High-yield astaxanthin vibrio natriureticus engineering bacterium and construction method thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an engineering bacterium capable of producing astaxanthin at high yield, a construction method and application of the engineering bacterium. According to the invention, a homologous recombination technology is utilized to construct a plasmid pVJtfoxampsc101 for expressing a tfox gene, the plasmid pVJtfoxampsc101 is converted into vibrio natriephalus ATCC14048, and then a plurality of optimized gene expression cassettes on natural astaxanthin synthesis routes are inserted into a vibrio natriephalus genome through the homologous recombination technology, so that a Vn92-ast engineering strain is successfully constructed, and the production of astaxanthin by heterologous fermentation is realized. The embodiment of the invention shows that the yield of the cultured and produced astaxanthin is high when the Vn92-ast strain is fermented by a feed supplement process, and the Vn92-ast strain has industrial production potential.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an engineering bacterium of Vibrio natrii with high astaxanthin production and a construction method thereof. Background Art

[0002] Astaxanthin (3,3'-dihydroxy-β,β'-carotene-4,4'-dione) is a ketocarotene with a molecular formula of C 40 H 52 O4, composed of 8 isoprene units. Astaxanthin is the strongest antioxidant discovered so far, with an antioxidant capacity 550 times that of vitamin E and 10 times that of β-carotene. It is widely used in pharmaceutical raw materials, cosmetic ingredients and colorant products. The global astaxanthin market is expected to reach approximately US$3.4 billion in 2027, with a compound annual growth rate of around 16.2%. Astaxanthin is an antioxidant with high application demand.

[0003] At present, there are three ways to produce astaxanthin: chemical synthesis, extraction, and biological fermentation. The chemical synthesis method produces a mixture of left-handed and right-handed isomers, which is complex and polluting. The extraction method uses physical and chemical methods to extract from aquatic waste, with low yields and also has the problem of large pollution. The biological fermentation method uses red Phaffia yeast or Haematococcus pluvialis to ferment and produce astaxanthin. The advantages are biological safety and low pollution. The disadvantages are that the astaxanthin produced by red Phaffia yeast is right-handed and the cultivation time takes about one week. Although Haematococcus pluvialis produces left-handed astaxanthin, the cultivation time is longer, usually more than 2 weeks.

[0004] With the continuous development of microbial expression systems, people often use prokaryotes to express heterologous genes to improve production technology. However, this method still has disadvantages. For example, when Escherichia coli is used as the host, whether it is electroporation or chemical transformation, the host cells need to be pre-treated, which takes a lot of time.

[0005] Therefore, there is an urgent need to use a strain that can quickly construct and express heterologous genes to improve astaxanthin production. Summary of the invention

[0006] The purpose of the present invention is to provide an engineered Vibrio natrii with high astaxanthin production, which can increase astaxanthin production and reduce the cost of industrial production of astaxanthin.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides an engineered bacterium with high astaxanthin production, wherein a gene expression cassette on a natural astaxanthin synthesis pathway is integrated into the genome of the engineered bacterium;

[0009] The gene expression cassette on the natural astaxanthin synthesis pathway includes an astaxanthin precursor synthesis pathway expression cassette sequence, an isopentenyl pyrophosphate isomerase sequence and a gene expression cassette sequence for synthesizing astaxanthin, and its nucleotide sequence is shown in SEQ ID NO8-10.

[0010] Preferably, the astaxanthin precursor synthesis pathway expression cassette sequence includes β-caratene synthase crtY, crtB, crtI, crtE gene clusters; the promoter is the Vibrio natriuresis constitutive promoter, and the terminator is BBa_B0010.

[0011] Preferably, the isopentenyl pyrophosphate isomerase sequence includes isopentenyl pyrophosphate isomerase, the promoter is a constitutive promoter of Vibrio natriuresis, and the terminator is BBa_B0010; the gene expression cassette sequence for synthesizing astaxanthin includes CrtW and CrtZ, the promoter is a constitutive promoter of Vibrio natriuresis, and the terminator is BBa_B0010.

