Delta12-fatty acid desaturase and application thereof

Abstract

The invention belongs to the technical field of biology, and provides a delta 12-fatty acid desaturase and application thereof, and the amino acid sequence of the delta 12-fatty acid desaturase is shown as SEQ ID NO: 1-4. The invention also provides a method for synthesizing linoleic acid by using yeast. The method comprises the following steps: fermenting yeast host cells for expressing the delta 12-fatty acid desaturase in a culture medium containing oleic acid, and then recovering the linoleic acid. The four delta 12-fatty acid desaturases provided by the invention can be used for constructing engineering bacteria for high-yield linoleic acid and downstream fatty acids and fatty acid derivatives thereof, and have wide application prospects in the fields of chemical engineering, energy, medicines, health care and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of a Δ12-fatty acid desaturase gene in improving the ability of oleaginous yeast to synthesize linoleic acid. Background Art

[0002] Linolenic acid (LA, C18:2) is a long-chain polyunsaturated essential fatty acid. Most of the linoleic acid ingested by the human body is metabolized through β-oxidation for energy, and the rest is mainly stored in the form of triglycerides, cholesterol esters and phospholipids. Adipose tissue is the main place where linoleic acid is stored in the form of triglycerides. Linoleic acid can lower blood cholesterol and prevent atherosclerosis, and is closely related to health and disease; its isomer, conjugated linoleic acid, has a positive effect on the alleviation of obesity, cardiovascular disease and certain cancers (Den Hartigh. Nutrients, 2019 11(2):370; Yang, et al. J Funct Foods, 2015, 15:314-325.). Linoleic acid can be used as a precursor for the synthesis of very long-chain unsaturated fatty acids such as arachidonic acid (ARA, C20:4), which affects the occurrence and development of various diseases such as obesity, cardiovascular disease, and inflammation (Zhou, et al. Int J Mol Sci, 2021, 22(21):12029.). Linoleic acid is also a precursor for the synthesis of oxygen-containing derivatives such as 9, 13-hydroxyoctadecadienoic acid (9, 13-HODE) and 9, 13-oxo-oxide (9, 13-oxoODE), and participates in various biological processes such as anti-inflammatory / anti-proliferation (Ziboh, et al. Prostaglandins Other Lipid Mediat, 2000, 63(1-2):3-13; Ramsden, et al. Prostaglandins LeukotEssent Fatty Acids, 2012, 87(4-5):135-141.).

[0003] Δ12-fatty acid desaturase (Δ12D) is a key enzyme in the dehydrogenation of oleic acid (OA, C18:1) to form linoleic acid, and is also a key upstream step in the synthesis of a large number of polyunsaturated fatty acids. At present, researchers have obtained the gene sequence of Δ12D from a variety of animals, plants and microorganisms, and verified the activity of exogenous enzymes in different chassis. Among them, Mortierella alpina ( Mortierella alpine ) combined with fatty acid desaturases from the Δ6 / Δ9 pathway from other sources in Yarrowia lipolytica ( Yarrowia lipolytica) has successfully constructed the synthesis pathway of γ-linolenic acid (GLA), realizing the industrial production of high-value fatty acids such as eicosapentaenoic acid (EPA) (Chuang, et al. N Biotechnol, 2010, 27(4):277-282; Xue, et al. Nat Biotechnol, 2013, 31(8):734-740.), but there is still a lot of room for improvement in production. In metabolic engineering, the accumulation of upstream linoleic acid is an effective way to strengthen metabolic pathways, which makes the importance of the Δ12D metabolic element more prominent. Although a variety of homologous sequences of Δ12D genes have been identified from animals, plants, and microorganisms, their biological functions in different hosts vary greatly due to species differences.

[0004] Yarrowia lipolytica is a typical oleaginous yeast, and the oil content of engineered bacteria can be as high as 80% of the cell dry weight (Xu, et al. Biotechnol Bioeng, 2017, 114(7): 1521-1530). At the same time, Yarrowia lipolytica is recognized as a "generally recognized as safe (GRAS)" by the US Food and Drug Administration (FAD), and is highly safe. Although Yarrowia lipolytica can synthesize a certain proportion of linoleic acid, a large amount of oleic acid cannot be used. At present, exogenous Δ12D resources that can efficiently convert oleic acid in Yarrowia lipolytica are still scarce. M. alpine Therefore, obtaining more Δ12D that can efficiently convert oleic acid in Yarrowia lipolytica and improving the ability of the base bacteria to convert oleic acid is expected to improve the ability of yeast to produce linoleic acid and its downstream derivatives by fermentation, and promote the application of a series of high-value fatty acid products. Summary of the invention

