Method for improving yield of astaxanthin by knocking out phaffia rhodozyma grease metabolism related genes

Abstract

The invention relates to the field of biotechnology and synthetic biology, in particular to a method for knocking out grease metabolism related genes to increase the yield of astaxanthin in phaffia rhodozyma. Recombinant strains are obtained by knocking out phaffia rhodozyma grease metabolism related genes, and the fermentation yield of astaxanthin in phaffia rhodozyma is increased. By knocking out the gene POX1 or GUT2, the astaxanthin content of the strain can be increased by 29% or above, the yield can be increased by 20% or above, and the method can be applied to construction of the astaxanthin high-yield strain.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and synthetic biology, and in particular to a method for knocking out oil metabolism genes to improve the astaxanthin production of Phaffia rhodozyma and a recombinant strain. Background Art

[0002] Astaxanthin is a carotenoid with strong antioxidant and free radical scavenging capabilities. It is widely used in aquaculture, cosmetics, health products and food industries (Gómez M, Campusano S, Gutiérrez MS, et al. J Lipid Res, 2020, 61: 1658-1674). The 2021 U.S. Grand View Research report (Report ID: GVR-1-68038-957-9) pointed out that the global market size of astaxanthin reached US$1.37 billion in 2020 and is expected to reach US$4.75 billion in 2028.

[0003] Phaffia rhodozyma (Xanthophyllomyces dendrorhous / Phaffia rhodozyma) is a basidiomycete yeast and one of the few microorganisms that can naturally synthesize astaxanthin. It has the advantages of rapid proliferation, short growth cycle, and high-density fermentation in fermentation tanks. The astaxanthin synthesized by Phaffia rhodozyma can account for 50% to 85% of the cell carotenoids and is considered to be a high-quality cell factory for producing natural astaxanthin. The astaxanthin content of wild-type Phaffia rhodozyma is low, and the astaxanthin content of the strain is usually not higher than 0.1 mg / g cell dry weight, so it cannot meet the requirements of industrial application. The astaxanthin content of the high-yield strain of Phaffia rhodozyma obtained by combining natural mutagenesis with metabolic engineering can reach 9 mg / g cell dry weight, but the fermentation cycle is long and the economy is poor (Gassel S, Breitenbach J, Sandmann G. Appl Microbiol Biotechnol, 2014, 98: 345-350). Therefore, developing a Phaffia rhodozyma strain that can efficiently synthesize astaxanthin is of great significance to accelerating the development of related industries.

[0004] Existing studies have shown that lipid structures in cells, especially lipid droplets, are important storage sites for hydrophobic carotenoids. Increasing oil production can improve the storage capacity of astaxanthin. In Saccharomyces cerevisiae, it has been reported that increasing oil content through metabolic engineering can increase lycopene content to 73.3 mg / g cell dry weight, and most of it is stored in lipid droplets (Ma T, Shi B, Ye Z, et al. Metab Eng, 2019, 52: 134-142). Larroude et al. (Larroude M, Celinska, E, Back A, et al. Biotechnol Bioeng, 2018, 115: 464-472) found that high-oil-yielding Yarrowia lipolytica strains can synthesize more β-carotene. Through metabolic engineering, they obtained a Yarrowia lipolytica strain with a β-carotene content of up to 90 mg / g, and the oil production can reach 42.6 g / L. The acyl-CoA oxidase encoded by the POX series genes (POX1-6) of Yarrowia lipolytica is involved in the β-oxidation of fatty acids. Knocking out these genes can promote the accumulation of oils and fats (Beopoulos A, Mrozova Z, Thevenieau F, et al. Appl Environ Microbiol, 2008, 74: 7779-7789). In addition, the Yarrowia lipolytica GUT2 encodes a glycerol-3-phosphate dehydrogenase isomer. Knocking out this gene can prevent the reduction of glycerol-3-phosphate to dihydroxyacetone phosphate, which is beneficial to the synthesis of triglycerides (Beopoulos A, Mrozova Z, Thevenieau F, et al. Appl Environ Microbiol, 2008, 74: 7779-7789). However, lipids compete with astaxanthin to synthesize precursor acetyl-CoA and coenzyme NADPH. Studies have shown that astaxanthin production is negatively correlated with lipid production in Phaffia rhodozyma (Miao LL, Chi SA, Tang Y, et al. FEMS Yeast Res, 2011, 11: 192-201; Xiao AF, Jiang XL, Ni H, et al. Electr J Biotec, 2015, 18: 148-153). Therefore, the relationship between POX and GUT2 genes and astaxanthin synthesis is still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide a method for knocking out oil metabolism genes to increase the astaxanthin production of Phaffia rhodozyma.

