Engineering strain for co-production of indigoidine and N-acetyl-indigoidine and fermentation method

Abstract

The invention relates to an engineering strain for co-production of indigoidine and N-acetul-indigoidine and a fermentation method, and the fermentation method comprises the following steps: in a fermentation culture medium with or without a precursor, carrying out fermentation culture on a gene engineering strain to produce indigoidine and N-acetul-indigoidine, and carrying out fermentation culture on the gene engineering strain to produce the indigoidine and N-acetul-indigoidine, and the indigoidine and N-acetul-indigoidine and N-acetul-indigoidine and N-acetul-indigoidine and N-acetul-indigoidine. According to the present invention, by controlling the endogenous N-acetylglutamate synthase of the bacterial strain, overexpressing different 4 '-phosphoric acid pantetheinyl transferase and adjusting the addition of the precursor, the purpose of co-producing the indigoidine and the N-acetyl-indigoidine through the microbial fermentation can be achieved;

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering of enzymes, and particularly relates to an engineered strain co-producing indigoidine and N-acetyl-indigoidine and a fermentation method thereof. Background Art

[0002] Indigoidine is a natural blue pigment formed by the action of natural blue pigment synthase on two glutamine molecules. It has a bright color and is harmless to the human body. It is a compound with great potential that can be widely used in the cosmetics, fabric printing and dyeing, and medical industries. N-acetyl-indigoidine is a derivative of indigoidine, which has a brighter color than indigoidine and has gradually attracted people's attention. The structural formulas of indigoidine and N-acetyl-indigoidine are as follows:

[0003]

[0004] Indigoidine and N-acetyl-indigoidine are simultaneously produced by engineered microbial fermentation technology. Since both are lipophilic substances, N-acetyl-indigoidine replaces one of the amino groups of indigoidine with an acetyl group, and the acetyl group has lower water solubility compared to the amino group. However, since only one group among many groups is changed, its impact on the overall solubility of the two is not significant. Therefore, the current indigoidine extraction technology cannot effectively remove N-acetyl-indigoidine, and the obtained dye product is usually a mixture of indigoidine and N-acetyl-indigoidine. Since indigoidine is much greater than N-acetyl-indigoidine in most of the currently disclosed engineered microbial strains, during decades of research and development, researchers usually use indigoidine to characterize the yield of engineered microbial strains.

[0005] With the successful construction of a high-yield N-acetyl-indigoidine strain, our company's researchers found that when the ratio of indigoidine and N-acetyl-indigoidine in the dye is different, although the color of the dye is still mainly blue, it also changes slightly. Therefore, how to regulate the ratio of indigoidine and N-acetyl-indigoidine in the fermentation broth as needed and ensure its stable production is crucial for the fine production of blue dyes. Summary of the Invention

[0006] The object of the present invention is to overcome the defects in the prior art, and provide a genetically engineered strain for co-producing indigoidine and N-acetyl-indigoidine and a fermentation method thereof. By controlling gene expression and the selection of precursors, the ratio of indigoidine and N-acetyl-indigoidine in the fermentation broth is controlled, so as to more precisely control the dyeing color.

[0007] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0008] A genetically engineered strain for co-producing indigoidine and N-acetyl-indigoidine, which is obtained by transforming the host bacterium by any one of the following A-C:

[0009] A: Free expression of the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene;

[0010] B: Knock out N-acetylglutamate synthase in the host bacterium, and free express the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene;

[0011] C: Free expression of the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene, free expression of the glutamine synthase gene and N-acetylglutamate synthase.

[0012] As a further technical solution, the natural blue pigment synthase gene uses the gene bpsA;

[0013] The 4'-phosphopantetheinyl transferase gene uses any one of the genes sfp2 and pptA;

[0014] As a further technical solution, the glutamine synthase gene uses the gene glnA, and the N-acetylglutamate synthase gene uses the gene argA.