[0005] In view of the problem of limited ability of yeast to produce linoleic acid by fermentation in the prior art, the present invention provides the use of four Δ12-fatty acid desaturase genes in yeast synthesis of linoleic acid. The expression of a single enzyme can effectively enhance the ability of Yarrowia lipolytica to synthesize linoleic acid.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] Application of Δ12-fatty acid desaturase in yeast synthesis of linoleic acid, wherein the amino acid sequences of the Δ12-fatty acid desaturase are shown in SEQ ID NOs: 1-4, which are respectively derived from Pleurotus ostreatus ( Pleurotus ostreatus ),sunflower( Helianthus annuus ),corn( Zea mays ) and Haematococcus pluvialis ( Haematococcus sp.).

[0008] The encoding nucleotides of the above-mentioned Δ12-fatty acid desaturase are shown in SEQ ID NOs: 5-8.

[0009] The method for synthesizing linoleic acid by using yeast comprises the following steps: The yeast host cells expressing the Δ12-fatty acid desaturase are fermented in a medium containing a substrate, and then the total oil of the cells is extracted.

[0010] The yeast is an oleaginous yeast selected from the genus Yarrowia, such as Yarrowia lipolytica ( Yarrowia lipolytica ).

[0011] Preferably, the above method may further comprise a step of purifying the linoleic acid.

[0012] The microbial oil obtained by the above method has a linoleic acid content of 65 wt % or more, preferably 70 wt % or more, and more preferably 75 wt % or more.

[0013] The present invention has the following advantages: The application of the Δ12-fatty acid desaturase provided by the present invention in enhancing yeast synthesis of linoleic acid, the Δ12-fatty acid desaturases of Pleurotus ostreatus, sunflower, corn and Haematococcus pluvialis are expressed in Yarrowia lipolytica respectively, which can catalyze the conversion of oleic acid into linoleic acid, and the expression of a single enzyme can effectively achieve the ability of yeast to enhance the synthesis of linoleic acid. Under unoptimized shake flask fermentation conditions, the proportion of linoleic acid in the total fatty acids in the four recombinant strains can reach 77.25%, 67.46%, 65.28% and 71.39%, respectively, which is 1.35 times, 1.06 times, 0.99 times and 1.18 times higher than the starting strains, respectively. The four Δ12-fatty acid desaturases provided by the present invention can be used to construct engineered bacteria with high production of linoleic acid and its downstream fatty acids and fatty acid derivatives, and have broad application prospects in the fields of chemical industry, energy, medicine and health care. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the phylogenetic tree of different Δ12-fatty acid desaturases; Figure 2 This is a comparison of the protein sequences of different Δ12-fatty acid desaturases; the boxes contain three conserved histidine motifs; Figure 3 It is the design map of the recombinant plasmid; Figure 4Diagnostic PCR and sequencing verification of recombinant strains; A: homologous recombination target sequence design and PCR primer design; BI: upstream and downstream PCR and sequencing results of different transformants of recombinant strains Po1g-ΔKu70-MaΔ12D (B), Po1g-ΔKu70-BeΔ12D-1 (C), Po1g-ΔKu70-BeΔ12D-2 (D), Po1g-ΔKu70-PoΔ12D (E), Po1g-ΔKu70-CtΔ12D (F), Po1g-ΔKu70-HaΔ12D (G), Po1g-ΔKu70-ZmΔ12D (H) and Po1g-ΔKu70-HsΔ12D (I); CK is the control strain Po1g-ΔKu70; Figure 5 It is the gas chromatography analysis of fatty acids in shake flask fermentation of recombinant strains; wherein, A: gas chromatography analysis of fatty acids; B: the proportion of C18:1 methyl esters of different strains in total oil; C: the proportion of C18:2 methyl esters of different strains in total oil. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0016] Example 1 Construction of recombinant Yarrowia lipolytica expressing Δ12-fatty acid desaturase The starting strain of Yarrowia lipolytica used in the examples is Y.lipolytica Po1g (MATa, leu2-270,ura3-302::URA3, xpr2-3), the Ku70 gene was knocked out by the CRISPR / Cas9 system to obtain the strain Po1g-ΔKu70, and then the uracil auxotrophic strain Po1g-ΔKu70-ΔURA was obtained by reverse screening using the 5-FOA method.

[0017] The culture medium used in the examples can be purchased commercially or obtained by formulas and methods known in the art.