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

[0007] A method for improving the astaxanthin production of Phaffia rhodozyma, comprising: knocking out genes encoding enzymes related to lipid metabolism of Phaffia rhodozyma to obtain a recombinant strain, further purifying the recombinant strain, and thereby improving the fermentation production of astaxanthin of Phaffia rhodozyma.

[0008] The gene sequence encoding Phaffia rhodozyma lipid metabolism-related enzyme CED83814.1 (POX1) is shown in SEQ ID NO.1; and / or,

[0009] The gene sequence encoding the enzyme CDZ96393.1 (GUT2) related to lipid metabolism of Phaffia rhodozyma is shown in SEQ ID NO.2.

[0010] Specifically

[0011] (1) Construction of homologous recombination plasmids: pUG6-TG-POX1-Zeocin-loxP and / or pUG6-TG-GUT2-Zeocin-loxP;

[0012] (2) using one or more of the homologous recombination plasmids obtained above as templates to amplify the knockout fragment, transforming Phaffia rhodozyma M1.3, performing gene homologous recombination knockout, and obtaining a gene recombinant strain;

[0013] The homologous recombination plasmid pUG6-TG-POX1-Zeocin-loxP construction method is to use pUG6-TG-Cbr.1-Zeocin-loxP plasmid as a template, use primers Backbone-POX-Zeocin-f and Backbone-POX-Zeocin-r to amplify the plasmid backbone, and use primers POX1-Zeocin-loxP-f and POX1-Zeocin-loxP-r to amplify the antibiotic screening marker fragment; use the genomic DNA of Phaffia rhodozyma CBS 6938 as a template, use primers POXup-f and POXup-r to amplify the upstream homology arm of POX1, and use primers POXdn-f and POXdn-r to amplify the downstream homology arm of POX1; finally, assemble the above four DNA fragments by Gibson assembly method to obtain the target plasmid;

[0014] The homologous recombination plasmid pUG6-TG-GUT2-Zeocin-loxP construction method is as follows: using pUG6-TG-Cbr.1-Zeocin-loxP plasmid as a template, using primers Backbone-GUT2-Zeocin-f and Backbone-GUT2-Zeocin-r to amplify the plasmid backbone, and using primers GUT2-Zeocin-loxP-f and GUT2-Zeocin-loxP-r to amplify the antibiotic screening marker fragment; using genomic DNA of Phaffia rhodozyma CBS 6938 as a template, using primers GUT2up-f and GUT2up-r to amplify the GUT2 upstream homology arm, and using primers GUT2dn-f and GUT2dn-r to amplify the GUT2 downstream homology arm; finally, the above four DNA fragments are assembled by Gibson assembly method to obtain the target plasmid.

[0015] A method for preparing a recombinant bacterium is described, wherein the method obtains a recombinant Phaffia rhodozyma bacterium in which the lipid metabolism gene is inactivated.

[0016] Application of the recombinant bacteria, application of the recombinant Phaffia rhodozyma with inactivated oil metabolism genes in the production of astaxanthin.

[0017] A gene for an enzyme related to lipid metabolism for improving the astaxanthin production of Phaffia rhodozyma, wherein the gene sequence encoding the enzyme CED83814.1 (POX1) related to lipid metabolism of Phaffia rhodozyma is shown in SEQ ID NO.1; and / or

[0018] The gene sequence encoding the enzyme CDZ96393.1 (GUT2) related to lipid metabolism of Phaffia rhodozyma is shown in SEQ ID NO.2.

[0019] A homologous recombination vector capable of inactivating oil metabolism genes, wherein the homologous recombination vector is a recombinant vector containing the oil metabolism-related genes of Phaffia rhodozyma.

[0020] The advantages of the present invention are:

[0021] The present invention achieves knockout of POX1 and / or GUT2 genes related to oil metabolism of Phaffia rhodozyma by homologous recombination, thereby significantly increasing the content of astaxanthin. The astaxanthin cell content of the POX1 single knockout strain is increased by 32.1% compared with the control strain, and the astaxanthin cell content of the GUT2 single knockout strain is increased by 29.2% compared with the control strain. The method can be applied to the construction of high-yield astaxanthin strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The cell dry weight after culturing the Phaffia rhodozyma oil metabolism gene knockout strain provided in the embodiment of the present invention. M1.3 is a wild-type strain, M1.3-Δpox1 and M1.3-Δgut2 are strains with POX1 and GUT2 gene knockouts, respectively; culture conditions: YM liquid medium (2% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract), 22°C, shaking speed of 220 rpm, time of 120 h.