[0015] As a further technical solution, the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene are respectively expressed under the initiation of the promoter Tac;

[0016] As a further technical solution, the plasmid vectors used for the free expression of the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene are pTrc99a or pXMJ19;

[0017] As a further technical solution, the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene are respectively expressed under the initiation of the promoter Tac;

[0018] As a further technical solution, the plasmid vector used for the free expression of the natural blue pigment synthase gene and the 4'-phosphopantetheine transferase gene is pCDFDuet;

[0019] The plasmid vector used for the free expression of the natural blue pigment synthase gene is pECXK99E.

[0020] As a further technical solution, the host bacterium includes any one of Escherichia coli BL21(DE3) and Corynebacterium glutamicum 13032.

[0021] As a further technical solution, the nucleotide sequence of the natural blue pigment synthase gene bpsA is as shown in SEQ ID NO.1 or has at least 98% identity with SEQ ID NO.1;

[0022] As a further technical solution, the nucleotide sequence of the 4'-phosphopantetheine transferase gene sfp2 is as shown in SEQ ID NO.2 or has at least 98% identity with SEQ ID NO.2;

[0023] As a further technical solution, the nucleotide sequence of the 4'-phosphopantetheine transferase gene pptA is as shown in SEQ ID NO.3 or has at least 98% identity with SEQ ID NO.3;

[0024] As a further technical solution, the nucleotide sequence of the glutamine synthetase gene glnA is as shown in SEQ ID NO.4 or has at least 98% identity with SEQ ID NO.4;

[0025] As a further technical solution, the nucleotide sequence of the N-acetylglutamate synthase gene argA is as shown in SEQ ID NO.5 or has at least 98% identity with SEQ ID NO.5;

[0026] As a further technical solution, the genetically engineered bacterium is a genetically engineered recombinant Escherichia coli, and the construction method of the genetically engineered recombinant Escherichia coli is as follows:

[0027] 1) The natural blue pigment synthase gene and the 4'-phosphopantetheine transferase-encoding gene are respectively amplified by PCR and linked to the plasmid pTrc99a-tac-tac to obtain recombinant plasmids A1 (including: pTrc99a-tac-bpsA-tac-sfp2, A2: pTrc99a-tac-bpsA-tac-pptA);

[0028] 2) The glutamine synthetase gene and N-acetylglutamate synthase gene were amplified by PCR and ligated into the plasmid pCDFDuet-1-tac-tac to obtain the recombinant plasmid B1 (pCDFDuet-1-tac-glnA-tac-argA);

[0029] 3) The recombinant plasmids A1 and A2 were respectively transformed into Escherichia coli BL21(DE3) and Escherichia coli BL21△argA to obtain the genetically engineered bacteria recombinant Escherichia coli (ecA1, ecA2, ecQA1, ecQA2);

[0030] Alternatively, the recombinant plasmids A1 and A2 were co-transformed with B1 into Escherichia coli BL21(DE3) respectively to obtain the genetically engineered bacteria recombinant Escherichia coli (ecAB1, ecAB2).

[0031] The method for co-producing indigoidine and N-acetyl-indigoidine using the said genetically engineered strains includes: fermenting and culturing the said genetically engineered bacteria in a fermentation medium with or without adding precursors to produce indigoidine and N-acetyl-indigoidine; the yield ratio of indigoidine and N-acetyl-indigoidine is 19 - 98% : 2 - 81%;

[0032] As a further technical solution, the precursors include one or more of L-glutamine, N-acetylglutamine, and N-acetylglutamate.

