Astaxanthin synthase mutant and its application

Abstract

The present invention discloses an astaxanthin synthase mutant SEQ ID NO: 5, which has significantly improved ability to promote astaxanthin synthesis compared with the wild-type astaxanthin synthase SEQ ID NO: 3, and can be used for the transformation of astaxanthin-producing bacteria such as Phaffia rhodozyma.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular, relates to an astaxanthin synthase mutant and its use in improving the astaxanthin production capacity of microbial production bacteria, especially Phaffia rhodozyma. Background Art

[0002] Astaxanthin, whose chemical name is 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene, is a fat-soluble keto-carotenoid widely found in shrimp, crab, fish, bird, some algae and fungi. Astaxanthin has a strong antioxidant capacity, which is 10 times that of β-carotene (Antioxidant activities of astaxanthinand related carotenoids.Journal of Agricultural and Food Chemistry 2000,48(4):1150-1154). It can remove nitrogen dioxide, disulfide, etc., and can also effectively inhibit lipid peroxidation caused by free radicals. It has a variety of biological activities, including anti-tumor effect, anti-inflammatory effect and immunity enhancement effect (Antitumoractivity of astaxanthin and its mode of action.Nutrition and Cancer-aninternational journal 2000,36(1):59-65). In addition, astaxanthin also has potential application value in the treatment of cardiovascular diseases, nervous system diseases and diabetes. In recent years, the demand for astaxanthin in health products, medicines, cosmetics, food additives and aquaculture has continued to rise, and the global market size is expected to reach US$2.57 billion in 2025 (Metabolic engineering of Escherichia coli for high-level astaxanthin production with high productivity. Metabolic Engineering 2018, 49: 105-115).

[0003] Chemically synthesized astaxanthin is mainly used as a feed additive and cannot be used by humans. Natural astaxanthin can be used by humans, but the yield is low, far from meeting market demand, and it is expensive. Waste from crustacean aquatic products such as shrimp and crab is one of the sources of astaxanthin, but there are certain difficulties in collecting raw materials in large quantities. At present, the synthesis of astaxanthin by algae or yeast fermentation is a research hotspot at home and abroad, and has excellent development prospects.

[0004] Phaffia rhodozyma or Xanthophyllomyces dendrorhous is one of the natural hosts for the synthesis of carotenoids, of which astaxanthin accounts for more than 85%, and is considered to be an important cell factory for the industrial production of astaxanthin. However, the astaxanthin content in wild-type Phaffia rhodozyma is less than 1% of the cell dry weight, and modification is needed to increase astaxanthin production. Summary of the invention

[0005] In order to improve the astaxanthin production capacity of Phaffia rhodozyma, the inventors transferred the wild astaxanthin synthase gene from Phaffia rhodozyma into Phaffia rhodozyma, and obtained a mutant strain with improved astaxanthin production by using random mutation and high-throughput screening methods. The study found that the astaxanthin synthase had mutated. By transferring the mutant gene through a backfilling experiment, the astaxanthin production of the transformant was significantly improved, confirming that the mutant enzyme can promote the production of astaxanthin by Phaffia rhodozyma. Specifically, the present invention includes the following technical solutions.

[0006] The first aspect of the present invention provides an astaxanthin synthase, which is a polypeptide selected from the group consisting of:

[0007] (1) a polypeptide having an amino acid sequence of SEQ ID NO: 5;

[0008] (2) A polypeptide having 90% or more homology, preferably 95% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, and more preferably 99% or more homology to SEQ ID NO: 5, and having an increased enzyme activity compared to SEQ ID NO: 5.

[0009] (SEQ ID NO:5).

[0010] The second aspect of the present invention provides a polynucleotide, which is a gene encoding the above polypeptide.

[0011] Preferably, the gene encoding SEQ ID NO:5 is a polynucleotide shown in SEQ ID NO:4, or a polynucleotide having more than 90%, preferably more than 95%, preferably more than 96%, preferably more than 97%, preferably more than 98%, and more preferably more than 99% homology with SEQ ID NO:4.

[0012] The third aspect of the present invention provides the use of the above-mentioned polypeptide or the above-mentioned gene in promoting the production of astaxanthin by microorganisms.

[0013] Specifically, the microorganism can produce astaxanthin by fermentation. The microorganism is an astaxanthin-producing bacterium, such as Phaffia rhodozyma.

[0014] The fourth aspect of the present invention provides an astaxanthin-producing bacterium, which expresses the above-mentioned gene, and preferably contains the above-mentioned gene in its genome.