[0023] Figure 2 The pigment titer of the Phaffia rhodozyma oil metabolism gene knockout strain after cultivation provided in the embodiment of the present invention. M1.3 is a wild-type strain, M1.3-Δpox1 and M1.3-Δgut2 are strains with POX1 and GUT2 gene knockouts, respectively; culture conditions: YM liquid medium (2% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract), 22°C, shaker speed of 220 rpm, time of 120 h.

[0024] Figure 3 The pigment content after cultivation of the Phaffia rhodozyma oil metabolism gene knockout strain provided in the embodiment of the present invention. M1.3 is a wild-type strain, M1.3-Δpox1 and M1.3-Δgut2 are wild-type strains with POX1 and GUT2 gene knockouts, respectively; culture conditions: YM liquid medium (2% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract), 22°C, shaking speed of 220 rpm, time of 120 h. DETAILED DESCRIPTION

[0025] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0026] The gene knockout of the present invention involves inactivating enzymes related to lipid metabolism of Phaffia rhodozyma, whose GenBank accession numbers are CED83814.1 and CDZ96393.1. According to their homology with Yarrowia lipolytica proteins, their corresponding genes are named POX1 (SEQ ID NO: 1) and GUT2 (SEQ ID NO: 1), respectively.

[0027] The method of the present invention can increase the astaxanthin content by knocking out genes related to oil metabolism of Phaffia rhodozyma. The present invention can provide methods and ideas for constructing high-yield strains to promote the industrialization of astaxanthin synthesis by Phaffia rhodozyma.

[0028] Example

[0029] 1. Materials and Methods

[0030] 1.1 Strains and culture conditions

[0031] Experimental strain: Phaffia rhodozyma M1.3, constructed with strain CBS 6938 (available from depository institutions such as CBS in the Netherlands or DSMZ in Germany) according to the method described in the patent (application number 201910788499.X). Pre-culture medium: YPD (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L). Astaxanthin production medium: YM (glucose 20 g / L, peptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L). Shake flask culture conditions: 22°C, 220 rpm.

[0032] 1.2 Vector construction

[0033] The vector was constructed by Gibson assembly method. The target fragment was amplified by high-fidelity Pfu (Quanshijin Biotechnology, Beijing) DNA polymerase, and the amplification system was 50 μL ( FastPfu PCR SuperMix: 25μL, 10μM upstream and downstream primers 1.5μL each, template 2μL, ddH2O: 20μL). The PCR amplification program was: 95℃ pre-denaturation for 3min; then 95℃ denaturation for 20s, 50-60℃ annealing for 20s, 72℃ extension (extension time calculated as 30s / kb), 35 cycles; finally 72℃ extension for 8min, 20℃ insulation. The size of the PCR amplified fragment was checked by agarose gel electrophoresis, and the DNA fragments with the expected size were purified and recovered using Gel Extraction Kit (Omega Bio-Tek, USA). The recovered DNA fragments were assembled into circular plasmids by Gibson assembly. The reaction system was 10 μL (2×Pro Ligation-free Cloning MasterMix: 5 μL, vector backbone 100 ng, the molar ratio of the remaining fragments to the vector backbone was 1:3, and ddH2O was added to 10 μL). The reaction was placed in a constant temperature metal bath at 50°C for 1 hour, and 5 μL was taken to transform Escherichia coli chemical competent cells Trans1-T1. After the colonies grew on the antibiotic plate, PCR was used for preliminary identification, and the positive clones were sequenced to confirm whether the sequence was correct. The primers used are shown in Table 1.

[0034] Construction of POX1 gene homologous knockout vector pUG6-TG-POX1-Zeocin-loxP: Using pUG6-TG-Cbr.1-Zeocin-loxP plasmid as template, primers Backbone-POX-Zeocin-f and Backbone-POX-Zeocin-r were used to amplify the plasmid backbone, and primers POX1-Zeocin-loxP-f and POX1-Zeocin-loxP-r were used to amplify the antibiotic selection marker fragment in the plasmid template; using the genomic DNA of Phaffia rhodozyma CBS 6938 as template, primers POXup-f and POXup-r were used to amplify the upstream homology arm of POX1, and primers POXdn-f and POXdn-r were used to amplify the downstream homology arm of POX1; finally, the target fragments obtained above were assembled by Gibson assembly method to obtain the target plasmid.

