Delta 12 fatty acid desaturase mutants and uses thereof

Abstract

The application discloses a kind of Δ12 fatty acid desaturase mutants and its application, belong to genetic engineering technical field.The Δ12 fatty acid desaturase mutant disclosed in the application is based on the amino acid sequence shown in SEQ ID NO.2, and the following mutations are carried out:K381P-H415Y.The K381P-H415Y double mutant of the application can significantly enhance the conversion capacity of oleic acid to linoleic acid, and can be used to construct a microbial cell factory with high yield of linoleic acid, and has good industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to a Δ12 fatty acid desaturase mutant and its applications. Background Technology

[0002] Linoleic acid is an important unsaturated fatty acid with applications in food, nutrition and health, pharmaceutical intermediates, daily chemicals, and fine chemicals. Traditionally, linoleic acid is mainly extracted from vegetable oils, but plant sources are greatly affected by crop variety, season, origin, climate, and oil composition, resulting in problems such as insufficient batch stability, long production cycle, and high separation and purification costs.

[0003] Microbial fermentation offers advantages such as short production cycles, controllable processes, ease of scale-up, and stable raw material sources. *Yarrowia lipolytica* possesses strong lipid metabolism capabilities, making it an excellent host for constructing unsaturated fatty acid cell factories. However, the efficient conversion of oleic acid to linoleic acid relies on high-performance Δ12 fatty acid desaturases. Existing enzymes may suffer from insufficient catalytic efficiency, limited expression adaptability, and low product accumulation capacity in heterologous hosts. Therefore, it is necessary to screen and modify more efficient Δ12 fatty acid desaturases.

[0004] Therefore, providing a Δ12 fatty acid desaturase mutant and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a Δ12 fatty acid desaturase mutant and its application.

[0006] This invention utilizes a protein screening algorithm to predict the function of candidate proteins derived from fungi, thereby screening out proteins derived from fungi. Horse-hair worm The hypothetical protein V5O48_015951, with NCBI ID KAL0566063.1, was predicted by an algorithm to possess Δ12 fatty acid desaturase function, potentially catalyzing the conversion of oleic acid to linoleic acid. Using this protein as the parent enzyme (starting protein McDes12), further rational design yielded an excellent double-point mutant, K381P-H415Y, which was used to improve the linoleic acid production capacity of *Yarrowia lipolytica*.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A Δ12 fatty acid desaturase mutant, based on the amino acid sequence shown in SEQ ID NO.2, is mutated as follows: K381P-H415Y.

[0009] Furthermore, the DNA molecule encoding the Δ12 fatty acid desaturase mutant.

[0010] Furthermore, a recombinant expression plasmid containing the DNA molecule.

[0011] Furthermore, a genetically engineered bacterium, in *Yarrowia lipophila* (… Yarrowia lipolytica The recombinant expression plasmid described above is expressed in Po1f.

[0012] Furthermore, the application of the Δ12 fatty acid desaturase mutant, the DNA molecule, the recombinant expression plasmid, or the genetically engineered bacteria in the production of linoleic acid.

[0013] Furthermore, the application of the Δ12 fatty acid desaturase mutant, the DNA molecule, the recombinant expression plasmid, or the genetically engineered bacteria in increasing linoleic acid production.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a Δ12 fatty acid desaturase mutant and its application. Horse-hair worm A Δ12 fatty acid desaturase McDes12 with highly efficient oleic acid to linoleic acid conversion was discovered in the hypothetical protein source, and a double mutant K381P-H415Y with significantly improved performance was obtained through rational design.

[0015] Compared with the parental enzyme, the double mutant K381P-H415Y significantly increased linoleic acid production in engineered Yersinia lipolytica, achieving a yield of 1519.05 mg / L in shake-flask fermentation, approximately 4.87 times that of the parental enzyme control. These results indicate that the K381P-H415Y double mutant can significantly enhance the conversion of oleic acid to linoleic acid and can be used to construct high-yield linoleic acid microbial cell factories, demonstrating good potential for industrial application. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1 This invention utilizes the mature Yersinia lipolyticis integrative expression vector pINA1269 to express the Δ12 fatty acid desaturase mutant K381P-H415Y. This vector contains the hp4d promoter, XPR2 terminator, and LEU2 selection marker, making it suitable for exogenous gene expression and SC-Leu selection in LEU-deficient Yersinia lipolyticis.

[0018] The target gene sequence shown in SEQ ID NO.1 or SEQ ID NO.3 was ligated to the PmlI and KpnI restriction enzymes of the pINA1269 vector, so that the target gene was operatively ligated between the hp4d promoter and the XPR2 terminator, resulting in the pINA1269-McDes12 or pINA1269-McDes12(K381P-H415Y) plasmid.

[0019] The McDes12 base sequence is shown in SEQ ID NO.1.

