing substrate C16 fatty acids.

In one embodiment of the invention, the C16 fatty acids include C16:0 and C16:1.

In one embodiment of the invention, the C _16/18 The elongase contains the amino acid sequence shown in SEQ ID NO. 2.

In one embodiment of the invention, the C _16/18 The elongase contains a nucleotide sequence shown in SEQ ID NO. 1.

A second object of the invention is to provide C _16/18 Use of elongase in the preparation of cis-oleic acid.

In one embodiment of the invention, the application is to connect C18:1 (Z) is converted into C18:1 (E).

A third object of the invention is a method for increasing the C18 fatty acid content of Saccharomyces cerevisiae, said method being overexpression of C _16/18 And (3) prolonging the enzyme.

In one embodiment of the invention, the C18 fatty acids include C18:0, C18:1.

In one embodiment of the invention, the vector pYES2-NT C, pCXS, pPIC9K, pYMIKP, pASN or pALDC is used to express C _16/18 And (3) prolonging the enzyme.

A fourth object of the present invention is to provide a recombinant Saccharomyces cerevisiae that overexpresses C _16/18 And (3) prolonging the enzyme.

In one embodiment of the invention, the Saccharomyces cerevisiae is Saccharomyces cerevisiae INVSc1.

In one embodiment of the present invention, pYES2-NT C, pCXS, pPIC9K, pYMIKP, pASN or pALDC is used as an expression vector.

In one embodiment of the present invention, pYES2-NT C is used as an expression vector.

The invention also provides application of the recombinant saccharomyces cerevisiae in preparation of products containing long-chain polyunsaturated fatty acid or cis-oleic acid, in particular to the preparation of C18:0 and C18:1 by taking C16:0 and C16:1 as substrates, wherein the C18:1 (Z) is converted into C18:1 (E).

[ advantageous effects ]

The invention relates to a C newly identified from mortierella alpina _16/18 The elongase is named as MaELO4, and after the elongase is expressed in saccharomyces cerevisiae, the ratio of C18/C16 fatty acid can be increased to 72.65% +/-0.2 percent, and the ratio is increased by about 0.66 times, so that the ratio is the C which is identified at present _16/18 The elongase is highest. Meanwhile, maELO4 also has the function of an isomerase, and can change trans oleic acid into cis oleic acid, namely, C18:1 (Z) is converted into C18:1 (E), providing a synthetic element for the production of cis-oleic acid by microorganisms. The optimized gene is used for constructing dominant strains of high-yield LC-PUFAs, and a foundation is laid for the transformation of oil-producing microorganisms for the subsequent industrialized production of the high-yield LC-PUFAs.

Drawings

FIG. 1 is a candidate gene and reported C _16/18 Multiple sequence alignment of elongases; wherein MaELO4 is Mortierella alpina C _16/18 A elongase amino acid sequence; maELO is Mortierella alpina C _16/18 A elongase amino acid sequence; YIELO is yarrowia lipolytica elongase amino acid sequence; egELO is the amino acid sequence of brown fine seaweed elongase; igELO is the amino acid sequence of dinoflagellate elongase such as ball; ppELO is the amino acid sequence of the Chinese sea-swelling bavus elongase; ctELO is the amino acid sequence of the Chinese golden algae elongase; the light blue region is a conserved amino acid residue, and the dark blue region is the same amino acid residue;

FIG. 2 is construction and verification of INVSc1 plasmid; (a) Constructing INVSc1 plasmid pYES2-MaELO based on restriction enzyme sites EcoRI and XbaI; (b) Agarose gel electrophoresis of Saccharomyces cerevisiae INVSc1 recombinant transformants; m, nucleic acid marker; n, negative control; lanes 1-5, pYES2-MaELO4 (1284 bp);

FIG. 3 is a Westernblot analysis of Saccharomyces cerevisiae INVSc1 transformant; m: protein marker; n: negative control protein pYES2-N; lane 1, pYES2-MaELO4; arrows indicate the position of the target protein.

Detailed Description

The invention will be further illustrated with reference to specific examples.

Mortierella alpina (Mortierella alpina) ATCC 32222, as referred to in the examples below, was purchased from the American type standard collection for organisms (ATCC); saccharomyces cerevisiae (Saccharomyces cerevisiae) INVSc1 and E.coli (Escherichia coli) Top10 and vector pYES2-NT C referred to in the examples below were purchased from Invitrogen corporation.