[0035] Construction of POX1 gene homologous knockout vector pUG6-TG-POX1-Zeocin-loxP: Using plasmid pUG6 (provided by EUROSCARF) as a template, primers Backbone-POX-Zeocin-f and Backbone-POX-Zeocin-r were used to amplify the plasmid backbone, and primers POX1-Zeocin-loxP-f and POX1-Zeocin-loxP-r were used to amplify the antibiotic gene. The antibiotic marker gene sequence used is shown in SEQ ID NO3. Using the genomic DNA of Phaffia rhodozyma CBS 6938 as a template, primers POXup-f and POXup-r were used to amplify the upstream homology arm of POX1, and primers POXdn-f and POXdn-r were used to amplify the downstream homology arm of POX1; finally, the target fragments obtained above were assembled by Gibson assembly method to obtain the target plasmid

[0036] Plasmid backbone amplification system: total volume 50 μL, including FastPfu PCR SuperMix 25μL, ddH2O 20μL, upstream primer Backbone-POX-Zeocin-f (10μM) 1.5μL, downstream primer Backbone-POX-Zeocin-r (10μM) 1.5μL, template is pUG6-TG-Cbr.1-Zeocin-loxP plasmid, the addition amount is 2μL, the sequences of the primers in the amplification system are shown in Table 1.

[0037] Plasmid backbone PCR amplification program: pre-denaturation at 95°C for 3 min; then denaturation at 95°C for 20 s, annealing at 51°C for 20 s, extension at 72°C for 1 min 15 s, 35 cycles; finally extension at 72°C for 8 min, and storage at 20°C.

[0038] POX1 upstream homology arm amplification system: total volume 50 μL, including FastPfu PCRSuperMix 25μL, ddH2O 20μL, upstream primer POXup-f (10μM) 1.5μL, downstream primer POXup-r (10μM) 1.5μL, template is genomic DNA of Phaffia rhodozyma CBS 6938, the addition amount is 2μL, the sequences of primers in the amplification system are shown in Table 1.

[0039] The PCR amplification procedure for the upstream homology arm of POX1 was as follows: pre-denaturation at 95°C for 3 min; followed by denaturation at 95°C for 20 s, annealing at 58°C for 20 s, extension at 72°C for 30 s, and 35 cycles; finally, extension at 72°C for 8 min and storage at 20°C.

[0040] POX1 downstream homology arm PCR amplification system: total volume 50 μL, including

[0041] FastPfu PCR SuperMix 25μL, ddH2O 20μL, upstream primer POXdn-f (10μM) 1.5μL, downstream primer POXdn-r (10μM) 1.5μL, template is genomic DNA of Phaffia rhodozyma CBS 6938, the addition amount is 2μL, the sequences of primers in the amplification system are shown in Table 1.

[0042] The PCR amplification procedure for the downstream homology arm of POX1 was as follows: pre-denaturation at 95°C for 3 min; followed by denaturation at 95°C for 20 s, annealing at 58°C for 20 s, and extension at 72°C for 30 s, for 35 cycles; and finally extension at 72°C for 8 min and storage at 20°C.

[0043] Antibiotic (Zeocin) screening marker fragment PCR amplification system: total volume 50 μL, including FastPfu PCR SuperMix 25μL, ddH2O 20μL, upstream primer POX1-Zeocin-loxP-f (10μM) 1.5μL, downstream primer POX1-Zeocin-loxP-r (10μM) 1.5μL, template is pUG6-TG-Cbr.1-Zeocin-loxP plasmid, the addition amount is 2μL, the sequences of the primers in the amplification system are shown in Table 1.

[0044] The PCR amplification procedure of the antibiotic selection marker fragment was as follows: pre-denaturation at 95°C for 3 min; followed by denaturation at 95°C for 20 s, annealing at 58°C for 20 s, extension at 72°C for 45 s, and 35 cycles; and finally extension at 72°C for 8 min and storage at 20°C.

[0045] Construction of GUT2 gene homologous knockout vector pUG6-TG-GUT2-Zeocin-loxP: Using pUG6-TG-Cbr.1-Zeocin-loxP plasmid as template, primers Backbone-GUT2-Zeocin-f and Backbone-GUT2-Zeocin-r were used to amplify the plasmid backbone, and primers GUT2-Zeocin-loxP-f and GUT2-Zeocin-loxP-r were used to amplify the antibiotic selection marker fragment in the plasmid template; using the genomic DNA of Phaffia rhodozyma CBS 6938 as template, primers GUT2up-f and GUT2up-r were used to amplify the upstream homology arm of GUT2, and primers GUT2dn-f and GUT2dn-r were used to amplify the downstream homology arm of GUT2; finally, the target fragments obtained above were assembled by Gibson assembly method to obtain the target plasmid.

