Recombinant schizochytrium limacinum with high yield of EPA (Eicosapentaenoic Acid) and construction method and application thereof

Abstract

The invention provides an application of a KSC gene for expressing beta-keto-acyl-ACP synthetase in improving EPA (eicosapentaenoic acid) produced by fermentation of recombinant schizochytrium limacinum, and further provides a beta-keto-acyl-ACP synthetase mutant capable of improving the EPA production capacity of the recombinant schizochytrium limacinum through fermentation. The invention also provides recombinant schizochytrium limacinum for high yield of EPA, a beta-keto acyl-ACP synthetase KSB gene of schizochytrium limacinum HSc-01 is knocked out, the beta-keto acyl-ACP synthetase mutant is homologous, recombined and integrated into a schizochytrium limacinum genome, and the number of acyl carrier proteins ACP is increased to 11-14. The total oil content of the recombinant schizochytrium limacinum obtained by the method for fermentation synthesis reaches 29.65 g / L, the EPA content reaches 2.45 g / L, and the total oil content is increased by 47 times on the basis of an original strain HSc-01.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a recombinant engineering strain with high EPA production, and also relates to a construction method of the recombinant strain and application of the recombinant strain in producing EPA. Technical Background

[0002] Eicosapentaenoic acid (EPA) is an important ω-3 long-chain polyunsaturated fatty acid. Its importance to higher animals and humans has been verified in clinical and epidemiological studies and is widely used in various fields such as food, medicine, and feed industries. The natural sources of EPA are marine animals or microalgae. Since the yield and quality of raw materials are uncontrollable to varying degrees, obtaining low-cost and sustainable EPA through genetic engineering has become a research focus.

[0003] There are two pathways for the synthesis of EPA. The traditional one is the fatty acid synthesis pathway. Foreign related studies have increased the EPA production in Yarrowia lipolytica to 56% of lipids. Xue Z, Sharpe PL, Hong SP, Yadav NS, Xie D, Short DR, Damude HG, Rupert RA, Seip JE, Wang J, Pollak DW, Bostick MW, Bosak MD, Macool DJ, Hollerbach DH, Zhang H, Arcilla DM, Bledsoe SA, Croker K, McCord EF, Tyreus BD, Jackson EN, Zhu Q. Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat Biotechnol. 2013Aug; 31(8):734-40. doi:10.1038 / nbt.2622. Epub 2013Jul 21.PMID:23873085. ", but the synthetic pathway is complex, with many reaction steps, and many genetic modifications of enzymes in the pathway, which leads to the problem of unstable strains and easy degeneration.

[0004] Schizochytrium is the main producer of DHA in the current industrialization. It has a fast growth rate, high oil content, and high DHA ratio. Since EPA and DHA have similar structures, similar synthesis pathways, and simple reaction steps, using Schizochytrium as the starting strain to synthesize EPA is an expected technical development direction in this field. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and to genetically modify Schizochytrium through a bioengineering method to achieve its EPA synthesis ability.

[0006] The purpose of the present invention is to increase the EPA content in Schizochytrium, and to obtain a high-yield EPA Schizochytrium recombinant engineering strain with simple synthesis steps, stable genetics and not easy to degenerate through a series of genetic modifications, as well as a construction method and application of the recombinant strain.

[0007] The idea of ​​the present invention is to replace the β-ketoacyl-ACP synthase KS in Schizochytrium by homologous recombination B gene, and increased the number of acyl carrier proteins (ACP) in the PKS gene cluster to increase the content of unsaturated fatty acids. Finally, by mutating the β-ketoacyl-ACP synthase KS C Further increase the content of EPA in Schizochytrium.

[0008] Based on this, the present invention provides a method for expressing β-ketoacyl-ACP synthase KS C Application of genes in improving the production of EPA by fermentation of recombinant Schizochytrium, the β-ketoacyl-ACP synthase KS C The gene comes from Shewanella oneidensis or Photobacterium profundum.

[0009] Particularly preferably, the expression of β-ketoacyl-ACP synthase KS from Shewanella oneidensis C gene for higher EPA production.

[0010] In particular, the recombinant Schizochytrium is a knockout of β-ketoacyl-ACP synthase KS B Among them, Schizochytrium sp. HSc-01 is a high-DNA-producing Schizochytrium sp., which is disclosed in Chinese invention patent application CN 202311718000.0 and its deposit number is CGMCC No.27700.

[0011] In the present invention, the β-ketoacyl-ACP synthase gene KS derived from Shewanella C The nucleic acid sequence is shown in SEQ ID No. 2, which is derived from the β-ketoacyl-ACP synthase gene KS of Photorhabdus spp. C The nucleic acid sequence is shown as SEQ ID No.3.