[0015] Preferably, the astaxanthin-producing bacteria is Phaffia rhodozyma.

[0016] In one embodiment, the above-mentioned microorganism is an engineered bacterium, that is, the original astaxanthin synthase encoding gene in the genome of the microbial host is replaced by the above-mentioned gene; or the above-mentioned gene is integrated into the genome of the microbial host.

[0017] The integration of the above-mentioned coding gene can be achieved by transforming a plasmid containing the gene into the microbial host bacteria, or directly cloning the gene into the host bacteria genome.

[0018] The above-mentioned genes are cloned into the genome of the microbial host bacteria by gene editing technology.

[0019] The above-mentioned gene editing technology can adopt homologous double exchange, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas related transposition system INTEGRATE system or CAST system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation).

[0020] The above-mentioned INTEGRATE system refers to the gene editing tool (Insertion of transposable elements by guide RNA-assisted targeting) developed by Sam Sternberg's research group; the CAST system refers to the gene editing tool (CRISPR-associated transposase) developed by Zhang Feng's research group.

[0021] Microorganisms with the above-mentioned astaxanthin synthase gene integrated into the genome, such as the genetically engineered strain of Phaffia rhodozyma, have the potential to be used in the industrial production of astaxanthin.

[0022] In one embodiment, when Phaffia rhodozyma is fermented to produce astaxanthin, the fermentation medium composition is: yeast extract powder 5g / L, malt extract powder 5g / L, peptone 5g / L, glucose 10g / L, tomato powder 20g / L, ammonium sulfate 3g / L, magnesium sulfate 2g / L, dipotassium hydrogen phosphate 1g / L, calcium chloride 0.5g / L, glycerol 1% (w / V), zinc sulfate 0.5mg / L, vitamin B6 100mg / L, vitamin B12 10mg / L, inositol 5mg / L, and defoaming agent 0.5‰.

[0023] Optionally, the feed medium is a 60% glucose solution (w / V).

[0024] The fermentation temperature may be 18-27°C, for example, about 21°C; the culture medium is controlled to have a pH of 5.0-6.5, for example, about pH 5.5-6.0.

[0025] It should be understood that when expressing numerical characteristics in this document, the term "approximately" or "around" means that the number represented may have an error range or floating range of ±10%, ±9%, ±8%, ±7%, ±6% or ±5%.

[0026] When Phaffia rhodozyma is fermented, a seed culture medium may be included. The seed culture medium consists of 2 g / L yeast extract powder, 3 g / L malt extract powder, 5 g / L peptone, and 2.5 g / L glucose. The pH value is adjusted to 6.0 using ammonia water or citric acid.

[0027] The astaxanthin synthase SEQ ID NO:5 screened by the present invention is a mutant of the wild-type astaxanthin synthase SEQ ID NO:3. Compared with the wild enzyme, the mutant can increase the astaxanthin synthesis level of the strain by about 5.5 times compared with the wild-type astaxanthin synthase, and can be used to promote the production of astaxanthin by Phaffia rhodozyma. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The map is of the plasmid pCambia1301-crtS constructed by the present invention for expressing the wild-type astaxanthin synthase SEQ ID NO: 3.

[0029] Figure 2 This is the HPLC spectrum for detecting astaxanthin produced by Phaffia rhodozyma. DETAILED DESCRIPTION

[0030] The activity of astaxanthin synthase is directly related to the astaxanthin production of Phaffia rhodozyma, so one of the focuses of the present invention is to improve the activity of the original astaxanthin synthase itself. The astaxanthin synthase SEQ ID NO:5 obtained by random mutation and high-throughput screening is a mutant in which five amino acids are replaced in the wild-type astaxanthin synthase SEQ ID NO:3 (crtS, NCBI accession number KR779667.1) from Phaffia rhodozyma. These five mutation sites are glutamine at position 143 mutated to lysine (Q143K), glutamic acid at position 368 mutated to lysine (E368K), alanine at position 459 mutated to glutamic acid (A459E), leucine at position 509 mutated to serine (L509S), and histidine at position 524 mutated to proline (H524P).

[0031] In this article, the terms "wild (type)", "wild enzyme", and "wild-type enzyme" have the same meaning, all referring to the wild-type astaxanthin synthase SEQ ID NO: 3:

[0032] (SEQ ID NO:3).