[0033] As a further technical solution, the fermentation process of the genetically engineered bacteria recombinant Escherichia coli is: in a fermentation medium, fermenting and culturing the said genetically engineered strain at 35 - 38 °C until OD 600 reaches 0.6 - 0.8, then adding IPTG with a final concentration of 0.05 - 1.0 mM, and inducing culture at 28 - 32 °C for at least 48 h to generate indigoidine and N-acetyl-indigoidine;

[0034] Or, in a fermentation medium, fermenting and culturing the said genetically engineered strain at 35 - 38 °C until OD 600 reaches 0.6 - 0.8, then adding IPTG with a final concentration of 0.05 - 1.0 mM, inducing culture at 28 - 32 °C for at least 24 h, then adding precursors, and continuing to culture for 24 h to generate indigoidine and N-acetyl-indigoidine.

[0035] As a further technical solution, the said genetically engineered bacteria adopt genetically engineered Corynebacterium glutamicum, and the construction method of the genetically engineered Corynebacterium glutamicum is as follows:

[0036] 1) The genes encoding natural blue pigment synthase and 4'-phosphopantetheinyl transferase were amplified by PCR respectively and ligated into the plasmid pXMJ19-tac-tac to obtain the recombinant plasmid C (pXMJ19-tac-bpsA-tac-sfp2).

[0037] 2) The gene encoding glutamine synthetase was amplified by PCR and ligated into the plasmid pECXK99E-1-tac-tac to obtain the recombinant plasmid D (pECXK99E-1-tac-glnA).

[0038] 3) The recombinant plasmids C and D were co-transformed into Corynebacterium glutamicum 13032 to obtain the genetically engineered bacterium recombinant Corynebacterium glutamicum (cg1).

[0039] As a further technical solution, the fermentation process of the genetically engineered bacterium recombinant Corynebacterium glutamicum is as follows: 1 mL was inoculated from the glycerol bacterial solution into 250 mL of BHIS medium, kanamycin and chloramphenicol with a final concentration of 10 mg / mL were added, and the culture conditions were 25 - 35 °C, 210 - 230 rpm, and cultured for 3 - 7 h until the OD of the bacterial solution 600 was not less than 2 to obtain the seed solution;

[0040] The seed solution of the genetically engineered bacterium recombinant Corynebacterium glutamicum was inoculated into a 50 L fermentation tank system. The fermentation temperature of the fermentation system was 30 °C, the stirring speed was 200 - 800 r / min, the ventilation volume was 1 - 5 vvm, the pH was 6.6 - 6.8, and the fermentation was carried out until OD 600 = 14 ± 1, IPTG with a final concentration of 0.2 ± 0.5 mM was added, and induced culture was carried out at 25 - 35 °C for not less than 72 h.

[0041] As a further technical solution, the ratio of indigoidine and N-acetyl-indigoidine in the fermentation broth was regulated by controlling the transformation of the host bacterium and the addition of precursors, as shown in Table 1 specifically;

[0042] Table 1

[0043]

[0044]

[0045] An engineered strain with high yield of N-acetyl-indigoidine, in which the N-acetylglutamate synthase gene argA of the host bacterium was knocked out, and the natural blue pigment synthase gene bpsA and 4'-phosphopantetheinyl transferase gene pptA were expressed freely.

[0046] A method for producing high-yield N-acetyl-indigoidine, in a fermentation medium with or without precursors, fermenting the engineering strain for producing high-yield N-acetyl-indigoidine to produce N-acetyl-indigoidine;

[0047] The precursors include one or more of L-glutamine, N-acetyl-glutamine, and N-acetyl-glutamic acid.

