A lysophosphatidylcholine acyltransferase, a nucleic acid molecule and their applications

Abstract

The present invention discloses a lysophosphatidylcholine acyltransferase, a nucleic acid molecule and their applications, belonging to the technical field of microbial synthetic biology or metabolic engineering. The present invention successfully applies a lysophosphatidylcholine acyltransferase (PtLPCAT) from Phaeodactylum tricornutum. By overexpressing it in recombinant cells, the transfer efficiency of fatty acyl groups between the PC pool and the CoA pool can be greatly improved, increasing the substrate source for the synthesis of long-chain polyunsaturated fatty acids and effectively increasing the relative content of long-chain polyunsaturated fatty acids in recombinant Yarrowia lipolytica. The lysophosphatidylcholine acyltransferase provided by the present invention can reduce the loss of intermediate metabolites and significantly increase the relative content of long-chain polyunsaturated fatty acids, especially eicosapentaenoic acid (C20:5n-3, EPA) in recombinant cells.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial synthetic biology or metabolic engineering, and in particular, to a lysophosphatidylcholine acyltransferase, a nucleic acid molecule, and their applications. Background Art

[0002] EPA, i.e., eicosapentaenoic acid, is an essential long-chain polyunsaturated fatty acid for the human body, which has functions such as reducing blood lipids, reducing the risk of cardiovascular diseases, anti-tumor, anti-inflammatory, antioxidant stress, and stabilizing cell membranes. It is an important raw material for adjuvant therapy drugs and health products [1, 2]. The production of eicosapentaenoic acid mainly comes from deep-sea fish oil. However, fish cells do not have the key genes for synthesizing polyunsaturated fatty acids such as EPA, and the polyunsaturated fatty acids in their fish oil actually come from marine microalgae. Microorganisms such as marine microalgae and fungi are the main producers of polyunsaturated fatty acids in nature. With the emergence of problems such as marine pollution and overfishing, the unique lipid metabolic pathways of microorganisms have become the focus and emphasis of related research. There are many important gene resources to be explored in the long-chain polyunsaturated fatty acid synthesis pathways of microorganisms such as algae and fungi.

[0003] Many microorganisms have been developed as engineering strains for fermentative production of long-chain polyunsaturated fatty acids. For example, Schizochytrium ( Schizochytrium sp. ) has been used for commercial production of docosahexaenoic acid [3], and Mortierella alpina has also been used for commercial production of arachidonic acid, etc. [4]. However, the lag in the metabolic research of algae and fungi and the bottleneck of biomass limit the potential of economic production. Selecting excellent chassis organisms as the starting point is another reliable metabolic engineering idea. Yarrowia lipolytica ( Yarrowia lipolytica ) as a non-conventional yeast has now received more attention in related research on metabolic engineering transformation, especially for the production of substances such as fatty acids and lipids. This is because Yarrowia lipolytica has a more efficient pentose phosphate pathway and acetyl-CoA metabolic ability compared to other yeasts, which can provide a large amount of NADPH and substrates for the synthesis of fatty acids. The oil content of Yarrowia lipolytica cells is high, reaching more than 30% of the dry weight [5, 6]. Moreover, Yarrowia lipolytica is a safe strain that can be industrially produced, has obtained the GRAS certification of the US FDA, and has mature fermentation production technology, making it an ideal chassis organism for the production of unsaturated fatty acids.

[0004] The traditional long-chain polyunsaturated fatty acid synthesis pathway is the aerobic desaturase and elongase pathway, that is, fatty acids (C16:0 / C18:0) are gradually synthesized into long-chain polyunsaturated fatty acids such as C18:1, C18:2, C18:3, C20:2, C20:3, C20:4, C20:5 through multiple desaturases and elongases. Yarrowia lipolytica itself can only synthesize up to C18:2, and additional expression of delta-9 elongase, delta-8 desaturase, delta-5 desaturase, delta-17 desaturase (or omega3-desaturase) in the cells is required to complete the pathway to EPA synthesis. At the same time, the synthesis of this kind of long-chain polyunsaturated fatty acid depends on the glycerol backbone of phospholipids, because most desaturases are lipid-linked and mainly introduce double bonds at the sn-2 position of phosphatidylcholine (PC) [7]. The catalytic substrate of the elongase is generally acyl-CoA, indicating that fatty acid acyl groups need to be repeatedly transferred between the PC pool and the CoA pool in the long-chain unsaturated fatty acid synthesis pathway [8]. Abbadi et al. [9] reconstructed the EPA synthesis pathway in flax, but the EPA yield was only 0.8%. This study believes that the substrate preference of the acyltransferase in the cells themselves limits the entry of a large amount of precursor fatty acids (C18:3, C18:4) into the acyl-CoA pool, resulting in a very low EPA synthesis amount.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a lysophosphatidylcholine acyltransferase, a nucleic acid molecule and their applications to solve the above technical problems.