KOD plus high-fidelity DNA polymerase referred to in the following examples was purchased from Toyobo Co., japan; taq DNA polymerase referred to in the examples below was purchased from CWBIO company; the reverse transcription kit (PrimeScript RT regent Kit with gDNA Eraser RR047A & R6110A) referred to in the following examples was purchased from the company Takara, japan; the plasmid extraction kit referred to in the following examples was purchased from beijing tiangen biochemical technology limited; restriction enzymes (EcoRI, xbaI), T4 ligase, trizol, PCR product purification kit, geneRuler DNA Ladder Mix, pageRuler Prestained Protein Ladder were purchased from Thermo Scientific; n-pentadecanoic acid (C15:0), 20% (w/w) methanol hydrochloride referred to in the examples below were purchased from Sigma company; DEPC water, ampicillin (Ampicillin), an amino-free yeast nitrogen source (YNB) and various amino acids referred to in the examples below were purchased from Shanghai Bioengineering Co., ltd;

the low adsorption type enzyme-free gun head, enzyme-free centrifuge tube, enzyme-free PCR tube, 2mL brown gas phase bottle and bottle cap referred in the following examples were purchased from Suzhou family QingBio Inc.; other reagents were purchased from the national drug group.

The vector construction and bacterial competent cell preparation referred to in the examples below are both referred to in the handbook of molecular cloning.

Primers referred to in the examples below were synthesized by the Suzhou Jin Wei intelligent company and sequencing work was done by the Suzhou Jin Weizhi company.

The following examples relate to the following media:

YPD medium (1000 mL): 20g glucose, 20g fish meal peptone, 10g yeast extract.

SC-U screening Medium (1000 mL): 6.7g yeast basal nitrogen source (YNB), 20g glucose, 0.1g L-arginine, 0.1g L-cysteine, 0.1g L-lysine, 0.1g L-threonine, 0.1g L-tryptophan, 0.1g L-leucine, 0.1g L-adenine, 0.05g L-aspartic acid, 0.05g L-isoleucine, 0.05g L-phenylalanine, 0.05g L-proline, 0.05g L-serine, 0.05 gL-tyrosine, 0.05g L-valine, 0.05g L-methionine, 0.05g L-histidine.

SC-U induction medium (1000 mL): 6.7g yeast basal nitrogen source (YNB), 20g galactose, 10g raffinose, 0.1g L-arginine, 0.1g L-cysteine, 0.1g L-lysine, 0.1g L-threonine, 0.1 gL-tryptophan, 0.1g L-uracil, 0.1g L-leucine, 0.1g L-adenine, 0.05g L-aspartic acid, 0.05g L-isoleucine, 0.05g L-phenylalanine, 0.05g L-proline, 0.05g L-serine, 0.05g L-tyrosine, 0.05g L-valine, 0.05g L-methionine, 0.05g L-histidine.

LB medium (1000 mL): 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 20g of agar. Ampicillin was added at 100. Mu.g/mL before use.

SOC resuscitation Medium (1000 mL): 20g of tryptone, 5g of yeast extract, 0.5g of sodium chloride, 0.186g of potassium chloride, 0.95g of magnesium chloride and 3.6g of glucose.

Example 1: c (C) _16/18 Screening of genes for elongases

The method comprises the following specific steps:

c according to the identified function in NCBI _16/18 The sequence of the elongase gene is used as a template, and BLAST comparison is carried out in a gene library of the M.alpina ATCC 32222 strain which has completed sequencing, so as to obtain an alternative target gene; and then carrying out secondary comparison screening on the candidate genes in NCBI library, and naming the finally obtained target gene as MaELO4 (the nucleotide sequence is shown as SEQ ID No. 1), and naming the corresponding protein as MaELO4 (the amino acid sequence is shown as SEQ ID No. 2).

The full length of the cDNA corresponding to MaELO4 is 948bp, and codes 315 amino acids. To further determine whether the screened MaELO4 belongs to C _16/18 Elongase, which is compared with the identified functional C in NCBI _16/18 Elongases were analyzed for amino acid homology and conserved structure.

