A recombinant Vibrio natriuresis and its fermentation method and application

Abstract

The present invention discloses a recombinant Vibrio natrii and its fermentation method and application. The recombinant Vibrio natrii contains exogenous genes; the exogenous genes include at least one blue pigment synthase gene; the blue pigment synthase gene is selected from any one of the genes with nucleotide sequences of SEQ ID NO.1-SEQ ID NO.4. The present invention utilizes the natural rapid growth characteristics of Vibrio natrii and obtains a recombinant expression strain with rapid synthesis of blue pigment by introducing exogenous genes related to blue pigment expression. The strain has a high yield of blue pigment, few by-products, and a simple subsequent preparation and purification method. Moreover, the production of blue pigment by the recombinant strain can achieve the completion of the entire process of blue pigment from fermentation to refining within 10 hours, which has great prospects for industrial application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant Vibrio natriureticus and a fermentation method and application thereof. Background Art

[0002] Gulan (CAS#: 2435-59-8) is a non-toxic, natural blue substance derived from microorganisms. Due to its natural occurrence and its origin in glutamic acid, it can be used as a colorant in foods, beverages, and cosmetics. Gulan's excellent color development and environmentally biodegradable properties have led to its increasing popularity in the textile industry. Gulan can also be used as a screening marker in molecular biology, such as in the blue-white screening system for gene expression. Gulan's molecular structure has a bipyridine ring structure, forming two large π bonds in the molecular space, giving it the potential to be used as an organic semiconductor material, with broad development and application prospects in solar energy, information recording, and liquid crystal materials.

[0003] Blue pigment exists in nature, but its content is rare, and natural strains are mostly pathogenic bacteria and marine bacteria, making it impossible to obtain through simple culture or collection and isolation. Some studies have used Escherichia coli, Pseudomonas, Corynebacterium glutamicum, etc. as expression systems for heterologous expression of blue pigment. However, the fermentation cycle of E. coli is long, the product yield is low, and it can easily lead to a high pH in the fermentation broth in the later stages, which is not conducive to the large-scale production of blue pigment. Pseudomonas requires the introduction of multiple sets of genetic information, and the substrate and sugar conversion efficiency is low, which does not have commercial application prospects. Corynebacterium glutamicum has a relatively high conversion efficiency, but the cultivation and post-processing process takes more than 72 hours, which will result in huge energy consumption if used for industrial production. For example, patent 201711027820.X reconstructed a Corynebacterium glutamicum that can produce blue pigment. However, when its fermentation culture reached a yield of 1.75g / L, it took nearly 48 hours, which is not efficient enough and is not suitable for industrial production.

[0004] At the same time, in addition to the need for efficient production of bacterial strains, the subsequent extraction process of indigo blue is also a difficulty in large-scale mass production: due to the non-water-soluble nature of indigo blue itself, after the bacterial strain heterologously expresses and synthesizes indigo blue in the cell, it cannot be effectively transported to the outside of the cell. This also requires a series of processes such as bacterial cell crushing and solvent extraction, which greatly increases production costs and production line pressure. For example, in patent 202110875041.5, the extraction of blue pigment is to first reduce the blue pigment, extract and purify it, and then use an oxidant to prepare the blue pigment. The extraction process uses a lot of related solvents, which can easily cause losses during the blue pigment extraction process; at the same time, the process is relatively complicated, which reduces the efficiency of the process production and is not suitable for industrial production.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a recombinant Vibrio natrii and its fermentation method and application. The production of indigo by the recombinant Vibrio natrii can shorten the time of microbial culture, extraction process and other processes, simplify the operation, increase the yield of indigo, and reduce the cost of biocatalytic manufacturing.

[0007] To achieve the above object, the present invention constructs a recombinant microorganism that can produce natural blue pigment-indigo blue. Among numerous microorganisms, the present invention selects Vibrio natriegens as the starting strain to construct a recombinant strain. Vibrio natriegens is the fastest growing microorganism known at present, and its doubling time in the logarithmic phase is less than 10min, and has substrate diversity, fast metabolic rate, no pathogenic harm to the human body, easy genetic manipulation, and advantages such as being able to express foreign proteins. But it itself can not synthesize indigo blue. The present invention first imports a vector containing a blue pigment synthase gene into Vibrio natriegens, and finds that the indigo blue output of the recombinant bacteria imported with the vector containing only the blue pigment synthase gene is higher. In order to obtain higher indigo blue output, the present invention creatively introduces an exogenous gene that can effectively promote indigo blue to be transported to the extracellular space, significantly improving the efficiency of indigo blue transport to the extracellular space in recombinant Vibrio natriegens, so that higher indigo blue output can be obtained with this.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a recombinant Vibrio natriuresis bacterium, wherein the recombinant bacterium contains an exogenous gene;

[0010] The above-mentioned foreign genes include at least one blue pigment synthase gene;

[0011] The above-mentioned blue pigment synthase gene is selected from any one of the genes with nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.4.