[0007] The present invention successfully applies a lysophosphatidylcholine acyltransferase (PtLPCAT) from Phaeodactylum tricornutum. By overexpressing in recombinant cells, it greatly improves the transfer efficiency of intermediate metabolites between the PC pool and the CoA pool, increases the substrate source for the synthesis of long-chain polyunsaturated fatty acids, and effectively increases the relative content of long-chain polyunsaturated fatty acids in recombinant Yarrowia lipolytica. The lysophosphatidylcholine acyltransferase provided by the present invention can reduce the loss of intermediate metabolites and significantly increase the synthesis amount of long-chain polyunsaturated fatty acids.

[0008] The present invention is implemented as follows:

[0009] The present invention provides a lysophosphatidylcholine acyltransferase, the amino acid sequence of which is shown in SEQ ID NO: 2, with a total of 499 amino acids. The lysophosphatidylcholine acyltransferase is specifically derived from oleaginous microalgae, specifically Phaeodactylum tricornutum. The inventors found that cloning the lysophosphatidylcholine acyltransferase of Phaeodactylum tricornutum into a recombinant vector and transforming it into recombinant cells can increase the production of long-chain polyunsaturated fatty acids in the recombinant cells, especially increase the relative content and production of EPA in the total fatty acids of the recombinant cells. Moreover, the wild-type strain of Yarrowia lipolytica can only synthesize up to C18:2 and needs to additionally express delta-9 elongase, delta-8 desaturase, delta-5 desaturase, delta-17 desaturase (or omega3-desaturase) or reconstruct the Δ6 synthetic pathway in the cells to complete the pathway to EPA synthesis. However, the lysophosphatidylcholine acyltransferase provided by the present invention can significantly increase the content and production of long-chain polyunsaturated fatty acids in the recombinant strain, especially the content and production of EPA.

[0010] The amino acid sequence is as follows:

[0011] MSLPEALTKVLDQSIEFVYALGPIFEPTVFLVPGVVREQVQALSDAIGFDVETLNYCLGLFLCYPLALIMNTIPYGQLRHLFSFLLGAFLLQFTLGVQWIHQLVTSLIAYGLLAILPRQTTSTLVPLFAMLYLVMGHLHRQYTNYLGYDLDFTGAQMVLTQKLYMIAYNLYDGEVLSQGKDSKAAKKCSEYALPQLPNLIEFLGYTFCFSNVLSGPVFEFSVYRDVCSGQILFDDAGKPRGKIPSNVWPTLRPLLTSLINMGLFVFLGGMFPLNDPSNPQGSTPVVLTADFLAKPWHVRYGYMWVGLLAVRQKYYFAWKNSEGANNLWYAGFQGFDEEGKPKGWENCSNMNIWGFETASNVQTLSKEWNKKTSVWLTRYVYIRTNGSLIAVYSMSAFWHGFYPGYYLFFMSIPMLTVCERLGRKKISPYFSKEKWSLYGIVTILFTSLAVEYMVSPFPLLALDRSWNNWKSHYFFGHIGCVVFYLIVSVLPTPKKEKKE.

[0012] In the case where the present invention provides the above amino acid sequence, those skilled in the art can easily obtain the nucleic acid sequence encoding the above lysophosphatidylcholine acyltransferase according to the degeneracy of codons. This is easily achievable for those skilled in the art.

[0013] The present invention also provides a nucleic acid molecule encoding a gene for lysophosphatidylcholine acyltransferase, the sequence of which is shown in SEQ ID NO: 1, with a full length of 1500 bp, as follows:

[0014]

[0015] The function of the PtLPCAT gene is to improve the transfer efficiency of intermediate fatty acid (acyl) metabolites between the phospholipid and acyl-CoA pools. In other embodiments, after obtaining the above gene sequence, those skilled in the art can optimize the codons according to the codon usage preference of the host cell or strain and perform whole gene synthesis. For example, the codons can be optimized according to the codon usage preference of Yarrowia lipolytica.

[0016] The present invention also provides an expression cassette or expression vector comprising the above nucleic acid molecule.

[0017] In a preferred embodiment of the application of the present invention, the above expression cassette or expression vector further comprises a promoter and a terminator.

[0018] In an alternative embodiment, the promoter is a strong constitutive promoter.

[0019] In an alternative embodiment, the strong constitutive promoter is selected from the TEFin strong promoter and the GPD strong promoter.

[0020] In an alternative embodiment, the above expression cassette is linked to regulatory sequences for regulating the expression of the above nucleic acid molecule, including but not limited to enhancers, signal peptide coding sequences, selectable marker genes, etc.

