Recombinant schizochytrium with high yield of epa, construction method and application thereof

Abstract

The application provides a β-ketoacyl-ACP synthase KS C The application also provides a β-ketoacyl-ACP synthase mutant capable of improving the EPA production capacity of the recombinant Schizochytrium sp. The application further provides a high-EPA-producing recombinant Schizochytrium sp. in which the β-ketoacyl-ACP synthase KS gene of Schizochytrium HSc-01 is knocked out. B The β-ketoacyl-ACP synthase mutant is homologously recombined and integrated into the Schizochytrium sp. genome to increase the number of acyl carrier proteins ACP to 11-14. The total oil content of the recombinant Schizochytrium sp. constructed by the method reaches 29.65 g / L, and the EPA content reaches 2.45 g / L, which is 47 times that of the original strain HSc-01.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a recombinant engineering strain for high-yield EPA, and further relates to a construction method of the recombinant strain and application of the recombinant strain in production of EPA. TECHNICAL BACKGROUND

[0002] Eicosapentaenoic acid (EPA) is an important omega-3 long-chain polyunsaturated fatty acid, and its importance to higher animals and human bodies has been verified in clinical and epidemiological studies and widely applied in various fields such as food, medicine, and feed industry. The natural source of EPA is marine animals or microalgae. Due to the uncontrollable yield and quality of raw materials, it has become a research focus to obtain low-cost and sustainable EPA through genetic engineering means.

[0003] There are two synthesis pathways of EPA. The traditional one is the fatty acid synthesis pathway. Foreign related research has increased the EPA yield in Yarrowia lipolytica to 56% of the lipid content (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. 2013 Aug; 31(8): 734-40. doi: 10.1038 / nbt.2622. Epub 2013 Jul 21. PMID: 23873085.). However, the synthesis pathway is complex, the reaction steps are many, and the genetic modification of enzymes in the pathway is much, resulting in unstable bacteria and the problem of easy degeneration.

[0004] Schizochytrium is the main production strain for current DHA industrialization, and has the advantages of fast growth, high oil content, and high DHA proportion. Since EPA and DHA have similar structures and synthesis pathways, and the reaction steps are simple, it is an expected technical development direction in the field to use Schizochytrium as a starting strain to synthesize EPA. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies of the prior art, and to genetically modify Schizochytrium by a bioengineering method to achieve its EPA synthesis ability.

[0006] The purpose of the present application is to improve the EPA content in Schizochytrium, through a series of genetic modifications, to obtain a high-yield EPA Schizochytrium recombinant engineering strain with simple synthesis steps and stable genetic inheritance, and a construction method and application of the recombinant strain.

[0007] The idea of the present application is to replace the beta-keto-ACP synthase KS B gene in Schizochytrium by homologous recombination, increase the number of acyl carrier proteins (ACP) in the PKS gene cluster to improve the content of unsaturated fatty acids, and finally further improve the EPA content in Schizochytrium by mutating the beta-keto-ACP synthase KS C gene.

[0008] Based on this, the present application provides the use of expressing the beta-keto-ACP synthase KS C gene in improving the EPA production of recombinant Schizochytrium fermentation, wherein the beta-keto-ACP synthase KS C gene is derived from Shewanella oneidensis or Photobacterium profundum.

[0009] In particular, the beta-keto-ACP synthase KS C gene derived from Shewanella oneidensis has higher EPA yield.

[0010] In particular, the recombinant Schizochytrium is Schizochytrium HSc-01 with a knockout beta-keto-ACP synthase KS B gene, wherein the Schizochytrium (Schizochytrium sp.) HSc-01 is a high-yield DNA Schizochytrium disclosed in Chinese invention patent application CN 202311718000.0, and its preservation number is CGMCC No.27700.

[0011] In the present application, the beta-keto-ACP synthase gene KS C derived from Shewanella oneidensis has a nucleic acid sequence as shown in SEQ ID No.2, and the beta-keto-ACP synthase gene KS C derived from Photobacterium profundum has a nucleic acid sequence as shown in SEQ ID No.3.

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

[0013] Particularly preferably, when the number of acyl carrier protein ACP is increased to 14, the recombinant Schizochytrium has the best EPA production capacity during fermentation.

