Natural blue pigment and biosynthesis method therefor

Abstract

The present application relates to the technical field of biocatalysis and biosynthesis, and specifically discloses a natural blue pigment and a biosynthesis method therefor. The present application uses metabolically engineered bacteria to express indigoidine synthetase and phosphopantetheinyl transferase, to catalyze glutamine and N-acetylglutamine to biosynthesize a natural blue pigment N-acetylindigoidine, and deduces its molecular structure by means of mass spectrometry, nuclear magnetic resonance and other methods. The present application achieves catalytic synthesis of N-acetylindigoidine from glutamine and N-acetylglutamine in Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae and Streptomyces. Compared with indigoidine, N-acetylindigoidine has a maximum absorption wavelength of 584 nm, possesses better color brightness, and has more stable coloration that is not easily faded, with a very broad application range and industrial production application prospects.

Description

Natural blue pigment and biosynthesis method thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biological catalysis and biosynthesis, in particular to a natural blue pigment and a biosynthesis method thereof. BACKGROUND

[0002] Indigoidine is a non-toxic bacterial natural blue product, which is synthesized by indigoidine synthase, a non-ribosomal peptide synthase (NRPS), by condensation of two molecules of L-glutamine. Indigoidine is bright and deep blue, similar to indigo, and can be applied to the dyeing, food and pharmaceutical industries as a new natural blue pigment and dye, and has been reported to be used for dyeing protein-based fiber fabrics.

[0003] The Chinese patent with the publication number CN 109722401 A discloses the co-expression of the coding gene of the indigoidine synthetase from Streptomyces lavendulae (bpsA) and the coding gene of phosphopantetheinyl transferase from Bacillus subtilis (sfp) in C. glutamicum. The substrate L-glutamine was added at a concentration of 11.68 g / L. After 48 h of induction with 0.8 mM IPTG at 18 °C, the yield of indigoidine was 1.75 g / L. The Chinese patent with the publication number CN 109722401 A discloses the co-expression of the coding genes of indigoidine synthetase, 4’-phosphopantetheinyl transferase and glutamine synthetase in E. coli. The substrate L-glutamate was used for the bioconversion of indigoidine. After 24 h of conversion, the yield of indigoidine reached 5.38 g / L. Wehrs, M. et al. integrated the coding gene of indigoidine synthetase from S. lavendulae (bpsA) and the coding gene of phosphopantetheinyl transferase from B. subtilis (sfp) into the genome of S. cerevisiae. Glucose was used as substrate in a 2 L fermenter for 72 h. The yield of indigoidine reached 980 mg / L (Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae. Microb. Cell Fact. 2018, 17, No. 193). Ming Zhao et al. established an efficient expression and regulation system in Streptomyces for economical and high-level production of the natural blue pigment indigoidine. The coding gene of indigoidine synthetase from S. albus J1704 (indC) was precisely regulated in S. lividans. Glycerol was used as substrate in a 4 L fermenter for 72 h. The yield of indigoidine reached 46.27 g / L (Establishment of an Efficient Expression and Regulation System in Streptomyces for Economical and High-Level Production of the Natural Blue Pigment Indigoidine. J Agric Food Chem Journal of Agricultural and Food Chemistry 2024 72(1), 483-492).

[0004] The above studies respectively use different sources of indigoidine synthetase and different substrates to realize the biosynthesis of indigoidine in C. glutamicum, E. coli, yeast and S. lividans, but N-acetylindigoidine is not found in the above documents.

[0005] SUMMARY

[0006] The present application aims to overcome the deficiencies of the prior art and provide a natural blue pigment and a biosynthesis method thereof. The natural blue pigment (N-acetylindigoidine) obtained by the present application has reduced polarity and water solubility due to the protection of the acetyl group, is not easy to fade, has brighter color, has very broad application range, and has the prospect of industrial production.

[0007] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0008] The present application provides a natural blue pigment, which has a chemical name of N-acetylindigoidine, a molecular formula of C 12 H 10 N4O5, and a chemical structure as shown in formula I.

