Fatty acid desaturase for heterologous synthesis of alpha-linolenic acid and application of fatty acid desaturase

Abstract

The invention discloses fatty acid desaturase for heterologous synthesis of alpha-linolenic acid and application of the fatty acid desaturase, and belongs to the technical field of biology. The purpose of the invention is to improve the yield and conversion rate of alpha-linolenic acid. The invention provides a fatty acid desaturase. The amino acid sequence of the fatty acid desaturase is as shown in SEQ ID NO. 4 or SEQ ID NO. 5. And a theoretical basis is provided for improving the yield of alpha-linolenic acid.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a fatty acid desaturase for heterologous synthesis of α-linolenic acid and its application. Background Technology

[0002] Alpha-linolenic acid (ALA, C18:3 ω-3) is an important polyunsaturated fatty acid (PUFA) belonging to the ω-3 fatty acid series. It offers numerous health benefits, such as supporting cardiovascular health, brain development, and anti-inflammation. Since the human body cannot synthesize ALA and it must be obtained through diet, efficient and sustainable production methods are of great interest. Traditionally, ALA is extracted from natural plant oils, but this method relies on agricultural cultivation, is limited by climate and land availability, has high extraction costs, and results in limited ALA content, making it difficult to meet large-scale demand. Therefore, a method for large-scale, stable production is urgently needed. Summary of the Invention

[0003] The purpose of this invention is to increase the yield and conversion rate of α-linolenic acid.

[0004] The present invention provides a fatty acid desaturase, characterized in that the amino acid sequence of the fatty acid desaturase is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0005] The present invention provides a gene encoding the above-mentioned fatty acid desaturase. The present invention provides a recombinant vector containing the above-mentioned genes.

[0006] The present invention provides a recombinant microbial cell containing the above-mentioned genes.

[0007] To further specify, the originating microbial cell is either a prokaryotic microbial cell or a eukaryotic microbial cell.

[0008] This invention provides the application of the above-mentioned fatty acid desaturase, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cells in the production of α-linolenic acid.

[0009] This invention provides a method for producing α-linolenic acid, wherein the above-mentioned gene is expressed in Saccharomyces cerevisiae cells to obtain recombinant Saccharomyces cerevisiae, which is then fermented to obtain linoleic acid.

[0010] To further specify, the brewing yeast is a brewing yeast that produces linoleic acid.

[0011] This invention provides a method for producing α-linolenic acid, wherein the above-mentioned gene is expressed in Saccharomyces cerevisiae cells to obtain recombinant Saccharomyces cerevisiae, and then fermented in a fermentation medium containing linoleic acid to obtain linolenic acid.

[0012] Beneficial effects: This invention utilizes bioinformatics to discover a series of candidate genes for fatty acid desaturases from Sichuan pepper, and verifies their function using a free plasmid expression system. GC analysis of the fermentation products showed that strains expressing ZaFAD3-1 and ZaFAD3-2 produced linolenic acid, indicating that ZaFAD3-1 and ZaFAD3-2 possess Δ-15 desaturase activity to convert linoleic acid to α-linolenic acid.

[0013] Further quantitative comparative analysis revealed significant differences in the activities of the two enzymes and their unique temperature-responsive characteristics. Under standard culture conditions of 30°C, the catalytic efficiency of ZaFAD3-1 was comparable to that of AtFAD3 from Arabidopsis thaliana, but its α-linolenic acid yield was approximately 3.0 times that of ZaFAD3-2, indicating that ZaFAD3-1 possesses higher catalytic activity in the yeast system. Notably, when the culture temperature was lowered to 22°C, the activities of all tested genes were upregulated, but the magnitude of the upregulation differed significantly: the yields of AtFAD3, ZaFAD3-1, and ZaFAD3-2 increased by approximately 1.5-fold, 3.92-fold, and 1.6-fold, respectively. Among them, ZaFAD3-1 exhibited the strongest low-temperature induction characteristics. Under these optimized conditions, the final α-linolenic acid yield of ZaFAD3-1 reached 1.42 times that of AtFAD3. Attached Figure Description