[0046] Plasmid backbone amplification system: total volume 50 μL, including FastPfu PCR SuperMix 25μL, ddH2O 20μL, upstream primer Backbone-GUT2-Zeocin-f (10μM) 1.5μL, downstream primer

[0047] Backbone-GUT2-Zeocin-f (10 μM) 1.5 μL, template is

[0048] pUG6-TG-Cbr.1-Zeocin-loxP plasmid, the addition amount is 2 μL, and the sequences of primers in the amplification system are shown in Table 1.

[0049] Plasmid backbone PCR amplification program: pre-denaturation at 95°C for 3 min; then denaturation at 95°C for 20 s, annealing at 52°C for 20 s, extension at 72°C for 1 min 15 s, 35 cycles; finally extension at 72°C for 8 min, and storage at 20°C.

[0050] GUT2 upstream homology arm amplification system: total volume 50 μL, including FastPfu PCRSuperMix 25μL, ddH2O 20μL, upstream primer GUT2up-f (10μM) 1.5μL, downstream primer GUT2up-r (10μM) 1.5μL, template is genomic DNA of Phaffia rhodozyma CBS 6938, the addition amount is 2μL, the sequences of primers in the amplification system are shown in Table 1.

[0051] The PCR amplification procedure for the upstream homology arm of GUT2 was as follows: pre-denaturation at 95°C for 3 min; followed by denaturation at 95°C for 20 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s, for 35 cycles; and finally extension at 72°C for 8 min and storage at 20°C.

[0052] GUT2 downstream homology arm PCR amplification system: total volume 50 μL, including FastPfu PCRSuperMix 25μL, ddH2O 20μL, upstream primer GUT2dn-f (10μM) 1.5μL, downstream primer GUT2dn-r (10μM) 1.5μL, template is genomic DNA of Phaffia rhodozyma CBS 6938, the addition amount is 2μL, the sequences of primers in the amplification system are shown in Table 1.

[0053] The PCR amplification procedure for the downstream homology arm of GUT2 was as follows: pre-denaturation at 95°C for 3 min; followed by denaturation at 95°C for 20 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s, for 35 cycles; and finally extension at 72°C for 8 min and storage at 20°C.

[0054] Antibiotic (Zeocin) screening marker fragment PCR amplification system: total volume 50 μL, including FastPfu PCR SuperMix 25μL, ddH2O 20μL, upstream primer Backbone-GUT2-Zeocin-f (10μM) 1.5μL, downstream primer

[0055] Backbone-GUT2-Zeocin-r (10 μM) 1.5 μL, template is

[0056] pUG6-TG-Cbr.1-Zeocin-loxP plasmid, the addition amount is 2 μL, and the sequences of primers in the amplification system are shown in Table 1.

[0057] The PCR amplification procedure of the antibiotic selection marker fragment was as follows: pre-denaturation at 95°C for 3 min; followed by denaturation at 95°C for 20 s, annealing at 59°C for 20 s, extension at 72°C for 45 s, and 35 cycles; and finally extension at 72°C for 8 min and storage at 20°C.

[0058] Table 1. PCR amplification primers used in the present invention

[0059]

[0060] 1.3 Extraction of genomic DNA

[0061] The genomic DNA of Phaffia rhodozyma was extracted by snail enzyme lysis method. The specific operation method is as follows: the yeast cells were cultured to the early stationary phase (OD 600=2~6), centrifuge and collect 2mL yeast cells of bacterial solution, wash once with ddH2O, add 200μLLET buffer (500mM EDTA-10mM Tris) and 40μL β-mercaptoethanol, and treat at 30℃ for 30min. Collect the cells by centrifugation at 5000g for 5min, resuspend in 300μL SECP buffer (1M sorbitol-0.1M EDTA-0.01M citrate-phosphate buffer, pH=5.8) containing 20mg / mL snail enzyme and 5mg / mL lytic enzyme, and incubate at 37℃ for 1.5~2.5h to digest and remove the cell wall to obtain protoplasts. Collect the protoplasts by centrifugation, add 600μL 0.05M Tris-0.05M EDTA and 30μL 20% SDS and resuspend, and incubate at 60℃ for 30min to lyse the cells. After cooling to room temperature, add 240 μL of 5M potassium acetate solution and shake well, then place on ice for 1 hour. Centrifuge at 12000g for 5 minutes, collect the supernatant into a new EP tube, add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and mix vigorously. Centrifuge at 12000g for 10 minutes, carefully transfer the upper aqueous phase to a new EP tube, add an equal volume of chloroform-isoamyl alcohol (24:1) and repeat the above operation once. Add 1 / 10 volume of 3M sodium acetate solution (pH=6.2) and 0.54 volume of 4℃ precooled isopropanol to the upper aqueous phase transferred to the new EP tube, and place at -20℃ for 10 minutes to fully precipitate DNA. Centrifuge at 12000g for 10 minutes, carefully aspirate the supernatant, and wash the precipitate twice with 70% ethanol precooled at -20℃. Open the EP tube cap to dry, and add 50 μL of deionized water containing RNase to dissolve the DNA.