[0012] The present invention also provides an application of overexpressing the acyl carrier protein gene ACP in improving the fermentation production of EPA by recombinant schizochytrium, wherein the recombinant schizochytrium knocks out the β-ketoacyl-ACP synthase KS of schizochytrium HSc-01. B The gene increases the number of acyl carrier protein ACP to 11-14, and the nucleic acid sequence of the acyl carrier protein ACP is shown in SEQ ID No.4.

[0013] Particularly preferably, when the number of acyl carrier proteins ACP is increased to 14, the recombinant Schizochytrium has the best ability to produce EPA by fermentation.

[0014] The present invention also provides a β-ketoacyl-ACP synthetase mutant, wherein the β-ketoacyl-ACP synthetase mutant is obtained by replacing the β-ketoacyl-ACP synthetase gene KS from Shewanella oneidensis with the β-ketoacyl-ACP synthetase gene KS C Mutation to KS C GLU344 >GLY / PHE440>LYS or KS C GLU344>GLY / PHE440>ARG , gene KS C The 344th glutamic acid is mutated to glycine and the 440th phenylalanine is mutated to lysine, or the gene KS C The glutamic acid at position 344 was mutated to glycine and the phenylalanine at position 440 was mutated to arginine.

[0015] The present invention also verifies the application of the above-mentioned β-ketoacyl-ACP synthase mutant in improving the production of EPA by recombinant Schizochytrium fermentation.

[0016] Among them, the mutant KS C GLU344>GLY / PHE440>LYS It has a more excellent effect of increasing EPA production.

[0017] According to a preferred embodiment, the recombinant Schizochytrium knocks out the β-ketoacyl-ACP synthase KS of Schizochytrium HSc-01. B genes and increase the number of acyl carrier protein ACP to 14.

[0018] On the other hand, the present invention also provides a recombinant Schizochytrium with high EPA production, wherein the recombinant Schizochytrium is a Schizochytrium HSc-01 β-ketoacyl-ACP synthase KS knocked out. B β-ketoacyl-ACP synthase mutant KS C GLU344 >GLY / PHE440>LYS or KS C GLU344>GLY / PHE440>ARGRecombinant Schizochytrium that was integrated into the genome of Schizochytrium by homologous recombination and increased the number of acyl carrier proteins ACP to 11-14.

[0019] The present invention also provides the use of the recombinant Schizochytrium in producing EPA by fermentation.

[0020] Furthermore, the present invention also provides a method for constructing a recombinant Schizochytrium that produces high EPA, the method comprising the following steps:

[0021] (1) KS B Construction of gene knockout plasmid

[0022] Obtaining β-ketoacyl-ACP synthase KS from the genome of Schizochytrium HSc-01 B Gene sequence, primers were designed to amplify KS B The upstream and downstream homology arms were connected to the pPICZαA vector to obtain the recombinant plasmid pPICZαA-KS B up-KS B down, and verified to be correct;

[0023] (2) Construction of recombinant plasmids containing multiple acyl carrier proteins ACP

[0024] The obtained acyl carrier protein ACP gene sequence was used to design primers to amplify the upstream and downstream homologous arms of the ACP gene of Schizochytrium HSc-01 and ligated to the pPICZαA vector to obtain the recombinant plasmid pPICZαA-ACPup-ACPdown, which was verified to be correct.

[0025] Primers were designed to amplify ACP fragments from the genomic DNA of Schizochytrium HSc-01, and sequences containing 11, 12, 13, and 14 repeated ACPs were artificially synthesized. The fragments containing 11, 12, 13, and 14 repeated ACPs were connected to the middle of the upstream and downstream homologous arms of the plasmid pPICZαA-ACPup-ACPdown by homologous recombination, and recombinant plasmids pPICZαA-ACPup-11ACP-ACPdown, pPICZαA-ACPup-12ACP-ACPdown, pPICZαA-ACPup-13ACP-ACPdown, and pPICZαA-ACPup-14ACP-ACPdown containing 11, 12, 13, and 14 repeated ACP fragments were constructed, respectively, and verified to be correct;

[0026] (3) Construction of recombinant Schizochytrium containing recombinant plasmids containing multiple acyl carrier proteins ACP

[0027] After the recombinant plasmid in step (3) is linearized by enzyme digestion, it is transformed into Schizochytrium competent cells, and then cultured at 28°C in the dark. Single exchange and double exchange single colonies are screened for colony PCR verification. The correct recombinant bacteria are Schizochytrium HSc-01 as the starting strain and β-ketoacyl-ACP synthase KS is knocked out. B Gene and increase the number of acyl carrier protein ACP to 11-14 recombinant Schizochytrium;