[0009] In the present application, a new natural blue pigment (N-acetylindigoidine) is newly obtained, and its molecular structure is deduced as shown in formula I by mass spectrometry, nuclear magnetic resonance, etc., and has a molecular formula of C 12 H 10 N4O5, a relative molecular mass of 289.9, a maximum absorption wavelength of 584 nm, 13 C CPMAS nuclear magnetic resonance spectrum δ = 172.23 ppm and δ = 24.90 ppm respectively appear characteristic peaks of -C=O and -CH3 on the acetyl group, and the color is more bright and bright compared with indigoidine.

[0010] The amino group in N-acetylindigoidine forms an amide structure with the acetyl group, reducing the polarity and hydrophilicity of N-acetylindigoidine. Compared with indigoidine, N-acetylindigoidine is more stable and not easy to fade, and at the same time, better color brightness is obtained, and has very broad application range and the prospect of industrial production.

[0011] The present application also provides a biosynthesis method of the above natural blue pigment, which utilizes metabolic engineering bacteria to express indigoidine synthetase and phosphopantetheinyl transferase to catalyze the biosynthesis of natural blue pigment from glutamine and N-acetylglutamine.

[0012] The present application is found through a large number of experiments that the metabolic engineering bacteria expressing the cyanophyll synthetase and the phosphopantetheinyi transferase catalyze the mixture of glutamine and N-acetyl glutamine as a substrate to biosynthesize the natural blue pigment (N-acetyl cyanophyll).

[0013] As a preferred embodiment of the method for biosynthesizing the natural blue pigment according to the present application, the coding gene of the cyanophyll synthetase comprises the cyanophyll synthetase coding gene bpsA or the cyanophyll synthetase coding gene indC.

[0014] The coding gene of the phosphopantetheinyi transferase comprises the phosphopantetheinyi transferase coding gene EntD or the phosphopantetheinyi transferase coding gene indB.

[0015] The present application newly finds that the metabolic engineering bacteria containing the cyanophyll synthetase coding gene bpsA and the phosphopantetheinyi transferase coding gene EntD can synthesize the new compound N-acetyl cyanophyll by using the mixture of glutamine and N-acetyl glutamine as a substrate. It is found through experiments that the metabolic engineering bacteria cannot synthesize N-acetyl cyanophyll by using single glutamine or single N-acetyl glutamine as a substrate.

[0016] The present application introduces the cyanophyll synthetase coding gene indC and the phosphopantetheinyi transferase coding gene indB into the host bacteria, which can also synthesize the new compound N-acetyl cyanophyll by using the mixture of glutamine and N-acetyl glutamine as a substrate.

[0017] In some specific embodiments, the nucleotide sequence of the cyanophyll synthetase coding gene bpsA is shown in SEQ ID NO: 1, the nucleotide sequence of the cyanophyll synthetase coding gene indC is shown in SEQ ID NO: 2, the nucleotide sequence of the phosphopantetheinyi transferase coding gene EntD is shown in SEQ ID NO: 3, and the nucleotide sequence of the phosphopantetheinyi transferase coding gene indB is shown in SEQ ID NO: 4.

[0018] The biosynthetic enzymes of the natural blue pigment according to the present application can be derived from any species, for example, the biosynthetic enzyme of the natural blue pigment can be derived from Streptomyces lavendulae, the nucleotide sequence of the gene bpsA encoding the biosynthetic enzyme of the natural blue pigment is shown in SEQ ID NO: 1, or the biosynthetic enzyme of the natural blue pigment can be derived from Streptomyces chromofuscus ATCC49982, the nucleotide sequence of the gene indC encoding the biosynthetic enzyme of the natural blue pigment is shown in SEQ ID NO: 2. For example, the phosphopantetheinyl transferase can be derived from Corynebacterium glutamicum, the nucleotide sequence of the gene entD encoding the phosphopantetheinyl transferase is shown in SEQ ID NO: 3, or the phosphopantetheinyl transferase can be derived from Streptomyces chromofuscus ATCC49982, the nucleotide sequence of the gene indC encoding the phosphopantetheinyl transferase is shown in SEQ ID NO: 4. The above are only examples, and the biosynthetic enzymes of the natural blue pigment derived from different sources are within the scope of the present application.