[0021] In a preferred embodiment of the application of the present invention, the copy number of the gene encoding lysophosphatidylcholine acyltransferase in the expression cassette or expression vector is 1-10. The inventors have found that increasing the copy number of the gene encoding lysophosphatidylcholine acyltransferase in the expression cassette or expression vector can further increase the content and yield of very long chain polyunsaturated fatty acids (such as C20:5) in the host organism after transformation of the host organism.

[0022] In an alternative embodiment, the copy number is 1-3.

[0023] In a preferred embodiment of the application of the present invention, the expression vector is selected from Yarrowia lipolytica integrative expression vectors. In other embodiments, as long as the expression of the gene encoding lysophosphatidylcholine acyltransferase can be achieved, it is within the protection scope of the present invention.

[0024] The present invention also provides a recombinant bacterium or recombinant cell comprising the above nucleic acid molecule, or the above expression cassette or expression vector.

[0025] In an alternative embodiment, the recombinant bacterium or recombinant cell is selected from yeast, Escherichia coli or yeast cells; the recombinant cell can be a competent cell, for example, selected from Escherichia coli or yeast competent cells.

[0026] In an alternative embodiment, the yeast is selected from Yarrowia lipolytica.

[0027] Use of the above-mentioned lysophosphatidylcholine acyltransferase, the above-mentioned nucleic acid molecule, the above-mentioned expression cassette or expression vector, or the above-mentioned recombinant bacterium or recombinant cell in the synthesis of long-chain polyunsaturated fatty acids.

[0028] In an alternative embodiment, the long-chain polyunsaturated fatty acids are selected from C20 unsaturated fatty acids and C22 unsaturated fatty acids.

[0029] In an alternative embodiment, the C20 long-chain polyunsaturated fatty acid is selected from C20:5 Δ5,8,11,14,17 , n-3, that is, eicosapentaenoic acid EPA.

[0030] In a preferred embodiment of the application of the present invention, the above application includes: causing the recombinant bacterium or recombinant cell to overexpress the nucleic acid molecule shown in SEQ ID NO: 1.

[0031] In an alternative embodiment, screening is carried out to obtain a recombinant bacterium or recombinant cell that overexpresses the nucleic acid molecule shown in SEQ ID NO: 1. By overexpression, the relative content of EPA in the total fatty acids of the recombinant bacterium or recombinant cell can be significantly increased, and the content or yield of other long-chain unsaturated fatty acids in the recombinant bacterium or recombinant cell can also be significantly increased.

[0032] The present invention also provides a method for producing long-chain polyunsaturated fatty acids, which includes: introducing a gene encoding the above-mentioned lysophosphatidylcholine acyltransferase into a recombinant bacterium or recombinant cell.

[0033] In an alternative embodiment, the introduction method is selected from genetic transformation methods, genome editing methods or gene mutation methods.

[0034] Genetic transformation methods are, for example, selected from PEG, electroporation, particle bombardment and lithium acetate.

[0035] For example, it is introduced into yeast cells by conventional biotechnology methods such as lithium acetate-mediated transformation method, PEG / lithium acetate-mediated method.

[0036] Genome editing methods or gene mutation methods refer to those that can be easily conceived by those skilled in the art to modify a target organism through conventional transgenic technologies and gene editing technologies in the art (such as through zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), or CRISPR / Cas9, etc.), so that it has a gene encoding lysophosphatidylcholine acyltransferase as described above, thereby increasing the content or yield of unsaturated fatty acids in recombinant bacteria or recombinant cells, especially the relative content of EPA in total fatty acids. Therefore, no matter which technology is adopted, as long as it utilizes the lysophosphatidylcholine acyltransferase provided by the present invention to endow the host organism with a relatively high content or yield of unsaturated fatty acids, it belongs to the protection scope of the present invention.

[0037] The present invention has the following beneficial effects:

[0038] The present invention provides a lysophosphatidylcholine acyltransferase. The lysophosphatidylcholine acyltransferase is derived from an oil-producing microalgae, specifically Phaeodactylum tricornutum. The inventors found that cloning the lysophosphatidylcholine acyltransferase of Phaeodactylum tricornutum into a recombinant vector and transforming it into a recombinant cell can increase the production of long-chain polyunsaturated fatty acids in the recombinant cell, especially increase the relative content and yield of EPA in the total fatty acids of the recombinant cell.

[0039] However, the fatty acids of the wild-type Yarrowia lipolytica strain can only be synthesized up to C18:2, and additional expression of delta-9 elongase, delta-8 desaturase, delta-5 desaturase, delta-17 desaturase (or omega3-desaturase) or reconstruction of the Δ6 pathway in the cell is required to complete the pathway to EPA synthesis. The lysophosphatidylcholine acyltransferase provided by the present invention can greatly increase the content and yield of long-chain polyunsaturated fatty acids in the recombinant strain, especially the content and yield of EPA.