[0028] (4) β-ketoacyl-ACP synthase KS C Mutation

[0029] The sequences shown in SEQ ID No. 5 and 7 were synthesized and connected to the restriction site Kpn I of plasmid pPICZαA to obtain mutant plasmids pPICZαA-KS C GLU344>GLY / PHE440>LYS and pPICZαA-KS C GLU344 >GLY / PHE440>ARG ;

[0030] After linearization of the mutant plasmid, 200 ng was taken to transform the recombinant Schizochytrium competent cells obtained in step (3), and cultured at 28°C in the dark to screen single colonies for colony PCR verification. The correct recombinant bacteria were recombinant Schizochytrium with high EPA production.

[0031] In the present invention, the KS of Schizochytrium B The sequence of is shown in SEQ ID No. 1, the sequence of ACP is shown in SEQ ID No. 4, and the β-ketoacyl-ACP synthase mutant KS C GLU344>GLY / PHE440>LYS The amino acid sequence of the mutant KS is shown in SEQ ID NO.5. C GLU344>GLY / PHE440>ILE The amino acid sequence of the mutant KS is shown in SEQ ID NO.6. C GLU344 >GLY / PHE440>ARG The amino acid sequence of the mutant KS is shown in SEQ ID NO.7. C GLU344>GLY / PHE440>LEU The amino acid sequence of the mutant KS is shown in SEQ ID NO.8. C GLU344>GLY / PHE440>TRP The amino acid sequence is shown in SEQ ID NO.9.

[0032] In the present invention, plasmid pPICZαA was purchased from Miaoling plasmid platform.

[0033] The acyl carrier protein (ACP) in the present invention is amplified and obtained from the genomic DNA of Schizochytrium HSc-01.

[0034] In the present invention, the transformation method of the recombinant strain is as follows: 200 ng of the linearized recombinant plasmid is added to 100 μl of Schizochytrium competent cells, mixed and ice-bathed for 30 minutes, and then transferred to a pre-cooled 0.1 cm gap electroporation cup for electric shock, the electric shock conditions are 1.5 KV, 200Q, 50uF, 2 times, and immediately after the end, 1 ml of seed culture medium is added, incubated at 28°C for 4 hours, and then centrifuged at 4000 rpm for 2 minutes, the supernatant is removed, the bacteria at the bottom of the EP tube are resuspended and spread on a plate containing 40 μg / mL zeocin (2% agar is added to the seed culture medium), and single exchange and double exchange single colonies are screened at 28°C in the dark for colony PCR verification.

[0035] In the present invention, the culture conditions of the recombinant strain are: culture in a seed culture medium in a constant temperature shaker at 200 rpm and 28° C. for 48 hours, then inoculated into a fermentation medium and cultured in a constant temperature shaker at 200 rpm and 28° C. for 120 hours.

[0036] The present invention knocks out KS in Schizochytrium B Gene, replaced with KS from Shewanella C , preventing the conversion of EPA to DHA in the synthesis pathway, achieving the purpose of EPA accumulation, and at the same time increasing the number of acyl carrier proteins ACP in the PKS gene cluster to 11-14, increasing the content of unsaturated fatty acids, and finally mutating β-ketoacyl-ACP synthase KS C The content of EPA in Schizochytrium is further increased, and a recombinant strain of Schizochytrium with high EPA production which is stable in inheritance and not easy to degenerate is obtained, and the process of synthesizing EPA by the recombinant strain is simple.

[0037] The Schizochytrium obtained by the method of the present invention has a total oil content of 29.65 g / L and an EPA content of 2.45 g / L in the fermentation synthesis of the recombinant strain AT-08, which is 47 times higher than that of the original strain HSc-01. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Gas phase analysis diagram of chassis bacteria HSc-01;

[0039] Figure 2 : Gas phase analysis of recombinant strain AT-08. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is explained below in a non-limiting manner in conjunction with the accompanying drawings and embodiments.

[0041] In the present invention, unless otherwise specified, "%" used to describe concentration means weight percentage, and ":" means weight ratio.

[0042] The present invention relates to the following culture medium:

[0043] The seed culture medium contained: glucose 40 g / L, yeast extract 2 g / L, sodium glutamate 10 g / L, KH2PO4 4 g / L, NaCl 15 g / L, MgCl2 3 g / L, CaCl2·2H2O 1 g / L, KCl 2 g / L, MgSO4·7H2O 5 g / L, FeCl3 0.1 g / L;

[0044] The fermentation medium contains: glucose 40 g / L, yeast extract 2 g / L, sodium glutamate 10 g / L, KH2PO4 4 g / L, NaCl 15 g / L, MgCl2 3 g / L, (NH4)2SO4 6 g / L, KCl 2 g / L, MgSO4·7H2O 5 g / L, and FeCl3 0.1 g / L.