[0019] As a preferred embodiment of the biosynthetic method of the natural blue pigment according to the present application, the metabolic engineering bacteria are:

[0020] The metabolic engineering bacteria are constructed by introducing the biosynthetic enzyme of the natural blue pigment encoding gene bpsA and the phosphopantetheinyl transferase encoding gene EntD into the host bacteria;

[0021] Alternatively,

[0022] The metabolic engineering bacteria are constructed by introducing the biosynthetic enzyme of the natural blue pigment encoding gene indC and the phosphopantetheinyl transferase encoding gene indB into the host bacteria.

[0023] As a preferred embodiment of the biosynthetic method of the natural blue pigment according to the present application, the method for constructing the metabolic engineering bacteria comprises the following steps:

[0024] The biosynthetic enzyme of the natural blue pigment encoding gene bpsA and the phosphopantetheinyl transferase encoding gene EntD are respectively amplified by PCR and ligated into a plasmid to obtain the metabolic engineering bacteria;

[0025] Alternatively, the biosynthetic enzyme of the natural blue pigment encoding gene indC and the phosphopantetheinyl transferase encoding gene indB are respectively amplified by PCR and ligated into a plasmid to obtain the metabolic engineering bacteria.

[0026] In some embodiments, the metabolically engineered bacteria is E. coli recombinant strain HG-N-Idg01, which is constructed by introducing expression vector pCDFDuet-bpsA-entD into E. coli BL21(DE3).

[0027] The expression vector pCDFDuet-bpsA-entD is obtained by PCR amplification, gene synthesis and ligation of the biosynthetic gene bpsA derived from S. lavendulae and the phosphopantetheinyl transferase gene EntD derived from C. glutamicum into expression vector pCDFDuet-1.

[0028] The construction method of the expression vector pCDFDuet-bpsA-entD is as follows:

[0029] (1) The biosynthetic gene bpsA and the phosphopantetheinyl transferase gene entD are synthesized by Genescript Biotechnology Co., Ltd.

[0030] (2) The bpsA and entD gene fragments are sequentially inserted into the two multiple cloning sites of pCDFDuet-1 plasmid using the Seamless Cloning Kit of Bao Biological Company to obtain recombinant plasmid pCDFDuet-bpsA-entD.

[0031] (3) The pCDFDuet-bpsA-entD is transformed into E. coli BL21(DE3) using chemical transformation method to obtain E. coli recombinant strain HG-N-Idg-01.

[0032] In some embodiments, the metabolically engineered bacteria is E. coli recombinant strain HG-N-Idg02 overexpressing biosynthetic gene indC derived from S. chromofuscus ATCC49982 and phosphopantetheinyl transferase gene indB, which is constructed by introducing expression vector pCDFDuet-indC-indB into E. coli BL21(DE3).

[0033] The expression vector pCDFDuet-bpsA-entD is obtained by PCR amplification, gene synthesis and ligation of the biosynthetic gene bpsA derived from S. lavendulae and the phosphopantetheinyl transferase gene EntD derived from C. glutamicum into expression vector pCDFDuet-1.

[0034] In some embodiments, the metabolically engineered bacteria is one of C. glutamicum recombinant strain HG-N-Idg03, S. cerevisiae recombinant strain HG-N-Idg04, S. lividans recombinant strain HG-N-Idg05.

[0035] The C. glutamicum recombinant strain HG-N-Idg03 is constructed by introducing the expression vector pXMJ19-bpsA-entD into C. glutamicum ATCC13032.

[0036] The expression vector pXMJ19-bpsA-entD is obtained by PCR amplification and ligation of the bpsA gene encoding the blue synthetase and the EntD gene encoding the phosphopantetheinyl transferase into the expression vector pXMJ19.