[0020]

[0021] The 12 amino acid sequence of McDes is shown in SEQ ID NO.2.

[0022] MTPDATDTVVEQGVYVVPDYTVKDLLSVIPPHCNQRSAIRSSLYIVMDVAVITTLFKVAQFADAQVLSGALTFPHDSLYYLARFAIWALYGFGAGLFATGLWVIAHECGHQAFSDSKFVNNTVGWVLHSALGVPYHSWRITHAKHHASTGHMTQDQVFVPPTRTEVGLKPLDSTKEDRLGARVSEAVKKELWEALGDSPIGATWGAASYLLGGWPAYLLTNASGQKRYPKGTNHFTPTAVMFTPKQYGQIILSDIGVVFWLAAVVSAIYYKGFVNVFAVYLVPYLWVNHWLVAITFLQHTDPLLPHYRAPEFTFPRGALATLDRSMLGNLGGFMGWLGAVSTHGIAETHILHHVASRIPHYHAWEASRALKQKLQEGGGMKVQGNPVSWGEIYRVYKECKFVEDEGDVVFFKNAHGLAQMRPAFNSESPSDSGIEINDK; SEQ ID NO.2.

[0023] The base sequence of the K381P-H415Y mutant is shown in SEQ ID NO.3.

[0024]

[0025] The amino acid sequence of the K381P-H415Y mutant is shown in SEQ ID NO.4.

[0026] MTPDATDTVVEQGVYVVPDYTVKDLLSVIPPHCNQRSAIRSSLYIVMDVAVITTLFKVAQFADAQVLSGALTFPHDSLYYLARFAIWALYGFGAGLFATGLWVIAHECGH QAFSDSKFVNNTVGWVLHSALGVPYHSWRITHAKHHASTGHMTQDQVFVPPTRTEVGLKPLDSTKEDRLGARVSEAVKKELWEALGDSPIGATWGAASYLLGGWPAYLLTN ASGQKRYPKGTNHFTPTAVMFTPKQYGQIILSDIGVVFWLAAVVSAIYYKGFVNVFAVYLVPYLWVNHWLVAITFLQHTDPLLPHYRAPEFTFPRGALATLDRSMLGNLGG SEQ IDNO.4.

[0027] Example 2: Plasmid transformation of Yersinia lipophila using chemical transformation method (1) Yeast extract ( Yarrowia lipolytica Po1f was inoculated into YPD liquid medium and cultured at 30°C and 200 rpm with shaking until the logarithmic growth phase (OD600 was 1.0) to obtain the logarithmic growth phase bacterial culture.

[0028] YPD liquid culture medium formula (1L): 10 g yeast extract, 20 g peptone, 20 g glucose, deionized water to 1 L.

[0029] (2) Take 3 mL of logarithmic phase bacterial culture, centrifuge at room temperature or 4°C to collect the bacterial cells, and discard the supernatant. Wash the bacterial cells twice with sterile water.

[0030] (3) Centrifuge and discard the supernatant. Resuspend the cells in 0.1 mol / L lithium acetate solution and incubate at 30°C for 15-30 min with static or gentle shaking to bring the cells into a suitable transformation state.

[0031] (4) Centrifuge and discard the supernatant. Add the following components to the bacterial pellet in sequence and mix thoroughly: 2.0 μg of the plasmid to be transformed, pINA1269-McDes12 or pINA1269-McDes12 (K381P-H415Y); 10 μL of 10 mg / mL heat-denatured salmon sperm DNA (heated at 95-100℃ for 10 min before use and then immediately cooled in an ice bath); approximately 70 μL of 50% PEG 4000; 10 μL of 1.0 mol / L lithium acetate; and sterile water to a final volume of 100 μL.

[0032] (5) Place the conversion system in a 39°C water bath for 60 min. Mix it once every 20 min during the process.

[0033] (6) After heat shock, centrifuge to remove the transformation solution and resuspend the cells in 500 μL of YPD liquid medium. Incubate at 30℃ for 2 h to improve the positive transformant yield.

[0034] (7) Spread the bacterial cells onto SC-Leu solid medium selective plates. Incubate upside down at 30°C for 2 days until single colonies are formed.

[0035] SC-Leu liquid medium formulation (1L): Yeast nitrogen base without amino acids, YNB 6.7 g; glucose 20 g; leucine-deficient amino acid mixture (Sigma-Aldrich Yeast Synthetic Drop-out Medium Supplements without leucine (catalog number Y1376)) 1.62 g; deionized water to 1 L. SC-Leu solid medium is prepared by adding 20 g / L agar to the SC-Leu liquid medium.

[0036] (8) Select single clones from the selection plate and perform sequencing verification to confirm that the target Δ12 fatty acid desaturase mutant gene has been successfully introduced into the host cell.