[0045] Solid seed culture medium: Add 1.5-2% agar by total weight to the seed culture medium, sterilize it, and pour it into a sterilized plate in a clean bench.

[0046] Bleomycin seed screening medium: After the solid seed culture medium is sterilized, when the culture medium is cooled to 50°C, add bleomycin (Z-cin) with a final concentration of 50 μg / ml, immediately pour the plate, and store it in a 4°C refrigerator away from light.

[0047] In the present invention, the following primers are also involved:

[0048] Table 1 Primers involved in the present invention

[0049]

[0050] In the present invention, each strain and the recombinant plasmid transferred are shown in Table 2:

[0051]

[0052]

[0053] In the present invention, the biomass detection method of the strain is as follows:

[0054] Accurately measure 200 ml of the evenly stirred fermentation liquid, pour it into a clean, water-free centrifuge tube, centrifuge at 4000 r / min for 10 min, take out the centrifuge tube, discard the supernatant, take out all the bacteria at the bottom of the centrifuge tube with a medicine spoon, put them into a weighing plate with constant weight, evenly flatten them, and divide them into regular squares; dry them in a drying oven at 105°C, turning the bacteria from time to time, take them out after 3 hours, weigh them at a constant weight and record them. The biomass is the mass of the dried bacteria (W) divided by the volume of the fermentation liquid (V) multiplied by 1000.

[0055] The calculation formula is:

[0056] The total oil content was determined as follows:

[0057] Determination of oil content: Use a measuring cylinder to measure 250ml of the evenly stirred fermentation liquid and pour it into a dry and clean single-necked flask. Add 250ml of concentrated hydrochloric acid with a mass concentration of 30% to the single-necked flask. Place the single-necked flask in a 75℃ water bath and acid hydrolyze at a constant temperature for 2h (shake the flask by hand 4 to 5 times during this period to ensure sufficient acid-heat wall breaking); after the fermentation liquid after wall breaking is cooled to room temperature, pour it into a 1L separatory funnel and add 400ml of n-hexane (the amount of n-hexane can be appropriately increased when the total oil yield is high). After sufficient oscillation and mixing, let it stand for stratification (if there is an emulsified layer, add an appropriate amount of alcohol or saturated brine for demulsification treatment), collect the lower aqueous phase, and pour the organic phase (dark yellow) into a pear-shaped flask from the upper opening of the separatory funnel. Repeat the extraction of the collected aqueous phase for about 2 to 4 times until there is no oil in the organic phase (the organic phase is colorless and transparent at this time). Use a rotary evaporator and a circulating water multi-purpose vacuum pump to evaporate and concentrate the organic phase under reduced pressure, and the water bath temperature is 60°C. When there is no n-hexane in the pear-shaped flask, raise the water bath temperature to 90°C to ensure that there is no water or ethanol residue in the extracted crude oil. Weigh the pear-shaped flask and the oil in it m2, subtract the mass of the pear-shaped flask m1, and the result is the crude oil yield contained in 250ml of fermentation liquid.

[0058] Total oil yield of fermentation broth (g / L) = (m2-m1) × 4

[0059] The detection method for EPA and DHA content in oils and fats refers to the national standard GB26400-2011.

[0060] Example 1 Construction of a protein containing β-ketoacyl-ACP synthetase (KS C )

[0061] 1. KS B Construction of gene knockout plasmid

[0062] Obtaining β-ketoacyl-ACP synthase KS in the genome of Schizochytrium HSc-01 by sequencing B Gene sequence, primer design KS B up-F and KS B up-R (add restriction site NdeⅠ at 3' end) and KS B down-F (add restriction site Spe I at the 5' end) and KS B down-R, amplification to obtain KS B The upstream and downstream homology arms were seamlessly connected between the EcoRⅠ and NotⅠ restriction endonucleases of the pPICZαA vector to obtain the recombinant plasmid pPICZαA-KSB up-KS B down, and used in subsequent experiments after being verified to be correct.

[0063] KS from Shewanella (GenBank: AE014299.2) and Photorhabditis elegans (GenBank: FR837668.1) were obtained from NCBI. C Gene sequence, add NdeⅠ at the 5' end of the sequence, add Spe I at the 3' end, and send it to the biotechnology for synthesis, as shown in SEQ ID No.2-3. Use NdeⅠ and Spe I to double-digest the target fragment and pPICZαA vector, and connect the recombinant plasmid pPICZαA-KS with T4 ligase. B up-KS B down, and obtained KS containing Shewanella and Photobacterium luminescens respectively. C pPICZαA-KS B up-SoKS C -KS B down and pPICZαA-KS B up-PpKS C -KS B down, and used in subsequent experiments after being verified to be correct.