[0033] Similarly, the terms "astaxanthin synthase mutant", "mutant astaxanthin synthase" and "mutant enzyme" mean the same thing and refer to the astaxanthin synthase SEQ ID NO:5.

[0034] Sometimes for the sake of convenience, the wild enzyme SEQ ID NO: 3 and its mutant SEQ ID NO: 5 may be collectively referred to as "astaxanthin synthase" herein.

[0035] After the astaxanthin synthase mutant SEQ ID NO: 5 is overexpressed in astaxanthin-producing Phaffia rhodozyma, the astaxanthin production capacity of Phaffia rhodozyma is increased by 5.5 times.

[0036] It can be expected that the astaxanthin synthase mutant SEQ ID NO: 5 can also be used in other astaxanthin-producing strains to improve the astaxanthin biosynthesis level of the engineered bacteria.

[0037] It is easy to understand that in order to express exogenous astaxanthin synthase in different host bacteria, the expression gene of the enzyme can be codon optimized. Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms usually show a special preference for one of some codons encoding the same amino acid due to mutation tendency and natural selection. For example, in fast-growing microorganisms such as Escherichia coli, the optimized codons reflect the composition of their respective genomic tRNA libraries. Therefore, in fast-growing microorganisms, low-frequency codons of amino acids can be replaced with high-frequency codons for the same amino acid. Therefore, the expression of the optimized DNA sequence is improved in fast-growing microorganisms.

[0038] For example, to express astaxanthin synthase in Phaffia rhodozyma, the gene encoding the codon-optimized wild astaxanthin synthase SEQ ID NO:3 can be SEQ ID NO:2; and the gene encoding the astaxanthin synthase mutant SEQ ID NO:5 can be SEQ ID NO:4.

[0039] The above gene sequences SEQ ID NO:2 and SEQ ID NO:4 contain a certain number of introns respectively. Alternatively, those skilled in the art can design a coding gene or expression cassette that does not include introns in the nucleotide sequence using computer software based on the amino acid sequence of the wild astaxanthin synthase with a clear structure SEQ ID NO:3 and the amino acid sequence of the astaxanthin synthase mutant SEQ ID NO:5. For different host microbial species, there can be codon-optimized coding gene sequences.

[0040] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0041] Example

[0042] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0043] Materials and methods

[0044] The whole gene synthesis, primer synthesis and sequencing in the examples were all completed by Suzhou Genewise Biotechnology Co., Ltd.

[0045] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0046] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0047] LB medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, pH 7.2. (LB solid medium plus 20g / L agar powder.)

[0048] YPD medium: peptone 20g / L, yeast extract 10g / L, glucose 5g / L, pH 6.0. (Add 20g / L agar powder to YPD solid medium.)

[0049] Astaxanthin extraction method:

[0050] Take 0.5-2mL of Phaffia rhodozyma fermentation broth and add it to a 50mL centrifuge tube. Centrifuge at 12,000rpm for 5min, discard the supernatant to obtain wet yeast mud, and place it in an oven at 80℃ for 15h to dry the water. Accurately weigh the bacteria, add 5mlDMSO to extract carotenoids in the bacteria, use a vortex mixer to fully mix, and place the centrifuge tube in a constant temperature water bath. The extraction time is 20min and the extraction temperature is 40℃. After the extraction is completed, centrifuge at 12,000rpm for 5min and collect the supernatant. Add 5mL DMSO to the extraction residue again, vortex and mix, and place it in a water bath for the second extraction. The extraction time is 20min and the extraction temperature is 40℃. After the extraction is completed, vortex and mix, centrifuge at 12,000rpm for 5min, and collect the supernatant. Mix the two extraction supernatants, centrifuge at 12,000rpm for 5min, remove the residual solid impurities and set aside.

[0051] Astaxanthin content detection method:

[0052] HPLC detection conditions: YMC Carotenoid C30 reverse phase chromatographic column (carotenoid analysis column); mobile phase A is a methanol-water solution containing 90% volume fraction, and mobile phase B is methyl tert-butyl ether; mobile phase A: mobile phase B = 9:1 for baseline; after injection, from 0 to 15 minutes, mobile phase A: mobile phase B linearly changes from 9:1 to 2:8, and from 15 to 30 minutes, mobile phase A: mobile phase B linearly changes from 2:8 to 9:1; flow rate 0.5 mL / min; detection wavelength is 450 nm.

[0053] Figure 2 The HPLC profile of detected astaxanthin is shown.