[0018] 1. Screening of Δ12-fatty acid desaturase Seven Δ12D potential target sequences were selected, which were derived from Boletus edulis ( Boletus edulis )、Oyster mushroom( Pleurotus ostreatus ),safflower( Carthamus tinctorius ),sunflower( Helianthus annuus ),corn( Zea mays ) and Haematococcus pluvialis ( Haematococcussp.), the coding gene sequences were named BeΔ12D-1, BeΔ12D-2, PoΔ12D, CtΔ12D, HaΔ12D, ZmΔ12D and HsΔ12D, and the corresponding amino acid sequences are shown in Table 1.

[0019] Table 1 Δ12-fatty acid desaturase from different sources

[0020] The NCBI database was used to search for other genes and amino acid sequences named as Δ12-fatty acid desaturases, including MaΔ12D (GenBank: AAF08684, from M. alpine ) has been shown to be able to catalyze the conversion of oleic acid to linoleic acid in Yarrowia lipolytica (Chuang, et al. N Biotechnol, 2010, 27(4):277-282.), and was used as a control. The protein sequences of the 19 selected Δ12D amino acid sequences were multiple aligned using ClusterW in MEGA5.1, and the neighbor-joining algorithm (self-introduction 1000 times) was used to draw the phylogenetic tree ( Figure 1 The amino acid sequences of eight Δ12Ds (MaΔ12D, BeΔ12D-1, BeΔ12D-2, PoΔ12D, CtΔ12D, HaΔ12D, ZmΔ12D, and HsΔ12D) were selected and compared using NCBI online domain structure analysis software (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) and DNAMAN software ( Figure 2 ). The results of preliminary similarity and homology analysis showed that the sequence similarity and homology of the seven Δ12D potential target sequences were low, but all contained three histidine motifs (framed parts) conserved in Δ12D.

[0021] 2. Preparation of recombinant yeast strains expressing exogenous Δ12D The genes of MaΔ12D and 7 target Δ12D were codon-optimized for Yarrowia lipolytica, and the sequences were shown in SEQ ID NOs: 9-11, SEQ ID NOs: 5, 12, and SEQ ID NOs: 6-8, respectively; after commercial synthesis, shuttle plasmids that can simultaneously express the Δ12D gene and the ampicillin resistance gene were prepared and named pUC57-MaΔ12D-AMP, pUC57-BeΔ12D-1-AMP, pUC57-BeΔ12D-2-AMP, pUC57-PoΔ12D-AMP, pUC57-CtΔ12D-AMP, pUC57-HaΔ12D-AMP, pUC57-ZmΔ12D-AMP and pUC57-HsΔ12D-AMP, respectively.

[0022] Using eight shuttle plasmids pUC57-Δ12D-AMP as templates, primer pairs Ma12-F / Ma12-R, Be12-F1 / Be12-R1, Be12-F2 / Be12-R2, Po12-F / Po12-R, Ct12-F / Ct12-R, Ha12-F / Ha12-R, Zm12-F / Zm12-R and Hs12-F / Hs12-R were used to amplify the MaΔ12D, BeΔ12D-1, BeΔ12D-2, PoΔ12D, CtΔ12D, HaΔ12D, ZmΔ12D and HsΔ12D gene fragments respectively.

[0023] Using homologous recombination plasmid pYL-D17-URA as template, primer pairs pYL-F / pYL2-R and pYL-F' / pYL-R were used to amplify the backbone DNA fragment with URA and AMP selection markers. The backbone was assembled with 8 Δ12D gene fragments by Gibson assembly method, wherein the upstream and downstream of Δ12D expression cassette were partial sequences of D17 gene of Yarrowia lipolytica (see SEQ ID NO: 13 and SEQ ID NO: 14). Then, the competent E. coli Trans-T1 was transformed, and the primers p-GPD-F and XPR2-R were used for colony PCR. The correct recombinant expression plasmids verified by sequencing were named pYL-D17-MaΔ12D-URA, pYL-D17-BeΔ12D-1-URA, pYL-D17-BeΔ12D-2-URA, pYL-D17-PoΔ12D-URA, pYL-D17-CtΔ12D-URA, pYL-D17-HaΔ12D-URA, pYL-D17-ZmΔ12D-URA and pYL-D17-HsΔ12D-URA. The design map of the recombinant plasmids is shown in the figure. Figure 3 The specific sequences of the primers are shown in Table 2: Table 2 Primers used for plasmid construction and strain PCR diagnosis

[0024] All PCR amplifications used PFU high-fidelity DNA polymerase, and the amplification system was 50 µL (2 × PFU mix, 25 µL; 1.5 µL of each 10 µM primer; 1 ng / 1 µL of template; add water to 50 µL). Amplification conditions were: 95 °C pre-denaturation for 3 min; 98 °C denaturation for 20 s, 60-72 °C annealing for 15 s, 72 °C extension (30 s / kb), 30 cycles; 72 °C extension for 10 min.

