Application of fas2 gene in regulating and controlling yield of mucor circinelloides lipid and beta-carotene

Abstract

The invention discloses application of a fas2 gene in regulating and controlling the yield of mucor circinelloides lipid and beta-carotene, and belongs to the technical field of gene engineering. The nucleic acid sequence of the fast 2 gene is as shown in SEQ ID NO: 1. After the fatty acid synthase gene fas2 in mucor circinelloides MU760 is knocked out through a homologous recombination gene engineering method, the fatty acid synthase gene fas2 is not expressed in the mucor circinelloides, and the mucor circinelloides fatty acid synthase gene fas2 knockout strain McFAS2 is obtained. Fermentation tests show that the yield of fatty acid and beta-carotene of the strain is remarkably increased, and the strain has important application value in the production of lipid and beta-carotene.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the application of fas2 gene in regulating the production of lipid and beta-carotene of Mucor circinelloides. Background Art

[0002] Mucor circinelloides is a diphasic fungus that can synthesize a large amount of γ-linolenic acid (cis-6,9,12-octadecatrienoic acid, GLA). It has the characteristics of rapid growth, easy cultivation and efficient production of specific metabolites.

[0003] Fatty acids and β-carotene are widely used in food, medicine, cosmetics and other fields. Fatty acids (FAs) are also important precursors of oils, biodiesel and high-value oil chemical products (such as fatty alcohols, aldehydes, olefins and waxes). β-carotene is one of the carotenoids, widely present in the leaves, flowers and roots of plants, and has antioxidant, anti-cancer and anti-skin photosensitivity effects.

[0004] In the traditional production process, fatty acids and β-carotene often need to be extracted and purified from different raw materials, which not only increases the difficulty and cost of production, but also may cause waste of resources. Through genetic engineering, Mucor circinelloides has been transformed into an engineered bacterium that can produce fatty acids and β-carotene at the same time, which can significantly improve the production efficiency of these two products. The construction of this engineered bacterium not only reduces production costs, but also makes the production of lipids and β-carotene more stable and controllable, thereby meeting the market's growing demand for these two products. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide an application of the fas2 gene in regulating the production of lipids and β-carotene in Mucor circinelloides.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The invention discloses an application of the fas2 gene in regulating the production of lipids and beta-carotene in Mucor circinelloides. The nucleic acid sequence of the fas2 gene is shown in SEQ ID NO: 1.

[0008] On the basis of the above scheme, the fatty acid synthase gene fas2 in Mucor circinelloides is knocked out by a genetic engineering method using homologous recombination, so that it is not expressed in Mucor circinelloides, thereby increasing the yield of lipid and β-carotene produced by Mucor circinelloides fermentation.

[0009] The invention relates to a Mucor circinelloides with high lipid and β-carotene production. The fatty acid synthase gene fas2 in Mucor circinelloides MU760 is knocked out by a genetic engineering method using homologous recombination so that the gene is not expressed in Mucor circinelloides, thereby obtaining a Mucor circinelloides fatty acid synthase gene fas2 knockout strain McFAS2△. The nucleic acid sequence of the fas2 gene is shown in SEQ ID NO: 1.

[0010] The method for constructing the above-mentioned Mucor circinelloides with high lipid and β-carotene production comprises the following steps:

[0011] The upstream gene sequence fas2 up and the downstream gene sequence fas2down of the fas2 gene in Mucor circinelloides WJ11 were used as homology arms, and the uracil synthesis gene pyrF was used as a screening resistance gene to construct a fas2 gene knockout frame fas2 up-pyrF-fas2down, and the fas2 up-pyrF-fas2 down was constructed into a pUC57 plasmid to obtain a knockout vector pCRC109; the knockout vector pCRC109 was transformed into the protoplasts of the MU760 strain by electroporation, and the uracil synthesis gene pyrF was used as a screening marker to screen positive clones, so as to obtain the Mucor circinelloides fatty acid synthase gene fas2 knockout strain McFAS2△.

