A method for biosynthesis of a linoleic acid-rich oil using yarrowia lipolytica

Abstract

The application provides a method for biosynthesis of linoleic acid-rich oil by Yarrowia lipolytica. The recombinant Yarrowia lipolytica is constructed by two levels of gene operation, the first level aims to improve the proportion of linoleic acid in oil, and the second level aims to improve the biosynthesis capacity of total oil. The construction method of the Yarrowia lipolytica provided by the application is simple, and the constructed Yarrowia lipolytica can biosynthesize linoleic acid-rich oil efficiently, on the one hand, the oil synthesis capacity is improved, and on the other hand, the proportion of linoleic acid in oil is also significantly improved, so that the Yarrowia lipolytica can replace the plant-derived linoleic acid oil and be applied in food and industry.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a method for biosynthesizing linoleic acid-rich oils using Yeast Extract. Background Technology

[0002] Linoleic acid (LA, C18:2Δ9,12) is an essential polyunsaturated fatty acid for humans. As an essential fatty acid, it cannot be synthesized by the human body and must be obtained from food; otherwise, deficiency can lead to illness. Therefore, consuming oils rich in linoleic acid can prevent or reduce the incidence of cardiovascular diseases, particularly hypertension, hyperlipidemia, angina pectoris, coronary heart disease, and atherosclerosis. Furthermore, linoleic acid is an ideal skin beautifying agent. A deficiency in linoleic acid can easily cause dry skin, thickened scales, stunted growth, and cholesterol deposits in blood vessels; hence, linoleic acid is also known as the "beauty acid."

[0003] Linoleic acid is naturally found in animal and plant fats, but its content is relatively low in animal fats, such as only 1.8% in butter and 6% in lard. In some plant oils, however, linoleic acid content is quite high; peanut oil contains 26%, soybean oil a remarkable 58%, and safflower oil an astonishing 70%. Therefore, plant-based linoleic acid has become the preferred dietary source. However, these methods of obtaining linoleic acid from plants not only consume large amounts of arable land but also are affected by seasons and geographical location, making year-round production impossible. The development of microbial oil resources is a powerful solution to this problem. Microbial oils are produced by yeasts, molds, and algae under certain conditions using carbohydrates, hydrocarbons, and ordinary oils as carbon and nitrogen sources, supplemented with inorganic salts. Microbial oil production not only has advantages such as high oil content, short production cycle, no seasonal impact, and no land occupation, but also allows for engineering modifications to enable microorganisms to produce highly nutritious oils rich in specific fatty acids. Microbial oils containing these components can not only effectively replace edible oils from plant sources, but also have a wider range of nutritional functions. Yeast extract (…) Yarrowia lipolyticaYersinia lipolytica is one of the most widely studied oil-producing microorganisms and is recognized as a food-grade safe microorganism. It has been modified to produce oils rich in polyunsaturated fatty acids (such as eicosapentaenoic acid, EPA) and monounsaturated fatty acids (such as nervonic acid, NA), which have high nutritional value. However, there are no reports on modifying Yersinia lipolytica to improve its ability to biosynthesize linoleic acid-rich oils. In fact, wild Yersinia lipolytica can naturally accumulate linoleic acid (accounting for about 10% of total oil), but the content is significantly lower than that of linoleic acid-rich oils from plant sources. This invention will provide a method based on genetic engineering to improve the biosynthesis of linoleic acid-rich oils in Yersinia lipolytica. Summary of the Invention

[0004] Purpose of the invention: This invention addresses the insufficient supply of linoleic acid oils from plant sources by providing a more efficient method for biosynthesizing linoleic acid-rich oils using the recognized and safe microorganism Yersinia lipolytica. Specifically, this is achieved by constructing recombinant Yersinia lipolytica, which on the one hand improves its ability to synthesize oils, and on the other hand increases the proportion of linoleic acid in the synthesized oils.

[0005] To address the aforementioned technical problems, this invention discloses a recombinant *Yarrowia lipolytica*, its construction method, and its applications. The constructed recombinant *Yarrowia lipolytica* can efficiently biosynthesize linoleic acid-rich oils, increasing the proportion of linoleic acid and the level of oil synthesis in *Yarrowia lipolytica*. Simultaneously, it increases the potential for food and industrial applications using oils derived from *Yarrowia lipolytica* to replace plant-derived linoleic acid oils. The technical solution of this invention is as follows: In a first aspect, the present invention provides a recombinant Yersinia lipolytica yeast, wherein the recombinant Yersinia lipolytica yeast expresses the gene encoding Δ12 fatty acid desaturase Fad2 (responsible for the conversion of oleic acid to linoleic acid), the gene encoding lysophosphatidylcholine acyltransferase Lpcat (EC 2.3.1.23), and the gene encoding choline phosphotransferase Cpt (EC 2.7.8.2); wherein Lpcat and Cpt are responsible for acyl editing pathways related to phosphatidyl accumulation.

[0006] The Fad2 encoding gene is derived from Yersinia lipophila (Yersinia lipophila). Yarrowia lipolytica ), Alpine spores ( Mortierella alpina ) or Rhodotorula buergerianum ( Rhodotorula toruloides Any one of the following, whose nucleotide sequences are shown in SEQ ID No. 1~3 respectively, and named respectively. Yl FAD2 (GRYC ID: YALI1_B13699g), Ma FAD2 (obtained from the amino acid sequence of GenBank No. AAF08684.1 after codon optimization), RtFAD2 (obtained by codon optimization of the amino acid sequence from GenBank No. AXG32174.1).

[0007] The Lpcat encoding gene is derived from pomegranate ( Punica granatum L.) or castor bean ( Ricinus communis L.), whose nucleotide sequences are shown in SEQ ID No. 4~5, and are named respectively. Pg LPCAT (obtained from the amino acid sequence of NCBI RefSeq: XP_031394089.1 after codon optimization) and Rc LPCAT (obtained by codon optimization of the amino acid sequence from GenBank No. AGO14581.1).

[0008] The Cpt encoding gene is derived from Yersinia lipophila ( Yarrowia lipolytica ) or brewer's yeast ( Saccharomyces cerevisiae Their nucleotide sequences are shown in SEQ ID No. 6~7, and are named respectively. Yl CPT (GRYC ID: YALI1_C15420g) and Sc CPT.