As shown in FIG. 1, the comparison results indicate that MaELO4 and NCBI have been identified for functional C _16/18 The elongase has high homology and has C _16/18 Prolongation of the conserved sequence of the enzyme, therefore, maELO4 was judged to be C _16/18 And (3) prolonging the enzyme.

Example 2: cloning of the Gene MaELO4

The method comprises the following specific steps:

total RNA of Mortierella alpina (Mortierella alpina) ATCC 32222 was extracted by the Trizol method, cDNA was obtained by reverse transcription according to the instructions of Takara reverse transcription kit, and MaELO4 was amplified by PCR in a cDNA library of Mortierella alpina (Mortierella alpina) ATCC 32222, and primers used for amplification of MaELO4 are shown in Table 1.

The PCR instrument is BIO-RAD T100 Thermal Cycler, KOD plus high-fidelity DNA polymerase is used, the reaction system is 50 mu L, and the system content is carried out according to the specification of the DNA polymerase; the reaction process is as follows: pre-denaturing at 95 ℃ for 5min, then denaturing at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extending at 68 ℃ for 1.5min, repeating the three steps for 30 times, fully extending at 68 ℃ for 5min, and finally cooling to 12 ℃ and keeping for 10min, and stopping.

After the reaction is finished, an amplification product is obtained, and after the amplification product is purified, the size of the band of the amplification product is verified by 1% agarose gel electrophoresis, so that the MaELO4 gene fragment is obtained.

TABLE 1 primer sequences and uses thereof

Example 3: construction and verification of recombinant expression vectors

The method comprises the following specific steps:

(1) Construction of recombinant expression vectors

The MaELO4 gene fragment obtained in example 2 and the expression vector pYES2-NT C were digested with restriction enzymes EcoRI and XbaI, and the digested and purified DNA was ligated using T4 ligase to obtain ligation products, the specific cleavage system (20. Mu.L) being as shown in Table 2.

TABLE 2 enzyme digestion system

After double cleavage with the corresponding endonucleases EcoRI and XbaI at 37℃for 2h, the cleaved products were purified using the SanPrep column PCR product purification kit, and the purification steps were carried out according to the procedures described in the specification.

In vitro ligation of DNA fragments: the purified DNA fragment and pYES2-NT C plasmid were added according to the ligation system of Table 3 (20. Mu.L):

table 3 connection system

The ligation system was ligated overnight at 4℃and transformed into E.coli Top10 competent cells as follows: taking 100 mu L of competent cells under the aseptic condition, adding 5-8 mu L of connection products, slightly stirring and uniformly mixing (not blowing and sucking) by using a gun head, and carrying out ice bath on the mixed system for 30min; placing the ice-bath mixed system in a water bath kettle at 42 ℃ for accurate heat shock for 90 seconds without shaking the centrifuge tube; rapidly transferring the centrifuge tube to ice after the heat shock is finished, and cooling for 2min; adding 800 mu L of precooled SOC resuscitating culture medium into each centrifuge tube, shake culturing at 37 ℃ and 150rpm for 1h to resuscitate bacteria and express antibiotic resistance genes on plasmids; 200. Mu.L of LB solid medium plate containing 100. Mu.g/mL ampicillin was used, and the plate was incubated at 37℃overnight in an inverted manner; and (3) picking up the positive transformant, extracting the plasmid, and obtaining the recombinant plasmid pYES2-MaELO4 by sequencing verification results, wherein the connection is successful.

(2) Screening and identification of transformants

Transformants grown on plates were picked, cultured overnight at 37℃in 5mL LB liquid medium containing Amp resistance, recombinant plasmids were extracted, and PCR-verified and sequenced (FIG. 2). The nucleic acid gel electrophoresis pattern showed a band identical to the position of the target gene, and the MaELO4 sequence was also identical to the measured sequence, indicating that the target gene had been successfully ligated into vector pYES2-NT C.