[0012] Based on the above scheme, the primers for amplifying the gene sequence fas2up upstream of the fas2 gene in the Mucor circinelloides WJ11 are shown as SEQ ID NO:2 and SEQ ID NO:3; the primers for amplifying the gene sequence fas2 down downstream of the fas2 gene in the Mucor circinelloides WJ11 are shown as SEQ ID NO:4 and SEQ ID NO:5.

[0013] On the basis of the above scheme, the gene sequence fas2 up upstream of the fas2 gene in the Mucor circinelloides WJ11 is shown as SEQ ID NO:6, and the gene sequence fas2 down downstream of the fas2 gene is shown as SEQ ID NO:7.

[0014] Based on the above scheme, the primers for amplifying the uracil synthesis gene pyrF are shown in SEQ ID NO:8 and SEQ ID NO:9.

[0015] Based on the above scheme, the sequence of the uracil synthesis gene pyrF is shown in SEQ ID NO:10.

[0016] The application of the high-yield lipid and beta-carotene Mucor circinelloides constructed by the above method in the production of lipid and beta-carotene.

[0017] A method for producing lipid and β-carotene by fermentation, wherein the seed liquid of Mucor circinelloides with high lipid and β-carotene yield constructed by the above method is inoculated into a fermentation medium, and fermentation is carried out at 25-30°C, 500-700rpm, and an air intake of 0.5-2.0v / v min -1 , pH maintained at 5.5-6.5, and the bacteria were collected after fermentation for 96-144 hours.

[0018] On the basis of the above scheme, during fermentation, defoaming agent polypropylene glycol 2000 was added to the fermentation liquid at a ratio of 1-3 mL / L.

[0019] On the basis of the above scheme, the composition of the 1L fermentation medium is: glucose 80g, MgSO4·7H2O1.5g, metal mother liquor 100μL, ammonium tartrate 2g, KH2PO4 7.0g, Na2HPO4 2g, yeast extract powder 1.5g, CaCl2·2H2O0.1g, leucine 0.6g, and the balance water;

[0020] The metal mother liquor is composed of: FeCl3·6H2O 0.8 mg, ZnSO4·7H2O 0.1 mg, CuSO4·5H2O 0.01 mg, Co(NO3)2·6H2O 0.01 mg, and MnSO4·5H2O 0.01 mg, and distilled water is added to prepare 100 mL.

[0021] Beneficial effects of the present invention:

[0022] The invention uses Mucor circinelloides WJ11 as a wild-type strain, constructs a FAS2 gene knockout vector pCRC109 in Escherichia coli using sequences at both ends of the FAS2 gene as homology arms and kanamycin as a screening marker, and introduces a functional fragment into the Mucor circinelloides genome through click transformation for homologous recombination, thereby obtaining a Mucor circinelloides fatty acid synthase gene knockout strain. Fermentation tests show that the outputs of fatty acids and beta-carotene of the strain are significantly improved, and the strain has important application value in the production of lipids and beta-carotene. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the gene knockout vector pUC57 of Mucor circinelloides;

[0024] Figure 2 Schematic diagram of the FAS2 gene knockout vector pCRC109 of Mucor circinelloides;

[0025] Figure 3 Schematic diagram of the construction process of the FAS2 gene knockout strain of Mucor circinelloides;

[0026] Figure 4Agarose gel electrophoresis of PCR identification of the recombinant strain McFAS2△ (wherein, lane 1 is the wild-type strain WT, lanes 2 and 3 are the McFAS2△ strain, and lane M is a marker);

[0027] Figure 5 Comparison of lipid content between the Mucor circinelloides knockout strain McFAS2△ and the control strain MU365.

[0028] Figure 6 Comparison of β-carotene content between the Mucor circinelloides knockout strain McFAS2△ and the control strain MU365. DETAILED DESCRIPTION

[0029] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0030] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The experimental materials, reagents, drugs, etc. used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0031] In the following embodiments,

[0032] The strain of Mucor circinelloides WJ11 is the Mucor circinelloides with a deposit number of CCTCC NO: M2014424;

[0033] The Mucor circinelloides MU760 strain is a leucine-uracil auxotrophic strain of Mucor circinelloides WJ11, donated by Professor V. Garre of Spain, and is currently stored in the Food Bioengineering and Technology Laboratory of Jinan University. This strain has been reported in existing literature: Improved SDA production in high lipid accumulating strain of Mucor circinelloides WJ11 by genetic modification.