[0064] 2. Preparation of Schizochytrium competent cells

[0065] Pick a single colony of activated Schizochytrium HSc-01 on the plate and place it in 50 ml seed culture medium and culture it at 28°C and 200 r / min for 24 h; transfer it to 50 ml seed culture medium again at 4% inoculum and culture it at 28°C and 200 r / min until the OD value is 7, collect the bacterial solution and centrifuge it (4000 rpm, 4°C, 2 min) to obtain bacterial cells, resuspend the bacteria with 25 ml of pretreatment agent (20 mM pH 6.5 phosphate buffer containing 25 mM DTT), and shake for 30 min to loosen the cell wall; then wash the bacteria twice with 20 ml of precooled sterile water and 1 M sterile precooled sorbitol solution, respectively, and centrifuge again (centrifugation conditions are the same as above); finally, resuspend the bacteria with 200 μL of sterile precooled sorbitol solution, divide it into 1.5 ml sterile centrifuge tubes, 100 μL per tube, and store at -80°C for later use.

[0066] 3. Build a KS C Recombinant engineering strains

[0067] The recombinant plasmid pPICZαA-KS B up-KS B down, pPICZαA-KS Bup-SoKS C -KS B down and pPICZαA-KS B up-PpKS C -KS B After down linearization, take 200ng and add 100μl Schizochytrium competent cells. After mixing, place in an ice bath for 30min and transfer to a pre-cooled 0.1cm gap electroporation cup for electroporation. The electroporation conditions are 1.5KV, 200Q, 50uF, 2 times. After the end, add 1ml seed culture medium immediately, incubate at 28℃ for 4h, and then centrifuge at 4000rpm for 2min. Remove the supernatant, resuspend the bacteria at the bottom of the EP tube and spread it on a plate containing 40μg / mL zeocin (seed culture medium with 2% agar), culture at 28℃ in the dark, screen single exchange and double exchange single colonies for colony PCR verification, and the correct recombinant bacteria are named Schizochytrium AT-01, AT-02, and AT-03, respectively.

[0068] 4. Screening of recombinant strains

[0069] The above-mentioned recombinant strains AT-01, AT-02, and AT-03 were cultured at 28°C and 200 rpm for 120 h, respectively, and their biomass, total oil content, EPA content, and DHA content were measured, and strains with higher biomass, total oil content, and EPA content were screened out. The results are shown in Table 3.

[0070] Table 3 Biomass, total oil content, EPA content and DHA content in the fermentation broth of each recombinant strain

[0071]

[0072] The results showed that compared with the bottom strain, KS knockout B The biomass, total oil content, EPA content, and DHA content of strain AT-01 expressing KS C The biomass and lipid content of the subsequent strains AT-02 and AT-03 were higher than those of the strain AT-01, indicating that β-ketoacyl-ACP synthase (KS B / KS C ) had a significant effect on the growth, lipid accumulation, and synthesis of EPA and DHA of Schizochytrium. C The EPA content of strain AT-02 reached 202.73 mg / L, and its biomass and oil content were 21.85 g / L and 14.83 g / L, respectively, which were significantly greater than those of the starting strain and significantly better than those of the strain expressing Photorhabdus spp. KS C strain AT-03, thus confirming that Shewanella is the gene KSC The best source of.

[0073] Example 2 Construction of recombinant strains containing multiple acyl carrier proteins (ACPs)

[0074] 1. Construction of recombinant plasmids containing multiple acyl carrier proteins (ACPs)

[0075] By sequencing the ACP gene sequence obtained from Schizochytrium HSc-01, primers ACPup-F and ACPup-R (with the restriction site NdeⅠ added at the 3' end) and ACPdown-F (with the restriction site Spe I added at the 5' end) and ACPdown-R were designed to amplify the upstream and downstream homologous arms of ACP in Schizochytrium HSc-01 and ligated them between the restriction endonuclease EcoR I of the pPICZαA vector to obtain the recombinant plasmid pPICZαA-ACPup-ACPdown, which was verified to be correct and used in subsequent experiments.