[0048] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0049] 1. It is found through research in the present invention that the addition or non-addition of precursors and the selection of precursors in the fermentation medium have an impact on the yield ratio of indigoidine and N-acetyl-indigoidine, but the impact is relatively small; in the construction of engineering strains, the selection of the 4'-phosphopantetheinyl transferase gene is the key factor affecting the yield ratio of indigoidine and N-acetyl-indigoidine, followed by the knockout and overexpression of the N-acetylglutamate synthase gene argA; when the gene sfp2 is selected, the yield of indigoidine is much greater than that of N-acetyl-indigoidine, and when the gene pptA is selected, compared with the selection of the gene sfp2, the yield of N-acetyl-indigoidine begins to increase, and at the same time the yield of indigoidine begins to decrease; when the genes glnA and argA are overexpressed, but the gene sfp2 is selected, compared with the non-overexpression of the genes glnA and argA, the ratio change of indigoidine and N-acetyl-indigoidine is very small, however, when the genes glnA and argA are overexpressed, but the gene pptA is selected, compared with the non-overexpression of the genes glnA and argA, indigoidine increases to a large extent, and at the same time the yield of N-acetyl-indigoidine decreases relatively; when the gene argA is knocked out without complementation, compared with the non-knockout of the gene argA, the yield of indigoidine decreases significantly, and the yield of N-acetyl-indigoidine increases correspondingly significantly, and when the selection is the gene pptA, compared with the selection of the gene sfp2, the yield change range of indigoidine and N-acetyl-indigoidine is greater.

[0050] In summary, the present invention can regulate the production ratio of indigoidine to N-acetyl-indigoidine by controlling the selection of the 4'-phosphopantetheine transferase gene, knocking out the gene argA, the free expression of the genes glnA and argA, and the addition and selection of precursors. Description of the Drawings

[0051] Figure 1 It is the color contrast at different ratios of indigoidine and N-acetyl-indigoidine in Example 2;

[0052] In Figure 1 A: 2:8; B: 9:1;

[0053] Figure 2 It is the liquid chromatogram of the fermentation broth of different test groups in Example 2;

[0054] In Figure 2 A: ecQAB2, without precursor; B: ecQA2, without precursor; C: ecQA1, without precursor; D: ecQA2, without precursor;

[0055] Figure 3 It is the liquid chromatogram of the fermentation broth at 48 h of fermentation culture in Example 3;

[0056] Figure 4 It is the LC-MS diagram of N-acetyl-indigoidine;

[0057] Figure 5 It is the nuclear magnetic 1 1H-NMR spectrum of N-acetyl-indigoidine;

[0058] Figure 6 It is the nuclear magnetic 13 13C-NMR spectrum of N-acetyl-indigoidine. Detailed Embodiments

[0059] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0060] In addition, it should be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or apparatus.

[0061] In the present invention,

[0062] 1. For all commercial products such as PCR amplification enzymes, restriction enzymes, plasmids, DNA gel extraction kits and column plasmid extraction kits used, the specific operations are all carried out according to the kit instructions. Conventional molecular biology experimental operations such as preparation of Escherichia coli competent cells, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation transformation, preparation of competent cells, colony PCR and extraction of bacterial genomes are carried out according to Molecular Cloing: A Laboratory Manua (Fourth Edition). The sequencing of plasmids and PCR amplification products is all completed by Suzhou Genewiz Co., Ltd.

[0063] 2. The strains constructed in the present invention are shown in Table 1;

[0064] Table 1: Strains

[0065]

[0066]

[0067] 3. The primers used for constructing the strains in the present invention are shown in Table 2;

[0068] Table 2: List of related primer sequences

[0069]

[0070]

[0071] 4. Unless otherwise specified, the raw materials used are all commercially available raw materials.

[0072] 5. The detection methods for natural blue pigment and N-acetyl natural blue pigment are as follows:

[0073] Sample preparation: Take 100 μL of the fermentation broth obtained by fermentation, dilute and dissolve it with DMSO by a certain multiple, filter it with a 0.22 μm filter membrane, and detect the sample by Agilent high performance liquid chromatography.

[0074] Detection method: through a high-performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II); chromatographic column: Agilent ZORBAX Eclipse Plus C18 column, 4.6×250mm, 5μm; mobile phase: pump A is H2O, pump B is methanol; gradient elution program: 80% A / 20% B at 0 min, 50% A / 50% B at 9 min, 80% A / 20% B at 13 min, 80% A / 20% B at 18 min; flow rate: 1.0 mL / min; column temperature: 35°C; injection volume: 10 μL.