[0034] The vector pUC57 was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0035] Mucor circinelloides MU365 is a double-deficient strain MU760 as the recipient strain, and the pyrF gene fragment is transformed into MU760 protoplasts. After multiple rounds of screening, a homokaryotic strain is obtained, which is the control strain constructed in this experiment. Compared with the MU760 strain, MU365 is a leucine single-deficient strain, so it can grow on MMC plates (plates without uracil), but additional leucine needs to be added during the growth process to ensure its normal growth.

[0036] The McFAS2△ strain constructed in the following examples is also a leucine single-deficient strain. Compared with the MU365 strain, the fas2 gene in the McFAS2△ strain is knocked out, while the fas2 gene in the MU365 strain is not knocked out. Both strains require additional leucine during their growth to ensure normal growth.

[0037] Seed liquid culture medium: Liquid A: glucose 30g / L, MgSO4·7H2O 1.5g / L, metal mother solution 100μL / L (FeCl3·6H2O, 8mg / L; ZnSO4·7H2O, 1mg / L; CuSO4·5H2O, 0.1mg / L; Co(NO3)2·6H2O, 0.1mg / L; MnSO4·5H2O, 0.1mg / L), CaCl2·2H2O 0.1g / L; Liquid B: ammonium tartrate 3.3g / L, KH2PO4·7g / L, Na2HPO42g / L, yeast extract powder 1.5g / L, leucine 0.6g / L. Liquid A and liquid B were sterilized at 121℃ for 20min and then mixed for use.

[0038] Fermentation medium: glucose 80g / L, ammonium tartrate 2g / L, and the contents of other ingredients are the same as those of seed culture medium.

[0039] MMC solid culture medium: divided into liquid A and liquid B, sterilized at 121℃ for 20 minutes and then mixed before use. Liquid A (MMC liquid culture medium): 20g / L anhydrous glucose, 10g / L casein hydrolysate, 0.5g / L YNB, adjusted to the required pH (2.8 or 3.2), add nicotinic acid and ammonium thiamine after sterilization (final concentration 1μg / mL). Liquid B: 16-20g / L agar powder.

[0040] MMCS medium: Add 91.1 g / L of sorbitol to solution A in MMC solid medium, and the rest is the same as MMC solid medium.

[0041] MMCU medium: Add 200 mg / L uracil to solution A in MMC solid medium, and the rest is the same as MMC solid medium.

[0042] YPG solid medium: divided into liquid A and liquid B, sterilized at 121℃ for 20 minutes and then mixed for use. Liquid A (YPG liquid medium): 20g / L anhydrous glucose, 3g / L yeast extract powder, 10g / L peptone, pH adjusted to 4.5. Liquid B: 16-20g / L agar powder.

[0043] YPGS liquid culture medium: 20 g / L anhydrous glucose, 3 g / L yeast extract powder, 10 g / L peptone, 91.1 g / L sorbitol, pH adjusted to 4.5.

[0044] YNBS liquid culture medium: glucose 10 g / L, YNB 0.5 g / L, glutamic acid 1.5 g / L, ammonium sulfate 1.5 g / L, sorbitol 91.1 g / L.

[0045] Example 1

[0046] The application of fas2 gene in regulating the production of lipids and β-carotene in Mucor circinelloides. The fatty acid synthase gene fas2 in Mucor circinelloides is knocked out by genetic engineering method of homologous recombination, so that it is not expressed in Mucor circinelloides, thereby increasing the production of lipids and β-carotene by fermentation of Mucor circinelloides.

[0047] The nucleic acid sequence of the fas2 gene is shown in SEQ ID NO:1.