[0040] The inventors found that by overexpressing the coding gene of lysophosphatidylcholine acyltransferase in a recombinant cell, the transfer efficiency between the PC pool and the CoA pool of intermediate metabolites can be greatly improved, increasing the substrate source for the synthesis of long-chain polyunsaturated fatty acids, and effectively increasing the relative content of long-chain polyunsaturated fatty acids, namely EPA, in the recombinant cell. The lysophosphatidylcholine acyltransferase provided by the present invention can reduce the loss of intermediate metabolites and significantly increase the relative content and yield of target products such as long-chain polyunsaturated fatty acids such as EPA. Description of the Drawings

[0041] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following accompanying drawings only show some embodiments of the present invention, and thus should not be regarded as a limitation of the scope. For those of ordinary skill in the art, without creative efforts, other related accompanying drawings can also be obtained based on these drawings.

[0042] Figure 1 It is the amplification result diagram of the candidate PtLPCAT gene of Phaeodactylum tricornutum;

[0043] Figure 2 It is the amplification result diagram of the PCR detection of the transformants of the PtLPCAT-007 vector;

[0044] Figure 3 It is the schematic diagram of the expression vector, gene and integration site used in the construction of the present invention;

[0045] Figure 4 It is the PCR detection result diagram of the insertion site of the target gene;

[0046] Figure 5 It is the PCR detection result diagram of the target gene after tag removal;

[0047] Figure 6 It is the result diagram of the gas chromatography detection of the fatty acid components in each strain;

[0048] Figure 7 It is the detection result diagram of the components and relative contents of the fatty acids in each strain. Detailed Embodiments

[0049] Reference to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0050] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those of ordinary skill in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir and C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller and M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991), each of which is hereby expressly incorporated by reference.

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0052] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0053] Example 1

[0054] In this example, cloning of the PtLPCAT gene, construction of an expression plasmid for synthesizing EPA in Yarrowia lipolytica, genetic transformation of Yarrowia lipolytica, and screening of positive clones were carried out.

[0055] 1. Cloning of the PtLPCAT gene encoding lysophosphatidylcholine acyltransferase from Phaeodactylum tricornutum

[0056] First, candidate LPCATs were screened in the Phaeodactylum tricornutum genome database

[0057] (http: / / protists.ensembl.org / Phaeodactylum_tricornutum / Info / Index / ) using the known gene and protein sequences of Arabidopsis thaliana and Nicotiana tabacum LPCATs through BLAST, and LPCAT was identified by complementation experiments with Saccharomyces cerevisiae LPCAT-deficient mutant strains. Finally, a LPCAT candidate gene (PtLPCAT) with a size of 1500 bp was screened, and then bioinformatics software was used for analysis, and primers were designed for reverse transcription PCR to amplify its coding sequence.

[0058] Phaeodactylum tricornutum cells were inoculated into fresh f / 2 medium and aerobically cultured to the logarithmic phase, and 40 mL of the lower precipitate was collected by centrifugation at 4000 rpm for 10 min for RNA extraction. The specific steps are as follows:

[0059] (1) Add 1 mL of TRIzol reagent to the EP tube containing the collected Phaeodactylum tricornutum cells, and let it stand at room temperature for 1 h, with vortexing for 1 min at regular intervals during this period.

[0060] (2) Add chloroform to the EP tube at a ratio of 200 μL of chloroform per 1 mL of TRIzol, then vortex for 15 s and let it stand at room temperature for 2 - 3 min. Centrifuge at 12000g at 4℃ for 15 min.

[0061] (3) Transfer the upper aqueous phase to a new 1.5 mL EP tube, add isopropanol to the EP tube at a ratio of 500 μL of isopropanol per 1 mL of TRIzol, let it stand at room temperature for 10 min, and then centrifuge at 12000g at 4℃ for 10 min.

[0062] (4) Remove the supernatant, add 75% ethanol at a ratio of 500 μL of 75% ethanol per 1 mL of TRIzol to wash the RNA precipitate, and centrifuge at 7500g at 4℃ for 5 min.

[0063] (5) Remove the upper ethanol, and then air-dry the RNA precipitate in a laminar flow hood. Add an appropriate amount of RNase-free ddH2O to the EP tube according to the amount of the precipitate.

[0064] Using the total RNA of *Phaeodactylum tricornutum* as a template, the cDNA was obtained using the PrimeScript RT reagent Kit with gDNAEraser kit, and the operation was carried out according to the following steps:

[0065] (1) Prepare the following system in a 50 μL EP tube to remove genomic DNA

[0066]

[0067] Place it in a PCR instrument for reaction: 42°C, 5 min → hold at 4°C. In this step of the reaction, gDNA Eraser is used to eliminate the contamination of genomic DNA in the total RNA of *Phaeodactylum tricornutum*.