[0075] Example 1: Construction of a genetically engineered bacterium for co-producing natural blue pigment and N-acetyl natural blue pigment

[0076] The genetically engineered bacterium for co-producing natural blue pigment and N-acetyl natural blue pigment includes any one of strain ecA1, strain ecA2, strain ecQA1, strain ecQA2, strain ecAB1, and strain ecAB2, where

[0077] Construction of strain ecA1: Using Escherichia coli BL21(DE3) as the starting strain, the genes bpsA initiated by the promoter tac and the gene sfpp2 initiated by the promoter tac are free-expressed using a plasmid vector to obtain strain ecA1;

[0078] Construction of strain ecA2: Using Escherichia coli BL21(DE3) as the starting strain, the genes bpsA initiated by the promoter tac and the gene pptA initiated by the promoter tac are free-expressed using a plasmid vector to obtain strain ecA1;

[0079] Construction of strain ecQA1: Using Escherichia coli BL21(DE3) as the starting strain, the gene argA of Escherichia coli BL21(DE3) is knocked out, and the genes bpsA initiated by the promoter tac and the gene sfpp2 initiated by the promoter tac are free-expressed using a plasmid vector to obtain strain ecQA1;

[0080] Construction of strain ecQA2: Using Escherichia coli BL21(DE3) as the starting strain, the gene argA of Escherichia coli BL21(DE3) is knocked out, and the genes bpsA initiated by the promoter tac and the gene pptA initiated by the promoter tac are free-expressed using a plasmid vector to obtain strain ecQA2;

[0081] Construction of strain ecAB1: Using Escherichia coli BL21(DE3) as the starting strain, the plasmid vector was used to freely express the gene bpsA initiated by the promoter tac and the gene sfp2 initiated by the promoter tac, and the plasmid vector pCDFDuet was used to freely express the gene glnA and the gene argA initiated by the promoter tac to obtain strain ecAB1;

[0082] Construction of strain ecAB2: Using Escherichia coli BL21(DE3) as the starting strain, the plasmid vector was used to freely express the gene bpsA initiated by the promoter tac and the gene pptA initiated by the promoter tac, and the plasmid vector pCDFDuet was used to freely express the gene glnA and the gene argA initiated by the promoter tac to obtain strain ecAB2.

[0083] The hosts of the above strains contain plasmids and the genotypes are shown in Table 1; the primers involved in constructing the above strains are shown in Table 2.

[0084] Example 2: Effects of 4'-phosphopantetheine transferase gene, glutamate synthase-encoding gene argA, types and presence or absence of precursors on the ratio of natural blue pigment and N-acetyl natural blue pigment in fermentation products

[0085] I. Test method

[0086] Take out the Escherichia coli strains ecA1, ecA2, ecQA1, ecQA2, ecAB1, and ecAB2 in the glycerol preservation tubes, streak them on the corresponding LB solid plates and culture overnight. Pick single colonies with normal colony morphology into 4 mL test tubes of LB culture liquid containing the corresponding antibiotics. After culturing for 10 - 12 h, transfer the seed liquid in the test tube to a shake flask containing 25 mL of fermentation medium with an initial pH of 7 (adding precursors according to requirements) at an inoculation amount of 2% (v / v), and culture with shaking at 37 °C and 200 rpm. When the cell OD 600 reaches 0.6 - 0.8, add IPTG to a final concentration of 0.2 mM, and ferment at 30 °C for 48 h. After fermentation, take the fermentation broth, prepare samples, and detect by HPLC. The results are shown in Table 3;

[0087] LB medium: 10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L peptone, and 15 g / L agar powder is additionally added to the LB solid medium.

[0088] Fermentation medium: 20 g / L glycerol, 3 g / L (NH4)2SO4, 2 g / L (NH4)2HPO4, 6.75 g / L KH2PO4, 0.85 g / L citric acid, 0.7 g / L MgSO4·7H2O, 5 mL trace metal solution, 5 g / L yeast extract.