[0062] 1.4 Transformation of Phaffia rhodozyma

[0063] The constructed pUG6-TG-POX1-Zeocin-loxP plasmid was used as a template to amplify the DNA fragment for POX1 knockout using pPOX-f and pPOX-r; the DNA fragment for GUT2 knockout was amplified from the plasmid pUG6-TG-GUT2-Zeocin-loxP using primers pGUT2-f and pGUT2-r. The amplified product was purified and recovered by Gel Extraction Kit (Omega Bio-Tek, USA) after agarose gel electrophoresis. The amount of DNA fragment required for knocking out each gene was about 10 μg. The recovered DNA fragment was concentrated to about 10 μL by vacuum centrifugation for use. The primers used are shown in Table 1.

[0064] DNA fragment PCR amplification system for POX1 knockout: total volume 50 μL, including FastPfu PCRSuperMix 25μL, ddH2O 20μL, upstream primer pPOX-f (10μM) 1.5μL, downstream primer pPOX-r (10μM) 1.5μL, template is pUG6-TG-POX1-Zeocin-loxP plasmid, the addition amount is 2μL, the sequences of primers in the amplification system are shown in Table 1.

[0065] The PCR amplification procedure of DNA fragment for POX1 knockout was as follows: pre-denaturation at 95°C for 3 min; followed by denaturation at 95°C for 20 s, annealing at 53°C for 20 s, extension at 72°C for 1 min 20 s, and 35 cycles; finally, extension at 72°C for 8 min and storage at 20°C.

[0066] GUT2 knockout DNA fragment PCR amplification system: total volume 50 μL, including FastPfu PCRSuperMix 25μL, ddH2O 20μL, upstream primer pGUT2-f (10μM) 1.5μL, downstream primer pGUT2-r (10μM) 1.5μL, template is

[0067] pUG6-TG-GUT2-Zeocin-loxP plasmid, the addition amount is 2 μL, and the sequences of primers in the amplification system are shown in Table 1.

[0068] The PCR amplification procedure of the DNA fragment for GUT2 knockout was as follows: pre-denaturation at 95°C for 3 min; followed by denaturation at 95°C for 20 s, annealing at 53°C for 20 s, and extension at 72°C for 1 min 20 s, for 35 cycles; and finally extension at 72°C for 8 min and storage at 20°C.

[0069] Streak the red yeast strain frozen in a glycerol tube on a YPD plate, culture it at 22°C for 3 days, and then inoculate it into a 250mL shake flask (30mL YPD medium). After culturing at 22°C and 250rpm for 48h, take an appropriate amount of the bacterial solution and inoculate it into a 500mL shake flask (50mL YPD medium) to make the initial OD 600 =0.04, 22°C, 250rpm. When the yeast cells grow to OD 600=1.2~1.5 (about 16h), centrifuge at 5000g for 5min to collect yeast cells, resuspend in 20mL PPB buffer (50mM KH2PO4-K2HPO4, pH 7.0) containing 25mM DTT, and stand at room temperature for 25min. Centrifuge at 5000g for 5min at 4℃, discard the supernatant, wash twice with 25mL ice-cold sucrose buffer STM buffer (270mM sucrose, 10mM Tris-HCl, pH 7.5, and 1mM MgCl2), and finally resuspend the cells in 60μL STM buffer, which is the yeast competent cells. Mix 60μL yeast competent cells with the DNA samples obtained by the above amplification, and then transfer them to the electroporation cup for electroporation, and the electroporation parameters are 1000Ω, 800V, 50μF. After the electroporation, 500 μL of ice-cold YPD was added for re-suspending, and the culture was shaken at 22°C and 250 rpm for 2.5 h, then spread on a YPD plate containing 200 μg / mL Zeocin and cultured at 22°C for 72 h to allow the transformants to grow. The resulting POX1 or GUT2 knockout strains were named M1.3-Δpox1 and M1.3-Δgut2, respectively.