[0054] Example 1: Construction of a plasmid expressing a wild astaxanthin synthase gene

[0055] The wild astaxanthin synthase gene (crtS, 961-4126 bp of NCBI accession number KR779667.1) derived from Phaffia rhodozyma is regulated by the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd), wherein the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase promoter is SEQ ID NO: 1, and the nucleotide sequence of the wild astaxanthin synthase gene is SEQ ID NO: 2 (including introns). Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize and cloned into the EcoRI and BamHI sites of plasmid pUC57 to obtain plasmid pUC-crtS, and the corresponding amino acid sequence of wild astaxanthin synthase is SEQ ID NO: 3.

[0056] Using plasmid pUC-crtS as a template, PCR technology was used to amplify the DNA fragment CRTS containing the gpd promoter-crtS gene. The following primer pairs were designed:

[0057] Forward primer CRTS-5:

[0058] 5'-CAGCTATGACCATGATTACCAAGTCGAGGGAACCCGAG-3',

[0059] Reverse primer CRTS-3:

[0060] 5'-GTAAAACGACGGCCAGTGCCTACCATGGTCAAGAGTCCCC-3'.

[0061] Using plasmid pCambia1301 as a template, PCR amplification was performed to obtain the pCambia1301 plasmid backbone. The following primer pairs were designed:

[0062] Cambia-5:5'-GGCACTGGCCGTCGTTTTAC-3',

[0063] Cambia-3: 5'-GTAATCATGGTCATAGCTGTTTC-3'.

[0064] Using Takara Biotechnology (Beijing) Co., Ltd. (Takara China) PCR amplification was performed using the GXL DNA polymerase kit. Reaction system: 10 μL of 5× Buffer, 10 mM dNTP Mixture, 1 μL each of forward primer and reverse primer, 0.5 μL of template, 2 μL of DNA polymerase, and ddH2O was added to make up to 50 μL.

[0065] PCR amplification conditions: denaturation at 95°C for 5 min; (denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1.2 min) for a total of 10 cycles; (denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1.2 min) for a total of 20 cycles; extension at 72°C for 7 min.

[0066] Directly add 1 μL of restriction endonuclease DpnI to digest the methylated template in the PCR reaction system. Perform electrophoresis on the PCR product and recover it on gel (Axygen DNA gel recovery kit AP-GX-50). Use the one-step cloning kit of Nanjing Novozyme Biotechnology Co., Ltd. to recombinantly form a circle of two homologous sequences of the linearized DNA (CRTS fragment and pCambia1301 plasmid backbone) to construct the plasmid pCambia1301-crtS expressing wild astaxanthin synthase. Figure 1 shown.

[0067] Example 2: Construction of a plasmid expressing wild astaxanthin synthase

[0068] Preparation of Phaffia rhodozyma competent state: Pick a single colony of Phaffia rhodozyma strain CBS 6938 (purchased from CBS (Centrares) Netherlands Microbial Culture Collection Center) in 20mL liquid YPD medium, incubate at 21°C, 180rpm for 48h, then take 5mL of bacterial solution and inoculate it into 100ml YPD liquid medium, and culture at 21°C, 180rpm until OD600 is about 1.2. Then centrifuge at 4°C, 4000rpm for 10min to collect the cells, resuspend them with 40mL potassium phosphate buffer (50mM, pH 7.0) containing 25mM dithiothreitol, and heat treat them at 25°C for 15min. Wash the cells twice with 40mL ice-cold sucrose buffer STM (270mM sucrose, 10mM Tris-HCl, 1mM MgCl2, pH7.5), and finally resuspend the cells with 0.1mL STM buffer and place them in a -80°C refrigerator for use.

[0069] The plasmid pCambia1301-crtS constructed in Example 1 was transformed into the rhodozyma strain CBS 6938. The specific operation steps were as follows: 5 μL of the plasmid pCambia1301-crtS was mixed with 0.1 mL of the competent cell suspension and placed in an ice-cold electroporation cup. The electroporation instrument parameters were adjusted to 1000Ω, 800V, 25 μF, and 0.5 mL of ice-cold YPD culture medium was quickly added after the electroporation. After incubation at 21°C for 3 hours, the culture was spread on a YPD plate containing antibiotics and incubated at 21°C for 48 hours.

[0070] The strain CBS-1 expressing wild astaxanthin synthase SEQ ID NO: 3 was obtained by the above transformation method.

[0071] Example 3: Construction of random mutation point library by error-prone PCR from round 1 to round 3

[0072] The random mutant library was constructed using error-prone PCR technology with plasmid pUC-crtS as template.