[0089] Among them: trace metal solution: 10 g / L FeSO4·7H2O, 2.25 g / L ZnSO4·7H2O, 1 g / L CuSO4·5H2O, 0.5 g / L MnSO4·5H2O, 0.23 g / L Na2B4O7, 2 g / L CaCl2·2H2O, 0.1 g / L (NH4)6Mo7O 24

[0090] In the above fermentation medium, according to different plasmid vectors, it contains 50 mg / L kanamycin antibiotic and 100 mg / L ampicillin antibiotic.

[0091] Inducer concentration: During the shake-flask fermentation process, the final added concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) is 0.2 mM.

[0092] II. Results and Analysis

[0093] The effects of different strains and precursor addition on the ratio of natural blue pigment and N-acetyl natural blue pigment are shown in Table 3 and Table 4; among them, the color differences when the ratio of natural blue pigment to N-acetyl natural blue pigment is 6:4 and 9:1 are shown in Figure 1 , and the HPLC chromatograms of Group 11, Group 16, Group 12, and Group 16 are shown in Figure 2 ;

[0094] Table 3: Effects of different 4'-phosphopantetheinyl transferases and substrate addition on the ratio of natural blue pigment and N-acetyl natural blue pigment

[0095]

[0096] It can be seen from the results in Table 3 that:

[0097] (1) The addition or not of precursors and the selection of precursors in the fermentation medium have no significant effect on the production ratio of indigoidine and N-acetyl-indigoidine; in the construction of engineering strains, the selection of the 4'-phosphopantetheinyl transferase gene is the key factor affecting the production ratio of indigoidine and N-acetyl-indigoidine, followed by the knockout and overexpression of the N-acetylglutamate synthase gene argA;

[0098] (2) When the gene sfp2 is selected, the production of indigoidine is much greater than that of N-acetyl-indigoidine, while when the gene pptA is selected, compared with the selection of the gene sfp2, the production of N-acetyl-indigoidine begins to increase, and at the same time the production of indigoidine begins to decrease;

[0099] Table 4: Influence of Strain Modification on the Ratio of Natural Blue Pigment and N-acetyl Natural Blue Pigment

[0100] Group number Strain name Precursor addition situation Indigoidine:N-acetyl-indigoidine 11 ecA1 None 95％:5％ 12 ecA2 None 75％:25％ 13 ecAB1 None 96％:4％ 14 ecAB2 None 95％:5％ 15 ecQA1 None 86％:14％ 16 ecQA2 None 68％:32％

[0101] It can be seen from the results in Table 4 that:

[0102] (1) When overexpressing genes glnA and argA and selecting sfp2, compared with not overexpressing genes glnA and argA, the ratio change of indigoidine and N-acetyl-indigoidine is very small. However, when overexpressing genes glnA and argA and selecting pptA, compared with not overexpressing genes glnA and argA, indigoidine increases to a large extent, while the yield of N-acetyl-indigoidine decreases relatively;

[0103] (2) When knocking out gene argA, compared with not knocking out gene argA, the yield of indigoidine decreases significantly, and the yield of N-acetyl-indigoidine increases significantly correspondingly. When the selected gene is pptA, compared with when the selected gene is sfp2, the yield change range of indigoidine and N-acetyl-indigoidine is larger.

[0104] In summary, the present invention can regulate the yield ratio of indigoidine and N-acetyl-indigoidine by regulating the selection of 4'-phosphopantetheinyl transferase gene, the knockout of gene argA, the overexpression of genes glnA and argA, and the addition and selection of precursors.