[0009] In some embodiments of the present invention, the Fad2 encoding gene is derived from *Morchella alpineensis* (…). Mortierella alpina Its nucleotide sequence is shown in SEQ ID No. 2; the Lpcat encoding gene is derived from castor bean ( Ricinus communis L., whose nucleotide sequence is shown in SEQ ID No. 5; the Cpt encoding gene is derived from Yersinia lipolytica (L.). Yarrowia lipolytica Its nucleotide sequence is shown in SEQ ID No. 6.

[0010] The recombinant Yersinia lipophila has also undergone any one or more of the following modifications (1) to (3): (1) It expresses the gene encoding phosphatidylglycerol acyltransferase Pdat (responsible for the conversion of phosphatidyl to triglycerides); (2) Genes related to the artificial uncarboxylated malonyl-CoA synthesis pathway were expressed; the artificial uncarboxylated malonyl-CoA synthesis pathway related genes include the gene encoding β-alanine-pyruvate transaminase BauA (EC 2.6.1.18) and / or the gene encoding malonyl-CoA reductase McrC (EC 1.2.1.75). (3) The gene encoding the citric acid transporter was knocked out.

[0011] The Pdat encoding gene is derived from Yersinia lipophila (Yersinia lipophila). Yarrowia lipolytica ) or brewer's yeast ( Saccharomyces cerevisiae Their nucleotide sequences are shown in SEQ ID No. 12~13, and are named respectively. Yl PDAT (GRYC ID: YALI1_E20049g) and Sc PDAT.

[0012] The BauA encoding gene is derived from Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) or Acinetobacter baumannii ( Acinetobacter baumannii Their nucleotide sequences are shown in SEQ ID No. 8~9, and are named respectively. Pa BAUA and Ab APTA.

[0013] The McrC encoding gene is derived from *Flexobacter orangeensis* (…). Chloroflexus aurantiacus ) or Headcodactylsulfuric acid fungus ( Sulfurisphaera tokodaii Their nucleotide sequences are shown in SEQ ID No. 10~11, and are named respectively. Ca MCR-C (obtained by codon optimization of the C-terminus of the amino acid sequence from NCBI RefSeq: WP_012258473.1) and St MCR (obtained by codon optimization of the amino acid sequence from NCBI RefSeq: WP_369610785.1).

[0014] The nucleotide sequence of the citrate transporter gene is shown in SEQ ID No. 14 (GRYC ID:YALI1_E32636g).

[0015] The Pdat encoding gene is derived from Yersinia lipophila (Yersinia lipophila). Yarrowia lipolytica Its nucleotide sequence is shown in SEQ ID No. 12; the BauA encoding gene is derived from Pseudomonas aeruginosa (…). Pseudomonas aeruginosa Its nucleotide sequence is shown in SEQ ID No. 8; the McrC encoding gene is derived from *Flexobacter orangeensis* (…). Chloroflexus aurantiacus Its nucleotide sequence is shown in SEQ ID No. 10.

[0016] In some embodiments of the present invention, the recombinant Yersinia lipolytica expresses the Fad2 encoding gene, the Lpcat encoding gene, and the Cpt encoding gene; it also expresses the Pdat encoding gene of phosphatidylglycerol acyltransferase and genes related to the artificial uncarboxylated malonyl-CoA synthesis pathway, and knocks out the citrate transport protein encoding gene. The Fad2 encoding gene is derived from *Morchella alpina* (a type of fungus). Mortierella alpina Its nucleotide sequence is shown in SEQ ID No. 2; the Lpcat encoding gene is derived from castor bean ( Ricinus communis L., whose nucleotide sequence is shown in SEQ ID No. 5; the Cpt encoding gene is derived from Yersinia lipolytica (L.). Yarrowia lipolytica Its nucleotide sequence is shown in SEQ ID No. 6; the Pdat encoding gene is derived from Yersinia lipolytica (Yersinia lipolytica). Yarrowia lipolytica Its nucleotide sequence is shown in SEQ ID No. 12; the genes related to the artificial non-carboxylated malonyl-CoA synthesis pathway include the gene encoding β-alanine-pyruvate transaminase BauA and the gene encoding malonyl-CoA reductase McrC, wherein the BauA encoding gene is derived from Pseudomonas aeruginosa ( Pseudomonas aeruginosa Its nucleotide sequence is shown in SEQ ID No. 8; the McrC encoding gene is derived from *Flexobacter orangeensis* (…). Chloroflexus aurantiacus The nucleotide sequence of the gene encoding the citrate transporter is shown in SEQ ID No. 10, and the nucleotide sequence of the gene encoding the citrate transporter is shown in SEQ ID No. 14.

[0017] The recombinant Yersinia lipolytica strain used as the starting strain is Yersinia lipolytica capable of synthesizing oleic acid. Yarrowia lipolytica ); preferably, Yersinia lipophila ( Yarrowia lipolytica )2Pg2E.

[0018] In some embodiments of the present invention, the present invention also provides a method for constructing the recombinant Yersinia lipolytica, wherein the target gene is expressed and the target gene is knocked out in the Yersinia lipolytica genome, and the method used is the gene editing method based on Ura-blaster reported in the literature (Green Chemistry, 2021, 23(2), 780-787; PLoS One, 2018, 13(3), e0194954; Applied and Environmental Microbiology, 2014, 80(5), 1660-1669), wherein the gene expression site is the neutral site of Yersinia lipolytica reported (BiotechnologyJournal, 2018, 13(9), 1700543).

[0019] Secondly, the present invention also provides the application of the recombinant Yersinia lipolytica described in the first aspect in the biosynthesis of linoleic acid-containing oils.

[0020] The linoleic acid-containing oil has a linoleic acid content of >70% w / w.