[0073] The following primer pair TB-5 / TB-3 was designed:

[0074] Forward primer TB-5: 5'-ATGTTCATCTTGGTCTTGCTCACAG-3',

[0075] Reverse primer TB-3: 5′-TCATTCGACCGGCTTGACCTGCAAA-3′.

[0076] Using plasmid pUC-crtS as a template, PCR amplification was performed to obtain an astaxanthin synthase mutant DNA sequence of approximately 3.2 kb.

[0077] The 50 μL error-prone PCR reaction system included: 10 ng plasmid (pUC-crtS) template, 50 pmol of a pair of primers TB-5 and TB-3, 1× Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM) MnCl2, and 2.5 units of Taq enzyme (Takara).

[0078] The PCR reaction conditions were: 95°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min / kbp, 30 cycles; 72°C for 10 min.

[0079] The PCR products were electrophoresed and recovered by gel (Axygen DNA gel recovery kit AP-GX-50). Using plasmid pCambia1301-crtS as template and the recovered product of about 3.2 kb (random mutation fragment) as large primer, MegaPrimer PCR was performed with KOD-plus DNA polymerase: 94℃5min; 98℃10s, 60℃30s, 68℃2min / kb, 25 cycles; 68℃10min. The plasmid template was digested with DpnI restriction endonuclease (Thermo Company), electroporated into Phaffia rhodozyma strain CBS 6938, and plated on YPD solid plates (ionone was added to the culture medium at a final concentration of 100 mg / L to reduce the background astaxanthin synthesis level), and a random mutation library of more than 2500 clones was obtained.

[0080] The astaxanthin content of each strain in the random mutation library was detected, and the strain with significantly improved astaxanthin content was selected to extract the plasmid, and Suzhou Jinweizhi Company was commissioned to perform nucleic acid sequencing, and the astaxanthin synthase-related fragments were compared with SEQ ID NO: 2 to determine the amino acid mutation site of astaxanthin synthase. The astaxanthin synthase mutant corresponding to the strain with the highest astaxanthin content was selected as the template for the next round of random mutant library construction, and three rounds were performed.

[0081] Example 4: High-throughput reaction screening of random mutation point libraries from round 1 to round 3

[0082] Pick a single colony with a reddish color and vigorous growth to a 96-well deep-well plate (each well contains 400 μL of liquid YPD medium, and the concentration of G418 antibiotics in the medium is 100 μg / mL). After incubation at 21°C and 400 rpm for 48 hours, take out 80 μL of bacterial solution from each well to a 96-well deep-well plate (each well contains 320 μL of liquid YPD medium, the medium contains 20 g / L tomato powder, and the concentration of G418 antibiotics is 100 μg / mL), and incubate at 21°C and 400 rpm for 48 hours. Centrifuge at 4°C and 4000 rpm for 10 minutes to collect the bacteria, and remove the supernatant culture solution. Subsequently, wash the bacteria with pre-cooled saline, centrifuge at 4°C and 4000 rpm for 10 minutes to collect the bacteria, and remove the supernatant. Add 300 μL of DMSO to each well to extract carotenoids from the bacteria, the extraction time is 20 minutes, and the extraction temperature is 40°C. After the extraction, centrifuge at 12,000 rpm for 5 minutes and collect the supernatant. Add 300 μL of DMSO to the extraction residue in each well for a second extraction, with an extraction time of 20 minutes and an extraction temperature of 40°C. After the extraction, centrifuge at 12,000 rpm for 5 minutes and collect the supernatant. Mix the two extraction supernatants, centrifuge at 12,000 rpm for 5 minutes, remove the residual solid impurities and set aside. See Table 1 for the screening results.

[0083] Table 1. Screening results of mutant strains and astaxanthin synthase mutants

[0084] Note: "+" represents that the astaxanthin content is greater than 0% and less than or equal to 50% relative to the respective starting strain; "++" represents that the astaxanthin content is greater than 50% and less than or equal to 100% relative to the respective starting strain; "+++" represents that the astaxanthin content is greater than 100% and less than or equal to 150% relative to the respective starting strain; "++++" represents that the astaxanthin content is greater than 150% relative to the respective starting strain.