[0068] (2) Add the following reagents to the reaction solution in (1)

[0069]

[0070] Place the above reaction solution in a PCR instrument for reaction: 37°C, 15 min → 85°C, 5 s → hold at 4°C. The obtained cDNA was amplified by PCR using the high-fidelity PCR enzyme 2 × Phanta Max Master Mix and the designed primers LPCAT-For:

[0071] AAGCTTATGAGTCTCCCCGAAGCC / LPCAT-Rev:TTCTTTCTTTTCTTTCTTGTCTAGA, and the system is as follows:

[0072]

[0073] The reaction program is: 95°C, 5 min → (95°C, 15 s → 56°C, 15 s → 72°C, 1 min) × 35 → 72°C, 5 min → hold at 12°C. The PCR liquid after the reaction was first subjected to agarose gel electrophoresis, and the amplification result was as Figure 1 shown, with a target band of approximately 1.5 kb. Then, the correct target fragment was recovered using the FastPure Gel DNA Extraction Mini Kit according to the corresponding instructions, and the concentration of the candidate PtLPCAT fragment was measured using a ultra-micro spectrophotometer. The recovered target fragment was ligated to the 007 vector (pClone007 Versatile Simple Vector Kit), and the reaction system is as follows:

[0074] 5×pClone007 Vector Mix, 2 μL; 50 - 100 ng of the target gene. Make up to 10 μL with ddH2O, react at room temperature for 10 - 20 min, and then transform all the reaction solution into DH5α competent cells. The transformation method is as follows:

[0075] (1) Place the DH5α competent cells on ice 5 - 10 min in advance.

[0076] (2) Add all the reaction solution to the competent cells, gently mix, and then heat shock at 42 °C for 45 s, and immediately place on ice for 2 min.

[0077] (3) Add 500 mL of antibiotic-free LB medium to the competent cells, and then incubate at 37 °C for 1 h for recovery.

[0078] (4) Spread an appropriate amount of the bacterial solution on the screening plate and culture overnight at 37 °C.

[0079] Pick monoclonal colonies on the screening plate for colony PCR. The detection results are as Figure 2 shown. There are basically target bands. Select 3 - 5 monoclonal colonies for sequencing. Compare the sequencing results with those in the database, and select the correct ones, that is, PtLPCAT has been cloned.

[0080] 2. Obtaining other target genes

[0081] The genes used to construct the polyunsaturated fatty acid synthesis pathway include unsaturated enzymes and elongases from various sources: The C16 / 18 elongase is from Mortierella alpina ( Mortierella alpina ); The delta-12 unsaturated enzyme is from Fusarium moniliforme ( Fusarium verticillioides ); The delta-9 elongase is from Physcomitrella patens ( Physcomitrium patens ); The delta-8 unsaturated enzyme is from Perkinsus marinus ( Perkinsus marinus ); The delta-5 unsaturated enzyme is from Phaeodactylum tricornutum ( Phaeodactylum tricornutum ); The omega3-unsaturated enzyme is from Thraustochytrium sp. ( Thraustochytrium sp. ).

[0082] The coding gene of C16 / 18 elongase is shown as SEQ ID NO: 3, the amino acid sequence of C16 / 18 elongase is shown as SEQ ID NO: 4, the coding gene of delta-12 desaturase is shown as SEQ ID NO: 5, the amino acid sequence of delta-12 desaturase is shown as SEQ ID NO: 6, the coding gene of delta-9 elongase is shown as SEQ ID NO: 7, the amino acid sequence of delta-9 elongase is shown as SEQ ID NO: 8, the coding gene of delta-5 desaturase is shown as SEQ ID NO: 9, the amino acid sequence of delta-5 desaturase is shown as SEQ ID NO: 10. The coding gene of delta-8 desaturase is shown as SEQ ID NO: 11, the amino acid sequence of delta-8 desaturase is shown as SEQ ID NO: 12. The coding gene of omega3-desaturase is shown as SEQ ID NO: 13, the amino acid sequence of omega3-desaturase is shown as SEQ ID NO: 14.

[0083] In the present invention, the function of the PtLPCAT gene is to improve the transfer efficiency of intermediate metabolites between the phospholipid and CoA pools, reduce the loss of intermediate metabolites, and increase the relative content and yield of target products such as long-chain polyunsaturated fatty acids.

[0084] After obtaining the above gene sequences, their codons are optimized according to the codon usage preference of Yarrowia lipolytica and the full genes are synthesized.