[0021] In some embodiments of the present invention, the method for biosynthesizing linoleic acid-containing oils using the recombinant *Yersinia lipolytica* yeast includes the following steps: After culturing the recombinant *Yersinia lipolytica* yeast to obtain a seed culture, it is inoculated into a fermentation medium. The seed culture is prepared as follows: The recombinant *Yersinia lipolytica* yeast is streaked on a YPD plate and cultured at 28-32 °C for 40-60 h. A single colony is picked from the YPD plate and inoculated into a shaker containing YPD liquid medium, and cultured at 28-32 °C with shaking for 20-24 h until OD (dose retardation) is reached. 600 The initial OD reached approximately 5.0. The primary seed culture was inoculated into Erlenmeyer flasks containing YPD medium. 600 Control the temperature to 0.5, 28–32 °C, and incubate with shaking for 20–24 h until OD is reached. 600 The initial OD reached approximately 5.0. The culture was then re-inoculated into Erlenmeyer flasks containing YPD medium. 600 Control the temperature to 0.5, 28–32 °C, and incubate with shaking for 20–24 h until OD is reached. 600 The pH reached approximately 5.0, completing the preparation of the tertiary seed culture for fed-batch fermentation. The fed-batch fermentation was carried out in a fermenter with an aeration rate of 3 vvm, pH controlled at 5–6, and temperature controlled at 28–32 °C. The stirring speed was automatically adjusted between 300–1000 rpm, linked to the dissolved oxygen (DO) concentration, to maintain DO at 20–40%. The tertiary seed culture was inoculated into the fermenter, with an initial OD... 600The concentration was controlled at 0.5–1.0. After culturing for 24 hours, glucose solution was continuously added to maintain a low glucose concentration. The total culturing time was 120–160 hours.

[0022] More preferably, the fermentation medium consists of: 7~8 g / L (NH4)2SO4, 14~15 g / L KH2PO4, 0.2~1 g / L MgSO4⋅7H2O, 10~40 g / L glucose, 1~3 mL / L metal ion mother liquor, and 0.5~2 mL / L vitamin mother liquor.

[0023] Beneficial effects: In this invention, in order to make Yersinia lipophila ( Yarrowia lipolytica The strain accumulated linoleic acid-rich oils through two levels of genetic manipulation. The first level aimed to increase the proportion of linoleic acid in the oils by overexpressing genes encoding Δ12 fatty acid desaturases that convert oleic acid to linoleic acid, and genes encoding acyl editing pathways related to phosphatidyl pool accumulation, including genes encoding plant-derived lysophosphatidylcholine acyltransferase and microbial-derived choline phosphotransferase. The second level aimed to improve the biosynthetic capacity of total oils by overexpressing genes encoding artificial non-carboxylated malonyl-CoA synthesis pathways, genes encoding genes responsible for converting phosphatidyl pools to triglyceride pools, and knocking out genes encoding citrate transport proteins. Specifically, the following five steps were taken to construct recombinant Yersinia lipolytica: (1) Overexpress the enzyme encoding gene related to the conversion of oleic acid to linoleic acid; (2) Overexpress the gene encoding the acyl editing pathway related to the accumulation of phosphatidyl pool, promote the synthesis of phosphatidyl, and provide a sufficient site for the conversion of oleic acid to linoleic acid; (3) Overexpress the gene encoding the phosphatidyl pool to the triglyceride pool, convert the phosphatidyl pool to the triglyceride pool in a timely manner and store it stably, thereby enhancing the synthesis of oils; (4) Overexpress the gene encoding the artificial non-carboxylated malonyl-CoA synthesis pathway, enhance the synthesis ability of malonyl-CoA, thereby enhancing the synthesis of oils; (5) Knock out the gene encoding the citric acid transport protein, alleviate the citric acid overflow phenomenon, and enhance the synthesis of oils.

[0024] Thus, the recombinant Yersinia lipolytica constructed through the above five steps can synthesize linoleic acid-rich oils using glucose as a substrate, with the linoleic acid content maintained at over 70% of the total oil. After fed-batch fermentation, the total oil content can reach over 30 g / L. The resulting microbial oil has the potential to replace plant-derived linoleic acid oils for food and industrial applications. Attached Figure Description

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] Figure 1 This is a schematic diagram illustrating the method for biosynthesizing linoleic acid-rich oils based on recombinant Yersinia lipolytica.

[0027] Figure 2 This study presents process curves for the production of linoleic acid-rich oils using recombinant *Yarrowia lipolytica* fermentation in shake flasks and fermenters. A compares the biomass (DCW) and lipid content (TAG) of the starting strain 2Pg2E and the recombinant strain during shake flask fermentation; B compares the citric acid content (a byproduct) of the starting strain 2Pg2E and the recombinant strain XJ-LA-14 during shake flask fermentation; C shows the feed-batch fermentation process curve for the starting strain 2Pg2E in the fermenter; D shows the feed-batch fermentation process curve for the recombinant strain XJ-LA-14 in the fermenter; E shows the fatty acid distribution of the lipids produced by the starting strain 2Pg2E at the end of the feed-batch fermentation in the fermenter; and F shows the fatty acid distribution of the lipids produced by the recombinant strain XJ-LA-14 at the end of the feed-batch fermentation in the fermenter. Wherein, Glucose: glucose; DCW: biomass; CIT: citric acid; TAG: triglycerides; C18:1Δ9: oleic acid; C18:2Δ9,12: linoleic acid. Detailed Implementation

[0028] The present invention will be further described below through specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0029] The chassis strain Yersinia lipophila described in the following examples ( Yarrowia lipolytica The strain 2Pg2E was previously constructed by the applicant. Its significant characteristic is its ability to accumulate oleic acid (a precursor fatty acid of linoleic acid) accounting for more than 80% of the total lipids, while linoleic acid accounts for 0%. This strain was previously constructed by the applicant, and the construction method is disclosed in the applicant's published literature (Engineering the Lipid and Fatty Acid Metabolism in...). Yarrowia lipolytica For Sustainable Production of High Oleic Oils, ACS Synthetic Biology, 2022, 11(4): 1542-1554). The genotype of this strain is (derived from Yarrowia lipolytica Po1f, KU70and FAD2 deleted, 2×PgSCD, MaELO2 and YlELO1 expressed, Δku70::HisG, Δfad2::HisG, Δscd::P TEFin-PgSCD-T CYC1 , Δlip1::P YAT1 -PgSCD-T CYC1 , Δscp2:: P FBA -MaELO2-T PEX10 , P GPD -YlELO1-T PEX20 HisG-URA3-HisG (URA3) + LEU2 - ));in Yarrowia lipolytica Po1f was purchased from the American Culture Collection (ATCC), catalog number ATCC MYA-2613.