[0070] 1.5 Shake flask fermentation of Phaffia rhodozyma

[0071] The Phaffia rhodozyma strain (in this example, Phaffia rhodozyma strain M1.3 was used as the experimental material to knock out the E3 ubiquitin ligase using the Phaffia rhodozyma strain CBS 6938 (purchased from the CBS Culture Collection of the Netherlands); the E3 ubiquitin ligase is one or more of the genes encoding CDZ97817.1, CDZ97878.1, CDZ97925.1, CED83156.1, CED83716.1, and CED83815.1, and in this example, it is GenBank CED83815.1) was streaked on a YPD plate and cultured at 22°C for 3 days. A single colony was picked and inoculated into a 250 mL shake flask (30 mL YPD medium). After activation at 22°C and 220 rpm for 48 h, the bacterial solution was transferred to 30 mL LYM medium (250 mL Erlenmeyer flask) to an initial OD of 1.5. 600 Each strain was inoculated in parallel into at least 3 shake flasks for fermentation and cultured at 22°C and 220 rpm for 5 days.

[0072] 1.6 Determination of dry cell weight of Phaffia rhodozyma and extraction of carotenoids

[0073] Take 2mL of the shake flask fermentation liquid obtained by the above 1.5 treatment, centrifuge at 12000rpm for 5min to collect the bacteria, wash once with an equal volume of deionized water, dry at 60-70℃ for at least 48h, and determine the cell dry weight. Take another 2mL of fermentation liquid from each shake flask and collect the bacteria according to the above method, add 1mL DMSO and treat at 60℃ for 1h, centrifuge to take the supernatant, and treat the precipitate again in the same way. All DMSO containing carotenoids is transferred to a glass centrifuge tube, and 1mL acetone, 1mL 20% (w / v) NaCl and 1mL petroleum ether are added and shaken. Centrifuge at 4000rpm to take the upper layer to a 2mL centrifuge tube and vacuum centrifuge to dry. Use petroleum ether to continue to extract the residual carotenoids in the lower layer, extract multiple times until the pigment is no longer dissolved, vacuum centrifuge to dry after each extraction, and freeze at -80℃ after the last drying.

[0074] 1.7 Determination of carotenoids in Phaffia rhodozyma by HPLC and calculation of their content

[0075] Each of the dried carotenoids obtained above was dissolved in 1 mL of acetone, and insoluble impurities were removed by filtration with a filter membrane with a pore size of 0.22 μm. The mobile phase was acetonitrile: methanol: isopropanol = 85:10:5 (v:v:v), the flow rate was 1 mL / min, the chromatographic column was Agilent Poroshell 120EC-C18 column (4.6×150 mm, 4 μm), the column temperature was 25°C, the sample load was 10 μL, and the detection wavelength of astaxanthin was 474 nm, and the detection wavelength of β-carotene was 453 nm. First, a standard curve was drawn with astaxanthin and β-carotene standard solutions, and the external standard method was used for quantification according to the standard curve. The pigment content (i.e., titer) of the unit volume of the shake flask fermentation broth was divided by the cell dry weight of the corresponding volume of the fermentation broth to obtain the carotenoid content in the yeast cells.

[0076] 2. Results and Analysis

[0077] After shake flask fermentation, the cell dry weight, cell content and yield of β-carotene and astaxanthin of the control strain M1.3, the POX1 knockout strain M1.3-Δpox1 and the GUT2 knockout strain M1.3-Δgut2 were measured. The results showed that the cell dry weight decreased slightly after POX1 and GUT2 knockout, by 14.6% and 15.1% respectively ( Figure 1 ), but the cell content and production of astaxanthin were significantly increased ( Figure 2 and Figure 3 Among them, after POX1 and GUT2 were knocked out, astaxanthin production increased by more than 20% ( Figure 2After POX1 was knocked out, the astaxanthin content in the cells reached 1.40 mg / g DCW (cell dry weight), which was 32.1% higher than that in the original strain, and the content of β-carotene did not change significantly ( Figure 3 ). After GUT2 was knocked out, the β-carotene content was 0.21 mg / g DCW, which was 25.0% lower than that of the original strain, but the astaxanthin content reached 1.37 mg / g DCW, which was 29.2% higher than that of the original strain ( Figure 3 ). The above results indicate that POX1 and GUT2 knockouts specifically increase the content and yield of astaxanthin in Phaffia rhodozyma and can be used as a method to increase astaxanthin production.