[0037] Example 3: Shake-flask fermentation (1) Selected positive transformants from SC-Leu selection plates and inoculated into 5 mL of SC-Leu liquid medium. The plates were then cultured at 30°C and 200 rpm for 24 h with shaking.

[0038] (2) The activated bacterial culture was transferred to fresh SC-Leu liquid medium at an inoculation rate of 1%, and cultured at 30℃ and 200 rpm until the logarithmic growth phase, with an OD600 of 1.5, as the seed culture.

[0039] (3) The seed culture was inoculated into a baffled Erlenmeyer flask containing SC-Leu fermentation medium at a 2% inoculation rate. 30 mL of SC-Leu fermentation medium was placed in a 250 mL Erlenmeyer flask.

[0040] SC-Leu fermentation medium formula (1L): Yeast nitrogen base without amino acids, YNB 6.7 g; glucose 40 g; leucine-deficient amino acid mixture 1.62 g; deionized water to 1 L.

[0041] (4) After inoculation, the shake flasks were placed in a constant temperature shaker at 30℃ and 200 rpm for 96 h.

[0042] Example 4 Sample methylation treatment: (1) Collecting bacterial cells: After fermentation, take 1 ml of fermentation broth, centrifuge at 12000 rpm for 3 min, discard the supernatant, and collect the bacterial cell precipitate.

[0043] (2) Washing the bacterial cells: Wash the bacterial cells twice with sterile water, centrifuge and discard the supernatant to remove culture medium residue.

[0044] (3) Alkali treatment / saponification: Add 0.5 mol / L sodium hydroxide-methanol solution to the sample, mix thoroughly, and react in a 70℃ water bath or metal bath for 20 min. During this period, the mixture can be shaken intermittently to ensure that the bacterial lipids are fully saponified.

[0045] (4) Acid-catalyzed methyl esterification: After cooling to room temperature, add 4% sulfuric acid-methanol solution, mix well, and continue the reaction at 70℃ for 30 min to convert fatty acids into fatty acid methyl esters.

[0046] (5) Hexane extraction: After cooling to room temperature, add 1 mL of hexane (containing 1 g / L of internal standard dodecane) to the reaction system, vortex vigorously for 10 min, and centrifuge at 12000 rpm for 3 min. Collect the upper hexane organic phase.

[0047] Fatty acid methyl ester samples were qualitatively and quantitatively analyzed using an Agilent gas chromatography-mass spectrometry (GC-MS) system. The n-hexane organic phase obtained after methylation was transferred to a vial. Chromatographic separation was performed using an HP-5MS capillary column with high-purity helium as the carrier gas at a flow rate of 1.0 mL / min; the injection volume was 1 μL, the injection port temperature was 250℃, and split injection was used with a split ratio of 10:1. The column temperature program was set as follows: initial temperature 80℃, held for 1-2 min, ramped up to 180℃ at 10℃ / min, then ramped up to 230℃ at 3℃ / min and held for 5-10 min. Mass spectrometry used an EI ionization source with an ionization energy of 70 eV, an ion source temperature of 230℃, a quadrupole temperature of 150℃, a transfer line temperature of 250-280℃, and a scan range of m / z 50-550. Qualitative analysis was performed by comparing the retention time and characteristic ions of the mass spectrometer with those of the methyl linoleate standard. Quantitative analysis was performed using the internal standard method, with a standard curve established by the ratio of the peak area of ​​methyl linoleate to that of the internal standard, and the linoleic acid content in the sample was calculated.

[0048] The experimental results showed that the linoleic acid yield of the control strain expressing the parental enzyme McDes12 was 311.71 mg / L; the yield of the engineered strain expressing the double-point mutant K381P-H415Y was 1519.05 mg / L, approximately 4.87 times that of the parental enzyme control. These results indicate that the K381P-H415Y double-point mutation significantly improved the ability of Δ12 fatty acid desaturase to convert oleic acid to linoleic acid in *Yarrowia lipolytica*.

[0049] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Δ12 fatty acid desaturase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.2, the following mutations were performed: K381P and H415Y.

2. A DNA molecule encoding the Δ12 fatty acid desaturase mutant of claim 1.

3. A recombinant expression plasmid comprising the DNA molecule of claim 2.

4. A genetically engineered bacterium, characterized in that, In Yersinia lipophila ( Yarrowia lipolytica The genetically engineered bacteria obtained by transforming the recombinant expression plasmid of claim 3 into Po1f.

5. The application of the Δ12 fatty acid desaturase mutant of claim 1, the DNA molecule of claim 2, the recombinant expression plasmid of claim 3, or the genetically engineered bacteria of claim 4 in the production of linoleic acid.

6. The application of the Δ12 fatty acid desaturase mutant of claim 1, the DNA molecule of claim 2, the recombinant expression plasmid of claim 3, or the genetically engineered bacteria of claim 4 in increasing linoleic acid production.