[0014] The application also provides a β-keto-acyl-ACP synthase mutant, which is a β-keto-acyl-ACP synthase gene KS C mutated into KS C GLU344 >GLY / PHE440>LYS or KS C GLU344>GLY / PHE440>ARG , i.e. glutamic acid at position 344 of the gene KS C is mutated into glycine and phenylalanine at position 440 is mutated into lysine, or glutamic acid at position 344 of the gene KS C is mutated into glycine and phenylalanine at position 440 is mutated into arginine.

[0015] The application also verifies the application of the above-mentioned β-keto-acyl-ACP synthase mutant in improving EPA production of recombinant Schizochytrium during fermentation.

[0016] , wherein the mutant KS C GLU344>GLY / PHE440>LYS has a more excellent effect of improving EPA production.

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

[0018] On the other hand, the application also provides a recombinant Schizochytrium with high EPA production, which is a recombinant Schizochytrium knocking out β-keto-acyl-ACP synthase KS B gene of Schizochytrium HSc-01, a β-keto-acyl-ACP synthase mutant KS C GLU344 >GLY / PHE440>LYS or KS C GLU344>GLY / PHE440>ARGRecombinant Schizochytrium were formed by homologous recombination and integration into the genome of Schizochytrium, with an additional 11-14 acyl carrier proteins (ACPs).

[0019] The present invention also provides the application of the above-mentioned recombinant Schizochytrium in the fermentation of EPA.

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

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

[0022] β-ketoyl-ACP synthase KS from the genome of Schizochytrium HSc-01 B Gene sequence, primers designed for amplification to obtain KS B The upstream and downstream homologous arms were ligated to the pPICZαA vector to obtain the recombinant plasmid pPICZαA-KS. B up-KS B The output was down and verified to be correct.

[0023] (2) Construct a recombinant plasmid containing multiple acyl carrier proteins ACP.

[0024] The obtained acyl vector 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 ligate them into the pPICZαA vector to obtain the recombinant plasmid pPICZαA-ACPup-ACPdown, which was verified to be correct.

[0025] Primers were designed to amplify the ACP fragment from the genomic DNA of Schizochytrium HSc-01. Sequences containing 11, 12, 13, and 14 repeating ACPs were artificially synthesized. Using homologous recombination, the fragments containing 11, 12, 13, and 14 repeating ACPs were ligated to the upstream and downstream homologous arms of the plasmid pPICZαA-ACPup-ACPdown, respectively. 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 repeating ACPs were constructed and verified to be correct.

[0026] (3) Constructing recombinant Schizochytrium containing a recombinant plasmid with multiple acyl carrier proteins ACP

[0027] After the recombined plasmid in step (3) is linearized by enzyme digestion, the linearized recombined plasmid is transformed into the Schizochytrium sp. competent cells, respectively, and then the Schizochytrium sp. is cultured at 28℃ in dark, and single exchange and double exchange single colonies are screened and verified by colony PCR, and the recombined Schizochytrium sp. with the Schizochytrium sp. HSc-01 as a starting strain, the β-keto-ACP synthase KS gene being knocked out and the number of acyl carrier protein (ACP) being increased to 11-14 is correct. B

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

[0029] The sequences shown in SEQ ID No. 5 and 7 are synthesized by genetic engineering, and are connected to the enzyme digestion site Kpn I of the plasmid pPICZαA, respectively, to obtain mutant plasmids pPICZαA-KS C GLU344>GLY / PHE440>LYS and pPICZαA-KS C GLU344 >GLY / PHE440>ARG .

[0030] After the obtained mutant plasmids are linearized, 200 ng of the linearized mutant plasmids are transformed into the recombined Schizochytrium sp. competent cells obtained in step (3), respectively, and then the Schizochytrium sp. is cultured at 28℃ in dark, and single colonies are screened and verified by colony PCR, and the recombined Schizochytrium sp. with high EPA yield is correct.

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

[0032] In the present application, the plasmid pPICZαA is purchased from Mingling Plasmid Platform.