[0085] 3. Construction of expression plasmids

[0086] The corresponding plasmids are constructed by combining the USER-clone and Cre-Loxp systems, which can achieve the marker-free integration of the gene expression vector into the genomic locus of Yarrowia lipolytica. The expression vector has specially selected genomic integration sites and uses expression elements (strong promoters and terminators) homologous to Yarrowia lipolytica, which can achieve high-level gene expression without affecting cell growth. An expression plasmid is constructed for every two genes in a group, and the completed expression plasmids are shown in Figure 3 and Table 1 in the appendix.

[0087] Figure 3 In the figure, A: YI_IntB-C16 / 18elo-PrTEFin-PrGPD-Δ12des; B YI_IntE_1-Δ9elo-PrTEFin-PrGPD-Δ8des;

[0088] C: YI_IntF_2 - Δ5des - PrTEFin - PrGPD - Ω3des;

[0089] D: YI_IntC_1 - PtLPCAT - PrTEFin - PrGPD - PtLPCAT.

[0090] Table 1 Plasmids used in the present invention.

[0091]

[0092] Taking the construction of the YI_IntB_C16 / 18elo_PrTEFin - PrGPD_Δ12des plasmid as an example, the specific method is as follows:

[0093] (1) The target gene and promoter fragment were amplified by PCR using the high - fidelity enzyme Phusion U Hot Start DNA Polymerase, and the primers are shown in Table 2.

[0094] Table 2 Primers used in the present invention.

[0095]

[0096] The system is as follows:

[0097]

[0098] The reaction program was: 98°C, 1 min → (98°C, 10 s → 54°C, 30 s → 72°C, 1 min) × 35 → 72°C, 5 min → 10°C. After the reaction, the PCR solution was subjected to agarose gel electrophoresis, and each correct target fragment was recovered to obtain the C16 / 18elo, PrTEFin, PrGPD, and Δ12des gene fragments.

[0099] (2) Digest the YI_IntB plasmid. First, digest it with AsiSI. The system is as follows:

[0100]

[0101] After reacting at 37°C for 2 h, gel recovery was carried out, and then it was digested with Nb.BsmI. The digestion system is as follows:

[0102]

[0103] React at 65°C for 1 h, and then gel recovery was carried out to obtain the end - modified linearized plasmid.

[0104] (3) Assemble each gene fragment onto the YI_IntB vector using the USER cloning method. The system is as follows:

[0105]

[0106] The reaction procedure is as follows: 37 °C for 25 min → 25 °C for 10 min → 4 °C for incubation. Transfer the above reaction solution into DH5α, and the specific steps refer to the USER instruction manual.

[0107] (3)Screening and detection of DH5α transformants

[0108] Pick monoclonal colonies and perform colony PCR detection using YI-Clone-F / YI-Clone-R primers. The reaction system is as follows:

[0109]

[0110] The reaction procedure is as follows: 98 °C for 3 min → (98 °C for 15 s → 56 °C for 15 s → 72 °C for 1 min) × 35 → 72 °C for 5 min → 12 °C for incubation. Then, detect by DNA gel electrophoresis and confirm by sequencing.

[0111] Construct other plasmids shown in Table 1 using the same method.

[0112] 4. Yeast transformation and screening

[0113] One day in advance, streak the transformed strain on a YPD plate for culture. Take 90 μL of PEG6000 and 5 μL of lithium acetate in a 1.5 mL sterilized EP tube, and then add 5 μL of salmon sperm treated at 100 °C for 5 min. Then pick a cell mass the size of a mung bean into the EP tube, shake for 10 - 15 s, and then add 500 ng of linearized expression plasmid; after shaking for 10 - 15 s, react at 30 °C for 10 min, then shake for 10 - 15 s again. Repeat this process 3 times, then react at 39 °C for 10 min. Finally, add 100 μL of sterilized ddH2O, mix well, and take 100 μL to coat on an SC-ura plate. Place it in an incubator at 28 °C for 2 - 4 days to grow monoclonal colonies. When picking the transformed yeast monoclonal colonies and streaking them on the SC-ura plate, inoculate them into the SC-ura medium at the same time, culture overnight, extract its genome, and use it as a template to perform PCR to detect whether the corresponding gene is integrated into the target site of the yeast genome.

[0114] After PCR detection, refer to Figure 4 as shown, M: marker; 1, 2 are wild types; 3 - 12 are transformants. It can be Figure 4 seen that the target gene is inserted into the target site of the yeast genome.

[0115] For the smooth transformation of the next plasmid and the screening of monoclonal colonies, it is necessary to first remove the URA tag in the positive clones obtained in the previous step. Since there are LoxP sites at both ends of URA on the constructed vector, the cre-loxp system is used to remove the URA tag. The specific method is to transfer the Cre plasmid into the positive clones obtained in the previous step, and after transformation, coat them on the SC-leu plate. Place them in an incubator at 28 °C and culture for 2 - 4 days until single colonies grow. Then, streak the grown monoclonal colonies on the YPD solid plate for two rounds of subculture, and then streak the clones in the second round on the YPD, SC-leu, and SC-ura plates. After about 5 - 6 rounds of continuous subculture, there will be monoclonal colonies that only grow on YPD, that is, the transformed yeast with the tag removed.