[0030] In the following examples, the formulation information of the YPD liquid culture medium, YPD plate, SD-URA plate, SD-LEU plate, and YPD plate of 5-fluoroorotic acid (5-FOA) can be found in Chinese Patent CN119875865A.

[0031] In the following examples, the bacterial biomass was determined using a gravimetric method. Yarrowia lipolyticaFor Sustainable Production of High Oleic Oils, ACS Synthetic Biology, 2022, 11(4): 1542-1554); The glucose concentration in the fermentation broth was determined by a biosensor analyzer; The methods for oil extraction, total oil content and fatty acid distribution are described in (Dynamic regulation combined with systematic metabolic engineering for high-level palmitoleic acid accumulation in oleaginous yeast, Metabolic Engineering, 2025, 89: 33-46); The citric acid content in the fermentation broth was determined by HPLC (AYarrowia lipolytica strain engineered for arachidonic acid production counteracts metabolic burden by redirecting carbon flux towards intracellular fatty acid accumulation at the expense of organic acids secretion, Biochemical Engineering Journal. 2017;128:201-209).

[0032] A schematic diagram of the method for biosynthesizing linoleic acid-rich lipids using *Yarrowia lipolytica* as described in this invention is shown below. Figure 1 As shown.

[0033] Example 1: Amplification and Integration of Gene Elements and Preparation of Knockout Plasmids (a) Amplification of gene elements According to information from NCBI, it comes from *Morchella alpina* (a type of fungus). Mortierella alpina Δ12 fatty acid desaturase ( Ma The nucleotide sequence encoding the Fad2 gene, after codon optimization, was synthesized by Suzhou Genewise Biotechnology Co., Ltd. (nucleotide sequence shown in SEQ ID No. 2) and inserted into plasmid pUC57, resulting in plasmid pUC57- Ma FAD2.

[0034] According to information from castor oil (as provided by NCBI) Ricinus communis L.) lysophosphatidylcholine acyltransferase (L.) RcThe nucleotide sequence encoding the Lpcat gene, after codon optimization, was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. (nucleotide sequence shown in SEQ ID No. 5) and inserted into plasmid pUC57, resulting in plasmid pUC57- Rc LPCAT.

[0035] According to information from Pseudomonas aeruginosa provided by NCBI ( Pseudomonas aeruginosa β-alanine-pyruvate transaminase ( Pa The nucleotide sequence encoding the BauA gene, after codon optimization, was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. (nucleotide sequence shown in SEQ ID No. 8) and inserted into plasmid pUC57, resulting in plasmid pUC57- Pa BAUA.

[0036] According to information from NCBI, it comes from *Flexobacterium orange-green* (… Chloroflexus aurantiacus The C-terminus of malonyl-CoA reductase ( Ca The nucleotide sequence encoding the McrC gene, after codon optimization, was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. (nucleotide sequence shown in SEQ ID No. 10) and inserted into plasmid pUC57, resulting in plasmid pUC57- Ca MCRC.

[0037] According to information from NCBI, it comes from Yersinia lipolytica ( Yarrowia lipolytica ) choline phosphotransferase ( Yl The nucleotide sequence of the gene encoding Cpt (GRYC ID: YALI1_C15420g, nucleotide sequence as shown in SEQ ID No. 6) was cloned by PCR and inserted into plasmid pUC57 to obtain plasmid pUC57- Yl CPT.

[0038] According to information from NCBI, it comes from Yersinia lipolytica ( Yarrowia lipolytica Phospholipid diglyceryl acyltransferase ( Yl The nucleotide sequence of the gene encoding Pdat (GRYC ID: YALI1_E20049g, nucleotide sequence as shown in SEQ ID No. 12) was cloned by PCR and inserted into plasmid pUC57 to obtain plasmid pUC57- Yl PDAT.

[0039] (II) Construction of integrated and knockout backbone plasmids The integrative plasmids of *Yarrowia lipolytica* described in the following examples were all constructed based on neutral sites reported in the literature (Biotechnology Journal, 2018, 13(9), 1700543). These neutral sites include A08, IntA, IntB, IntC1, IntC2, and IntC3, as shown in Table 1. The construction methods of the parent plasmids pUC-LEU and pUC-HUH, as well as the A08 site integrative plasmid pUC-LEU-A08 and the IntA site integrative plasmid pUC-HUH-IntA, are detailed in Chinese Patent CN119875865A.

[0040] Table 1. Neutral sites and their integrative plasmids used in this invention

[0041] (1) IntB site integration plasmid pUC-HUH-IntB The IntB site integration plasmid pUC-HUH-IntB inserts a 1402 bp sequence (upstream homologous arm) upstream of the IntB site (YALI1_B07043g) on ​​chromosome B of the *Yalexinia lipolytica* genome into the pUC-HUH vector. EcoR At the I restriction site, a 1396 bp sequence downstream of the IntB stop codon (downstream homologous arm) is inserted into the pUC-HUH vector. [[ID=6 Obtained at the III restriction site.

[0042] (2) InC1 site integration plasmid pUC-HUH-IntC1 The IntC1 site integration plasmid pUC-HUH-IntC1 inserts a 1402 bp sequence (upstream homologous arm) upstream of the IntC1 site (YALI1_C25990g) on ​​chromosome C of the Yersinia lipolyticis genome into the pUC-HUH vector. ​ At the I restriction site, a 1396 bp sequence (downstream homologous arm) downstream of the IntC1 stop codon is inserted into the pUC-HUH vector. ​ Obtained at the III restriction site.

[0043] (3) IntC2 site integration plasmid pUC-HUH-IntC2 The IntC2 site integration plasmid pUC-HUH-IntC2 inserts a 1402 bp sequence (upstream homologous arm) upstream of the IntC2 site (YALI1_C25998g) on ​​chromosome C of the *Yalexinia lipolytica* genome into the pUC-HUH vector. ​At the I restriction site, a 1396 bp sequence (downstream homologous arm) downstream of the IntC2 stop codon is inserted into the pUC-HUH vector. ​ Obtained at the III restriction site.