[0025] use Not The recombinant expression plasmids pYL-D17-MaΔ12D-URA, pYL-D17-BeΔ12D-1-URA, pYL-D17-BeΔ12D-2-URA, pYL-D17-PoΔ12D-URA, pYL-D17-CtΔ12D-URA, pYL-D17-HaΔ12D-URA, pYL-D17-ZmΔ12D-URA and pYL-D17-HsΔ12D-URA were digested with restriction endonuclease I to obtain linear plasmids, which were confirmed by agarose gel electrophoresis. The transformation fragments were recovered from the gel and transformed into Yarrowia lipolytica Po1g-ΔKu70-ΔURA using the LiAc transformation method. The transformation system is shown in Table 3.

[0026] Table 3 Yarrowia lipolytica transformation system

[0027] The transformation system was vortexed after being incubated in a 30 ℃ water bath for 1 h and heat shocked at 39 ℃ for 10 min. The mixed solution of the transformation system was then spread on a screening plate of YNB-Δura medium and cultured at 28 ℃ for 2-4 d to obtain the recombinant strains Po1g-Δku70-MaΔ12D, Po1g-Δku70-BeΔ12D-1, Po1g-Δku70-BeΔ12D-2, Po1g-Δku70-PoΔ12D, Po1g-Δku70-CtΔ12D, Po1g-Δku70-HaΔ12D, Po1g-Δku70-ZmΔ12D and Po1g-Δku70-HsΔ12D.

[0028] Diagnostic PCR verification was performed on different transformants of each recombinant strain obtained above: a single colony of the recombinant strain was picked from the screening plate and plated on a YNB-Δura plate. After growing for about 24 h, the bacteria were picked and the genomic DNA of the recombinant strain was extracted using a commercial fungal genome quick extraction kit. Using the genomic DNA as a template, the upstream part of the introduced gene was verified by PCR using primers D17-UF and XPR2-R1, the downstream part was verified by primers XPR2-F and D17-DR, and the full length was verified by PCR using primers D17-UF and D17-DR. Po1g-ΔKu70 was used as a control. The specific sequences of the primers are shown in Table 4: Table 4 Primers used for PCR diagnosis of strains

[0029] PCR amplification used EasyTaq DNA polymerase, and the amplification system was 20 µL (2 × EasyTaq Mix, 10 µL; 0.8 µL of each 10 µM primer; 1 ng / µL of template; water was added to make up to 20 µL), and the amplified fragment was confirmed by agarose gel electrophoresis. Amplification conditions were: 94 °C pre-denaturation for 3 min; 94 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension (60 s / kb), 30 cycles; 72 °C extension for 10 min.

[0030] Agarose gel electrophoresis of PCR products is shown in Figure 4 As shown: There is no full-length PCR product in the control group Po1g-ΔKu70 ( Figure 4 C). The transformants with positive results for upstream, downstream and full-length PCR are shown in Table 5. Based on the brightness of the upstream and downstream PCR products, 3 transformants of each type (underlined in Table 5) were selected for subsequent experiments.

[0031] Table 5 Positive transformants (underlined transformants with high brightness bands) .

[0032] Example 2 Fermentation of recombinant Yarrowia lipolytica to produce linoleic acid The recombinant strain selected in Example 1 was inoculated into 5 mL of seed culture medium (4% glucose, (NH 4 ) 2 SO 4 1%, peptone 2%, yeast extract 1%, corn steep liquor 0.3%, KH 2 PO 4 1%,MgSO 4 7H 2O 0.5%, the balance is water), 28 ℃, 220 rpm for about 24 h to obtain the fermentation seed solution. The seed solution was inoculated into 30 mL of fermentation medium (glucose 10%, (NH 4 ) 2 SO 4 1%, peptone 0.3%, yeast extract 0.2%, corn steep liquor 0.2%, KH 2 PO 4 0.5%, MgSO 4 7H 2 O0.3%, the balance was water) and cultured at 28 ℃, 220 rpm for 7 d; the Po1g-Δku70 strain was used as the control, and three parallel groups were set up for each group.

[0033] After the fermentation, 3 mL of fermentation culture medium was taken to extract fatty acids: 3 mL of fermentation culture medium was taken to a glass centrifuge tube, and centrifuged at 4000 rpm for 10 min to collect the bacteria; 3 mL of 4 M HCl was added, and the mixture was shaken and mixed, and shaken at 28 °C for 1.5-2 h; boiling water bath was placed for 10 min, and cooled at -20 °C for 30 min; 6 mL of chloroform / methanol (1:1v / v) solution was added, and the mixture was shaken and mixed, and centrifuged at 4000 rpm for 10 min; the lower layer of chloroform was taken to a new centrifuge tube, and 3 mL of 0.15% NaCl was added, and the mixture was centrifuged at 4000 rpm for 10 min after mixing; the lower layer of chloroform was taken to a new glass centrifuge tube that had been dried and weighed in advance, and the chloroform was evaporated by nitrogen blowing and heating in a fume hood to obtain oil.

