Recombinant saccharomyces cerevisiae strain for producing 17, 18-EEQ as well as construction method and application of recombinant saccharomyces cerevisiae strain

Abstract

The invention provides a recombinant saccharomyces cerevisiae strain for producing 17, 18-EEQ as well as a construction method and application of the recombinant saccharomyces cerevisiae strain. According to the invention, the BM3 gene is introduced into the saccharomyces cerevisiae by using a synthetic biology method, meanwhile, the sEH gene in the saccharomyces cerevisiae is knocked out, the high-activity 17, 18-EEQ is produced by using the saccharomyces cerevisiae in a heterologous manner, the relative yield of the 17, 18-EEQ produced by the strain can be increased from 1.65392 to 9.65042 only through simple induction, and meanwhile, the absolute yield of the 17, 18-EEQ in a recombinant yeast strain reaches 32.058 mu g / mL. In addition, the invention also proves that the yield of 17, 18-EEQ can be increased by 47% by knocking out the sEH gene. In conclusion, the method overcomes the defects that in-vitro enzymatic synthesis of 17, 18-EEQ in the prior art is tedious in step, rigorous in reaction condition, high in time consumption and cost of subsequent extraction and purification and the like, and provides a new thought and method for in-vitro efficient synthesis of 17, 18-EEQ.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and in particular relates to a recombinant saccharomyces cerevisiae strain for producing 17,18-EEQ, and a construction method and application thereof. Background Art

[0002] EEQ (epoxyeicosatetraenoic acid) is a bioactive lipid metabolite with cardioprotective effects. It can reduce the risk of cardiovascular diseases (such as atherosclerosis) by regulating the function of the cardiovascular system (Lauterbach et al., 2002; McDougle et al., 2017; Zhou et al., 2024). At the same time, EEQ plays an important role in regulating inflammatory immune responses and has a significant effect on the control of allergic and inflammatory diseases, such as asthma (Morin et al., 2010; Miyata and Arita, 2015), retinal vascular inflammation (Capozzi et al., 2016) and peritonitis (Zhang and Spite, 2012; Kubota et al., 2014; Nagatake et al., 2018). In addition, EEQ, as a second messenger of various hormones, growth factors and cytokines, is involved in regulating cardiovascular and renal function, tumor suppression and other physiological processes (Hasegawa et al., 2017; Roy et al., 2018). These effects make EEQ an important factor in preventing and treating a variety of core diseases such as cardiovascular and cerebrovascular diseases and inflammation. However, the in vitro synthesis of EEQ is complex and the currently available high-purity products are difficult to obtain and are expensive, which makes the efficient production of EEQ an issue that needs to be urgently addressed.

[0003] Current studies have shown that heterologously expressed purified BM3 enzyme (a bacterial CYP450 enzyme originally isolated from Bacillus megaterium) epoxidizes eicosapentaenoic acid (EPA) to produce epoxyeicosatetraenoic acid (EEQ), and obtains 17,18-EEQ after esterification (Woodman et al., 2019). However, this method is cumbersome, the reaction conditions are harsh, and the subsequent extraction and purification are time-consuming and costly. In addition, it has been reported that oatmeal peroxidase was used to successfully achieve the selective epoxidation of EPA (eicosapentaenoic acid) ethanolamide to obtain the chain-end epoxide 17,18-EEQ-EA, and the use of acetone as a co-solvent can also improve its regioselectivity and productivity (Sanfilippo and Patti, 2021). Although this enzyme preparation is cheap and easy to obtain, some regioisomers of epoxides cannot be produced by wild-type BM3 enzymes and require large and expensive purification (Sanfilippo and Patti, 2021). Summary of the invention

[0004] In view of the shortcomings of the prior art in vitro synthesis of 17,18-EEQ, such as cumbersome steps, harsh reaction conditions, time-consuming and costly subsequent extraction and purification, the object of the present invention is to provide a recombinant Saccharomyces cerevisiae strain for producing 17,18-EEQ, and a construction method and application thereof.

[0005] In a first aspect of the present invention, a method for constructing a recombinant Saccharomyces cerevisiae strain producing 17,18-EEQ is provided, characterized in that it comprises: introducing the BM3 gene into a Saccharomyces cerevisiae host strain to obtain a recombinant Saccharomyces cerevisiae strain producing 17,18-EEQ.

[0006] Furthermore, the amino acid sequence of the protein encoded by the BM3 gene is shown in SEQ ID NO.1.

[0007] Furthermore, the construction method also includes the steps of optimizing the BM3 gene for Saccharomyces cerevisiae codon and synthesizing it onto a vector before introduction.