[0033] The acyl carrier protein (ACP) in the present application is obtained by amplification from the genomic DNA of the Schizochytrium sp. HSc-01. ​​

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

[0035] In the present application, the culture conditions of the recombinant strain are as follows: the seed medium is cultured in a constant temperature shaker at 200 rpm and 28°C for 48 h, and then transferred to a fermentation medium, and then cultured in a constant temperature shaker at 200 rpm and 28°C for 120 h.

[0036] The present application knocks out the KS B gene in Schizochytrium thurensis and replaces it with the KS C gene from Shewanella, thereby preventing the conversion of EPA to DHA in the synthesis pathway, achieving the purpose of accumulating EPA, increasing the number of acyl carrier protein ACP in the PKS gene cluster to 11-14, increasing the content of unsaturated fatty acids, and finally further improving the content of EPA in Schizochytrium thurensis by mutating the β-keto-ACP synthase KS C , thereby obtaining a recombinant strain of Schizochytrium thurensis with stable heredity and high EPA yield, and the synthesis process of the recombinant strain is simple.

[0037] The total oil content of the Schizochytrium thurensis constructed by the method of the present application reaches 29.65 g / L, and the EPA content reaches 2.45 g / L, which is 47 times higher than that of the original strain HSc-01. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 : Gas chromatogram of the chassis strain HSc-01;

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

[0040] The technical solutions of the present application are explained below in combination with the drawings and examples, but are not limited thereto.

[0041] In the present application, unless otherwise specified, "%" used to indicate concentration is weight percent, and ":" is weight ratio.

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

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

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

[0045] Solid seed culture medium: on the basis of the seed culture medium, 1.5-2% agar is added, sterilized, and poured into sterilized flat plates in a super-clean bench.

[0046] Z-cin is added to the solid seed culture medium after sterilization, with a final concentration of 50μg / ml, and the medium is immediately poured into flat plates and stored in a 4℃ refrigerator in the dark.

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

[0048] Table 1 Primers involved in the present application

[0049]

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

[0051]

[0052]

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

[0054] Accurately take 200ml of the well-stirred fermentation broth, pour it into a clean and dry centrifuge tube, centrifuge at a speed of 4000r / min for 10min, 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 it into a weighing dish that has been weighed, evenly spread it out and draw a regular square; dry it in a 105℃ drying oven, move the bacteria from time to time during the process, take it out after 3h, weigh it and record it, and the biomass is the dried bacterial mass (W) divided by the fermentation broth volume (V) multiplied by 1000.

[0055] The calculation formula is:

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

[0057] The oil content was determined as follows: 250 ml of the well-mixed fermentation broth was measured with a measuring cylinder and poured into a dry and clean single-neck flask. 250 ml of 30% concentrated hydrochloric acid was added to the single-neck flask. The single-neck flask was placed in a 75°C water bath for 2 hours of constant temperature acid hydrolysis (the flask was shaken by hand 4 to 5 times during the process to ensure that the acid heat broke the wall sufficiently); after the broken wall fermentation broth was cooled to room temperature, it was poured into a 1L separatory funnel, and 400 ml of n-hexane was added (the amount of n-hexane added can be appropriately increased when the total oil yield is high). After being fully shaken and mixed, it was allowed to stand and separate, (when there is an emulsion layer, a proper amount of alcohol or saturated brine can be added for demulsification treatment), the lower water phase was collected, and the organic phase (dark yellow) was poured into a pear-shaped flask from the upper opening of the separatory funnel. The collected water phase was repeatedly extracted for about 2 to 4 times until there was no oil in the organic phase (at this time, the organic phase was colorless and transparent). The organic phase was concentrated by vacuum evaporation using a rotary evaporator and a circulating water multi-purpose vacuum pump, and the water bath temperature was 60°C. When there was no n-hexane in the pear-shaped flask, the water bath temperature was increased to 90°C to ensure that there was no water or ethanol residue in the extracted crude oil. The pear-shaped flask and the oil therein were weighed m2, and the mass of the pear-shaped flask m1 was subtracted. The result was the yield of crude oil contained in 250 ml of fermentation broth.

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

[0059] The EPA and DHA content in the oil was determined according to the national standard GB26400-2011.