[0044] (4) IntC3 site integration plasmid pUC-HUH-IntC3 The IntC3 site integration plasmid pUC-HUH-IntC3 inserts a 1402 bp sequence (upstream homologous arm) upstream of the IntC3 site (YALI1_C26027g) on ​​chromosome C of the *Yalexinia lipolytica* genome into the pUC-HUH vector. ​ At the I restriction site, a 1396 bp sequence (downstream homologous arm) downstream of the IntC3 stop codon is inserted into the pUC-HUH vector. ​ Obtained at the III restriction site.

[0045] (III) Construction of integration and knockout plasmids (1) Integrating plasmid pUC-LEU-A08- ​ Construction of FAD2 Using the *Yarrowia lipophila* 2Pg2E genome as a template, the TEFin promoter and XPR2 terminator for expressing MaFAD2 were amplified by PCR using the primers A08-MaFAD2-TEFin-F / R and A08-MaFAD2-XPR2-F / R as described in Table 2, respectively; the plasmid pUC57- ​ Using FAD2 as a template and the primers A08-MaFAD2-Cassette-F / R listed in Table 2, PCR amplification was performed to obtain the coding gene required for the introduction of the MaFAD2 expression cassette at the A08 site. ​ FAD2. This involves setting the TEFin promoter and the XPR2 terminator. ​ FAD2 encoding gene and through ​ The framework plasmid pUC-LEU-A08, digested with enzyme I, was mixed proportionally using a one-step cloning kit from Novizan and assembled into the integrative plasmid pUC-LEU-A08-MaFAD2 in one step. Linearization and transformation of this plasmid can then convert P... TEFin - ​ FAD2-T XPR2 - The LEU framework replaces the position of the neutral site in Yersinia lipophila A08.

[0046] Table 2 Integrative plasmid pUC-LEU-A08- ​ FAD2 construction primer sequences

[0047] (2) Integral plasmid pUC-HUH-IntA- ​ Construction of LPCAT Using the Yersinia lipophila 2Pg2E genome as a template, primers IntA- as described in Table 3 were used respectively. ​ LPCAT-EXP-F / R, IntA- ​ LPCAT-XPR2-F / R, PCR amplification to obtain the expression ​ LPCAT's EXP promoter and XPR2 terminator; using plasmid pUC57- ​ LPCAT is the template and the primers IntA- described in Table 3. ​ LPCAT-Cassette-F / R, PCR amplification yielded samples intended for introduction at the IntA site. ​ The coding gene required for the LPCAT expression cassette ​ LPCAT. This will include the EXP starter, XPR2 terminator, and... ​ The LPCAT encoding gene and the framework plasmid pUC-HUH-IntA digested with Kpn I were mixed in a specific ratio using a one-step cloning kit from Novizan and assembled into an integrative plasmid pUC-HUH-IntA in one step. ​ LPCAT. This plasmid, after linearization and transformation, can convert P... EXP - ​ LPCAT-T XPR2 - The HUH framework replaces the position of the neutral site in Yersinia lipophila IntA.

[0048] Table 3 Primer sequences for constructing the integrative plasmid pUC-HUH-IntA-RcLPCAT

[0049] (3) Integrating plasmid pUC-HUH-IntB- Yl CPT Construction Using the *Yerithiopsis lipolyticis* 2Pg2E genome as a template, the TEFin promoter and CYC1 terminator for YlCPT expression were amplified by PCR using primers IntB-YlCPT-TEFin-F / R and IntB-YlCPT-CYC1-F / R as described in Table 4. The coding gene YlCPT, required for the introduction of the YlCPT expression cassette at the IntB site, was amplified by PCR using plasmid pUC57-YlCPT as a template and primers IntB-YlCPT-Cassette-F / R as described in Table 4. The TEFin promoter, CYC1 terminator, YlCPT coding gene, and the framework plasmid pUC-HUH-IntB digested with Kpn I were mixed in proportion using a one-step cloning kit from Novizan and assembled into the integrative plasmid pUC-HUH-IntB-YlCPT in one step. This plasmid, after linearization and transformation, can be used to express P... TEFin -YlCPT-T CYC1 - The HUH framework replaces the position of the neutral site of Yersinia lipophila IntB.

[0050] Table 4 Primer sequences for constructing the integrative plasmid pUC-HUH-IntB-YlCPT

[0051] (4) Integral plasmid pUC-HUH-IntC1- Yl PDAT Construction Using the *Yerithia lipolytica* 2Pg2E genome as a template, the H3 promoter and LIP2 terminator for expressing YlPDAT were amplified by PCR using the primers IntC1-YlPDAT-H3-F / R and IntC1-YlPDAT-LIP2-F / R described in Table 5, respectively. Using plasmid pUC57-YlPDAT as a template and the primers IntC1-YlPDAT-Cassette-F / R described in Table 5, the encoding gene YlPDAT, required for the introduction of the YlPDAT expression cassette at the IntC1 site, was amplified by PCR. The H3 promoter, LIP2 terminator, YlPDAT encoding gene, and the primers in Table 5 were then used to amplify the expression cassette. ​ The framework plasmid pUC-HUH-IntC1, digested with enzyme I, was mixed proportionally using a one-step cloning kit from Novizan and assembled into the integrative plasmid pUC-HUH-IntC1-YlPDAT in one step to obtain the integrative plasmid. After linearization and transformation, this plasmid can be used to transform P... H3 -YlPDAT-T LIP2 - The HUH framework replaces the position of the neutral site in Yersinia lipophila IntC1.