[0012] The present invention found that recombinant Vibrio natrii containing any one of the above-mentioned blue pigment synthase genes can produce cyanobacteria, but the recombinant Vibrio natrii containing two or more pigment synthase genes has a higher cyanobacteria yield.

[0013] The indigoidine synthase gene with the nucleotide sequence of SEQ ID NO. 1 is derived from the genus Pseudoalteromonas rubra, specifically the gene indigoidine synthase IndC of DSM 6842 (NCBI.Gene.ID: 61359656).

[0014] The blue-pigment synthetase gene with the nucleotide sequence of SEQ ID NO. 2 is derived from the genus Streptomyces fulvissimus, specifically the gene Blue-pigment synthetase of DSM 40593 (NCBI.Gene.ID: 15402259).

[0015] The above-mentioned indigoidine synthase gene with the nucleotide sequence of SEQ ID NO.3 is derived from the genus Burkholderiagladioli, specifically the gene putative indigoidine synthase IndC of BSR3 (NCBI.Gene.ID: 10467378).

[0016] The above-mentioned indigoidine synthase gene with the nucleotide sequence of SEQ ID NO.4 is derived from the genus Burkholderiagladioli, specifically the gene putative indigoidine synthase IndC of BSR3 (NCBI.Gene.ID: 56652119).

[0017] In order to further improve the indigo production of the recombinant Vibrio natriuresis, the present invention further connects a transporter gene to the plasmid.

[0018] Specifically, the above-mentioned transporter gene can be an ABC transporter gene, and its nucleotide sequence is SEQ ID NO.5.

[0019] The ABC superfamily transport system exists in many eukaryotic and prokaryotic organisms, participates in various physiological activities of organisms, and completes the transmembrane transport of a variety of molecules in an active transport manner. The substrates it transports include sugars, amino acids, peptides, proteins, metal ions, drug molecules, etc.

[0020] The transporter gene with the nucleotide sequence of SEQ ID NO. 5 is derived from the genus Pseudomonas aeruginosa, specifically the gene NCGM2.S1 (NCBI.Gene.ID: 12576080).

[0021] Based on the above metabolic engineering improvements, the present invention further optimizes the above recombinant Vibrio natriuresis. As a preferred embodiment of the present invention, the exogenous genes in the recombinant Vibrio natriuresis are: blue pigment synthase genes with nucleotide sequences of SEQ ID NO. 1 and SEQ ID NO. 4, respectively, and a transporter gene with a nucleotide sequence of SEQ ID NO. 5.

[0022] In some embodiments, the strain used to construct the recombinant Vibrio natriuresis is Vibrio natriuresis Bio-85355.

[0023] In some embodiments, the expression vectors used in the construction of the recombinant Vibrio natriuresis include pET28a(+) and pEC-XK99E.

[0024] In the present invention, regarding the mode of introducing a plasmid carrying genes encoding the above-mentioned enzymes, these encoding genes may be present on the same plasmid or on different plasmids.

[0025] In some embodiments, the cyanin synthase gene and the transporter gene are placed on a plasmid for expression.

[0026] In some embodiments, the method for constructing a recombinant Vibrio natriureticus includes: connecting two or more blue pigment synthase genes to an expression vector, and then introducing the recombinant expression vector into the host Vibrio natriureticus to obtain the recombinant Vibrio natriureticus.

[0027] As a preferred construction scheme of the present invention, the above-mentioned expression vector is further connected to a nucleotide sequence of a transporter protein.

[0028] In a second aspect, the present invention provides a fermentation and culture method for the above-mentioned recombinant Vibrio natriuresis, which comprises inoculating a suspension of the recombinant Vibrio natriuresis into a culture medium for activation, then expanding and culturing the activated bacterial liquid, and then continuing to ferment the expanded culture liquid until the strain in the fermentation liquid reaches an appropriate concentration, adding an inducer, and then continuing to culture.

[0029] In the present invention, the inoculation amount of the mother liquor in the activation, expansion culture and fermentation steps is 0.5-2%; the culture medium used is conventional yeast extract culture medium.