[0012] Preferably, the engineered bacteria are obtained by introducing a recombinant plasmid into a host bacterium and inserting an astaxanthin precursor synthesis pathway expression cassette sequence, an isopentenyl pyrophosphate isomerase sequence and a gene expression cassette sequence for synthesizing astaxanthin into the genome of Vibrio natriuresis, and the recombinant plasmid is constructed by seamlessly cloning and connecting the coding genes of the tfox recombinant protein gene, the ampicillin resistance gene and the temperature-sensitive replicon SC101 after overlapping extension PCR.

[0013] More preferably, the host bacteria is Vibrio natriuresis ATCC14048.

[0014] The present invention also provides a process for constructing a high-yield astaxanthin engineering bacterium, comprising transforming a recombinant plasmid heterologously expressing tfox protein into a host cell, and then inserting the gene expression box on the above-mentioned natural astaxanthin synthesis pathway into the host bacteria genome to construct a high-yield astaxanthin engineering bacterium.

[0015] Preferably, the construction of the recombinant plasmid for heterologously expressing the tfox protein comprises the following steps:

[0016] S1. Amplify the AmpR sequence using plasmid pKD46 as a template to obtain the product AmpR-a; amplify the temperature-sensitive promoter SC101 sequence using plasmid pKD46 as a template to obtain the product SC101-a;

[0017] S2, artificially synthesize tfox sequence (Organism: Vibrio cholerae (strain: RFB16), Locus tag: F0316_RS08345), use the synthesized tfox sequence as a template for amplification to obtain product tfox-a, and use the tfox-a sequence as a template for amplification to obtain product tfox-b;

[0018] S3, using AmpR-a and SC101-a as templates for amplification to obtain the product AmpR-SC101;

[0019] S4. One-step cloning method was used to construct a recombinant plasmid of AmpR-SC101 and tfox-b.

[0020] Preferably, the engineered bacteria is Vibrio natriuresis; in the process of constructing the Vibrio natriuresis engineered bacteria, 500 bp upstream and downstream of the dns, wbfF, and pta genes of Vibrio natriuresis are used as homology arms, and the astaxanthin precursor synthesis pathway expression cassette sequence, the isopentenyl pyrophosphate isomerase sequence, and the gene expression cassette sequence for synthesizing astaxanthin are inserted into the Vibrio natriuresis genome.

[0021] The present invention also provides application of the above-mentioned engineering bacteria or the above-mentioned construction process in high-yield astaxanthin.

[0022] Preferably, the application process of high-yield astaxanthin also includes fermenting the engineered bacteria by a fed-batch process to culture and produce astaxanthin.

[0023] Beneficial effects of the present invention:

[0024] 1. The present invention uses Vibrio natrii as a fermentation base, designs a new metabolic pathway combination strategy, and achieves the purpose of rapidly fermenting Vibrio natrii to produce astaxanthin by introducing the synthetic genes of β-carotene and L-astaxanthin.

[0025] 2. The present invention uses homologous recombination technology to transfer multiple optimized gene expression cassettes on the natural astaxanthin synthesis pathway into the genome of Vibrio natriuresis, successfully constructs the Vn92-ast engineering strain, and realizes the heterologous fermentation production of astaxanthin.

[0026] 3. The present invention ferments the Vn92-ast strain through a fed-batch process, and the maximum astaxanthin yield generated after 68 hours of culture is 732 mg / L, which fully demonstrates that the strain constructed by the present invention increases the astaxanthin yield and reduces the cost of industrial production of astaxanthin. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0028] Figure 1 For the constructed plasmid map;

[0029] Figure 2This is a bacterial image of the engineered strain of Vibrio natrii under a 1600X microscope;

[0030] Figure 3 For the production of astaxanthin;

[0031] Figure 4 This is the liquid chromatography detection spectrum of astaxanthin. DETAILED DESCRIPTION

[0032] The present invention uses Vibrio natriuresis as a fermentation chassis, designs a new metabolic pathway combination strategy, and utilizes the designed plasmid to enable Vibrio natriuresis to carry out natural transformation under the condition of heterologous expression of tfox protein. By introducing the synthetic genes of β-carotene and L-astaxanthin, the purpose of rapidly fermenting L-astaxanthin in Vibrio natriuresis is achieved.