[0052] Table 5 Primer sequences for constructing the integrative plasmid pUC-HUH-IntC1-YlPDAT

[0053] (5) Integral plasmid pUC-HUH-IntC2- ​ BAUA Construction Using the Yersinia lipophila 2Pg2E genome as a template, and employing the primers IntC2-PaBauA-FBA-F / R and IntC2-PaBauA-CYC1-F / R described in Table 6, PCR amplification was performed to obtain the genome for expression. ​ BAUA's FBA promoter and CYC1 terminator; using plasmid pUC57- ​ Using BAUA as a template and the primers IntC2-PaBauA-Cassette-F / R listed in Table 6, PCR amplification was performed to obtain the sample to be introduced at the IntC2 site. ​ The coding gene required for the BAUA expression cassette ​ BAUA. This will include the FBA starter, CYC1 terminator, and... ​ BAUA encoding gene and process ​ The framework plasmid pUC-HUH-IntC2, digested with enzyme I, was mixed in the specified proportions using a one-step cloning kit from Novizan and assembled into the integrative plasmid pUC-HUH-IntC2 in one step. ​ BAUA. This plasmid, after linearization and transformation, can convert P... FBA -PaBAUA-T CYC1 - The HUH framework replaces the position of the neutral site in Yersinia lipophila IntC2.

[0054] Table 6 Integral plasmid pUC-HUH-IntC2- ​ BAUA construction primer sequence

[0055] (6) Integral plasmid pUC-HUH-IntC3- ​ Construction of MCR-C Using the Yersinia lipophila 2Pg2E genome as a template, and using the primers IntC3- described in Table 7... ​ MCR -C-FBA-F / R, IntC3- ​ MCR-C-MIG1-F / R, PCR amplification to obtain the expression ​ MCR-C's FBA promoter and MIG1 terminator; using plasmid pUC57- ​ Using MCR-C as the template and the primers IntC3-CaMCR-C-Cassette-F / R listed in Table 7, PCR amplification was performed to obtain the primers intended to be introduced at the IntC3 site. ​ The gene encoding the MCR-C expression cassette​ MCR-C. This involves the FBA starter, MIG1 terminator, ​ MCR-C encoding gene and through ​ The framework plasmid pUC-HUH-IntC3, digested with enzyme I, was mixed in the specified proportions using a one-step cloning kit from Novizan and assembled into the integrative plasmid pUC-HUH-IntC3 in one step. ​ MCR-C. This plasmid, after linearization and transformation, can convert P... FBA - ​ MCR-CT MIG1 - The HUH framework replaces the position of the neutral site in Yersinia lipophila IntC3.

[0056] Table 7 Integral plasmid pUC-HUH-IntC3- ​ MCR-C construction primer sequences

[0057] (7) Construction of the knockout plasmid pUC-HUH-CEX1 Using the *Yarrowia lipolyticis* 2Pg2E genome as a template, and with primers CEX1-Up-F / R and CEX1-Dw-F / R as described in Table 8, PCR amplification was performed to obtain the upstream and downstream homologous arms (900 bp each) of the gene encoding the citrate transporter CEX1 (GRYC ID: YALI1_E32636g, nucleotide sequence as shown in SEQ ID No. 14). Using plasmid pUC-HUH as a template and primers CEX1-HUH-F / R as described in Table 8, PCR amplification was performed to obtain the HUH expression cassette. The upstream and downstream homologous arms of CEX1, the HUH expression cassette, and the... ​ The framework plasmid pUC57, digested with enzyme I, was mixed proportionally using a one-step cloning kit from Novizan and assembled into the integration plasmid pUC-HUH-CEX1 in one step. This plasmid was linearized and transformed to knock out the CEX1 gene.

[0058] Table 8 Primer sequences for constructing the knockout plasmid pUC-HUH-CEX1

[0059] Example 2: Construction of a synthetic lipophilic yeast strain rich in linoleic acid and with increased oil yield. The linearized recombinant plasmid pUC-LEU-A08- constructed in Example 1 was used. ​ FAD2, pUC-HUH-IntA- ​ LPCAT, pUC-HUH-IntB- Yl CPT, pUC-HUH-IntC1- YlPDAT, pUC-HUH-IntC2- ​ BAUA、pUC-HUH-IntC3- ​ MCR-C and pUC-HUH-CEX1 were sequentially converted into Yersinia lipolytica (Yersinia lipolytica). ​ In 2Pg2E, homologous recombination was performed, with the MaFAD2 expression cassette inserted at site A08 and at site IntA. ​ LPCAT expression cassette, IntB site insertion Yl CPT expression cassette, IntC1 site insertion Yl PDAT expression cassette, IntC2 site insertion ​ BAUA expression cassette, IntC3 insertion site ​ The MCR-C expression cassette was used to knock out the CEX1 gene, resulting in recombinant Yersinia lipophila.

[0060] The specific method is as follows: (1) Yarrowia lipolytica Competent cells were prepared by overnight culturing 2Pg2E in YPD liquid medium. Linearized pUC-LEU-A08- cells were then cultured using the Zymogen Frozen EZ Yeast Transformation Kit II. Ma FAD2 conversion to Yarrowia lipolytica Homologous recombination occurs in 2Pg2E competent cells.

[0061] (2) Positive clones were screened using SD-LEU plates, and single colonies were identified by PCR.

[0062] (3) After culturing the positive clone identified by PCR in step (2) (i.e., the intermediate strain 2Pg2E-MaFAD2) overnight in YPD liquid medium, competent cells were prepared. Linearized pUC-HUH-IntA- was then used in the Zymogen Frozen EZ Yeast Transformation Kit II. Rc LPCAT was transformed into competent cells of the positive clones identified by PCR in step (2) for homologous recombination.

[0063] (4) Positive clones were screened using SD-URA plates and identified by PCR. Positive clones that were correctly identified by PCR were plated on YPD plates containing 5-fluoroorotic acid and incubated at 30 ℃ for 3 days. Single colonies were streaked simultaneously on both YPD and SD-URA plates containing 5-fluoroorotic acid, and the growth of the bacteria was observed. Single colonies that grew on YPD plates containing 5-fluoroorotic acid but not on SD-URA plates were selected for PCR identification.

[0064] (5) Prepare competent cells by culturing the positive clone identified by PCR in step (4) (i.e., the intermediate strain 2Pg2E-MaFAD2-RcLPCAT) overnight in YPD liquid medium. Linearized pUC-HUH-IntB- Yl CPT is converted into competent cells of the positive clones identified by PCR in step (4) for homologous recombination.