[0048] Example 2

[0049] The Mucor circinelloides with high lipid and β-carotene production is obtained by knocking out the fatty acid synthase gene fas2 in Mucor circinelloides MU760 through a genetic engineering method using homologous recombination so that the gene is not expressed in Mucor circinelloides, thereby obtaining the Mucor circinelloides fatty acid synthase gene fas2 knockout strain McFAS2△.

[0050] The nucleic acid sequence of the fatty acid synthase gene fas2 is shown in SEQ ID NO:1.

[0051] Example 3

[0052] The method for constructing Mucor circinelloides with high lipid and β-carotene production comprises the following steps:

[0053] 1. Construction of knockout vector pCRC109

[0054] (1) Based on the gene sequence of 1000 bp upstream and downstream of the fas2 gene in Mucor circinelloides WJ11, homology arm amplification primers fas2 up-F / R and fas2 down-F / R (as shown in SEQ ID NO:2-5) were designed. Using the genomic DNA of WJ11 as a template, the two pairs of primers were used to perform PCR amplification on the gene fragments of 1000 bp upstream and downstream of the fas2 gene, respectively, to obtain 1000 bp target fragments, namely the upstream homology arm sequence of the fas2 gene in the WJ11 genome (SEQ ID NO:6) and the downstream homology arm sequence of the fas2 gene in the WJ11 genome (SEQ ID NO:7).

[0055] (2) Primers pyrF-F / R (as shown in SEQ ID NOs: 8-9) were designed based on the sequence of the pyrF gene (uracil biosynthesis gene) in Mucor circinelloides WJ11, and PCR amplification was performed to obtain the pyrF gene fragment (SEQ ID NO: 10).

[0056] In the above steps (1) and (2), PCR amplification used PrimeSTAR HSDNA Polymerase high-fidelity enzyme, and the amplification conditions were set as follows: 95°C for 10 min; (95°C for 30 s, 52-55°C for 30 s, 72°C for 1 min / kb) × 35 cycles, 72°C for 10 min, and then cooled to 4°C for storage.

[0057] (3) Using the 1 kb upstream and downstream fragments of the fas2 gene of Mucor circinelloides obtained in step (1) and the cloned fragment of the pyrF gene obtained in step (2) as templates, using the up-F / down-R of the fas2 gene as primers, and using Herculase II fusion enzyme to perform a fusion PCR reaction, the reaction conditions are: 95°C for 5 min, (95°C for 30 s, 70°C for 1 s, 55°C for 30 s, 68°C for 2 min) × 12 cycles, (95°C for 30 s, 70°C for 1 s, 55°C for 30 s, 68°C for 350 s) × 23 cycles, 68°C for 5 min, and finally storing at 4°C to obtain the fusion fragment fas2 up-pyrF-fas2 down.

[0058] (4) The knockout vector pUC57 ( Figure 1 ) and the fusion fragment fas2 up-pyrF-fas2down were digested at 37°C for 3 h.

[0059] (5) Use T4 ligase to ligate the plasmid pUC57-Kan purified by enzyme digestion in step (4) with the fusion fragment fas2up-pyrF-fas2down, and react at 16°C for 3 h.

[0060] (6) The ligation product of step (5) was transformed into E. coli DH5α competent cells, positive transformants were selected, plasmids were extracted, and after successful PCR verification and sequencing verification, the fas2 gene knockout vector pCRC109 ( Figure 2 ).

[0061] 2. Construction of an engineered strain of Mucor circinelloides with fas2 gene knockout

[0062] (1) The fas2 gene knockout vector pCRC109 was digested with restriction endonuclease SnaBⅠ at 37°C for 3 h to obtain the fas2 up-pyrF-fas2 down gene fragment, and the required concentration was determined by NanoDrop2000.