[0085] After three rounds of screening, the astaxanthin production of the mutant strain CBS-13 expressing the mutant crtS-13 was significantly improved compared to the strain CBS-1 expressing the wild astaxanthin synthase SEQ ID NO: 3. Gene sequencing confirmed that the nucleotide sequence of the mutant crtS-13 gene was SEQ ID NO: 4, and the corresponding amino acid sequence was SEQ ID NO: 5, which had (Q143K, E368K, A459E, L509S, H524P) mutations relative to the wild astaxanthin synthase crtS.

[0086] Example 5: Complementation experiment to verify the function of mutant crtS-13

[0087] The plasmid pCambia1301-crtS-13 expressing the mutant crtS-13 gene was constructed according to the method of Example 1, except that the nucleotide sequence SEQ ID NO:2 was replaced by the nucleotide sequence SEQ ID NO:4.

[0088] According to the method of Example 2, the plasmid pCambia1301-crtS-13 was transformed into Phaffia rhodozyma strain CBS 6938 to construct a genetically engineered Phaffia rhodozyma strain, numbered SHBL0413.

[0089] The preserved Phaffia rhodozyma SHBL0413 glycerol bacteria were spread on solid YPD medium plates and incubated upside down at 21°C for 48 hours. A single colony was picked and transferred to 500 mL of seed medium for culture. The liquid volume was 100 mL and incubated at 21°C and 180 rpm for 48 hours until the OD 600nmThe value reaches 6 or above. Two bottles of seed liquid were inoculated into a 5L bioreactor containing fermentation medium at a volume ratio of 10% v / v. The volume of fermentation medium was 1.8L, the pH value was controlled to be 5.5 (pH value was controlled by ammonia and citric acid), the temperature was 21°C, the glucose concentration was 5-10g / L, and the DO was above 50%. After 168h of fermentation, 2mL of fermentation liquid was sampled and added to a 50mL centrifuge tube, centrifuged at 12,000rpm for 5min, the supernatant was discarded to obtain wet yeast mud, and placed in an oven at 80°C for 15h to dry the water. Accurately weigh the cell weight, add 5mL DMSO to extract the carotenoids in the cell, use a vortex mixer to fully mix, and place the centrifuge tube in a constant temperature water bath. The extraction time was 20min and the extraction temperature was 40°C. After the extraction, centrifuge at 12,000rpm for 5min and collect the supernatant. 5 mL of DMSO was added to the extraction residue again, and the mixture was vortexed and placed in a water bath for a second extraction. The extraction time was 20 min and the extraction temperature was 40°C. After vortexing, the mixture was centrifuged at 12,000 rpm for 5 min and the supernatant was collected. The two extraction supernatants were mixed, centrifuged at 12,000 rpm for 5 min, and the residual solid impurities were removed and HPLC analysis was performed. The results showed that the astaxanthin content of strain SHBL0413 accounted for 0.437% of the cell dry weight (e.g. Figure 2 As shown in Figure 3 , while under the same fermentation conditions, astaxanthin in strain CBS-1 accounted for only 0.079% of the cell dry weight.

[0090] The experimental results show that the astaxanthin synthase mutant SEQ ID NO: 5 obtained by screening the present invention effectively promotes the production of astaxanthin by Phaffia rhodozyma, can be used for the genetic modification of astaxanthin-producing bacteria, and has development and application prospects.

Claims

1. An astaxanthin synthase, It is characterized in that The amino acid sequence is SEQ ID NO:

5.

2. A gene encoding the astaxanthin synthase according to claim 1.

3. The gene according to claim 2, It is characterized in that The nucleotide sequence of the gene is SEQ ID NO:

4.

4. Use of the astaxanthin synthase according to claim 1 or the gene according to any one of claims 2 to 3 in promoting the production of astaxanthin by Phaffia rhodozyma.

5. An astaxanthin-producing bacterium, It is characterized in that The genome of the astaxanthin-producing bacterium comprises the gene according to claim 2 or 3, and the astaxanthin-producing bacterium is Phaffia rhodozyma.

6. The astaxanthin-producing bacteria according to claim 5, It is characterized in that The original astaxanthin synthase encoding gene in the Phaffia rhodozyma host genome is replaced by the gene according to claim 2 or 3; or the gene according to claim 2 or 3 is integrated into the Phaffia rhodozyma host genome.

7. The astaxanthin-producing bacteria according to claim 5, It is characterized in that The integration of the gene refers to the process of transforming a plasmid containing the gene into a Phaffia rhodozyma host bacterium, or directly cloning the gene into the host bacterium genome.

8. Use of the astaxanthin-producing bacteria according to any one of claims 5 to 7 in producing astaxanthin.

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