[0014] Figure 1 The graph shows the relative content of α-linolenic acid. Figure 2 The graph shows the relative content of α-linolenic acid. Figure 3 The graph shows the relative content of α-linolenic acid. Detailed Implementation

[0015] Example 1. Method for obtaining the gene encoding fatty acid desaturase To mine FAD3 gene family members from the genomic data of the target species *Zanthoxylum bungeanum*, this study employed a BLASTp1 strategy based on a local server for homologous sequence retrieval. The specific steps are as follows: 1. Local database construction Download / obtain the transcriptomic protein sequence data of *Zanthoxylum bungeanum*, and use the makeblastdb program in the NCBI BLAST+ software package (version number: v2.12.0) to build a local protein database. Set the parameter to: -dbtype prot (protein database type).

[0016] 2. Query sequence selection The model plant selected was Arabidopsis thaliana ( Arabidopsis thalianaThe AtFAD3 protein sequence that has been functionally validated in the database, as well as FAD3 protein sequences from other closely related species, are used as query sequences.

[0017] 3. Homologous Alignment Retrieval The BLASTP program was used to perform homology comparisons in the local database constructed above. To ensure the specificity and comprehensiveness of the search results, the key parameters were set as follows: Expected value threshold (E-value): set to 1e-5 (or 1e-10) to filter out randomly matched false positive sequences; Alignment mode: Standard protein-protein alignment mode is used.

[0018] 4. Candidate sequence screening and validation The BLASTp output was initially screened, retaining sequences with amino acid sequence identity greater than 40% and coverage greater than 50%. The target gene was then obtained. ZaFAD3-1 and ZaFAD3-2 The full-length encoded protein sequence.

[0019] ZaFAD3-1: (SEQ ID NO.1) ZaFAD3-2:(SEQ ID NO.2) Protein sequence AtFAD3: (SEQ ID NO.3) MVVAMDQRTNVNGDPGAGDRKKEERFDPSAQPPFKIGDIRAAIPKHCWVKSPLRSMSYVVRDIIAVAALAIAAVYVDSWFLWPLYWAAQGTLFWAIFVLGHDCGHGSFSDIPLLNSVVGHILHSFILVPYHGWRISHRTHHQNHGHVENDESWVPLPERVYKKLPHSTRMLRYTVPLPMLAYPLYLCYRSPGKEGSHFNPYSSLFAPSERKLIATSTTCWSIMFVSLIALSFVFGPLAVLKVYGVPYIIFVMWLDAVTYLHHHGHDEKLPWYRGKEWSYLRGGLTTIDRDYGIFNNIHHDIGTHVIHHLFPQIPHYHLVDATKAAKHVLGRYYREPKTSGAIPIHLVESLVASIKKDHYVSDTGDIVFYETDPDLYVYASDKSKIN; ZaFAD3-1: (SEQ ID NO.4) MEKERKLRNEVNVNGVVEKVMNGGGGFFDPAAPPPFKIGEIRAAIPKHCWVKNPWRSLSYVLRDLLVVLALLAAATHFNTWFFWPLYWVAQGTMFWAIFVLGHDCGHGSFSDSPLLNSIVGHILHSSILVPYNGWRISHRTHHQNHGNVEHDESWVPLPEKIYKKLDSSTRILRFSLPLPMLAYPLYLWYRSPGKDGSHFNPRSDLFAPEERKQVVISTACWTSMFVLLVCLSFAFGPITMFKLYLVPYLIFVMWLDFVTYLHHHGHDEQKLPWYRGKEWSYLRGGLTTVDRDYGIFNNIHHDIGTHVIHHLFPQIPHYHLVEATKAAKPVLGKYYREPKKSGPIPLHLINNLVRSVSQDHFVSDTGEIVYYQTDPQLQKISATKFE; ZaFAD3-2: (SEQ ID NO.5) MEQERKLKNEVNVNGVVEKVVDDGGFFDPAAPPPFKIGEIRAAIPKHCWVKNPWRSLSYVLRDLLLVLALLAAATYFNTWFFWPLYWFAQGTVFWAIFSLGHDCGHGSFSDSPFLNSFVGHILHSLILVPFNGWKISHRTHHQNHGNVENDESWVPLTEKIYKKLDNSTKFMRFTLPLPMLAYPLYLWYRSPG KDGSFNPYSDLFAPEERKAVVISTACWILMFAMLVCLCFVFGPITMFKFYGVPYLIFVMWLDCVTYLHHHGHDEHKLPWYRGKEWSYLRGGLTTVD RDYGIFNNIHHDIGTHVIHHLFPQIPHYHLVEATKAAKPVLGKYYHEPKKSGLIPLHLIKNLVRSISQDHFVSDTGDIVFYQTDTQLQKFLATKFE.