[0008] Furthermore, the nucleotide sequence of the codon-optimized BM3 gene is shown in SEQ ID NO.2.

[0009] Furthermore, the vector comprises pUC57.

[0010] Furthermore, the Saccharomyces cerevisiae host strain includes Saccharomyces cerevisiae BY4742.

[0011] Furthermore, the construction method also includes the step of knocking out the sEH gene of the Saccharomyces cerevisiae host strain.

[0012] Furthermore, the amino acid sequence of the protein encoded by the sEH gene is shown in SEQ ID NO.5.

[0013] Furthermore, the nucleotide sequence of the sEH gene is shown in SEQ ID NO.6.

[0014] Furthermore, the knockout step uses the CRISPR / CAS9 system to knock out the sEH gene in the Saccharomyces cerevisiae host strain.

[0015] Furthermore, the upstream primer sequence and downstream primer sequence of the sgRNA of the CRISPR / CAS9 system used in the knockout step are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0016] In a second aspect of the present invention, a recombinant Saccharomyces cerevisiae strain producing 17,18-EEQ is provided, characterized in that it is constructed by the construction method described in the first aspect of the present invention.

[0017] In the third aspect of the present invention, a method for producing 17,18-EEQ is provided, which comprises: culturing the recombinant Saccharomyces cerevisiae strain described in the second aspect of the present invention to obtain a fermentation product, namely, obtaining 17,18-EEQ.

[0018] In a fourth aspect of the present invention, provided is the use of the recombinant Saccharomyces cerevisiae strain described in the second aspect of the present invention in the production of 17,18-EEQ.

[0019] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0020] Compared with the prior art, the present invention has the following advantages and progress:

[0021] The cerevisiae used in the present invention is a single-cell model organism, which has unique advantages in the expression and modification of post-transcriptional proteins. In addition, it has a clear genetic background, simple genetic manipulation, and a short culture cycle, which is conducive to large-scale fermentation and product extraction. As one of the most widely used cell factories, cerevisiae has been widely used to synthesize a variety of products. The present invention uses a synthetic biology method to introduce the BM3 gene into cerevisiae and knock out the sEH gene in cerevisiae, and uses cerevisiae to heterologously produce highly active 17,18-EEQ. Only a simple induction is required to increase the relative yield of 17,18-EEQ produced by the strain from 1.65392 to 9.65042. At the same time, 17,18-EEQ in BM3-sEH KO -EEQ absolute production in the recombinant yeast strain reached 32.058ug / mL. In addition, the present invention also proves that knocking out the sEH gene can also increase the production of 17,18-EEQ by 47%. In summary, the present invention overcomes the shortcomings of the prior art of in vitro enzyme-catalyzed synthesis of 17,18-EEQ, such as cumbersome steps, harsh reaction conditions, and time-consuming and costly subsequent extraction and purification, and provides a new idea and method for the efficient in vitro synthesis of 17,18-EEQ. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 :The expression plasmid map of BM3 gene in Saccharomyces cerevisiae;

[0024] Figure 2 :BM3-sEH before and after galactose induction KO- Comparison of EEQ production of recombinant Saccharomyces cerevisiae strains;

[0025] Figure 3 :BM3-sEH KO -Detection mass spectrum of EEQ products produced by EEQ recombinant Saccharomyces cerevisiae strain;

[0026] Figure 4 :BM3-EEQ recombinant yeast strain and BM3-sEH KO - Comparison of EEQ production by EEQ recombinant Saccharomyces cerevisiae strains. DETAILED DESCRIPTION

[0027] The present invention provides a recombinant Saccharomyces cerevisiae strain for producing 17,18-EEQ, and a construction method and application thereof. The present invention is specifically described below in conjunction with the embodiments to facilitate further understanding of the present invention by those skilled in the art. However, the embodiments described below are only part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be pointed out that adjustments and improvements made by ordinary technicians in this field based on the concept of the present invention should be regarded as the scope of protection of the present invention. The specific technical operation steps and operators are not indicated in the embodiments, and they are all carried out in accordance with the general technical conditions described in the literature in the field or the relevant product instructions.

[0028] Example 1 Construction of BM3-EEQ recombinant Saccharomyces cerevisiae

[0029] The BM3 enzyme gene sequence was optimized by Jiutian Gene Technology (Tianjin) Co., Ltd. for Saccharomyces cerevisiae codons and synthesized into the pUC57 vector. Codon optimization can increase the expression of BM3 enzyme in host cells, which is very important for industrial production. This is because codon optimization can enhance mRNA stability and reduce translation interruptions, making the BM3 enzyme purer.