Example 4: yeast transformation and validation of recombinant expression vectors

(1) Preparation of Saccharomyces cerevisiae competent cells

Taking a defective saccharomyces cerevisiae strain INVSc1 from the temperature of minus 80 ℃, streaking on a YPD plate, and standing and culturing for 24 hours at the temperature of 28 ℃ until colonies are formed; a single colony is picked, transferred into 50mLYPD liquid medium and cultured overnight at 28 ℃ in a shaking table at 200 rpm; taking 1mL of overnight culture solution to 100mLYPD liquid culture medium according to 1% (v/v), culturing at 28 ℃ with a shaking table at 200rpm for about 5 hours, and measuring OD600 every 20-30 min until the OD600 value is approximately equal to 0.5-0.8; transferring the culture solution into a 50mL centrifuge tube (sterilized in advance and precooled), and centrifuging at 4 ℃ for 10min at 5000g to collect thalli; removing the supernatant under aseptic operation, re-suspending the thallus with 25mL of ice sterile water, placing on ice for 10min, and centrifuging at 4 ℃ for 10min at 5000g to collect thallus; removing the supernatant under aseptic operation, re-suspending the thalli with 40mL of pre-cooled 1 xTE solution, and centrifuging at 4 ℃ for 10min at 5000g to collect thalli; the supernatant was removed under aseptic conditions, the cells were resuspended in 2mL of a pre-chilled 1 XLIAc/0.5 XSTE solution, and the cells were left at room temperature for 10min and then sub-packed into 100. Mu.l/tube for use.

(2) Saccharomyces cerevisiae transformation

The prepared salmon sperm DNA is cooled rapidly after 5min of boiling water bath, 5 μl of salmon sperm DNA and 1 μg of pYES2 series carrier are added into each 100 μl of competence, and water bath is carried out at 30deg.C for 5min; 700 μl of 1 XLIAc/40% PEG/1 XTE (ready to use, sterilization at 121℃for 20min before use) was added, and the mixture was subjected to a water bath at 30℃for 5min; adding 88 μl DMSO, heat-shocking in a water bath at 42deg.C for 5min, and rapidly ice-bathing for 5min; centrifugation at 3000g for 10min to collect the thalli, removal of supernatant under aseptic operation, resuspension of the thalli with 1mL of 1×te solution pre-chilled with ice; centrifugation at 3000g for 10min to collect the thalli, removal of supernatant under aseptic operation, resuspension of the thalli with 1mL of 1×te solution pre-chilled with ice; removing the supernatant under aseptic operation, reserving about 200 μl of liquid, blowing with a gun, and coating SC-U screening solid medium; the plate was inverted and incubated in an incubator at 28℃until transformants developed (about 2-3 days).

(3) Saccharomyces cerevisiae protein induced expression

Inoculating Saccharomyces cerevisiae transformant into a 50mL centrifuge tube which is sterilized and contains 10mL of SC-U screening culture medium, packaging sterilized gauze on a tube orifice, and shake culturing in a shaker at 28 ℃ and 200rpm for about 24 hours; inoculating into SC-U screening culture medium according to 1% (v/v) inoculum size, culturing at 28deg.C in shaking table of 200rpm for about 24 hr, centrifuging for 5min at 5000g, removing supernatant under aseptic operation, adding sterile water to rinse thallus, and centrifuging again under the above conditions; resuspension of the thalli with 10mL of SC-U induction culture medium, so that the final OD600 is about 0.4, the temperature is 28 ℃, and the induction culture is carried out at 200rpm for 24 hours; separating thallus and culture solution by centrifugation at 5000g for 5min, and storing in-20deg.C refrigerator.