[0034] The extracted oil was methylated: 2.6 mL of methanol: sulfuric acid = 98: 2 (v / v) solution was added to a glass tube, mixed and placed at 85 °C for 3 h, oscillated once every 30 min during the reaction until the oil droplets disappeared; then 1 mL of saturated NaCl solution and 1 mL of n-hexane were added, oscillated and mixed, centrifuged at 4000 rpm for 5 min, 800 µL of the supernatant was aspirated into a clean 1.5 mL EP tube, centrifuged at 12000 rpm for 5 min, and 200 µL of the supernatant was gently aspirated and sealed for storage in the next step of gas chromatography analysis.

[0035] The methyl esterified fatty acids were analyzed by gas chromatography (Agilent 7890B-GC). Chromatographic detection conditions: chromatographic column: HP-INOWAX (30 m × 0.32 mm × 0.5 μm); injection temperature: 250 ℃; detector temperature: 250 ℃; injection volume: 1 μL; initial column temperature was 140 ℃, maintained for 1 min, then increased to 180 ℃ at 10 ℃ / min, maintained for 2 min, increased to 210 ℃ at 5 ℃ / min, maintained for 4 min, then increased to 250 ℃ at 5 ℃ / min, maintained for 4 min; the relative content of each fatty acid component was obtained by area normalization method.

[0036] like Figure 5 As shown in the figure, in the control strain Po1g-Δku70, the proportion of linoleic acid accounted for only 32.76% of the total fatty acids. Similar to the strain Po1g-Δku70-MaΔ12D (68.87%), the proportion of linoleic acid in the total fatty acids of the recombinant strains Po1g-Δku70-PoΔ12D, Po1g-Δku70-HaΔ12D, Po1g-Δku70-ZmΔ12D, Po1g-Δku70-HaΔ12D and Po1g-Δku70-HsΔ12D were 77.25%, 67.46%, 65.28% and 71.39%, respectively, which were 1.35 times, 1.06 times, 0.99 times and 1.18 times higher than those of the control group, respectively; they were able to convert almost all of the substrate oleic acid, and the remaining oleic acid accounted for 4.27%, 10.19%, 12.35% and 6.97% of the total fatty acids, respectively. Other fatty acids are mainly palmitic acid (C16:0) and palmitoleic acid (C16:1), but the content of each is less than 10%.

[0037] Compared with MaΔ12D derived from Mortierella alpina, the enzymes involved in the present invention are derived from three plants (Pleurotus ostreatus, sunflower and corn) and a new resource food (Haematococcus pluvialis), and are all safer than molds. The present invention enriches the resources of Δ12-fatty acid desaturase, provides enzyme elements for better utilization and transformation of the Yarrowia lipolytica chassis, and is expected to enhance the ability of yeast to produce linoleic acid and its downstream derivatives by fermentation, and promote the application of a series of high-value fatty acid products.

Claims

1. The use of Δ12-fatty acid desaturase in yeast synthesis of linoleic acid, characterized in that: The amino acid sequence of the Δ12-fatty acid desaturase is shown in SEQ ID NO: 1-4.

2. The use according to claim 1, characterized in that: The nucleotide sequences encoding the Δ12-fatty acid desaturase are shown in SEQ ID NOs: 5-8.

3. A method for synthesizing linoleic acid using yeast, characterized in that: The following steps are involved: The yeast host cells expressing the Δ12-fatty acid desaturase are fermented in a medium containing a substrate, and then the total oil of the cells is extracted.

4. The method according to claim 3, characterized in that The yeast is an oleaginous yeast; preferably a yeast of the genus Yarrowia.

5. The method according to claim 3, characterized in that: The yeast is Yarrowia lipolytica ( Yarrowialipolytica ).

6. The method according to claim 3, characterized in that The method also comprises a purification step to obtain linoleic acid.

7. The microbial grease obtained by the arbitrary method of claim 3-6.

8. The microbial oil according to claim 7, characterized in that The content of linoleic acid is above 65 wt%.

9. The microbial oil according to claim 7, characterized in that The content of linoleic acid is above 70wt%.

10. The microbial oil according to claim 7, characterized in that The content of linoleic acid is above 75wt%.

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