[0065] (6) Positive clones were screened using SD-URA plates and identified by PCR, using the same method as step (4).

[0066] (7) Prepare competent cells by culturing the positive clone identified by PCR in step (6) (i.e., the intermediate strain 2Pg2E-MaFAD2-RcLPCAT-YlCPT) overnight in YPD liquid medium. Transform linearized pUC-HUH-IntC1-YlPDAT into the competent cells of the positive clone identified by PCR in step (6) using Zymogen Frozen EZ YeastTransformation Kit II for homologous recombination.

[0067] (8) Positive clones were screened using SD-URA plates and identified by PCR, using the same method as step (4).

[0068] (9) Prepare competent cells by culturing the positive clone identified by PCR in step (8) (i.e., the intermediate strain 2Pg2E-MaFAD2-RcLPCAT-YlCPT-YlPDAT) overnight in YPD liquid medium. Transform linearized pUC-HUH-IntC2-PaBAUA into the competent cells of the positive clone identified by PCR in step (8) using Zymogen Frozen EZYeast Transformation Kit II for homologous recombination.

[0069] (10) Positive clones were screened using SD-URA plates and identified by PCR, using the same method as step (4).

[0070] (11) Prepare competent cells by culturing the positive clone identified by PCR in step (10) (i.e., the intermediate strain 2Pg2E-MaFAD2-RcLPCAT-YlCPT-YlPDAT-PaBAUA) overnight in YPD liquid medium. Transform linearized pUC-HUH-IntC3-CaMCR-C into the competent cells of the positive clone identified by PCR in step (10) using ZymogenFrozen EZ Yeast Transformation Kit II for homologous recombination.

[0071] (12) Positive clones were screened using SD-URA plates and identified by PCR, using the same method as step (4).

[0072] (13) Prepare competent cells by culturing the positive clone identified by PCR in step (12) (i.e., the intermediate strain 2Pg2E-MaFAD2-RcLPCAT-YlCPT-YlPDAT-PaBAUA-CaMCR-C) overnight in YPD liquid medium. Transform linearized pUC-HUH-CEX1 into the competent cells of the positive clone identified by PCR in step (12) using Zymogen Frozen EZ Yeast Transformation Kit II for homologous recombination.

[0073] (14) Positive clones were screened using SD-URA plates and identified by PCR, using the same method as step (4). The correctly identified strain was named recombinant Yersinia lipophila XJ-LA-14.

[0074] Example 3: Production of linoleic acid-rich oils using recombinant Yersinia lipolytica fermentation. (I) Preparation of Seed Liquid The starting strain and the recombinant Yersinia lipophila obtained above were streaked onto YPD plates and cultured at 30 °C for 48 h. Single colonies were picked from the YPD plates and inoculated into sterile shake tubes containing 5 mL of YPD liquid medium, and cultured at 30 °C with shaking at 200 rpm for 20–24 h until OD (dose retardation) was achieved. 600 The initial seed culture was prepared by reaching approximately 5.0. The initial seed culture was then inoculated into 500 mL Erlenmeyer flasks containing 100 mL of YPD medium. 600 Incubate at 0.5°C, 30 °C, and 200 rpm with shaking for 20–24 h until OD is reached. 600 The initial OD reached approximately 5.0. The culture was then re-inoculated into a 500 mL Erlenmeyer flask containing 100 mL of YPD medium. 600 Incubate at 0.5°C, 30 °C, and 200 rpm with shaking for 20–24 h until OD is reached.600 Reaching approximately 5.0, the preparation of the third-stage seed culture is completed and used for subsequent fermentation culture in the fermenter.

[0075] (II) Shake-flask fermentation culture The above primary seed culture was transferred to a 250 mL shake flask containing 50 mL of fermentation medium to allow its initial OD to be determined. 600 The culture medium was controlled at 0.5 g / L and fermented at 30 °C and 200 rpm for 60 h in a shaker. The fermentation medium consisted of 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4⋅7H2O, 60 g / L glucose, 2.0 mL / L metal ion stock solution, and 1.0 mL / L vitamin stock solution. The vitamin stock solution was an aqueous solution containing the following components: biotin 0.05 g / L, calcium pantothenate 1 g / L, thiamine 1 g / L, pyridoxine 1 g / L, nicotinic acid 1 g / L, para-aminobenzoic acid 0.2 g / L, and inositol 25 g / L. The metal ion mother liquor is an aqueous solution containing the following components: EDTA 15 g / L, zinc sulfate 4.5 g / L, cobalt chloride 0.3 g / L, manganese chloride 1 g / L, copper sulfate 0.1 g / L, calcium chloride 4.5 g / L, ferrous sulfate 3 g / L, sodium molybdate 0.4 g / L, boric acid 1 g / L, and potassium iodide 0.1 g / L.

[0076] The results of shake-flask fermentation are as follows Figure 2 As shown in A, compared to the starting strain Yersinia lipophila 2Pg2E, the dry weight of the recombinant strain XJ-LA-14 remained almost unchanged, but the lipid content increased significantly (by nearly 2 times), and the content of the byproduct citric acid in the fermentation broth also decreased significantly. Figure 2 (B in the middle).

[0077] In this invention, the intermediate strains prepared in Example 2 were cultured according to the shake-flask fermentation method described above, as shown in Table 9.

[0078] Table 9. Shake-flask fermentation results of the starting strain and the recombinant strain

[0079] (III) Fermentation culture in fermenter Fed-batch fermentation was carried out in a 5 L fermenter with an initial volume of 3 L (the fermentation medium formula was the same as that for shake flask fermentation, except that glucose was adjusted to 20 g / L). The aeration rate was 3 vvm, the pH was controlled at 5.5 by automatically adding 10 M NaOH, and the temperature was controlled at 30 °C. The stirring speed was correlated with the dissolved oxygen (DO) concentration and was automatically adjusted between 300 and 1000 rpm to maintain the DO at 30%. The three-stage seed culture prepared above was inoculated into the fermenter, and the initial OD was... 600 The concentration was controlled at 0.5. After 24 hours of cultivation, 800 g / L glucose solution was continuously added to maintain a low glucose concentration. Throughout the fermentation cycle, samples were taken every 12 hours to determine glucose concentration, cell biomass, total lipids, and fatty acid distribution. After 144 hours of fermentation, as... Figure 2 According to the CF, compared with the original strain Yersinia lipophila 2Pg2E, the content of citric acid byproduct in the fermentation broth of recombinant strain XJ-LA-14 was significantly reduced, while the content of lipids was significantly increased (from 12 g / L to 35 g / L; its proportion of cell dry weight increased from 21% to 53%), and the proportion of linoleic acid in the lipids also increased from 0% to 71%.