(4) Westernbolt analysis

First, a gel plate was mounted, 5mL of 12% separation gel (1.6 mL of deionized water, 2mL of 30% acrylamide stock solution, 1.3mL of separation gel buffer (pH 8.8), 50. Mu.L of 10% SDS, 50. Mu.L of APS, 2. Mu.L of TEMED) was prepared and mixed in a small beaker. Slowly adding the prepared separating gel into a gel preparation tank to a position which is 1.5cm away from the top end of the front glass plate, lightly covering a layer of double-distilled water sealing gel on the separating gel solution, and standing for half an hour. After pouring off the water, the gel was blotted with filter paper, 2mL of concentrated gel (1.4 mL of deionized water, 330. Mu.l of 30% acrylamide stock solution, 250. Mu.l of separation gel buffer (pH 6.8), 20. Mu.l of 10% SDS, 20. Mu.l of APS, 2. Mu.l of TEMED) was prepared, mixed well in a small beaker, poured onto the separation gel, and a comb was inserted into the gel and allowed to stand for half an hour. Taking 2mL of the induced culture solution, centrifuging to remove the supernatant, adding 160 mu L of yeast wall breaking Buffer (50 mM sodium phosphate, 5% glycerol, 1mM PMSF) and an equal volume of glass beads, breaking the wall by using a FastPrep rapid nucleic acid extractor, adding 40 mu L of 5×loading Buffer, boiling water bath for 5-10 min, and placing on ice. The gel plate is put into an electrophoresis tank, 1 Xelectrophoresis buffer solution is added into the upper electrophoresis tank and the lower electrophoresis tank until the upper end and the lower end of gel can be soaked in the buffer solution, and a comb is gently pulled out. The sample was centrifuged briefly, 10 μl of the treated sample was loaded, and standard protein was added to another well. And (3) switching on a power supply with the voltage of 120V, and switching off the power supply when the front edge of bromophenol blue reaches the bottom of the electrophoresis tank, and taking out the glass plate.

Samples were separated by SDS-PAGE and subjected to Westernbolt analysis. Cutting and activating the PVDF film, paving the filter paper, the gel and the PVDF film according to a sandwich model, and putting the PVDF film into a film transfer groove. The transfer tank is placed on ice, film transfer is carried out at constant pressure overnight, the voltage is 20-30V, and the current is not more than 80mA. After the transfer, the membrane was removed, a corner was marked, rinsed 3 times with TBST solution, 5 min/time, slowly shaken 45min with TBST solution containing 5% nonfat milk powder at 4 ℃, rinsed 3 times with TBST solution, 5 min/time, 6 XHis-tagged murine antibody was added at 1:5000, incubated 1h, primary antibody was recovered, rinsed 3 times with TBST solution, 5 min/time, HRP-tagged goat anti-rabbit IgG was added at 1:2000, slowly shaken 45min, rinsed 3 times with TBST solution, 5 min/time. Equal volumes of A, B were mixed and PVDF membranes were attached back to A, B the mixture and observed on a fluorochem FC3 imager. The molecular weight of the target bands was analyzed using a gel image processing system (FIG. 3).

The analysis shows that a distinct band appears at the 40kD position, which is similar to the position of the positive protein MaELO4, and the recombinant protein of the MaELO4 Saccharomyces cerevisiae is successfully expressed. The protein gray scale scanning result also shows that the expression quantity of the candidate gene MaELO4 protein is not obviously different from that of the positive control protein.

The above results demonstrate that recombinant s.cerevisiae overexpressing MaELO4 was successfully constructed and was able to successfully express the protein MaELO4.

Example 5: fatty acid composition of recombinant Saccharomyces cerevisiae

The method comprises the following specific steps:

inoculating the recombinant Saccharomyces cerevisiae constructed in example 4 into a 50mL centrifuge tube which is sterilized and contains 10mL of SC-U screening medium, wrapping sterilized gauze on a tube orifice, and shake-culturing in a shaking table at 28 ℃ and 200rpm for about 24 hours; inoculating into fresh SC-U screening culture medium according to 1% (v/v) inoculum size, culturing in a shaking table at 28 ℃ and 200rpm for about 24 hours, centrifuging at 5000g for 5min, removing supernatant under aseptic operation, adding sterile water to rinse thalli, and centrifuging again under the conditions described above; re-suspending the thallus with 10mL SC-U inducing culture medium to reach final OD600 of about 0.4, inducing culture at 28 deg.c and 200rpm for 24 hr, centrifuging to collect precipitate, preparing 20mL of induced recombinant Saccharomyces cerevisiae, vacuum freeze drying to constant weight, grinding thallus into powder, weighing 20mg, adding 100 μL of C15:0 as internal standard, adding 2mL of 4mol/L hydrochloric acid, and mixing; water bath at 80 ℃ for 1h and standing at-80 ℃ for 15min; repeating for 3 times, cooling to room temperature, adding 1mL of methanol and 1mL of chloroform, mixing, and shaking for 2min; centrifuging for 10min at 3000 g; collecting chloroform layer in new lipid extraction bottle; this step was repeated twice; mixing chloroform layers, nitrogen-blowing and drying, adding 1mL 10% hydrochloric acid-methanol, and performing methyl esterification treatment in water bath at 60 ℃ for 3 h; then adding 1mL of saturated sodium chloride and 1mL of normal hexane into the methyl esterification treatment system, uniformly mixing, centrifuging for 10min at 3000g, and repeating the steps twice; collecting n-hexane layer in a new bottle, adding 1mL of n-hexane into the rest liquid, shaking and mixing for 1min, and centrifuging at 3000g for 10min; mixing n-hexane layers, drying by nitrogen, and adding 1mL of n-hexane for redissolution to obtain fatty acid methyl ester; the composition and content of fatty acids in the cells were measured by GC-MS, and the measurement results are shown in Table 4.