[0037] The construction method of the expression vector pXMJ19-bpsA-entD includes the following specific steps:

[0038] (1) The bpsA gene encoding the blue synthetase and the EntD gene encoding the phosphopantetheinyl transferase are obtained by PCR amplification using the plasmid pCDF-bpsA-entD as a template.

[0039] (2) The bpsA and entD gene fragments are inserted into the multiple cloning site of the pXMJ19 plasmid using the seamless cloning kit of Bao Biological Company to obtain the recombinant plasmid pXMJ19-bpsA-entD.

[0040] (3) The recombinant plasmid pXMJ19-bpsA-entD is transformed into C. glutamicum ATCC13032 by electroporation to obtain the C. glutamicum recombinant strain HG-N-Idg03.

[0041] The S. cerevisiae recombinant strain HG-N-Idg04 is constructed by introducing the expression vector pRS425-bpsA-entD into S. cerevisiae INVSC1.

[0042] The expression vector pRS425-bpsA-entD is obtained by PCR amplification, gene synthesis and ligation of the bpsA gene encoding the blue synthetase from S. lavendulae and the EntD gene encoding the phosphopantetheinyl transferase from C. glutamicum into the expression vector pRS425.

[0043] The construction method of the expression vector pRS425-bpsA-entD expression vector is specifically as follows:

[0044] (1) The bpsA gene encoding the cyanophyll synthetase and the EntD gene encoding the phosphopantetheinyl transferase are synthesized by Genesky Biotechnology Co., Ltd.

[0045] (2) The bpsA and entD gene fragments are inserted into the multiple cloning site of the pRS425 plasmid to obtain the recombinant plasmid pRS425-bpsA-entD.

[0046] (3) The recombinant plasmid and the pRS425-bpsA-entD are transformed into Saccharomyces cerevisiae INVSC1 by using the lithium acetate transformation method to obtain the recombinant strain HG-N-Idg-04.

[0047] The S. lividans recombinant strain HG-N-Idg05 is constructed by introducing the expression vector pKCH-PkasO-bpsA-entD into S. lividans TK24.

[0048] The expression vector pKCH-PkasO-bpsA-entD is obtained by, respectively, PCR amplification, gene synthesis, and connection to the plasmid pKC1139 of the hyg r ), the PkasO promoter, the bpsA gene encoding the cyanophyll synthetase derived from S. lavendulae, the EntD gene encoding the phosphopantetheinyl transferase derived from C. glutamicum, and the terminator.

[0049] The construction method of the expression vector pKCH-PkasO-bpsA-entD expression vector is specifically as follows:

[0050] (1) The PermE-SlglnA-SlargA gene sequence, the hygB resistance gene sequence, and the PkasO-bpsA-entD gene sequence are synthesized by Genesky Biotechnology Co., Ltd.

[0051] (2) The hygB resistance gene fragment and the PkasO-bpsA-entD gene fragment are inserted into the multiple cloning site of the pKC1139 plasmid to obtain the recombinant plasmid pKCH-PkasO-bpsA-entD.

[0052] (3) The recombinant plasmid pKCH-PkasO-bpsA-entD is sequentially transformed into S. lividans TK24 by using the conjugation transformation method to obtain the S. lividans recombinant strain HG-N-Idg05.

[0053] The E. coli recombinant strains HG-N-Idg01, HG-N-Idg02, C. glutamicum recombinant strain HG-N-Idg03, S. cerevisiae recombinant strain HG-N-Idg04, and S. lividans recombinant strain HG-N-Idg05 constructed in the present application can express the orsellinate synthase and 4'-phosphopantetheinyl transferase through plasmid high-efficiency expression, and all of them can realize the biosynthesis of N-acetylorcellin with glutamine and N-acetylglutamine as substrates.

[0054] The metabolic engineering bacteria of the present application can be used as a cell catalyst to catalyze the synthesis of a new compound, N-acetylorcellin, from substrates.

[0055] As a preferred embodiment of the biosynthesis method of the natural blue pigment described in the present application, the plasmid I comprises at least one of pCDFDuet, pXMJ19, pRS425, and pKC1139 plasmids.