[0116] The PCR detection results of the target gene after tag removal are shown in Figure 5 as follows. M: marker; 1 - 10 are transformants. After multiple rounds of transformation screening, the target recombinant yeast strain was finally obtained.

[0117] Example 2

[0118] In this example, the recombinant yeast obtained by screening was fermented and fatty acid analysis was carried out.

[0119] First, the recombinant yeast strains transformed with foreign genes were activated overnight at 28 °C, and then their OD 600 was measured. After adjusting their OD 600 to be the same, the same amount of bacterial liquid was taken and inoculated into 100 mL of YPD liquid medium, and cultured at 28 °C and 220 rpm for 3 days. Then, the cell bacterial liquid was collected and stored in a -80 °C refrigerator for 1 day, and then placed in a vacuum freeze dryer to be completely dried. Finally, the bacterial powder of each strain was ground. Then, the fatty acid components of the corresponding strains were detected by gas chromatography. The method is as follows:

[0120] (1) Weigh 8 mg of each bacterial powder and place it in a 4 mL white-capped bottle. Add 0.75 mL of sulfuric acid:methanol = 5:95 (V / V) solution and 20 μL of C21:0 (1 mg / ml) internal standard (Sigma, product number: 51535 - 1G), and mix well with a vortex mixer until no large particles can be seen.

[0121] (2) Place the white-capped bottle in a metal bath and heat it at 90 °C for 1.5 h.

[0122] (3) After the reaction, cool the white-capped bottle to room temperature, add 500 μl of 0.9% sodium chloride (W / V) and 300 μL of n-hexane, and mix well again. Centrifuge at 3000 rpm for 10 min.

[0123] (4) Use a longer and thinner pipette tip to draw the supernatant into a new 1.5 mL EP tube, centrifuge at 12,000 rpm for 5 min, take 300 μL of the supernatant and add it to the inner liner tube, seal it, and load it for analysis.

[0124] Gas chromatography detection conditions: hydrogen flame ionization detector; Agilent7890A gas chromatograph; HP-FFAP column (30m×250μm×0.25μm). The carrier gas is high-purity nitrogen, and the gas flow rates of nitrogen, hydrogen, air, and tail gas are 18mL / min, 28mL / min, 380mL / min, and 18mL / min, respectively. The injection port temperature is 250℃, and the detector temperature is 280℃.

[0125] The results of gas chromatography detection of fatty acid components in each strain refer to Figure 6 As shown, A shows the GC test results of the Po1f:EPA strain, B shows the GC test results of the Po1f:EPA-PtLPCAT strain (i.e., a recombinant yeast strain with a single copy of the target gene), and Figure C shows the GC test results of the Po1f:EPA-PtLPCAT×2 strain (i.e., a recombinant yeast strain with two copies of the target gene). Figure 6 As shown in A, the strain Po1f:EPA that transformed the exogenous gene to achieve the reconstruction of the EPA synthesis pathway did synthesize EPA. On this basis, in the strains Po1f:EPA-PtLPCAT and Po1f:EPA-PtLPCAT×2 that further transformed the PtLPCAT gene, GC detection found that the EPA content was greatly increased ( Figure 6 B and C).

[0126] The composition and relative content of fatty acids in each strain were detected. Figure 7 As shown, Figure 7 The EPA (C20:5) content in the Po1f:EPA-PtLPCAT strain and the Po1f:EPA-PtLPCAT×2 strain increased from 7.25% in the Po1f:EPA strain to 12.06% and 18.23%, respectively ( Figure 7 ); It shows that overexpression of PtLPCAT can significantly increase the relative content of EPA in total fatty acids in recombinant Yarrowia lipolytica. Moreover, the content of C18:3 unsaturated fatty acids was also significantly increased after overexpression.

[0127] References

[0128] [1] Zhang Zhaoqian, Liu Fei, Zhang Jinhua, et al. Research progress on the biological activity and pharmacological effects of eicosapentaenoic acid[J]. Food and Drug, 2020, 22(4):5.

[0129] [2] Zhang T T, Xu J, Wang Y M, et al. Health benefits of dietary marine DHA / EPA-enriched glycerophospholipids [J]. Progress in Lipid Research, 2019, 75:100997.

[0130] [3] Barclay W R, Meager K M, Abril J R. Heterotrophic production of long chain omega-3 fatty acids utilizing algae and algae-like microorganisms [J]. Journal of Applied Phycology, 1994, 6(2):123-129.

[0131] [4] Kikukawa H, Sakuradani E, Ando A, et al. Arachidonic acid production by the oleaginous fungus Mortierella alpina 1S-4: A review [J]. Journal of Advanced Research, 2018, 11.