[0030] Then, the synthesized plasmid was amplified using primer pair F1 / R1 to obtain the amplified target gene fragment. The yeast expression vector pESC-ura plasmid was double-digested with BamHI and NheI, and the linearized vector was recovered. The above-mentioned amplified product and the recovered linearized vector were mixed at a molar ratio of 3:1, and the volume was controlled at 10ul. 10ul of 2Xseamless Cloning PreMix recombinase (Jiutian Gene Technology (Tianjin) Co., Ltd., JT201-20) was added to the mixed DNA fragment system, mixed well, reacted at 50℃ for 30min, and transformed into the Escherichia coli Top10 strain, plated and cultured overnight, and the correct clones were screened to obtain the pESC-ura-BM3 expression plasmid (see Figure 1). The correctly constructed expression plasmid was transformed into Saccharomyces cerevisiae BY4742 to obtain a yeast strain that can express the BM3 gene, named BM3-EEQ.

[0031] BM3 protein sequence:

[0032]

[0033] The gene sequence of BM3 enzyme after codon optimization:

[0034]

[0035] Primer sequences:

[0036] F1:TACTTTAACGTCAAGGAGAAAAAACCCCGGATCCATGACTATCAAGGAAA TGCCACAACC;

[0037] R1:ACTCCTTCCTTTTCGGTTAGAGCGGATCTTAGCTAGCTTAACCAGCCCAAACATCTTTAGCATATC.

[0038] Example 2BM3-sEH KO Construction of -EEQ recombinant Saccharomyces cerevisiae

[0039] The present invention knocks out the sEH gene in the BM3-EEQ engineering strain of Saccharomyces cerevisiae that heterologously expresses the BM3 enzyme, in order to increase the yield of the target product 17,18-EEQ in the engineering strain.

[0040] First, the CDS sequence of the sEH gene of Saccharomyces cerevisiae is obtained from databases such as NCBI. Then, according to the sequence of the target gene, an online tool such as CRISPR direct or a website (such as the Zhang Feng Laboratory website) is used to design a specific sgRNA to ensure that a sequence with high cutting efficiency and targeting efficiency is selected. The length of the sgRNA should generally be about 20 nucleotides, and it is necessary to contain a PAM (Protospacer Adjacent Motif) sequence (usually NGG). At the same time, the target site of the sgRNA should be close to the N-terminus of the protein coding region to increase the possibility of gene disruption. The sgRNA sequence information used in the present invention is as follows.

[0041] sgRNA sequence:

[0042] The upstream primer is GACTTTATATACCAAAGGGGTAAAGA (SEQ ID NO. 3);

[0043] The downstream primer is AAACTCTTTACCCCTTTGGTATATAA (SEQ ID NO. 4).

[0044] Then, a suitable vector Ptys0006 plasmid was selected to construct a co-expression system of sgRNA and Cas9, and the constructed co-expression vector was introduced into BM3-EEQ recombinant Saccharomyces cerevisiae cells. Then, the corresponding screening markers (such as fluorescent protein or antibiotic resistance gene, etc.) were used to screen yeast clones that successfully integrated the knockout vector. Finally, PCR, sequencing and other methods were used to verify that the strain in which the sEH gene was successfully knocked out was named BM3-sEH. KO-EEQ。

[0045] sEH protein sequence:

[0046] MQPFDSGHDDLVHDVVYDFYGRHVATCSSDQHIKVFKLDKDTSNWELSDSWRAHDSSIVAIDWASPEYGRIIASASYDKTVKLWEEDPDQEECSGRRWNKLCTLNDSKGSLYSVKFAPAHLGLKLACLGNDGILRLYDALEPSDLRSWTLTSEMKVLSIPPANHLQSDFCLSWCPSRFSPEKLAVSALEQAIIYQRGKDGKLHVAAKLPGHKSLIRSISWAPSIGRWYQLIATGCKDGRIRIFKITEKLSPLASEESLTNSNMFDNSADVDMDAQGRSDSNTEEKAELQSNLQVELLSEHDDHNGEVWSVSWNLTGTILSSAGDDGKVRLWKATYSNEFKCMSVITAQQ (SEQ ID NO.5).

[0047] sEH gene sequence:

[0048]

[0049] Example 3 Application of recombinant Saccharomyces cerevisiae in the production of 17,18-EEQ

[0050] 1. Chelation of substrate EPA

[0051] Weigh 1g fat-free BSA and dissolve it in 10ml 1×PBS solution, then filter it with a 0.22μm filter membrane. Take a certain amount of EPA stock solution and add it to 10% BSA for full chelation. When chelating, first insert the gun tip to the bottom of the tube, and move it up while pumping until it is completely pumped out. When pumping, precipitation will appear inside the gun tip and adhere to the inner wall. At this time, blow it repeatedly with force until it is completely clean. In this process, you must also ensure that the fatty acids in the BSA are completely dissolved, then vortex it slightly, and incubate it at 37 degrees for 5-10 minutes. For full chelation, you can see that the BSA with added fatty acids should be significantly yellower than the BSA with added ethanol.