[0060] Example 1: Construction of a recombinant engineering strain containing β-ketoacyl-ACP synthase (KS C )

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

[0062] The β-ketoacyl-ACP synthase KS B gene sequence in the genome of Schizochytrium HSc-01 was obtained by sequencing, and primers KS B up-F and KS B up-R (3' end adding enzyme cutting site Nde I) and KS B down-F (5' end adding enzyme cutting site Spe I) and KS B down-R were designed, and the upstream and downstream homologous arms of KS B were amplified and connected to the EcoR I and Not I restriction endonucleases of the pPICZαA vector by seamless connection to obtain the recombinant plasmid pPICZαA-KSB up-KS B down, which was verified to be correct and used in subsequent experiments.

[0063] KS genes from Shewanella sp. (GenBank: AE014299.2) and Photobacterium profundum (GenBank: FR837668.1) were obtained from NCBI, respectively, and the 5' end of the sequence was added with Nde I and the 3' end was added with Spe I, and then sent to a synthetic company for synthesis, as shown in SEQ ID No. 2-3. The target fragment and the pPICZαA vector were double-digested with Nde I and Spe I, and the recombinant plasmid pPICZαA-KS C B up-KS B down, respectively, to obtain the recombinant plasmids pPICZαA-KS C containing KS genes of Shewanella sp. and Photobacterium profundum, respectively. B up-SoKS C -KS B down and pPICZαA-KS B up-PpKS C -KS B down, which was verified to be correct and used in subsequent experiments.

[0064] 2, Preparation of Schizochytrium limacinum competent cells

[0065] A single colony of Schizochytrium limacinum HSc-01 that had been activated on a plate was picked into 50 ml of seed culture medium and cultured at 28°C and 200 r / min for 24 h; 4% of the inoculum was transferred again into 50 ml of seed culture medium and cultured at 28°C and 200 r / min until the OD value was 7, and the bacterial liquid was collected by centrifugation (4000 rpm, 4°C, 2 min) to obtain bacterial cells. The bacterial cells were resuspended with 25 ml of pretreatment agent (20 mM phosphate buffer containing 25 mM DTT, pH 6.5), and shaken for 30 min to loosen the cell wall. Then the bacterial cells were washed twice with 20 ml of pre-cooled sterile water and 1 M of pre-cooled sterile sorbitol solution, and centrifuged again (the centrifugation conditions were the same as above). Finally, the bacterial cells were resuspended with 200 μL of pre-cooled sterile sorbitol solution, and 100 μL of each was dispensed into 1.5 ml sterile centrifuge tubes, and stored at -80°C for standby use.

[0066] 3, Construction of recombinant engineering strain containing KS C

[0067] The aforementioned recombinant plasmid pPICZαA-KS B up-KS B down, pPICZαA-KS B ​​up-SoKS C -KS B down and pPICZαA-KS B up-PpKS C -KS B down Linearization 200 ng was added to 100 μl of Schizochytrium sp. competent cells, mixed, and then placed on ice for 30 min before being transferred to a pre-chilled 0.1 cm gap cuvette for electroporation. The electroporation conditions were 1.5 KV, 200 Q, 50 uF, 2 times, and 1 ml of seed medium was added immediately after the end of the electroporation. The mixture was incubated at 28°C for 4 h, then centrifuged at 4000 rpm for 2 min, and the supernatant was removed. The bottom of the EP tube was resuspended and plated on a plate containing 40 μg / mL zeocin (seed medium added with 2% agar), and incubated at 28°C in the dark. Single exchange and double exchange single colonies were screened and verified by colony PCR. The correct recombinant strains were named Schizochytrium sp. AT-01, AT-02, and AT-03.

[0068] 4. Screening of recombinant strains

[0069] The above recombinant strains AT-01, AT-02, and AT-03 were cultured at 28°C and 200 rpm for 120 h, and their biomass, total oil content, EPA content, and DHA content were determined. The strains with higher biomass, total oil content, and EPA content were screened, and 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 show that, compared with the chassis strain, the biomass, total oil content, EPA content, and DHA content of the strain AT-01 with the KS B gene knocked out all decreased significantly, while the strains AT-02 and AT-03 expressing KS C after AT-01 had higher biomass and oil content, indicating that the β-keto-ACP synthase (KS B / KS C ) in Schizochytrium sp. has a significant impact on the growth, oil accumulation, and synthesis of EPA and DHA. Among them, the strain AT-02 expressing KS C from Shewanella sp. had the highest EPA content of 202.73 mg / L, and its biomass and oil content were 21.85 g / L and 14.83 g / L, respectively, which were significantly higher than those of the original strain and significantly better than those of the strain AT-03 expressing KS C from Photobacterium profundum, so Shewanella sp. was determined to be the gene KSC The best source.