[0132] [5] Barth G, Gaillardin C, Wolf K. Nonconventional Yeasts in Biotechnology[M]. 1996. (pp. 313–388). Berlin, Heidelberg: Springer Berlin Heidelberg.

[0133] [6] Ledesma-Amaro R, Nicaud J M. Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids[J]. Progress in Lipid Research, 2016, 61:40-50.

[0134] [7] Domergue, F. Acyl Carriers Used as Substrates by the Desaturasesand Elongases Involved in Very Long-chain Polyunsaturated Fatty AcidsBiosynthesis Reconstituted in Yeast[J]. Journal of Biological Chemistry,2003, 278(37):35115-35126.

[0135] [8] Petrie J R, Shrestha P, Mansour M P, Nichols P D, Liu Q, Singh SP. Metabolic engineering of omega-3 long-chain polyunsaturated fatty acids inplants using an acyl-CoA Delta6-desaturase with omega3-preference from themarine microalga Micromonas pusilla.[j]. Metabolic engineering, 2010, 12(3):233-240.

[0136] [9] Abbadi, A. Biosynthesis of Very-Long-Chain Polyunsaturated FattyAcids in Transgenic Oilseeds: Constraints on Their Accumulation[J]. The PlantCell, 2004, 16(10):2734-2748.

[0137] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of lysophosphatidylcholine acyltransferase, nucleic acid molecule encoding lysophosphatidylcholine acyltransferase, expression cassette or expression vector in enhancing the synthesis of EPA by Yarrowia lipolytica; characterized in that, The amino acid sequence of the lysophosphatidylcholine acyltransferase is as shown in SEQ ID NO: 2; the expression cassette or expression vector comprises the nucleic acid molecule; the Yarrowia lipolytica further comprises: the coding gene of C16 / 18 elongase, the coding gene of delta-12 desaturase, the coding gene of delta-9 elongase, the coding gene of delta-5 desaturase, the coding gene of delta-8 desaturase and the coding gene of omega3-desaturase; The amino acid sequence of C16 / 18 elongase is as shown in SEQ ID NO: 4, the amino acid sequence of delta-12 desaturase is as shown in SEQ ID NO: 6, the amino acid sequence of delta-9 elongase is as shown in SEQ ID NO: 8, the amino acid sequence of delta-5 desaturase is as shown in SEQ ID NO: 10, the amino acid sequence of delta-8 desaturase is as shown in SEQ ID NO: 12, and the amino acid sequence of omega3-desaturase is as shown in SEQ ID NO:

14.

2. The application according to claim 1, characterized in that, The expression cassette or expression vector further comprises a promoter and a terminator.

3. The application according to claim 2, wherein The promoter is a strong constitutive promoter.

4. The application according to claim 3, characterized in that, The strong constitutive promoter is selected from the TEFin strong promoter and the GPD strong promoter.

5. The application according to claim 1, wherein The copy number of the gene encoding lysophosphatidylcholine acyltransferase in the expression cassette or expression vector is 1-10.

6. The application according to claim 5, wherein The copy number is 1-3.

7. The application according to claim 1, characterized in that The expression vector is selected from Yarrowia lipolytica integrative expression vectors.

8. The application according to claim 1, characterized in that, The sequence of the nucleic acid molecule is as shown in SEQ ID NO:

1.

9. The application according to claim 1, wherein It comprises: making the recombinant bacterium or recombinant cell overexpress the nucleic acid molecule as shown in SEQ ID NO:

1.

10. A method for improving the synthesis of EPA by Yarrowia lipolytica, characterized in that, It comprises: introducing into Yarrowia lipolytica a gene encoding lysophosphatidylcholine acyltransferase, the amino acid sequence of the lysophosphatidylcholine acyltransferase being as shown in SEQ ID NO: 2; The Yarrowia lipolytica further comprises: the coding gene of C16 / 18 elongase, the coding gene of delta-12 desaturase, the coding gene of delta-9 elongase, the coding gene of delta-5 desaturase, the coding gene of delta-8 desaturase and the coding gene of omega3-desaturase; The amino acid sequence of C16 / 18 elongase is as shown in SEQ ID NO: 4, the amino acid sequence of delta-12 desaturase is as shown in SEQ ID NO: 6, the amino acid sequence of delta-9 elongase is as shown in SEQ ID NO: 8, the amino acid sequence of delta-5 desaturase is as shown in SEQ ID NO: 10, the amino acid sequence of delta-8 desaturase is as shown in SEQ ID NO: 12, and the amino acid sequence of omega3-desaturase is as shown in SEQ ID NO:

14.

11. The method according to claim 10, wherein The introduction method is selected from genetic transformation methods or genome editing methods.

Images