[0020] Example 2. Obtaining the recombinant vector The original p426GPD plasmid was sent to a biotechnology company, which then synthesized the gene into the p426GPD vector to obtain the p426GPD-ZaFAD3-1 and p426GPD-ZaFAD3-2 plasmids.

[0021] Example 3. Obtaining recombinant microbial cells The constructed expression plasmids (such as p426GPD-ZaFAD3-1 and p426GPD-ZaFAD3-2 plasmids) and empty vector plasmids were transformed into Saccharomyces cerevisiae strains (URA deficient strains that also contain plasmids carrying HIS selection tags and are genetically modified to produce linoleic acid) using lithium acetate-mediated chemical transformation.

[0022] 1. Preparation of competent cells: Saccharomyces cerevisiae strains were streaked onto SC-His plates for activation. Single colonies were picked and inoculated into liquid SC-His medium and cultured overnight at 30°C with shaking. The next day, they were transferred to fresh YPD liquid medium and cultured until the optical density reached a certain value. OD600 reaches 0.6-0.8 (logarithmic growth phase).

[0023] 2. Cell pretreatment: The bacterial culture was centrifuged (3000 rpm, 5 min) to collect the bacterial cells, washed once with sterile deionized water, and then resuspended in 0.1M lithium acetate (LiAc) solution to prepare competent cells.

[0024] 3. Construction of the transformation premix system: Take an appropriate amount of competent cell suspension and add the following components: 240 μL of 50% PEG4000, 36 μL of 1M lithium acetate (LiAc), 10 μL of carrier DNA (denatured salmon sperm DNA, which needs to be pre-boiled for 5 min), 400 ng of the plasmid DNA to be transformed, and an equal amount of empty vector plasmid added to the control group.

[0025] 4. Thoroughly mix the above mixture, incubate at 30°C for 30 minutes, then heat shock in a 42°C water bath for 40 minutes. 5. Resuscitation and screening: After heat shock, centrifuge and discard the supernatant. Resuspend the bacterial cells in sterile water and spread them on SC-His-Ura screening plates. Incubate the plates upside down at 30°C for 2-4 days until single colonies grow.

[0026] Construction of a linoleic acid-producing Saccharomyces cerevisiae cell factory: Basic strain: The basic strain SLA01 used in this study is a Ura-deficient Saccharomyces cerevisiae strain carrying the CRISPR-Cas9 system (URA-deficient, and also containing a plasmid carrying the HIS selection tag, derived from Qin, J., et al., Engineering yeast metabolism for the discovery and production of polyamines and polyamine analogues. Nature Catalysis, 2021.4(6): p. 498-509.), which was previously constructed and preserved in our laboratory.