[0078] >SEQ ID NO.1

[0079] POX1 gene sequence

[0080]

[0081] >SEQ ID NO.2

[0082] GUT2 gene sequence

[0083]

[0084] >SEQ ID NO3

[0085] Antibiotic gene sequence

[0086]

Claims

1. A method for increasing the astaxanthin production of Phaffia rhodozyma, characterized in that: The recombinant strain obtained by knocking out the genes encoding enzymes related to oil metabolism of Phaffia rhodozyma can improve the fermentation yield of astaxanthin from Phaffia rhodozyma.

2. The method for increasing the astaxanthin production of Phaffia rhodozyma according to claim 1, characterized in that: The gene sequence encoding Phaffia rhodozyma lipid metabolism-related enzyme CED83814.1 (POX1) is shown in SEQ ID NO.1; and / or, The gene sequence encoding the enzyme CDZ96393.1 (GUT2) related to lipid metabolism of Phaffia rhodozyma is shown in SEQ ID NO.

2.

3. The method for increasing the astaxanthin production of Phaffia rhodozyma according to claim 1 or 2, characterized in that: (1) Construction of homologous recombination plasmids: pUG6-TG-POX1-Zeocin-loxP and / or pUG6-TG-GUT2-Zeocin-loxP; (2) Using one or more of the homologous recombination plasmids obtained above as templates to amplify the knockout fragment, transform the fragment into Phaffia rhodozyma M1.3, perform gene homologous recombination knockout, and obtain a gene recombinant strain.

4. The method for increasing the astaxanthin production of Phaffia rhodozyma according to claim 3, characterized in that: The homologous recombination plasmid pUG6-TG-POX1-Zeocin-loxP construction method is to use pUG6-TG-Cbr.1-Zeocin-loxP plasmid as a template, use primers Backbone-POX-Zeocin-f and Backbone-POX-Zeocin-r to amplify the plasmid backbone, and use primers POX1-Zeocin-loxP-f and POX1-Zeocin-loxP-r to amplify the antibiotic screening marker fragment; use the genomic DNA of Phaffia rhodozyma CBS6938 as a template, use primers POXup-f and POXup-r to amplify the upstream homology arm of POX1, and use primers POXdn-f and POXdn-r to amplify the downstream homology arm of POX1; finally, assemble the above four DNA fragments by Gibson assembly method to obtain the target plasmid; The homologous recombination plasmid pUG6-TG-GUT2-Zeocin-loxP construction method is as follows: using pUG6-TG-Cbr.1-Zeocin-loxP plasmid as a template, using primers Backbone-GUT2-Zeocin-f and Backbone-GUT2-Zeocin-r to amplify the plasmid backbone, and using primers GUT2-Zeocin-loxP-f and GUT2-Zeocin-loxP-r to amplify the antibiotic screening marker fragment; using genomic DNA of Phaffia rhodozyma CBS 6938 as a template, using primers GUT2up-f and GUT2up-r to amplify the GUT2 upstream homology arm, and using primers GUT2dn-f and GUT2dn-r to amplify the GUT2 downstream homology arm; finally, the above four DNA fragments are assembled by Gibson assembly method to obtain the target plasmid.

5. A recombinant bacterium prepared by the method of claim 1, characterized in that: The method of claim 1 is used to obtain a recombinant Phaffia rhodozyma with inactivated lipid metabolism genes.

6. The use of the recombinant bacteria according to claim 5, characterized in that: The invention discloses an application of the recombinant Phaffia rhodozyma with inactivated oil metabolism gene in the production of astaxanthin.

7. A gene for an enzyme related to lipid metabolism for increasing the astaxanthin production of Phaffia rhodozyma, characterized in that: The gene sequence encoding Phaffia rhodozyma lipid metabolism-related enzyme CED83814.1 (POX1) is shown in SEQ ID NO.1; and / or The gene sequence encoding the enzyme CDZ96393.1 (GUT2) related to lipid metabolism of Phaffia rhodozyma is shown in SEQ ID NO.

2.

8. A homologous recombination vector capable of inactivating lipid metabolism genes, characterized in that: The homologous recombination vector is a recombinant vector containing the gene related to lipid metabolism of Phaffia rhodozyma as claimed in claim 7.

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