[0052] 2. Fermentation of Saccharomyces cerevisiae

[0053] 2.1 24-well deep-well plate fermentation (for qualitative detection of small-scale fermentation)

[0054] Streak the activated single colony of Saccharomyces cerevisiae onto the SC-Ura solid culture medium plate, culture at 30°C for 2 days, pick an appropriate amount of cells and inoculate them into a 24-well deep-well plate, each well is filled with 2mL SC-Ura liquid culture medium, 30°C, 250rpm, culture for 16 hours, after OD600 rises to about 8-12, centrifuge at 4000rpm for 2 minutes, and collect the seed cells of OD600. Wash with sterile water 3 times to remove the residual glucose in the culture medium. Resuspend the cells with 200μL sterile water, and then transfer them to a new 24-well deep-well plate with an initial concentration of OD600=0.5, each well is filled with 2mL SC-Ura+Gal (with galactose as the only carbon source) liquid culture medium. Add the above-mentioned chelated EPA according to the agreed concentration and culture at 30°C for 24h.

[0055] 2.2 Shake flask fermentation

[0056] Inoculate a single colony of activated Saccharomyces cerevisiae into a test tube containing 5 mL of SC-Ura liquid culture medium, and culture at 220 rpm in a 30°C shaker for 16 hours until OD600 rises to about 8-12. Centrifuge at 4000 rpm for 2 minutes to collect seed cells of OD600. Wash 3 times with sterile water to remove residual glucose in the culture medium. Resuspend the cells with 500 μL of sterile water and transfer to a 250 mL wide-mouth shake flask containing 30 mL of SC-Ura+Gal (with galactose as the only carbon source) liquid culture medium (starting density OD600 = 0.5, use sealing film to maintain oxygen), 30°C, 250 rpm, shaker culture for 24 hours. If the shake flask fermentation condition is a culture medium with glucose as the carbon source, after preparing the seeds in the test tube, directly transfer the shake flask according to the starting density OD600 = 0.5 for fermentation.

[0057] 3. Extraction method of 17,18-EEQ

[0058] Take 10mL of fermentation broth, centrifuge and collect the bacteria in a 2mL centrifuge tube. Wash once with 2mL sterile water and place on ice for use. Prepare 2-3 mixed extraction reagents (480μL 100% methanol + 10μL 10% BHT + 10μL acetic acid + 1μL eico internal reference each) according to the number of samples, and pre-cool them at -20℃. Then resuspend the bacteria with 500μL mixed extraction reagent, add 200μL quartz sand and vortex for 15 minutes. Repeat the above shaking until the bacterial solution is uniform. Centrifuge at 4℃, 12000rpm for 10 minutes, transfer the supernatant to a new 2mL centrifuge tube, add 500μL deionized water and 700μL ethyl acetate, and vortex for 5 minutes to extract. Centrifuge at 4℃, 12000rpm for 10 minutes, and collect the upper organic phase into a new 2mL centrifuge tube. At the same time, add 700 μL of ethyl acetate to the old centrifuge tube (containing quartz sand and the remaining unextracted bacterial liquid), repeat the above oscillation, and transfer the extract to the same 2 mL centrifuge tube. After nitrogen blowing, vortex and oscillate 100 μL of 30% acetonitrile for 10 minutes to re-dissolve 17,18-EEQ. After a short centrifugation, filter the prepared sample with a 0.22 filter membrane to remove impurities, and complete the sample analysis as soon as possible.

[0059] 4. UPLC and UPLC-MS detection methods

[0060] UPLC conditions: chromatographic column ACQUITY UPLC BEH C18 (2.1 mm×50 mm, 1.7 μm, Waters); mobile phase A: 0.1% formic acid in water; mobile phase B: methanol; flow rate: 0.3 mL / min; column temperature: 25°C; automatic sampler temperature: 5°C; injection volume: 5 μL.

[0061] The gradient elution program is as follows:

[0062]

[0063] Mass spectrometry conditions: electrospray ion source (ESI), negative ion scanning mode, capillary voltage: 1.0 kV; cone voltage: 25 V, cone gas flow: 150 L / Hr, ion source temperature: 150 ° C, desolvation gas temperature: 600 ° C, desolvation gas flow: 1000 L / Hr, collision gas flow rate: 0.18 mL / min; nebulizer pressure: 7.00 bar. Using multiple ion reaction monitoring (MRM) scanning mode, EPA ion pairs are: 302.167>105.931, EEQ ion pairs are 318.157>256.116.