[0076] Primers ACP-F and ACP-R were designed to amplify the ACP fragment from the genomic DNA of Schizochytrium HSc-01, and the fragment was sent for sequencing verification, as shown in SEQ ID No.4. It was then spliced ​​into sequences containing 11, 12, 13, and 14 repeated ACPs, respectively, and the restriction site NdeⅠ was added to the 5' end of the sequence and the restriction site Spe I was added to the 3' end. The fragments were sent for synthesis at Bioengineering, as shown in SEQ ID No.10-13, respectively. The synthetic sequence and plasmid pPICZαA-ACPup-ACPdown were double-digested with NdeⅠ and Spe I, and ligated with T4 ligase to obtain recombinant plasmids pPICZαA-ACPup-11ACP-ACPdown, pPICZαA-ACPup-12ACP-ACPdown, pPICZαA-ACPup-13ACP-ACPdown and pPICZαA-ACPup-14ACP-ACPdown containing 11, 12, 13 and 14 repeated ACP fragments, respectively, which were used in subsequent experiments after verification.

[0077] 2. Construction of recombinant strains containing multiple acyl carrier proteins (ACPs)

[0078] The above-verified recombinant plasmids pPICZαA-ACPup-11ACP-ACPdown, pPICZαA-ACPup-12ACP-ACPdown, pPICZαA-ACPup-13ACP-ACPdown and pPICZαA-ACPup-14ACP-ACPdown were linearized and 200 ng was added to 100 μl Schizochytrium AT-02 competent cells. After mixing, the mixture was placed in an ice bath for 30 min and then transferred to a pre-cooled 0.1 cm gap electroporation cup for electroporation at 1.5 KV, 200 Q, 50 uF, 2 times. After the electroporation, 1 ml of seed culture medium was immediately added and the mixture was incubated at 28 °C for 4 h. The mixture was then centrifuged at 4000 rpm for 2 min. The supernatant was removed and the bacteria at the bottom of the EP tube were resuspended and spread on a plate containing 40 μg / mL The single exchange and double exchange single colonies were screened on zeocin plates (seed medium supplemented with 2% agar) at 28°C in the dark and colony PCR was performed to verify the correct recombinant bacteria, and the correct recombinant bacteria were named Schizochytrium AT-04, AT-05, AT-06, and AT-07, respectively.

[0079] 3. Screening of recombinant strains

[0080] The above recombinant strains were cultured at 28°C and 200 rpm for 120 h, and their biomass, total oil content, EPA content and DHA content were measured respectively. The strains with higher biomass, total oil content and EPA content were screened out. The results are shown in Table 4.

[0081] Table 4 Biomass, total oil content, EPA content and DHA content in the fermentation broth of each recombinant strain

[0082]

[0083] The results showed that the biomass, total oil content, EPA content and DHA content of all recombinant strains were increased, and increased with the increase of the number of ACP fragment repeats. Among them, the EPA content of the recombinant strain AT-07 reached a maximum of 419.40 mg / L, which was 836% of the EPA production of the chassis strain, significantly increasing the EPA production. It also reflected that the number of acyl carrier proteins significantly affected the growth of Schizochytrium, oil accumulation, and the synthesis of EPA and DHA. The more the number of ACP fragment repeats, the more oil accumulated, and the more EPA and DHA were synthesized.

[0084] Example 3 Recombinant β-ketoacyl-ACP synthase KS in Schizochytrium AT-07 C Mutation

[0085] KS from Shewanella spp. was analyzed by SWISS MODEL CThe protein sequence was modeled to obtain the 3D structure of β-ketoacyl-ACP synthase protein. KS C The existing ligands in the protein structure were deleted. The substrates malonyl-CoA (PubChem CID: 644066) and octadecapentaenoic acid (PubChem CID: 5312510) were downloaded from PubChem. Other groups in the substrate structure were deleted using pymol. C The protein model is the receptor, malonyl-CoA and octadecapentaenoic acid are the ligands, the receptor and ligand are hydrogenated and balanced using Autoduck Vina 1.2.5, the docking algorithm is used to complete the molecular docking, and the grid box range is determined according to the docking results and docked again. The results show that the receptor molecule and the ligand binding pocket have good spatial complementary characteristics. With the ligand as the center, select the ligand molecule There are 16 amino acids in the range (PHE240, ALA242, PHE300, LEU302, SER303, PRO304, GLN309, HIS333, ALA334, THR335, THR337, LEU339, GLY340, GLU344, PHE440, PHE442), and these 16 amino acids are used as candidate amino acids for mutation. The 16 candidate amino acids were first subjected to alanine scanning mutagenesis using Discovery Studio. According to the changes in mutation energy, it was found that the mutation of 2 amino acids to alanine would lead to an increase in the affinity between the protein and the substrate and an enhancement of the interaction relationship, the mutation of 5 amino acids to alanine would lead to a decrease in the affinity between the protein and the substrate and a weakened interaction relationship, and the mutation of the remaining 9 amino acids to alanine had no significant effect on the affinity between the protein and the substrate. Discovery Studio was used to perform single-point amino acid mutations on two affinity-increasing candidate genes, mutated to 19 amino acids respectively, and the changes in mutation energy were observed. Mutations that would increase affinity were selected as mutations with positive effects. A total of 5 combination mutations were selected, including mutating glutamate at position 344 to glycine, and phenylalanine at position 440 to lysine, isoleucine, arginine, leucine or tryptophan, namely GLU344>GLY / PHE440>LYS, GLU344>GLY / PHE440>ILE, GLU344>GLY / PHE440>ARG, GLU344>GLY / PHE440>LEU and GLU344>GLY / PHE440>TRP, respectively. The screened mutants were verified by sequence synthesis experiments, and the amino acid sequences of these β-ketoacyl-ACP synthase mutants are shown in SEQ ID NO.5-9, respectively.