[0063] (2) Protoplasts of Mucor circinelloides leucine-uracil double auxotrophic strain MU760 were prepared by the following steps:

[0064] ① Take the preserved MU760 spores and streak them on YPG (pH = 4.5) solid medium supplemented with uracil (final concentration 200μg / mL) and leucine (final concentration 0.6mg / mL), and culture them in a constant temperature incubator at 28℃ for 24h to activate the strain. Take a single clone of hyphae and plant them on a plate of YPG medium. Culture them at 28℃ for 3-4 days until the spores grow well. After sufficient spores grow, collect them with sterile water, centrifuge at 3000rpm for 5min at room temperature, discard the supernatant, and add 5mL of sterile dH2O to resuspend the spores. Take 1.25×10 7 The spores were centrifuged at 2500 rpm for 5 min, the supernatant was discarded, and YPG liquid medium was added to resuspend.

[0065] ② Transfer all the suspension containing spores to a sterilized Erlenmeyer flask, add 20mL YPG liquid culture medium, 50μL leucine (10mg / mL) and 25μL uracil (200mg / mL). Place in a 4℃ refrigerator overnight to allow the spores to fully absorb water and swell. Place the Erlenmeyer flask in a 250rpm shaker at 28℃ for incubation. After 5-6 hours, take a small amount of spores and observe under a microscope. When the length of most spores is less than twice the length of the spores, the incubation is complete.

[0066] ③ Transfer the incubated spores to a sterilized 50mL centrifuge tube, centrifuge at 1500rpm for 5min at room temperature, discard the supernatant, slowly add 5mL PS Buffer and centrifuge at 2000rpm for 5min to wash the spores twice. After washing, add 4mL PS Buffer with a pH of 6.5 [18.22g sorbitol and 20mL PBS buffer (NaCl 137mM, KCl 2.7mM, Na2HPO4 10mM, KH2PO42mM)] to resuspend the spores, and add a final concentration of 4mg / mL of lysase and 0.06U / mL of chitosanase dissolved in 1mL PS Buffer to prepare a mixed enzyme solution and add it to the spore solution. Shake at 60rpm and 30℃ to enzymatically hydrolyze the germinated spores for 80-100min. Observe the enzymatic hydrolysis under a microscope during this period.

[0067] ④ After the enzymatic hydrolysis is complete, place the spores on ice, add 10 mL of pre-cooled 0.5 mol / L sorbitol solution, centrifuge at 1100 rpm for 5 min at 4°C, discard the supernatant, and repeat this step twice. Add the required amount of pre-cooled 0.5 mol / L sorbitol solution to resuspend the spores to obtain protoplasts, and dispense into 100 μL / tube for use.

[0068] (3) Electroporation of target fragment into MU760 protoplasts:

[0069] ① Add 20 μL of 100 ng / μL target gene fragment digested with sorbitol to the aliquoted protoplasts.

[0070] ② After gently stirring, add the mixture into the pre-cooled electroporation cup, and perform electroporation at a voltage of 800 V, a capacitance of 25 μF, and a resistance of 400 Ω. After the electroporation, quickly remove the electroporation cup and immediately add 1 mL of pre-cooled YPGS liquid medium. Aspirate the mixture into a 1.5 mL centrifuge tube and place it on ice.

[0071] ③ Incubate at 28℃ for 1h on a shaker at 100rpm. After incubation, centrifuge at 1500rpm at 4℃ for 5min, discard the supernatant, add 300-500μL YNBS liquid culture medium with a pH of 4.5, and gently stir to resuspend.

[0072] ④ Take 200-300 μL of the sample from step ③ and gently spread it on MMCS solid culture medium with a pH of 3.2, and culture it in a 28°C incubator away from light for 3-4 days.

[0073] (4) Screening of positive transformants

[0074] ① Transfer the single colony on the MMCS solid culture medium to the MMC plate with a pH of 3.2 and culture under light at 28°C for 3 days. Collect the spores and evenly spread the same number of spores on the screening medium MMC and MMCU plates with a pH of 2.8. After culturing at 28°C for 36 hours, observe the number of colonies on the two plates, pick a single colony on the MMC plate with a similar number of colonies and continue to transfer it to the MMC plate with a pH of 3.2 for culture. Repeat the screening for 3-4 generations to verify the stability of the transformants. When the number of colonies on the MMC and MMC (with uracil added) plates is roughly the same, collect the spores on the MMC plate and store them at -20°C, and collect the mycelium for genome extraction and PCR verification.