[0105] Example 3: Method for Fermenting and Co-producing Natural Blue Pigment and N-acetyl Natural Blue Pigment Using Engineered Corynebacterium glutamicum

[0106] 1. Construction of engineered Corynebacterium glutamicum cg1: Using Corynebacterium glutamicum ATCC13032 as the starting strain, plasmid vector pXMJ19 was used to overexpress gene bpsA initiated by promoter tac and gene sfp2 initiated by promoter tac, and plasmid vector pECXK99E was used to overexpress gene glnA initiated by promoter tac to obtain engineered Corynebacterium glutamicum cg1.

[0107] 2. Fermentation culture:

[0108] (1) Take out the Corynebacterium glutamicum strain cg1 from the glycerol preservation tube, streak it on the corresponding BHIS solid plate and culture it overnight. Pick a single colony with normal colony morphology into a 4 mL test tube of BHIS culture medium containing the corresponding antibiotic. After culturing for 10 - 12 h, transfer the seed liquid in the test tube to a shake flask containing a fermentation medium with an initial pH of 7 at an inoculation amount of 4% (v / v) to obtain the activated bacterial liquid;

[0109] (2) Add CGXII fermentation medium to a 50 L fermenter, and then perform steam sterilization. After the sterilization is completed, cool it down to 30 °C. Inoculate the activated bacterial liquid obtained in (1) into the fermenter at an inoculation amount of 5% for cultivation. During this period, control the pH = 7, the temperature is controlled at 30 °C, the initial rotation speed is 200 rpm, stir and correlate the dissolved oxygen DO, and control the dissolved oxygen at 50%;

[0110] (3) During the fermentation process, sample and monitor the yields of the two blue pigments (by HPLC method). When the fermentation reaches 72 h, the yields of the two blue pigments basically stop increasing, and the fermentation ends. For the fermentation process monitoring, when the fermentation reaches 48 h, the HPLC chromatogram results are shown in Figure 3 ;

[0111] Among them, BHIS medium: 38.5 g / L BHI, 91 g / L sorbitol;

[0112] CGXII fermentation medium: 1 g / L K2HPO4, 1 g / L KH2PO4, 42 g / L MOPS, 80 g / L glucose, 20 g / L (NH4)2SO4, 5 g / L urea, 0.25 g / L MgSO4·7H2O, 0.01 g / L MnSO4·H2O, 0.01 g / L FeSO4·7H2O, 2 mg / L CuSO·5H2O, 20 mg / L NiSO4·6H2O, 10 mg / L CaCl2, 0.31 mg / L CoCl2·6H2O.

[0113] In the CGXII fermentation medium, according to different plasmid vector situations, it contains 10 mg / L kanamycin antibiotic and 10 mg / L chloramphenicol antibiotic.

[0114] Inducer concentration: During the fermentation process, the final added concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) is 0.2 mM.

[0115] 3. Results and analysis

[0116] From Figure 3It can be seen that when Corynebacterium glutamicum ATCC13032 is used as the host, the genes bpsA and sfpp2 are expressed freely, and the gene glnA is expressed freely at the same time, the co-production of indigoidine and N-acetyl-indigoidine can also be achieved.

[0117] Example 4: Collection, purification and identification of N-acetyl-indigoidine sample

[0118] Take the fermentation broth obtained in Example 3, remove the supernatant after centrifugation at 10,000 rpm to obtain a cell precipitate. The precipitate is washed 3 times with clear water, then ultrasonically disrupted, and then washed 2 times with methanol, ethyl acetate and n-hexane respectively. The obtained solid is dried in an oven at 60 °C for 24 h to obtain the purified sample of N-acetyl-indigoidine. The purified sample is detected by LC-MS and NMR in the Analysis and Testing Center of Jiangnan University. Figure 4 It is the LC-MS detection chart of N-acetyl-indigoidine. Figure 5 For N-acetyl-indigoidine 1 1H-NMR chart; Figure 6 For N-acetyl-indigoidine 13 13C-NMR chart.

[0119] After LC-MS detection, the relative molecular mass of N-acetyl-indigoidine is 290.01, which is in line with the theoretical value. 1 1H-NMR and 13 13C-NMR can infer that the structure is consistent with the structure of N-acetyl-indigoidine.