[0073] Example 2: Construction of a recombinant strain containing multiple acyl carrier proteins (ACP)

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

[0075] The ACP gene sequence obtained from Schizochytrium HSc-01 was sequenced. Primers ACPup-F and ACPup-R (with NdeⅠ restriction site added at the 3' end) and ACPdown-F (with Spe I restriction site 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 to the restriction endonuclease EcoRI of the pPICZαA vector to obtain the recombinant plasmid pPICZαA-ACPup-ACPdown. After verification, the plasmid was used for subsequent experiments.

[0076] Primers ACP-F and ACP-R were designed to amplify the ACP fragment from the genomic DNA of Schizochytrium HSc-01. The amplification was verified by sequencing, as shown in SEQ ID No. 4. The amplified fragments were then spliced ​​into sequences containing 11, 12, 13, and 14 repeats of ACP, respectively. An enzyme restriction site NdeⅠ was added to the 5' end of the sequence, and an enzyme restriction site Spe I was added to the 3' end. The amplified fragments were then sent to bioengineering for synthesis, as shown in SEQ ID No. 10-13, respectively. The sequence and plasmid pPICZαA-ACPup-ACPdown were synthesized by double digestion with NdeI and SpeI. 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 repeating ACP fragments, respectively, were obtained using T4 ligase. After verification, they were used in subsequent experiments.

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

[0078] The above-mentioned verification correct recombinant plasmid pPICZ alpha A-ACPup-11 ACP-ACPdown, pPICZ alpha A-ACPup-12 ACP-ACPdown, pPICZ alpha A-ACPup-13 ACP-ACPdown and pPICZ alpha A-ACPup-14 ACP-ACPdown were linearized, 200 ng of which was added to 100 μl of Schizochytrium sp. AT-02 competent cells, mixed, and then transferred to a pre-cooled 0.1 cm gap electroporation cup for electroporation under the conditions of 1.5 KV, 200 Q, 50 uF, 2 times, and then 1 ml of seed medium was immediately added after the end of the electroporation, and the mixture was incubated at 28°C for 4 h, followed by centrifugation at 4000 rpm for 2 min, removal of the supernatant, resuspension of the bottom of the EP tube, and then plating on a plate containing 40 μg / mL zeocin (seed medium added with 2% agar), and then the plate was cultured at 28°C in the dark to screen single exchange and double exchange single colonies for colony PCR verification, and the correct recombinant bacteria were named as Schizochytrium sp. AT-04, AT-05, AT-06 and AT-07, respectively.

[0079] 3. Screening of recombinant strains

[0080] The above-mentioned recombinant strains were cultured at 28°C and 200 rpm for 120 h, and then the biomass, total oil content, EPA content and DHA content were determined, and a strain with high biomass, total oil content and EPA content was screened, and 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 show that the biomass, total oil content, EPA content and DHA content of all the recombinant strains are increased, and increase with the increase of the number of ACP fragments, and the EPA content of the recombinant strain AT-07 is the highest, reaching 419.40 mg / L, which is 836% of the fermentation yield of EPA of the chassis strain, significantly increasing the yield of EPA, and at the same time, reflecting that the number of acyl carrier protein significantly affects the growth of Schizochytrium sp., oil accumulation and synthesis of EPA and DHA, and the more the number of ACP fragments, the more the oil accumulation and the more the synthesis of EPA and DHA.