[0080] This invention provides a method for biosynthesizing linoleic acid-rich oils using *Yarrowia lipolytica*. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A recombinant lipophilic yeast, characterized in that, The recombinant Yersinia lipolyticis expressed the gene encoding Δ12 fatty acid desaturase Fad2, the gene encoding lysophosphatidylcholine acyltransferase Lpcat, and the gene encoding choline phosphotransferase Cpt.

2. The recombinant lipophilic yeast according to claim 1, characterized in that, The Fad2 encoding gene is derived from Yersinia lipophila ( Yarrowia lipolytica ), Alpine spores ( Mortierella alpina ) or Rhodotorula buergerianum ( Rhodotorula toruloides Any one of the following, whose nucleotide sequences are shown in SEQ ID No. 1~3 respectively; The Lpcat encoding gene is derived from pomegranate ( Punica granatum L.) or castor bean ( Ricinus communis L.), whose nucleotide sequences are shown in SEQ ID No. 4~5 respectively; The Cpt encoding gene is derived from Yersinia lipophila ( Yarrowia lipolytica ) or brewer's yeast ( Saccharomyces cerevisiae Their nucleotide sequences are shown in SEQ ID No. 6~7.

3. The recombinant lipophilic yeast according to claim 2, characterized in that, The Fad2 encoding gene is derived from *Morchella alpineensis* (…). Mortierella alpina Its nucleotide sequence is shown in SEQ ID No. 2; The Lpcat encoding gene is derived from castor bean ( Ricinus communis L.), whose nucleotide sequence is shown in SEQ ID No. 5; The Cpt encoding gene is derived from Yersinia lipophila ( Yarrowia lipolytica Its nucleotide sequence is shown in SEQ ID No.

6.

4. The recombinant lipophilic yeast according to claim 1, characterized in that, The recombinant Yersinia lipophila was modified in any one or more of the following ways (1) to (3): (1) The gene encoding the phosphatidylglycerol acyltransferase Pdat was expressed; (2) Genes related to the artificial uncarboxylated malonyl-CoA synthesis pathway were expressed; the artificial uncarboxylated malonyl-CoA synthesis pathway related genes include the gene encoding β-alanine-pyruvate transaminase BauA and / or the gene encoding malonyl-CoA reductase McC. (3) The gene encoding the citric acid transporter was knocked out.

5. The recombinant lipophilic yeast according to claim 4, characterized in that, The Pdat encoding gene is derived from Yersinia lipophila ( Yarrowia lipolytica ) or brewer's yeast ( Saccharomyces cerevisiae The nucleotide sequences of these organisms are shown in SEQ ID No. 12-13, respectively; the BauA encoding gene is derived from *Pseudomonas aeruginosa* (…). Pseudomonas aeruginosa ) or Acinetobacter baumannii ( Acinetobacterbaumannii The nucleotide sequences of the bacteria are shown in SEQ ID No. 8-9, respectively; the McrC encoding gene is derived from *Flexobacterium orangeense* (…). Chloroflexus aurantiacus ) or Headcodactylsulfuric acid fungus ( Sulfurisphaera tokodaii The nucleotide sequences of the gene encoding the citrate transport protein are shown in SEQ ID No. 10-11, respectively; the nucleotide sequence of the gene encoding the citrate transport protein is shown in SEQ ID No.

14.

6. The recombinant lipophilic Yersinia according to claim 5, characterized in that, The Pdat encoding gene is derived from Yersinia lipophila ( Yarrowia lipolytica Its nucleotide sequence is shown in SEQ ID No. 12; the BauA encoding gene is derived from Pseudomonas aeruginosa (…). Pseudomonas aeruginosa Its nucleotide sequence is shown in SEQ ID No. 8; the McrC encoding gene is derived from *Flexobacter orangeensis* (…). Chloroflexus aurantiacus Its nucleotide sequence is shown in SEQ ID No.

10.

7. The recombinant lipophilic yeast according to claim 3, characterized in that, The recombinant Yersinia lipolytica expressed the gene encoding phosphatidylglycerol acyltransferase Pdat and genes related to the synthesis pathway of artificial uncarboxylated malonyl-CoA, and knocked out the gene encoding citrate transport protein. The Pdat encoding gene is derived from Yersinia lipophila (Yersinia lipophila). Yarrowia lipolytica Its nucleotide sequence is shown in SEQ ID No. 12; the genes related to the artificial non-carboxylated malonyl-CoA synthesis pathway include the gene encoding β-alanine-pyruvate transaminase BauA and the gene encoding malonyl-CoA reductase McrC, wherein the BauA encoding gene is derived from Pseudomonas aeruginosa ( Pseudomonas aeruginosa Its nucleotide sequence is shown in SEQ ID No. 8; the McrC encoding gene is derived from *Flexobacter orangeensis* (…). Chloroflexus aurantiacus The nucleotide sequence of the gene encoding the citrate transporter is shown in SEQ ID No. 10, and the nucleotide sequence of the gene encoding the citrate transporter is shown in SEQ ID No.

14.

8. The recombinant lipophilic yeast according to claim 1, characterized in that, The recombinant Yersinia lipolytica strain is a Yersinia lipolytica strain capable of synthesizing oleic acid. Yarrowia lipolytica ); preferably, Yersinia lipophila ( Yarrowia lipolytica )2Pg2E.

9. The application of the recombinant Yersinia lipolyticis according to any one of claims 1 to 8 in the biosynthesis of linoleic acid-containing oils.

10. The application according to claim 9, characterized in that, The linoleic acid-containing oil has a linoleic acid content >70% w / w.

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