[0075] ② The genome of the mycelium in step ① was extracted using a quick plant genome extraction kit. The genome quality was tested by agarose gel electrophoresis, and its concentration was determined using a NanoDrop 2000 spectrophotometer. The genome was stored at -20°C for subsequent experiments.

[0076] ③Use the genomic DNA extracted in step ② above as a template (concentration of 100-200ng / μL), use fas2-out-F / R as a verification primer (SEQ ID NO:11-12), and use ApexHF HSDNA Polymerase CL enzyme for PCR amplification. Reaction conditions: 94℃ pre-denaturation for 1min, (98℃ denaturation for 10s, 60℃ annealing for 15s, 68℃ extension for 5min) × 35 cycles, cool to 4℃, take out and store. The PCR product was then verified by agarose gel electrophoresis to verify whether the actual size was consistent with the theoretical value, thereby obtaining a recombinant transformant with successful knockout. Schematic diagram of the construction process of the FAS2 gene knockout strain of Mucor circinelloides is shown in the figure. Figure 3 As shown, the agarose gel electrophoresis diagram of genetically engineered strain identification is as follows Figure 4 As shown, the results showed that the FAS2 gene had been successfully knocked out. Finally, the recombinant strain of Mucor circinelloides McFAS2△ was obtained. The successfully constructed genetic engineering strain of Mucor circinelloides with the fas2 gene knocked out was stored at -80°C.

[0077] Example 4

[0078] The method for fermenting and producing lipids and β-carotene using the recombinant strain McFAS2△ of Mucor circinelloides, the steps are as follows:

[0079] (1) Activation of strains: Spores of the recombinant strain McFAS2△ of Mucor circinelloides stored at -80°C were streaked on YPG solid medium and cultured at 28°C for 48 h.

[0080] (2) Seed culture: 100 μL of the recombinant strain spores (1.0×10 7 / mL) was inoculated into a sterilized baffled flask containing 100mL of liquid seed culture medium (containing 0.6g / L leucine) and fermented in a shaking incubator at 130rpm and 28°C for 24h as seed culture medium for subsequent fermentation.

[0081] (3) Fermentation tank culture: The inoculum was inoculated at 10% (w / w) into a 1.5 L fermentation tank containing 1 L of fermentation medium (containing 0.6 g / L leucine), and the defoaming agent polypropylene glycol 2000 was added at 2 mL / L; the fermentation conditions were 28°C, 600 rpm, and the air intake was 1 v / v min -1 , pH was maintained at around 6.0. According to the oil production law of Mucor circinelloides, fermentation samples were collected at 6, 12, 24, 36, 48, 60, 72, 84, and 96 h, the fermentation broth and mycelium were separated, the fermentation supernatant was stored in a -20°C refrigerator for later use, the mycelium was filtered, washed with distilled water 3 to 5 times, and then freeze-dried for later use.

[0082] Mucor circinelloides MU36 was used as the control strain group, and the culture method was the same as that of the recombinant Mucor circinelloides strain McFAS2△.

[0083] Lipids and β-carotene were extracted from the mycelium of the recombinant strain McFAS2△ of Mucor circinelloides and the control group Mucor circinelloides MU365 after fermentation, and their contents were determined. The determination method of lipids and β-carotene is as follows:

[0084] Extraction and determination of fatty acid content: refer to the method (Folch J, Lees M, Sloane-Stanley G, et al. A simple method for the isolation and purification of total lipids from animal tissues. Biol Chem, 1957, 226, 497-509), and make appropriate modifications. The specific method is as follows: accurately weigh 10-15 mg of freeze-dried mycelium into a 5 mL glass lipid extraction bottle, add 2 mL of 4 mol / L hydrochloric acid, and place in an 80°C water bath for 3-6 hours. During this period, vortex and shake for 30 seconds every 30 minutes to fully lyse the bacteria; after the water bath, cool to room temperature, add 2 mL of chloroform, 1 mL of methanol and 100 μL of 2 mg / L C15:0 internal standard, place in a mixer for rotation extraction for 0.5 hours, and centrifuge at 3000 rpm for 5 minutes. , the solution was separated into layers, the lower chloroform layer was transferred to a new fat extraction bottle, and dried with nitrogen; 1 mL of 10% sulfuric acid methanol solution was added, and the mixture was placed in a 60°C water bath for 3-6 h, during which time the mixture was vortexed for 30 sec every 30 min to allow the solidified oil droplets to fully react with the solution; after the water bath was completed, the mixture was cooled to room temperature, 2 mL of n-hexane (chromatographically pure) and 1 mL of saturated NaCl solution were added, the mixture was mixed by inversion for 15-20 min, and the mixture was centrifuged at 3000 rpm for 5 min, and the n-hexane layer of the upper liquid in the fat extraction bottle was transferred to a gas phase bottle to obtain a fatty acid methyl ester solution, and gas phase detection was performed.

[0085] The extraction and determination of β-carotene were carried out according to the method (Papp, T.; Velayos, A.; Bartók, T.; Eslava, A. P.; C.; Iturriaga, EA Heterologous expression of astaxanthinbiosynthesis genes in Mucor circinelloides. Appl. Microbiol. Biotechnol. 2006, 69, 526–531). Take 100-200 mg of freeze-dried mycelium of Mucor circinelloides and freeze it quickly in liquid nitrogen. Immediately grind it thoroughly in a pre-cooled mortar. Transfer the ground powder to a 10 mL centrifuge tube and accurately weigh the mass of the mycelium powder. Add 1 mL of n-hexane to the centrifuge tube and vortex for 1 min. Repeat 4 times to obtain 4 mL of mixed liquid. Transfer the mixed liquid to a 15 mL centrifuge tube, add an equal volume of 10% ether petroleum ether and then add 2 mL of ddH2O to separate the layers. Centrifuge at 4°C and 3200 rpm for 8 min. Transfer the colored liquid on the upper layer to a new 10 mL centrifuge tube. Use a nitrogen blower to blow dry the petroleum ether fraction to obtain a dried extract.

[0086] Analysis of β-carotene content by HPLC: The dried extract was resuspended in 700 μL of tetrahydrofuran supplemented with butylated hydroxytoluene (BHT) (100 μg / mL), and 10 μL was subjected to HPLC analysis on an infinity LabProsell120EC-C18 column (4.6×150, ODS 4 μm). Two solvents A (96% methanol) and B (100% methyl tert-butyl ether) were used as mobile phases. These two solvents were used to analyze our samples with the following gradient: min / solvent A% / solvent B% was 0 / 99 / 1; 8 / 60 / 40; 13 / 46 / 54; 15 / 0 / 100; 18 / 0 / 100; 21 / 99 / 1. The flow rate was set to 1 mL min -1 The detection wavelength was 450 nm, the column temperature was 35°C, and a diode array detector (Agilent Technologies, Santa Clara, CA, USA) was used.

[0087] The results are as follows Figure 5 and Figure 6 As shown, McFAS2Δ lipid accumulation was 24.82% CDW, which was 18.4% higher than the control strain MU365 (20.96% CDW). The β-carotene content of MU365 and McFAS2Δ was 85.5±2.87μg / g and 295.9±6.93μg / g, respectively. The β-carotene content of McFAS2Δ strain was 246% higher than that of the control strain MU365.

[0088] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. Application of fas2 gene in regulating lipid and β-carotene production of Mucor circinelloides, characterized in that: The nucleic acid sequence of the fas2 gene is shown in SEQ ID NO:

1.

2. The use of the fas2 gene according to claim 1 in regulating the production of lipids and β-carotene in Mucor circinelloides, characterized in that: The fatty acid synthase gene fas2 in Mucor circinelloides is knocked out by a genetic engineering method using homologous recombination, so that it is not expressed in Mucor circinelloides, thereby increasing the yield of lipid and beta-carotene produced by fermentation of Mucor circinelloides.