[0033] The engineered bacteria constructed by the present invention are inserted with an astaxanthin precursor synthesis pathway expression cassette sequence, isopentenyl pyrophosphate isomerase and a gene expression cassette sequence for synthesizing astaxanthin, wherein isopentenyl pyrophosphate isomerase (idi) can catalyze the isomerization reaction of isopentenyl pyrophosphate (IPP) and convert it into dimethylacrylic acid (DMAPP), which is an important reaction step for the organism to synthesize the isoprenoid skeleton. The isoprenoid skeleton is the structural basis of astaxanthin, and astaxanthin is composed of 8 isoprene units. Therefore, isopentenyl pyrophosphate isomerase plays a vital role in high-yield astaxanthin.

[0034] The recombinant plasmid (pVJ_tfox_amp_sc101) of the present invention is constructed by seamlessly cloning and connecting the plasmid pKD46 as a template, combining the ampicillin resistance gene (AmpR), the temperature-sensitive replicon (pSC101), the IPTG-inducible promoter Ptac and the tfox natural transformation-inducing gene through overlapping extension PCR.

[0035] Gene splicing by overlap extension PCR (SOE PCR) is a technique that uses primers with complementary ends to form overlapping chains in PCR products, so that in the subsequent amplification reaction, the amplified fragments from different sources are overlapped and spliced ​​together through the extension of the overlapping chains.

[0036] The plasmid pVJ_tfox_amp_sc101 was transformed into competent Vibrio natrii, and positive strains were screened on LBv2 resistance plates containing ampicillin. Then, 500 bp of the upstream and downstream of the dns, wbfF, and pta genes of Vibrio natrii were used as homologous arms, and the astaxanthin precursor synthesis pathway expression cassette sequence (SEQ ID NO.8) from Vibrio campbellii, the sequence containing ipp / dmapp isomerase (idi) and promoter and terminator (SEQ ID NO.9) and the gene expression cassette sequence (SEQ ID NO.10) for synthesizing astaxanthin from Haematococcus pluvialis were inserted into the genome of Vibrio natrii by homologous recombination to construct the Vn92-ast strain. A single colony was picked from the constructed Vn92-ast strain, inoculated into a liquid LBv2 medium, and cultured and fermented.

[0037] The expression cassette sequence of the astaxanthin precursor synthesis pathway includes the β-caratene synthase crtY, crtB, crtI, and crtE gene clusters; ipp / dmapp isomerase (idi); and the gene expression cassette sequence for synthesizing astaxanthin includes CrtW and CrtZ.

[0038] The fermented bacterial liquid was detected by high performance liquid chromatography with a C18 column. The astaxanthin concentration in the fermented liquid after 68 hours of fermentation was 732 mg / ml.

[0039] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0041] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0042] Example 1 Construction of plasmid pVJ_tfox_amp_sc101

[0043] Using primers primer 1 and primer 2, the AmpR sequence was amplified using plasmid pKD46 as a template to obtain the product AmpR-a; using primers primer 3 and primer 4, the thermosensitive promoter SC101 sequence was amplified using plasmid pKD46 as a template to obtain the product SC101-a; the tfox sequence was artificially synthesized; using primers primer 5 and primer 6, the synthesized tfox sequence was amplified using the template to obtain the product tfox-a; using primers primer 5 and primer 7, the tfox-a sequence was amplified using the template to obtain the product tfox-b.

[0044] Table 1 Primer sequences used

[0045]

[0046]

[0047] Primers primer 1 and primer 4 were used to amplify AmpR-a and SC101-a as templates to obtain the product AmpR-SC101.

[0048] A one-step cloning method was used to make AmpR-SC101 and tfox-b form a circular plasmid, numbered pVJ_tfox_amp_sc101, and the nucleotide sequence is shown in the sequence table SEQ ID NO.11. Figure 1 As shown, the pVJ_tfox_amp_sc101 recombinant plasmid was successfully constructed.