[0027] To stably integrate the Δ-12 desaturase gene into a specific site XII-5 of the Saccharomyces cerevisiae genome, CRISPR-Cas9-mediated homologous recombination technology was used. The specific construction process is as follows: (1) Component amplification and purification Homologous arm amplification: Using the genomic DNA of the laboratory-preserved Saccharomyces cerevisiae strain SLA01 as a template, the sequences 500 bp upstream and 500 bp downstream of the XII-5 site in the genome were amplified by PCR using a high-fidelity DNA polymerase (Primerstar enzyme) as the 5' and 3' homologous arms. Primer sequences are shown in Table 1 (e.g., XII-upstream-fw / rv). Regulatory element amplification: Using the same genomic DNA as a template, the required promoters TEF1p and PGK1p and terminators PRM9t and CYC1t were amplified by PCR using the primers shown in Table 1.

[0028] All RCR amplification products were separated by 1.0% agarose gel electrophoresis, and the target bands were purified by gel extraction using a gel extraction kit for later use.

[0029] (2) Assembly of the expression box The purified DNA fragments were sequentially assembled into a complete homologous recombination expression cassette using overlap extension PCR technology. For the expression cassette TEF1p-ZaFAD2-PRM9t: the three fragments TEF1p, ZaFAD2 and PRM9t were mixed in sequence, and a second round of PCR was performed using the outer primer to amplify the complete linear expression cassette.

[0030] Construction of the CRISPR-Cas9 system and homologous recombination template: To stably integrate the Δ-12 desaturase gene into the specific site XII-5 of the Saccharomyces cerevisiae genome, regulatory elements TEF1p, PGK1p, PRM9t, and CYC1t with 500 bp homologous arms upstream and downstream of XII-5 were first cloned from the Saccharomyces cerevisiae genome using amplification primers, and the ZaFAD2 gene was cloned from the p426GPD plasmid. The primer sequences used for further amplification to the complete expression cassette TEF1p-ZaFAD2-PRM9t via overlap extension PCR are shown in Table 1. Table 1 Primer sequences

[0031] Table 2

[0032] Yeast transformation and positive clone screening: The obtained gene cassette and the plasmid carrying sgRNA (containing the URA selection marker, described in Rewiring carbon metabolism in yeast for high level production of aromatic chemicals. 2019 Oct 31;10(1):4976. doi: 10.1038 / s41467-019-12961-5.) were selected and cultured on SC-His-Ura plates using the lithium acetate transformation method. Correct positive single clones were obtained by colony PCR verification. The plasmid carrying sgRNA was removed on 5-FOA plates using selection pressure to obtain the engineered strain SLA03 expressing ZaFAD2.

[0033] ZaFAD2 gene: (SEQ ID NO.25) Table 3

[0034] The recombinant vector obtained in Example 2 was transferred into Saccharomyces cerevisiae SLA03, which is capable of synthesizing linoleic acid (engineered Saccharomyces cerevisiae SLA03 can synthesize linoleic acid), to obtain recombinant yeast cells of engineered strain ZaFAD3-1, recombinant Saccharomyces cerevisiae cells of engineered strain ZaFAD3-2, and blank control Saccharomyces cerevisiae cells, respectively.

[0035] Example 4. Method for obtaining α-linolenic acid Fermentation of engineered strains: Engineered strains ZaFAD3-1, ZaFAD3-2, and AtFAD3 obtained in Example 3 were picked from plates and inoculated into 2 mL of SC-His-Ura liquid medium. The culture was incubated overnight at 30°C / 22°C with shaking at 250 rpm to prepare a seed culture. Subsequently, the culture was transferred to 50 mL of SC-His-Ura liquid medium at an initial inoculum size of 0.1 and fermented for 4 days.

[0036] Fatty acid extraction from yeast: Pretreatment: Transfer 50 mL of fermentation broth to a 50 mL centrifuge tube and centrifuge at 6000 rpm for 10 min, discarding the supernatant. Resuspend the bacterial pellet in 30 mL of sterile deionized water, centrifuge again, and repeat the washing once more. Pre-freeze the collected bacterial cells overnight at -80°C, seal the tube openings with a membrane and puncture the membrane, then freeze-dry in a vacuum freeze dryer for 48 hours until constant weight. Grind the bacterial cells into powder using a mortar and pestle, and store in 1.5 mL EP tubes for later use.