Wherein, the fatty acid methyl ester analysis adopts GC2010 (Shimadzu Co., japan) and the chromatographic column is DB-Waxetr (30 m multiplied by 0.32m,0.22 μm); detecting by a hydrogen flame ion detector, wherein the temperatures of a vaporization chamber and the detector are 240 ℃ and 260 ℃, the split-flow mode is adopted for feeding 1uL, the split-flow ratio is 10:1, and the carrier gas is nitrogen; programming temperature: the initial temperature is kept at 120 ℃ for 3min, is increased to 190 ℃ at 5 ℃/min, is increased to 220 ℃ at 4 ℃/min, and is kept for 20min; the fatty acid composition of the samples was analyzed qualitatively and quantitatively by mass comparison with commercial fatty acid methyl ester standards (37 fatty acid methyl ester blends, supelco, USA) and with the addition of an internal standard c15:0, and the total fatty acid content was expressed as mass of total fatty acids per cell.

TABLE 4 fatty acid component identification results

From the above, it was found that after the MaELO4 gene was overexpressed, the conversion rate of C18/C16 fatty acid of Saccharomyces cerevisiae increased from 43.86% to 72.65.+ -. 0.2% by 0.66-fold, wherein the C18:1 (Z) content was increased from 2.47% to 39.90.+ -. 0.3% in the present inventionThe conversion rate of the 6 fatty acid is improved better than the reported effects of rELO2, mortierella alpina MALCE1 and marine microalgae NSFAE. The invention provides a good C for the genetic modification of other oleaginous microorganisms _16/18 The elongase element also provides the possibility for high C18:1 (Z) yields from Saccharomyces cerevisiae.

Conversion of c18/C16= (c18:0+c18:1)/(c16:0+c16:1+c18:0+c18:1);

while the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1.C _16/18 Use of a elongase for the catalytic synthesis of long chain polyunsaturated fatty acids, characterized in that said use is a catalytic substrate, C16 fatty acid, said C _16/18 The elongase contains the amino acid sequence shown in SEQ ID NO. 2.

2. The use according to claim 1, wherein the c16 fatty acids comprise c16:0 and c16:1.

3.C _16/18 Use of a elongase for the preparation of cis-oleic acid, characterized in that said C _16/18 The elongase contains the amino acid sequence shown in SEQ ID NO. 2.

4. A method for increasing C18 fatty acid content in yeast is characterized by over-expressing C _16/18 Elongase, the C _16/18 The elongase contains the amino acid sequence shown in SEQ ID NO. 2.

5. The method of claim 4, wherein the c18 fatty acids comprise c18:0, c18:1.

6. The method according to claim 4 or 5, wherein the vector pYES2-NTC is used,pCXS, pPIC9K, pYMIKP, pASN or pALDC expressing C _16/18 And (3) prolonging the enzyme.

7. A recombinant saccharomyces cerevisiae is characterized in that the recombinant saccharomyces cerevisiae overexpresses C _16/18 Elongase, the C _16/18 The elongase contains the amino acid sequence shown in SEQ ID NO. 2.

8. The recombinant s.cerevisiae according to claim 7, wherein pYES2-NTC, pCXS, pPIC9K, pYMIKP, pASN or pALDC is used as the expression vector.

9. The recombinant saccharomyces cerevisiae according to claim 7 or 8, wherein said saccharomyces cerevisiae is saccharomyces cerevisiae INVSc1.

10. Use of the recombinant s.cerevisiae according to any of claims 7 to 9 for the preparation of a product comprising long chain polyunsaturated fatty acids or cis-oleic acid.