[0049] Example 2 Construction of Vibrio natriuresis containing pVJ_tfox_amp_sc101 plasmid

[0050] Pick a single colony of Vibrio natrii, inoculate it into 20mL LBv2 liquid medium, culture overnight (16-18h), transform the plasmid pVJ_tfox_amp_sc101 into competent Vibrio natrii (ATCC 14048), apply it to LBv2 resistance plate with 100μg / mL ampicillin, and culture overnight. The LBv2 formula is: tryptone 10g / L, yeast powder 5g / L, sodium chloride 15g / L, magnesium chloride 2.2g / L, potassium chloride 0.31g / L.

[0051] Ten colonies were randomly selected from the resistance plate as positive clones and used as test strains for the next step.

[0052] Example 3 Construction and cultivation of Vn92-ast strain

[0053] Using 500 bp upstream and downstream of the dns, wbfF, and pta genes of Vibrio natrii as homology arms, the astaxanthin precursor synthesis pathway expression cassette sequence from Vibrio campbellii (SEQ ID NO.8), a sequence containing ipp / dmapp isomerase idi and a promoter and terminator (SEQ ID NO.9), and a gene expression cassette sequence for synthesizing astaxanthin from Haematococcus pluvialis (SEQ ID NO.10) were inserted into the genome of Vibrio natrii by homologous recombination. The bacterial cell was observed under a 1600X microscope. Figure 2 shown.

[0054] Pick a single colony of the constructed Vn92-ast strain and inoculate it into 50 mL of liquid LBv2 medium. Cultivate it at 30°C and 220 rpm. Take samples every hour to measure OD. 600 , wait for OD 600 When the OD reaches about 30, feed. Continue to measure OD 600 , wait for OD 600 When there is no longer a significant increase, the fermentation is stopped and the astaxanthin fermentation liquid is Figure 3 shown.

[0055] The color of astaxanthin fermentation liquid is dark red. This is because there are a large number of double bonds and heteroatoms in the molecular structure of astaxanthin, which is easy to undergo oxidation reaction with oxygen in the air, thus making its color darker.

[0056] Example 4 Detection of astaxanthin concentration in fermentation broth

[0057] The fermented bacterial solution was homogenized at 900 bar and the cell wall was checked under a microscope until more than 95% of the cells were broken. The precipitate was collected after centrifugation at 3000 rpm for 10 min and anhydrous ethanol was added at a ratio of 1:1 (v / w).

[0058] Centrifuge at 3000 rpm for 10 min and collect the supernatant into a separate container.

[0059] Repeat the above extraction operation on the precipitate until the supernatant has no obvious red color. Combine the supernatants.

[0060] A liquid chromatograph with a C18 column and a 476 nm detector was used. The mobile phase was methanol: water = 9:1, the flow rate was 0.8 mL / min, the column temperature was 30°C, and the injection volume was in accordance with the requirements of the chromatograph.

[0061] Table 2 Chromatographic instrument detection process

[0062] Time / min A / ％ B / ％ 0～4 5 95 4～7 10 90 7～23 15 85 23～32 18 82 32～45 25 75 45～60 5 95

[0063] Preparation of standard curve. Accurately weigh 10 mg of astaxanthin and place it in a 100 mL brown volumetric flask; first dissolve it in anhydrous ethanol, then dilute it to the scale with acetonitrile, shake it upside down several times to obtain a 0.1 mg / mL mother solution standard. The mother solution is stored in a dark place and at low temperature, and is valid for one week. Use mobile phase to dilute the mother solution to prepare 0.001, 0.002, 0.004, 0.006, 0.008, and 0.01 mg / mL standard solutions, take 10 μL respectively and detect at a wavelength of 476 nm, use peak area as the ordinate and standard concentration as the abscissa, perform linear regression analysis, and obtain the relationship between peak area and astaxanthin concentration as shown below Figure 4 shown.