[0037] Preparation of yeast fatty acid methyl esterification: Approximately 50 mg of yeast powder was placed in a 2 mL EP tube, and 100 μL of hexane solution containing 2 g / L methyl nonadecanoate (as internal standard) was added, followed by 1 mL of 1 M NaOH in methanol solution. The sample was vortexed at 25°C and 1200 rpm for 2 hours. Subsequently, 80 μL of 98% concentrated sulfuric acid was added to neutralize the alkalinity, followed by 400 μL of hexane, and the mixture was vortexed at 1200 rpm for 10 minutes. After centrifugation at 8000 rpm for 2 minutes, the supernatant was filtered through a 0.22 μm filter membrane, and the resulting sample was used for gas chromatography detection.

[0038] GC-FID analysis method: GC analysis was performed using Agilent instruments. Table 4 Chromatographic conditions

[0039] Under the above chromatographic conditions, a mixed standard of 37 fatty acid methyl esters and a sample for determination were injected into a gas chromatograph, and quantification was performed based on the chromatographic peak area.

[0040] The results are shown in Tables 5 and 6. Figures 1-3 As shown, the expression ZaFAD3-1 and ZaFAD3-2 α-Linolenic acid production was detected in all recombinant strains, confirming that... ZaFAD3-1 and ZaFAD3-2 The gene-encoded products all possess Δ-15 fatty acid desaturase activity, which converts linoleic acid into α-linolenic acid. The engineered Saccharomyces cerevisiae SLA03 can synthesize linoleic acid.

[0041] Quantitative analysis results showed that under 30°C culture conditions, ZaFAD3-1 Its catalytic activity is significantly higher than ZaFAD3- 2 Its product accumulation was approximately 3.0 times that of the latter, compared to the positive control derived from Arabidopsis thaliana. AtFAD3 The skill levels are comparable.

[0042] Further temperature gradient experiments showed that low temperature conditions significantly promoted enzyme activity. When the culture temperature dropped to 22°C, the product yield of all tested strains showed an increasing trend. ZaFAD3-1 It exhibited a significant response to low temperature, with the largest increase in α-linolenic acid production, approximately 3.92 times, significantly higher than the control group. AtFAD3 (1.5 times) and ZaFAD3-2 (1.6 times). At this optimized temperature (22°C), ZaFAD3-1 The final α-linolenic acid yield reached AtFAD3 1.42 times.

[0043] Table 5

[0044] Table 6

[0045] Formula for relative content: Since the control group does not produce linolenic acid, the linolenic acid content of the control group is set to 0.01. Multiply all contents by 100% to express the relative percentage content.

Claims

1. A fatty acid desaturase, characterized in that, The amino acid sequence of the fatty acid desaturase is shown in SEQ ID NO.4 or SEQ ID NO.

5.

2. A gene encoding the fatty acid desaturase of claim 1.

3. A recombinant vector containing the gene described in claim 2.

4. A recombinant microbial cell containing the gene described in claim 2.

5. The recombinant microbial cell according to claim 4, characterized in that, The starting microbial cells are either prokaryotic or eukaryotic microbial cells.

6. The use of the fatty acid desaturase of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, or the recombinant microbial cell of claim 5 or 6 in the production of α-linolenic acid.

7. A method for producing α-linolenic acid, characterized in that, Recombinant Saccharomyces cerevisiae was obtained by expressing the gene described in claim 2 in Saccharomyces cerevisiae cells, and then fermented to obtain linoleic acid.

8. The method according to claim 7, characterized in that, The brewer's yeast is a type of yeast that produces linoleic acid.

9. The method according to claim 8, characterized in that, The yeast Saccharomyces cerevisiae expresses the gene shown in SEQ ID NO.

25.

10. A method for producing α-linolenic acid, characterized in that, Recombinant Saccharomyces cerevisiae was obtained by expressing the gene of claim 2 in Saccharomyces cerevisiae cells, and then fermented in a fermentation medium containing linoleic acid to obtain linolenic acid.

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