[0084] Example 3 Mutation of β-ketoacyl-ACP synthase KS in recombinant Schizochytrium sp. AT-07 C

[0085] KS from Shewanella sp. was mutated by SWISS MODEL C ​Protein sequence modeling was performed to obtain the 3D structure of β-ketoacyl-ACP synthase protein, and KS was analyzed using PyMOL. C Existing ligands in the protein structure were deleted. Substrates malonyl-CoA (PubChem CID: 644066) and octadecanoic acid (PubChem CID: 5312510) were downloaded from PubChem. Other groups in the substrate structure were then deleted using PyMOL, with KS... C The protein model was used as the receptor, and malonyl-CoA and octadecanoic acid (CVA) as ligands. Autoduck Vina 1.2.5 was used to hydrogenate and equilibrate the receptor and ligands. Molecular docking was performed using the docking algorithm. After determining the grid box range based on the docking results, docking was performed again. The results showed that the receptor molecule and ligand binding pocket exhibited good spatial stereocomplementation. Ligand molecules were selected centered on the ligand. A total of 16 amino acids within the specified range (PHE240, ALA242, PHE300, LEU302, SER303, PRO304, GLN309, HIS333, ALA334, THR335, THR337, LEU339, GLY340, GLU344, PHE440, and PHE442) were selected as candidate amino acids for mutation. These 16 candidate amino acids were first subjected to alanine-scanning mutagenesis using Discovery Studio. Based on the changes in mutation energy, it was found that mutations of two amino acids to alanine increased the protein-substrate affinity and enhanced the interaction; mutations of five amino acids to alanine decreased the protein-substrate affinity and weakened the interaction; and mutations of the remaining nine amino acids to alanine had no significant effect on the protein-substrate affinity. Single-point amino acid mutations were performed on two candidate genes for increased affinity using Discovery Studio, resulting in 19 different amino acids. Changes in the mutation energy were observed, and mutations leading to increased affinity were selected as those with a positive effect. Five combined mutations were chosen, including mutating glutamic acid at position 344 to glycine and phenylalanine at position 440 to lysine, isoleucine, arginine, leucine, or tryptophan, respectively: GLU344>GLY / PHE440>LYS, GLU344>GLY / PHE440>ILE, GLU344>GLY / PHE440>ARG, GLU344>GLY / PHE440>LEU, and GLU344>GLY / PHE440>TRP. Sequence synthesis experiments were performed to verify the selected mutants. The amino acid sequences of these β-ketoacyl-ACP synthase mutants are shown in SEQ ID NO. 5-9.

[0086] The above sequence after mutation was sent to synthetic and connected to the restriction site Kpn I of plasmid pPICZαA, to obtain mutant plasmid. 200 ng of the linearized mutant plasmid was added to 100 ul of Schizochytrium AT-07 competent cells, mixed, and then transferred to a pre-cooled 0.1 cm gap electroporation cup for electroporation under the conditions of 1.5 KV, 200 Q, 50 uF, 2 times, and then 1 ml of seed medium was immediately added after the end of electroporation, and incubated at 28°C for 4 h, followed by centrifugation at 4000 rpm for 2 min, removal of the supernatant, resuspension of the bottom of the EP tube, and then plated on plates containing 40 μg / mL zeocin (seed medium added with 2% agar), and cultured at 28°C in the dark to screen single colonies for colony PCR verification. The correct recombinant bacteria were named as Schizochytrium AT-08, AT-09, AT-10, AT-11, and AT-12, respectively.

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

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

[0089]

[0090]

[0091] The results show that the biomass, total oil content, EPA content and DHA content of all recombinant strains are increased, among which the recombinant strains AT-08 and AT-10 have a significant increase, especially the recombinant strain AT-08, which has the highest EPA content of 985.03 mg / L( Figure 2 ), which is 235% of the EPA yield of the recombinant strain AT-07, and is 18.6 times higher than the chassis strain H Sc-01( Figure 1 ), significantly increasing the yield 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 the biomass, total oil content and EPA content were determined.

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

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

[0096]

[0097] For those skilled in the art, without departing from the scope of the technical solutions of the present application, the technical solutions of the present application can be changed and modified in various possible ways or be replaced by equivalent embodiments. Therefore, equivalent changes and modifications made to the above embodiments according to the technical essence of the present application, without departing from the scope of the technical solutions of the present application, still belong to the scope of the technical solutions of the present application. The embodiments of the present application 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. A β-ketoacyl-ACP synthase mutant, wherein the β-ketoacyl-ACP synthase mutant is derived from the β-ketoacyl-ACP synthase gene KS from Shewanella oneidensis. C Mutation to KS C GLU344>GLY / PHE440>LYS or KS C GLU344>GLY / PHE440>ARG mutant KS C GLU344>GLY / PHE440>LYS The amino acid sequence is shown in SEQ ID NO.5, mutant KS C GLU344>GLY / PHE440>ARG The amino acid sequence is shown in SEQ ID NO.