[0064] The astaxanthin concentration in the fermentation broth was calculated according to the standard curve to be 732 mg / mL, indicating that the engineered Vibrio natriureticus bacteria constructed in the present application were successfully cultured and produced astaxanthin, and the yield of astaxanthin was high, which has the potential for industrial production.

[0065] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An engineered bacterium with high astaxanthin production, characterized in that: A gene expression cassette on the natural astaxanthin synthesis pathway is integrated into the genome of the engineered bacteria; The gene expression cassette on the natural astaxanthin synthesis pathway includes an astaxanthin precursor synthesis pathway expression cassette sequence, an isopentenyl pyrophosphate isomerase sequence and a gene expression cassette sequence for synthesizing astaxanthin, and the nucleotide sequences thereof are shown in SEQ ID NOs 8-10.

2. The engineered bacteria according to claim 1, characterized in that: The astaxanthin precursor synthesis pathway expression box sequence includes β-caratene synthase crtY, crtB, crtI, and crtE gene clusters; the promoter is a constitutive promoter of Vibrio natriuresis, and the terminator is BBa_B0010.

3. The engineered bacteria according to claim 1, characterized in that: The isopentenyl pyrophosphate isomerase sequence includes isopentenyl pyrophosphate isomerase, the promoter is a constitutive promoter of Vibrio natriuresis, and the terminator is BBa_B0010; the gene expression box sequence for synthesizing astaxanthin includes CrtW and CrtZ, the promoter is a constitutive promoter of Vibrio natriuresis, and the terminator is BBa_B0010.

4. The engineered bacteria according to claim 1, characterized in that: The engineered bacteria are obtained by introducing a recombinant plasmid into a host bacterium, and the recombinant plasmid is constructed by seamlessly cloning and connecting the coding gene of the tfox recombinant protein gene, the ampicillin resistance gene and the temperature-sensitive replicon SC101 through overlapping extension PCR and subsequent seamless cloning.

5. The engineered bacteria according to claim 3, characterized in that: The host bacteria is Vibrio natriuresis ATCC14048.

6. A process for constructing a high-yield astaxanthin engineering bacterium, characterized in that: The method comprises transforming a recombinant plasmid heterologously expressing tfox protein into a host cell, and then inserting the gene expression box on the natural astaxanthin synthesis pathway described in claim 1 into the host bacterial genome to construct a high-yield astaxanthin engineering bacterium.

7. The construction process according to claim 6, characterized in that: The construction of the recombinant plasmid for heterologous expression of tfox protein comprises the following steps: S1. Amplify the AmpR sequence using plasmid pKD46 as a template to obtain the product AmpR-a; amplify the temperature-sensitive promoter SC101 sequence using plasmid pKD46 as a template to obtain the product SC101-a; S2, artificially synthesize tfox sequence (Organism: Vibrio cholerae (strain: RFB16), Locus tag: F0316_RS08345), use the synthesized tfox sequence as a template for amplification to obtain product tfox-a, and use the tfox-a sequence as a template for amplification to obtain product tfox-b; S3, using AmpR-a and SC101-a as templates for amplification to obtain the product AmpR-SC101; S4. One-step cloning method was used to construct a recombinant plasmid of AmpR-SC101 and tfox-b.

8. The construction process according to claim 6, characterized in that: The engineered bacteria is Vibrio natriuresis; in the process of constructing the Vibrio natriuresis engineered bacteria, 500 bp upstream and downstream of the dns, wbfF, and pta genes of Vibrio natriuresis are used as homology arms, and the astaxanthin precursor synthesis pathway expression box sequence, the isopentenyl pyrophosphate isomerase sequence, and the gene expression box sequence for synthesizing astaxanthin are inserted into the genome of Vibrio natriuresis.

9. Use of the engineered bacteria according to any one of claims 1 to 5 or the construction process according to claim 6 in high production of astaxanthin.

10. The use according to claim 9, characterized in that: The method comprises fermenting the engineered bacteria constructed according to claim 6 by a fed-batch process to culture and produce astaxanthin.

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