[0030] In some embodiments, the culture conditions in the activation, expansion culture and fermentation steps are: a temperature range of 22-28° C. and a rotation speed of 150-250 rpm.

[0031] In some embodiments, the inducing agent comprises isopropylthiogalactoside.

[0032] In some embodiments, the final concentration of the isopropylthiogalactoside solution added is 180-220 uM.

[0033] In some embodiments, the continuous culture time after adding the inducer is 1-6 hours.

[0034] In a third aspect, the present invention also provides the use of the recombinant Vibrio natriuresis in the fermentation production of indigo.

[0035] The steps of producing indigo blue using the recombinant Vibrio natriuresis include: removing cells after the first centrifugation of the fermentation broth containing the recombinant Vibrio natriuresis, obtaining a crude extract after a second centrifugation, removing organic matter in the crude extract, and obtaining purified indigo blue after multiple washings.

[0036] In some embodiments, the first centrifugation has a rotation speed of 3000-5000 rpm and a time of 3-6 min.

[0037] In some embodiments, the second centrifugation has a rotation speed of 6000-10000 rpm and a time of 8-12 min.

[0038] In some embodiments, the reagent in the step of removing organic matter from the crude extract comprises methanol.

[0039] In some embodiments, the reagents in the washing step include ethyl acetate and n-hexane.

[0040] In a fourth aspect, the present invention also provides applications of the recombinant Vibrio natriuresis in the fields of food, beverages, cosmetics and textiles.

[0041] The present invention has the following beneficial effects:

[0042] The present invention utilizes the natural rapid growth characteristics of Vibrio natriuresis and introduces exogenous genes related to indigo indigo expression to obtain a recombinant expression strain capable of rapidly synthesizing indigo indigo. The strain has a high indigo indigo yield, few by-products, and a simple subsequent preparation and purification method. Moreover, the production of indigo indigo by the recombinant strain achieves the completion of the entire process from fermentation to refining within 10 hours, thus having great prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 is the infrared spectrum of the indigo plant product in Example 6;

[0045] Figure 2 This is the standard curve of the absorbance of glutamic blue in Experimental Example 1;

[0046] Figure 3 This is a curve chart showing the change of the indigo plant content over time in Experimental Example 1. DETAILED DESCRIPTION

[0047] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0048] Example 1

[0049] This embodiment provides a recombinant Vibrio natriuresis and a method for constructing the same.

[0050] Among them, the exogenous genes in the recombinant Vibrio natriuresis are: blue pigment synthase genes with nucleotide sequences of SEQ ID NO.1 and SEQ ID NO.4, and a transporter gene with a nucleotide sequence of SEQ ID NO.5; the host is: Vibrio natriuresis Bio-85355; and the vector is pET28a(+).

[0051] The construction method is:

[0052] (1) Inoculate 100 μL of seed solution into 50 mL of LB3 liquid medium and incubate at 37°C and 250 rpm until the OD600 reaches approximately 0.4. Transfer the bacterial solution to a pre-chilled 50 mL centrifuge tube and place on ice for 5 min.

[0053] (2) Then centrifuge at 4°C and 2,000×g for 5 min, discard the supernatant, and gently resuspend the bacterial pellet in 1 mL of pre-cooled 1 mol / L sorbitol buffer and centrifuge at 4°C and 5,000×g for 5 min.

[0054] (3) Wash the bacterial pellet twice (3 times in total) with 1 mL of pre-cooled 1 mol / L sorbitol buffer. Gently resuspend the bacterial pellet in 250 μL of pre-cooled 1 mol / L sorbitol buffer and aliquot 50 μL per tube. If not used immediately, store in a refrigerator at -80°C.

[0055] (4) Add approximately 100 ng of plasmid to the competent cells, mix gently, transfer to a pre-cooled 1 mm electroporation cuvette, let it stand for 3 minutes, and then perform transformation. The transformation voltage is 0.7–0.9 kV (Bio-Rad, MicroPulser™).

[0056] (5) Immediately after electroporation, add 1 mL of pre-chilled LB3 medium and transfer the bacterial solution to a 2 mL centrifuge tube. Resuscitate the culture at 37°C, 250 rpm for 45 min or at 30°C, 250 rpm for 1.5 h.

[0057] (6) Spread the bacterial solution on LB3 solid medium of corresponding resistance and culture at 30℃ overnight.

[0058] Example 2

[0059] This embodiment provides a recombinant Vibrio natriuresis and a method for constructing the same.