[0086] The above mutant sequences were sent to Bioengineering for synthesis and connected to the restriction site Kpn I of plasmid pPICZαA to obtain mutant plasmids. After linearization of the above mutant plasmids, 200 ng was added to 100 ul Schizochytrium AT-07 competent cells, mixed and ice-bathed for 30 min, and then transferred to a pre-cooled 0.1 cm gap electroporation cup for electroporation. The electroporation conditions were 1.5 KV, 200 Q, 50 uF, 2 times, and 1 ml of seed culture medium was added immediately after the end. The cells were incubated at 28°C for 4 h, and then centrifuged at 4000 rpm for 2 min. The supernatant was removed, the bacteria at the bottom of the EP tube were resuspended and spread on a plate containing 40 μg / mL zeocin (2% agar was added to the seed culture medium), and single colonies were screened at 28°C in the dark for colony PCR verification. The correct recombinant bacteria were named Schizochytrium AT-08, AT-09, AT-10, AT-11, and AT-12.

[0087] The above recombinant strains were cultured at 28°C and 200 rpm for 120 h, and their biomass, total oil content, EPA content and DHA content were measured respectively. The strains with higher biomass, total oil content and EPA content were screened out. The results are shown in Table 5.

[0088] Table 5 Biomass, total oil content, EPA content and DHA content in the fermentation broth of each recombinant strain

[0089]

[0090]

[0091] The results showed that the biomass, total oil content, EPA content and DHA content of all recombinant strains were increased, among which the recombinant strains AT-08 and AT-10 increased significantly, especially the recombinant strain AT-08, which had a maximum EPA content of 985.03 mg / L ( Figure 2 ), which is 235% of the EPA production of the recombinant strain AT-07, and in the chassis strain H Sc-01 ( Figure 1 ) was increased by 18.6 times, significantly increasing the production of EPA.

[0092] Example 4 Application of recombinant Schizochytrium AT-08 in EPA production

[0093] The obtained recombinant strain AT-08 was fermented in a 5L fermenter for 150 h, and then samples were taken to determine its biomass, total oil content and EPA content.

[0094] The results are shown in Table 6. After fermentation in a fermenter, the biomass of the recombinant strain AT-08 reached 38.45 DWg / L, the total oil content reached 29.65 g / L, and the EPA content reached 2.45 g / L, which was 22 times higher than that of the original strain HSc-01.

[0095] Table 6 Biomass, total oil content and EPA content in the fermentation broth of the recombinant strains

[0096]

[0097] For those skilled in the art, without departing from the scope of the technical solution of the present invention, the above-disclosed technical content can be used to make various possible changes and modifications to the technical solution of the present invention, or to change it into an equivalent embodiment with equivalent changes. Therefore, without departing from the content of the technical solution of the present invention, the equivalent changes and modifications made to the above-mentioned embodiments according to the technical essence of the present invention still belong to the scope of the technical solution of the present invention. The embodiments of the present invention should not be limited to the exemplary embodiments described below, but should be controlled by the limitations set forth in the claims and any equivalent forms thereof.

Claims

1. Expression of β-ketoacyl-ACP synthase KS C Application of genes in improving the production of EPA by fermentation of recombinant Schizochytrium, the β-ketoacyl-ACP synthase KS C The gene comes from Shewanella oneidensis or Photobacterium profundum.

2. The use according to claim 1, characterized in that The recombinant Schizochytrium is a knockout of β-ketoacyl-ACP synthase KS B Gene of Schizochytrium sp. HSc-01.

3. The use according to claim 1, characterized in that β-Ketoacyl-ACP synthase gene KS from Shewanella C The nucleic acid sequence is shown in SEQ ID No. 2, which is derived from the β-ketoacyl-ACP synthase gene KS of Photorhabdus spp. C The nucleic acid sequence is shown as SEQ ID No.