[0064] 5. Results Analysis

[0065] As a single-cell model organism, Saccharomyces cerevisiae has unique advantages in the expression and modification of post-transcriptional proteins, and its genetic background is clear, genetic manipulation is simple, and the culture cycle is short, which is conducive to large-scale fermentation and product extraction. As one of the most widely used cell factories, Saccharomyces cerevisiae has been widely used to synthesize a variety of products. The purpose of the present invention is to use synthetic biology methods to use Saccharomyces cerevisiae to heterologously produce highly active 17,18-EEQ.

[0066] BM3-sEH constructed in Example 2 KO After the recombinant yeast strain was activated, the shake flask fermentation culture and EPA bioconversion method in this example were used to induce the strain with galactose. After 24 hours of fermentation, the detection method in this example was used for detection. The relative yield of 17,18-EEQ in 1 mL of fermentation broth increased from 1.65392 to 9.65042, and the yield increased by 483%. The results are shown in Table 1. Figure 2 At the same time, based on the standard curve made with the gradient concentration of 17,18-EEQ standard, the concentration of 17,18-EEQ in BM3-sEH was calculated. KO -EEQ recombinant yeast strains reached 32.058ug / mL, and the mass spectrometry of the product showed the results. Figure 3 .

[0067] In addition, the present invention uses CRISPR / Cas9 gene knockout technology to knock out the sEH gene in Saccharomyces cerevisiae, in order to increase the yield of the target product 17,18-EEQ in the engineered strain. The BM3-EEQ recombinant yeast strain constructed in Example 1 and the BM3-sEH constructed in Example 2 were KOAfter the activation of the BM3-EEQ recombinant yeast strains, the strains were induced with galactose according to the shake flask fermentation and EPA bioconversion methods in this example. After 24 hours of fermentation, the strains were tested using the detection method of this example. It was found that the yield of the target product 17,18-EEQ produced by the BM3-EEQ recombinant yeast strain constructed in Example 1 was 21.804 ug / mL, and the yield of the target product 17,18-EEQ produced by the BM3-EEQ recombinant yeast strain constructed in Example 2 was 21.804 ug / mL. KO The yield of the target product 17,18-EEQ produced by the -EEQ recombinant yeast strain was 32.058ug / mL. It can be seen that the knockout of the sEH gene increased the yield by 47%. Figure 4 shown.

[0068] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modification and change made to the above embodiment according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for constructing a recombinant Saccharomyces cerevisiae strain for producing 17,18-EEQ, characterized in that: include: The BM3 gene was introduced into the host strain of Saccharomyces cerevisiae to obtain a recombinant Saccharomyces cerevisiae strain that produces 17,18-EEQ.

2. The method for constructing a recombinant Saccharomyces cerevisiae strain according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the BM3 gene is shown in SEQ ID NO.

1.

3. The method for constructing a recombinant Saccharomyces cerevisiae strain according to claim 1 or 2, characterized in that: The construction method also includes the steps of optimizing the BM3 gene for Saccharomyces cerevisiae codon and synthesizing it onto a vector before introduction.

4. The method for constructing a recombinant Saccharomyces cerevisiae strain according to claim 3, characterized in that: The nucleotide sequence of the codon-optimized BM3 gene is shown in SEQ ID NO.

2.

5. The method for constructing a recombinant Saccharomyces cerevisiae strain according to claim 1, characterized in that: The construction method also includes the step of knocking out the sEH gene of the Saccharomyces cerevisiae host strain.

6. The method for constructing a recombinant Saccharomyces cerevisiae strain according to claim 5, characterized in that: The amino acid sequence of the protein encoded by the sEH gene is shown in SEQ ID NO.

5.

7. The method for constructing a recombinant Saccharomyces cerevisiae strain according to claim 5 or 6, characterized in that: The knockout step uses the CRISPR / CAS9 system to knock out the sEH gene in the Saccharomyces cerevisiae host strain.

8. A recombinant Saccharomyces cerevisiae strain producing 17,18-EEQ, characterized in that: The method is constructed by the construction method of claims 1-7.

9. A method for producing 17,18-EEQ, characterized in that: include: Cultivate the recombinant Saccharomyces cerevisiae strain according to claim 8 to obtain a fermentation product, namely, obtain 17,18-EEQ.

10. Use of the recombinant Saccharomyces cerevisiae strain according to claim 8 in the production of 17,18-EEQ.

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