7.

2. The use of the β-ketoacyl-ACP synthase mutant of claim 1 in increasing the production of EPA by fermentation of a recombinant Schizochytrium sp.

3. A recombinant Schizochytrium sp. that produces high levels of EPA, characterized in that The recombinant Schizochytrium is a Schizochytrium HSc-01 in which a β-ketoacyl-ACP synthase KS B gene, the β-ketoacyl-ACP synthase mutant KS according to claim 1 C GLU344 >GLY / PHE440>LYS or KS C GLU344>GLY / PHE440>ARG The recombinant Schizochytrium in which the homologous recombination is integrated into the Schizochytrium genome and the number of acyl carrier proteins ACP is increased to 14, wherein the Schizochytrium HSc-01 has a preservation number of CGMCC No. 27700, the sequence of the β-ketoacyl-ACP synthase KS B gene is shown as SEQ ID No. 1, and the nucleic acid sequence of the acyl carrier protein ACP is shown as SEQ ID No.

4.

4. The use of the recombinant Schizochytrium sp. of claim 3 in the production of EPA by fermentation.

5. The method for constructing the recombinant Schizochytrium sp. of claim 3 with high EPA production, comprising the following steps: (1) KS B Construction of gene knockout plasmid Obtaining β-ketoacyl-ACP synthetase KS in genome of Schizochytrium H Sc-01 B Gene sequence, designing primer to amplify KS B up and down homologous arms of KS and connecting to pPICZαA vector to obtain recombinant plasmid pPICZαA-KS B up-KS B down, and verifying correctness; (2) Constructing a recombinant plasmid containing multiple acyl carrier protein (ACP) genes The ACP gene sequence obtained was used to design primers to amplify the upstream and downstream homologous arms of the ACP gene of Schizochytrium sp. HSc-01 and ligate them to the pPICZαA vector to obtain the recombinant plasmid pPICZαA-ACPup-ACPdown, which was verified to be correct. The ACP fragment was amplified from the genomic DNA of Schizochytrium sp. HSc-01 using primers, and a sequence containing 14 repeats of ACP was artificially synthesized. The sequence containing 14 repeats of ACP was ligated into the recombinant plasmid pPICZαA-ACPup-ACPdown using homologous recombination to construct the recombinant plasmid pPICZαA-ACPup-14ACP-ACPdown containing 14 repeats of ACP, which was verified to be correct. (3) Constructing a recombinant Schizochytrium sp. containing multiple acyl carrier protein (ACP) genes After the recombinant plasmid in step (2) is linearized by enzyme digestion, the Schizochytrium sp. competent cells are transformed, and then cultured at 28°C in the dark. Single exchange and double exchange single colonies are screened and verified by colony PCR. The correct recombinant bacteria are the recombinant Schizochytrium sp. with Schizochytrium sp. HSc-01 as the starting strain, the β-keto-ACP synthase KS B gene is knocked out, and the number of acyl carrier protein ACP is increased to 14. (4) a beta-ketoacyl-ACP synthase KS C mutated The sequences shown in SEQ ID No. 5 and 7 were synthesized by gene synthesis to obtain the corresponding nucleic acid sequences, which were respectively connected to the restriction site Kpnl of plasmid pPICZαA to obtain mutant plasmids pPICZαA-KS C GLU344>GLY / PHE440>LYS and pPICZαA-KS C GLU344 >GLY / PHE440>ARG ; The mutant plasmid obtained was linearized, and 200 ng of the linearized plasmid was used to transform the competent cells of the recombinant Schizochytrium sp. obtained in step (3). The cells were cultured at 28°C in the dark, and single colonies were selected and subjected to colony PCR to verify the correct recombinant bacteria, which were the recombinant Schizochytrium sp. with high EPA production.

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