[0060] Among them, the exogenous genes in the recombinant Vibrio natriuresis are: blue pigment synthase genes with nucleotide sequences of SEQ ID NO.2 and SEQ ID NO.4, and a transporter gene with a nucleotide sequence of SEQ ID NO.5; the host is: Vibrio natriuresis Bio-85355; and the vector is pET28a(+).

[0061] The construction method is the same as that of Example 1.

[0062] Example 3

[0063] This embodiment provides a recombinant Vibrio natriuresis and a method for constructing the same.

[0064] Among them, the exogenous genes in the recombinant Vibrio natriuresis are: blue pigment synthase genes with nucleotide sequences of SEQ ID NO.3 and SEQ ID NO.4, and a transporter gene with a nucleotide sequence of SEQ ID NO.5; the host is: Vibrio natriuresis Bio-85355; and the vector is pET28a(+).

[0065] The construction method is the same as that of Example 1.

[0066] Example 4

[0067] This embodiment provides a recombinant Vibrio natriuresis and a method for constructing the same.

[0068] The exogenous genes in the recombinant Vibrio natriuresis are: blue pigment synthase genes with nucleotide sequences of SEQ ID NO.1 and SEQ ID NO.4; the host is: Vibrio natriuresis Bio-85355; and the vector is pET28a(+).

[0069] The construction method is the same as that of Example 1.

[0070] Example 5

[0071] This example provides a fermentation method for the recombinant Vibrio natriuresis constructed in Examples 1-4, and the specific steps are as follows:

[0072] (1) The recombinant Vibrio natriuresis constructed in Examples 1-4 was inoculated into 5 mL of the above-mentioned yeast extract medium (purchased from Beijing Biobo Biotechnology Co., Ltd.) at a 1% inoculum size and activated for 12 h at a temperature of 25° C. and a shaking speed of 200 rpm;

[0073] (2) The bacterial solution activated once in step (1) was then inoculated into 50 mL of yeast extract culture medium at a 1% inoculation rate, and a secondary activation culture was performed under the same culture conditions.

[0074] (3) Take the secondary activated bacterial liquid and inoculate it into 1 L of yeast extract medium at a 1% inoculum size. Use a spectrophotometer to detect the absorbance of the fermentation liquid at a wavelength of 600 nm. When the absorbance of the fermentation liquid at a wavelength of 600 nm reaches 0.6, add IPTG at a final concentration of 200 μM to the above fermentation liquid and place it in a shaker at 25°C and 200 rpm for continuous culture for 1-6 hours.

[0075] Example 6

[0076] This example provides a method for producing indigo using the recombinant Vibrio natriuresis fermentation broth constructed in Examples 1-4. The specific steps are as follows:

[0077] (1) After the fermentation is completed for 6 h, 200 mL of the fermentation broth is taken and centrifuged at 4000 rpm for 5 min to remove the cells of the recombinant Vibrio natriureticus constructed in Example 1-4.

[0078] (2) The fermentation broth after cell removal was centrifuged at 8000 rpm for 10 min to obtain a crude extract.

[0079] (3) The crude extract was suspended in 1 mL of methanol by oscillation, then centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. This process was repeated 2-3 times to remove organic matter in the crude extract; the crude extract was then washed 2-3 times with ethyl acetate and n-hexane respectively using the same method to obtain the target compound.

[0080] The target compound obtained by the above method was identified by liquid chromatography, mass spectrometry and infrared spectroscopy. The results of product identification are as follows: Figure 1 From the identification results, it can be determined that the target compound is Gulan.

[0081] The purity of the obtained indigo product was tested, and the test data are shown in Table 1:

[0082] Table 1 Purity data of the blue produced by different recombinant Vibrio natrium fermentation broths

[0083] plan purity Example 1 99.1％ Example 2 95.3％ Example 3 97.9％ Example 4 96.7％

[0084] As can be seen from Table 1, the purity of the indigo plant obtained by the production method of the present invention is extremely high, reaching more than 95%.

[0085] Experimental Example 1

[0086] The yield of indigo blue produced by fermentation in Example 5 was determined by the following method: 1 mL of fermentation broth was aspirated every 1 h for absorbance measurement, and the content of indigo blue in 1 mL of fermentation broth of recombinant Vibrio natrii was obtained according to the standard curve, and the total yield of indigo blue in 1 L of fermentation broth of recombinant Vibrio natrii was calculated at different times.