3. Mucor circinelloides with high lipid and β-carotene production, characterized in that: The fatty acid synthase gene fas2 in Mucor circinelloides MU760 is knocked out by a genetic engineering method using homologous recombination so that it is not expressed in Mucor circinelloides, thereby obtaining the Mucor circinelloides fatty acid synthase gene fas2 knockout strain McFAS2△, wherein the nucleic acid sequence of the fas2 gene is shown in SEQ ID NO:

1.

4. The method for constructing the Mucor circinelloides with high lipid and β-carotene production according to claim 3, characterized in that: Here are the steps: The upstream gene sequence fas2 up and the downstream gene sequence fas2 down of the fas2 gene in Mucor circinelloides WJ11 were used as homology arms, and the uracil synthesis gene pyrF was used as a screening resistance gene to construct a fas2 gene knockout frame fas2 up-pyrF-fas2down, and the fas2 up-pyrF-fas2 down was constructed into a pUC57 plasmid to obtain a knockout vector pCRC109; the knockout vector pCRC109 was transformed into the protoplasts of the MU760 strain by electroporation, and the uracil synthesis gene pyrF was used as a screening marker to screen positive clones, so as to obtain the Mucor circinelloides fatty acid synthase gene fas2 knockout strain McFAS2△.

5. The method for constructing Mucor circinelloides with high lipid and β-carotene production according to claim 4, characterized in that: The primers for amplifying the gene sequence fas2 up upstream of the fas2 gene in the Mucor circinelloides WJ11 are shown in SEQ ID NO:2 and SEQ ID NO:3; the primers for amplifying the gene sequence fas2 down downstream of the fas2 gene in the Mucor circinelloides WJ11 are shown in SEQ ID NO:4 and SEQ ID NO:5; the primers for amplifying the uracil synthesis gene pyrF are shown in SEQ ID NO:8 and SEQ ID NO:

9.

6. The method for constructing Mucor circinelloides with high lipid and β-carotene production according to claim 5, characterized in that: The gene sequence fas2 up upstream of the fas2 gene in the Mucor circinelloides WJ11 is shown in SEQ ID NO:6, and the gene sequence fas2 down downstream of the fas2 gene is shown in SEQ ID NO:7; the sequence of the uracil synthesis gene pyrF is shown in SEQ ID NO:

10.

7. Use of the high lipid and β-carotene-producing Mucor circinelloides constructed by the method according to any one of claims 3 to 6 in the production of lipids and β-carotene.

8. A method for producing lipids and β-carotene by fermentation, characterized in that: The seed liquid of Mucor circinelloides with high lipid and β-carotene production constructed by the method according to any one of claims 3 to 6 is inoculated into a fermentation medium for fermentation culture under the conditions of 25-30° C., 500-700 rpm, and an air intake of 0.5-2 v / v min -1 , pH maintained at 5.5-6.5, and the bacteria were collected after fermentation for 96-144 hours.

9. The method for producing lipid and β-carotene by fermentation according to claim 8, characterized in that: During fermentation, add defoaming agent polypropylene glycol 2000 to the fermentation broth at a ratio of 1-3 mL / L.

10. The method for producing lipid and β-carotene by fermentation according to claim 8, characterized in that: The composition of 1L fermentation medium is: glucose 80g, MgSO4·7H2O 1.5g, metal mother liquor 100μL, ammonium tartrate 2g, KH2PO4 7.0g, Na2HPO4 2g, yeast extract 1.5g, CaCl2·2H2O 0.1g, leucine 0.6g, and the balance water; The metal mother liquor is composed of: FeCl3·6H2O 0.8 mg, ZnSO4·7H2O 0.1 mg, CuSO4·5H2O 0.01 mg, Co(NO3)2·6H2O 0.01 mg, and MnSO4·5H2O 0.01 mg, and distilled water is added to prepare 100 mL.

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