3.

4. Application of overexpression of acyl carrier protein gene ACP in improving EPA production by fermentation of recombinant Schizochytrium, wherein the recombinant Schizochytrium knocks out β-ketoacyl-ACP synthase KS of Schizochytrium HSc-01 B The gene increases the number of acyl carrier protein ACP to 11-14, and the nucleic acid sequence of the acyl carrier protein ACP is shown in SEQ ID No.

4.

5. A β-ketoacyl-ACP synthetase mutant, wherein the β-ketoacyl-ACP synthetase mutant is obtained by replacing the β-ketoacyl-ACP synthetase gene KS from Shewanella oneidensis with the C Mutation to KS C GLU344>GLY / PHE440>LYS or KS C GLU344>GLY / PHE440>ARG .

6. Use of the β-ketoacyl-ACP synthase mutant according to claim 5 in improving the production of EPA by recombinant Schizochytrium fermentation.

7. The use according to claim 6, characterized in that The recombinant Schizochytrium knocks out the β-ketoacyl-ACP synthase KS of Schizochytrium HSc-01 B genes and increase the number of acyl carrier protein ACP to 14.

8. A recombinant Schizochytrium that produces high EPA, characterized in that The recombinant Schizochytrium is a Schizochytrium HSc-01 β-ketoacyl-ACP synthase KS knocked out B β-ketoacyl-ACP synthase mutant KS C GLU344>GLY / PHE440>LYS or KS C GLU344 >GLY / PHE440>ARG Recombinant Schizochytrium that was integrated into the genome of Schizochytrium by homologous recombination and increased the number of acyl carrier proteins ACP to 11-14.

9. Use of the recombinant Schizochytrium according to claim 8 in producing EPA by fermentation.

10. A method for constructing a recombinant Schizochytrium that produces high EPA, the method comprising the following steps: (1) KS B Construction of gene knockout plasmid Obtaining β-ketoacyl-ACP synthase KS from the genome of Schizochytrium HSc-01 B Gene sequence, designed primers to amplify KS B The upstream and downstream homology arms were connected to the pPICZαA vector to obtain the recombinant plasmid pPICZαA-KS B up-KS B down, and verified to be correct; (2) Construction of recombinant plasmids containing multiple acyl carrier proteins ACP The obtained acyl carrier protein ACP gene sequence was used to design primers to amplify the upstream and downstream homologous arms of the ACP gene of Schizochytrium HSc-01 and ligated to the pPICZαA vector to obtain the recombinant plasmid pPICZαA-ACPup-ACPdown, which was verified to be correct. Primers were designed to amplify ACP fragments from the genomic DNA of Schizochytrium HSc-01, and sequences containing 11, 12, 13, and 14 repeated ACPs were artificially synthesized. The fragments containing 11, 12, 13, and 14 repeated ACPs were connected to the middle of the upstream and downstream homologous arms of the plasmid pPICZαA-ACPup-ACPdown by homologous recombination, and recombinant plasmids pPICZαA-ACPup-11ACP-ACPdown, pPICZαA-ACPup-12ACP-ACPdown, pPICZαA-ACPup-13ACP-ACPdown, and pPICZαA-ACPup-14ACP-ACPdown containing 11, 12, 13, and 14 repeated ACP fragments were constructed, respectively, and verified to be correct; (3) Construction of recombinant Schizochytrium containing recombinant plasmids containing multiple acyl carrier proteins ACP After the recombinant plasmid in step (3) is linearized by enzyme digestion, it is transformed into Schizochytrium competent cells, and then cultured at 28°C in the dark. Single exchange and double exchange single colonies are screened for colony PCR verification. The correct recombinant bacteria are Schizochytrium HSc-01 as the starting strain and β-ketoacyl-ACP synthase KS is knocked out. B Gene and increase the number of acyl carrier protein ACP to 11-14 recombinant Schizochytrium; (4) β-ketoacyl-ACP synthase KS C Mutation The sequences shown in SEQ ID No. 5 and 7 were synthesized and connected to the restriction site Kpn I of plasmid pPICZαA to obtain mutant plasmids pPICZαA-KS C GLU344>GLY / PHE440>LYS and pPICZαA-KS C GLU344>GLY / PHE440>ARG ; After linearization of the mutant plasmid, 200 ng was taken to transform the recombinant Schizochytrium competent cells obtained in step (3), and cultured at 28°C in the dark to screen single colonies for colony PCR verification. The correct recombinant bacteria were recombinant Schizochytrium with high EPA production.

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