[0087] The standard curve is as follows: Figure 2 As shown; the obtained data of the change of the content of indigo plant with time, with the content of indigo plant as the vertical axis and time as the horizontal axis, a linear fitting curve of the change of the content of indigo plant with time is obtained, as shown Figure 3 The corresponding measurement results are shown in Table 2:

[0088] Table 2: The yield data of the recombinant Vibrio natrii fermented in Example 1-4

[0089]

[0090] As can be seen from Table 2, the recombinant Vibrio natrii constructed by the present invention can obtain a good yield of indigo after 6 hours of fermentation. Compared with the prior art, the indigo produced by the recombinant Vibrio natrii fermentation of the present invention not only has a higher yield, but also significantly improves production efficiency.

[0091] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A recombinant Vibrio natriuresis, characterized in that The recombinant Vibrio natriuresis contains two exogenous blue pigment synthase genes; The two exogenous blue pigment synthases are proteins represented by NCBI Gene ID: 61359656 and NCBI Gene ID: 56652119; or The two exogenous blue pigment synthases are proteins represented by NCBI Gene ID: 15402259 and NCBI Gene ID: 56652119; or The two exogenous blue pigment synthases are proteins represented by NCBI Gene ID: 10467378 and NCBI Gene ID: 56652119.

2. The recombinant Vibrio natriuresis according to claim 1, wherein Foreign genes also include transporter genes.

3. The recombinant Vibrio natriuresis according to claim 2, characterized in that The transporter gene is an ABC transporter gene.

4. The recombinant Vibrio natriuresis according to claim 3, characterized in that The sequence of the transporter gene is shown in NCBI Gene ID: 12576080.

5. The recombinant Vibrio natriuresis according to claim 4, characterized in that The recombinant Vibrio natrii expresses proteins shown by NCBI Gene ID: 61359656 and NCBI Gene ID: 56652119, and a transporter protein shown by NCBI Gene ID: 12576080.

6. The recombinant Vibrio natriuresis according to claim 1, characterized in that The recombinant Vibrio natriuresis uses Vibrio natriuresis Bio-85355 as a host.

7. The recombinant Vibrio natriuresis according to claim 1, characterized in that The recombinant Vibrio natriuresis contains an expression vector, which includes pET28a(+) and pEC-XK99E.

8. The recombinant Vibrio natriuresis according to any one of claims 1 to 7, characterized in that The method for constructing the recombinant Vibrio natriureticus comprises: connecting the blue pigment synthase gene to an expression vector, and then introducing the recombinant expression vector into Vibrio natriureticus to obtain the recombinant Vibrio natriureticus.

9. The recombinant Vibrio natriuresis according to claim 8, characterized in that The expression vector is also connected to the nucleotide sequence of the transporter protein.

10. The fermentation culture method of the recombinant Vibrio natriuresis according to claim 9, wherein The fermentation culture method comprises: inoculating the suspension of the recombinant Vibrio natrii into a culture medium for activation, then expanding the activated bacterial liquid for culture, then continuing to ferment the expanded culture liquid until the strain in the fermentation liquid reaches an appropriate concentration, adding an inducer, and then continuing to culture.

11. The fermentation culture method according to claim 10, characterized in that: The culture conditions in the activation, expansion culture and fermentation steps are: temperature range of 22-28° C. and rotation speed of 150-250 rpm.

12. The fermentation culture method according to claim 10, characterized in that: The inducer includes isopropylthiogalactoside.

13. The fermentation culture method according to claim 12, characterized in that: The final concentration of the isopropylthiogalactoside solution is 180-220 μM.

14. The fermentation culture method according to claim 10, characterized in that: The continuous culture time is 1-6 hours.

15. Use of the recombinant Vibrio natriuresis according to any one of claims 1 to 9 in the fermentation production of indigo plant, characterized in that: The steps of producing indigo blue by using the recombinant Vibrio natriuresis include: removing cells after a first centrifugation of a fermentation broth containing the recombinant Vibrio natriuresis, obtaining a crude extract after a second centrifugation, removing organic matter in the crude extract, and obtaining purified indigo blue after multiple washings.

16. The use according to claim 15, characterized in that The first centrifugation has a rotation speed of 3000-5000 rpm and a time of 3-6 min.

17. The use according to claim 15, characterized in that The second centrifugation has a rotation speed of 6000-10000 rpm and a time of 8-12 min.

18. The use according to claim 15, characterized in that The reagent in the step of removing organic matter from the crude extract includes methanol.

19. The use according to claim 15, characterized in that The reagents in the cleaning step include ethyl acetate and n-hexane.

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