[0120] The above-described embodiments are only the preferred embodiments of the present invention, and are not an exhaustive list of the feasible embodiments of the present invention. For those of ordinary skill in the art, any obvious changes made without departing from the principle and spirit of the present invention should be considered to be included within the protection scope of the claims of the present invention.

Claims

1. A genetically engineered strain for co-producing indigoidine and N-acetyl-indigoidine, characterized in that, It is obtained by performing any one of the following modifications A - C on the host bacterium: A: Free expression of the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene; B: Knockout of N-acetylglutamate synthase in the host bacterium, and free expression of the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene; C: Free expression of the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene, free expression of the glutamine synthase gene and N-acetylglutamate synthase.

2. The genetically engineered strain according to claim 1, wherein The natural blue pigment synthase gene uses the gene bpsA; The 4'-phosphopantetheinyl transferase gene uses any one of the genes sfp2 and pptA; The glutamine synthase gene uses the gene glnA, and the N-acetylglutamate synthase gene uses the gene argA.

3. The genetically engineered strain according to claim 1, characterized in that, The natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene are respectively expressed under the initiation of the promoter Tac; The plasmid vectors used for the free expression of the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene are pTrc99a or pXMJ19; The natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene are respectively expressed under the initiation of the promoter Tac; The plasmid vector used for the free expression of the natural blue pigment synthase gene and the 4'-phosphopantetheinyl transferase gene is pCDFDuet; The plasmid vector used for the free expression of the natural blue pigment synthase gene is pECXK99E.

4. The genetically engineered strain according to claim 1, characterized in that, The host bacterium includes any one of Escherichia coli BL21(DE3) and Corynebacterium glutamicum 13032.

5. A method for co-producing indigoidine and N-acetyl-indigoidine using the genetically engineered strain according to any one of claims 1-4, characterized in that, It includes: In a fermentation medium with or without the addition of precursors, the genetically engineered bacterium is fermented and cultured to produce indigoidine and N-acetyl-indigoidine; the yield ratio of indigoidine and N-acetyl-indigoidine is 19 - 98%:2 - 81%; The precursors include one or several of L-glutamine, N-acetylglutamine, and N-acetylglutamate.

6. The method according to claim 5, characterized in that, The fermentation process is as follows: in the fermentation medium, the genetically engineered strain is fermented and cultured at 35-38 °C until OD 600 reaches 0.6-0.8, then IPTG with a final concentration of 0.05-1.0 mM is added, and the culture is induced at 28-32 °C for at least 48 h to produce indigoidine and N-acetyl-indigoidine; Alternatively, in the fermentation medium, the genetically engineered strain is fermented and cultured at 35-38 °C until OD 600 reaches 0.6-0.8, then IPTG with a final concentration of 0.05-1.0 mM is added, and after inducing culture at 28-32 °C for at least 24 h, precursors are added and the culture is continued for 24 h to produce indigoidine and N-acetyl-indigoidine.

7. The method according to claim 5, wherein The ratio of indigoidine and N-acetyl-indigoidine in the fermentation broth is regulated by controlling the modification of the host bacterium and the addition of precursors, as shown in Table 1 specifically; Table 1 8. An engineered strain with high-yield N-acetyl-indigoidine, characterized in that, Knockout the N-acetylglutamate synthase gene argA of the host bacterium, and free express the natural blue pigment synthase gene bpsA and the 4'-phosphopantetheinyl transferase gene pptA.

9. A method for producing high-yield N-acetyl-indigoidine, characterized in that, In a fermentation medium with or without precursors, the engineered strain with high yield of N-acetyl-indigoidine according to claim 8 is fermented to produce N-acetyl-indigoidine; The precursors include one or several of L-glutamine, N-acetylglutamine, and N-acetylglutamate.

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