[0056] As a preferred embodiment of the biosynthesis method of the natural blue pigment described in the present application, the PCR amplification is performed using primers with sequences as shown in SEQ ID NOs: 5-30.

[0057] As a preferred embodiment of the biosynthesis method of the natural blue pigment described in the present application, the host bacteria comprise at least one of E. coli, C. glutamicum, S. cerevisiae, and S. lividans.

[0058] Preferably, the E. coli comprises E. coli BL21 (DE3); the C. glutamicum comprises C. glutamicum ATCC13032; the S. cerevisiae comprises S. cerevisiae INVSC1; and the S. lividans comprises S. lividans TK24.

[0059] The original Escherichia coli BL21 (DE3), Corynebacterium glutamicum ATCC13032, Saccharomyces cerevisiae INVSc1, Streptomyces lividans TK24 cannot utilize glutamine and N-acetylglutamine to synthesize N-acetylindigo, and thus it is necessary to introduce the related enzyme genes into the metabolic engineering bacteria to realize the biological catalysis of N-acetylindigo synthesis.

[0060] As a preferred embodiment of the biosynthesis method of the natural blue pigment, the biosynthesis method comprises: centrifuging to collect the bacteria induced to culture the metabolic engineering bacteria, resuspending the bacteria in a catalytic liquid containing a phosphate buffer, glutamine and N-acetylglutamine, obtaining a conversion liquid after catalysis, and centrifuging to collect the natural blue pigment in the conversion liquid.

[0061] As a preferred embodiment of the biosynthesis method of the natural blue pigment, the concentration of glutamine in the catalytic liquid is 0.1-10 g / L, and the concentration of N-acetylglutamine is 0.1-10 g / L.

[0062] Preferably, the concentration of glutamine is 1 g / L, and the concentration of N-acetylglutamine is 1 g / L.

[0063] The application also provides a purification method of N-acetylindigo, which comprises centrifuging to collect the solid precipitate in the above-mentioned catalytic liquid, extracting N-acetylindigo in the precipitate by using dimethylformamide (DMF), evaporating dimethylformamide by using freeze vacuum drying, sequentially washing the solid by using ultrapure water, methanol, ethyl acetate and n-hexane, and obtaining an N-acetylindigo sample by freeze vacuum drying again.

[0064] The application also provides a cell catalyst, which contains the above-mentioned metabolic engineering bacteria.

[0065] In some specific embodiments, the application provides a cell catalyst containing the recombinant Escherichia coli strain HG-N-Idg01, and the preparation method of the cell catalyst comprises the following specific steps: picking a single colony of the recombinant strain HG-N-Idg01 and transferring it into 5 mL of LB culture medium containing 50 μg / mL of streptomycin, culturing at 37°C and 220 rpm for 16 h, inoculating into 50 mL of ZYM culture medium at a 1% inoculation amount, adding streptomycin at a final concentration of 50 μg / mL and an inducer IPTG at a final concentration of 0.1 mM, culturing at 30°C and 220 rpm for 24 h, and centrifuging to collect the bacteria, wherein the obtained bacteria are the cell catalyst.

[0066] In some embodiments, the present application provides a cell catalyst containing C. glutamicum recombinant strain HG-N-Idg03, and the preparation method of the cell catalyst comprises the following specific steps: picking a single colony of the recombinant strain HG-N-Idg03 and inoculating it into 5 mL of BHISG medium containing 15 μg / mL chloramphenicol and 50 μg / L biotin, and culturing at 30°C and 220 rpm for 16 h; inoculating the culture into 50 mL of GAP medium with a 1% inoculation amount, and adding 15 μg / mL chloramphenicol and 50 μg / L biotin to the medium to a final concentration; culturing at 30°C and 220 rpm for 3 h, then adding 1 mM IPTG to a final concentration, and continuing to culture for 24 h; and centrifuging to collect the bacterial cells, which are the cell catalyst.

[0067] In some embodiments, the present application provides a cell catalyst containing S. cerevisiae recombinant strain HG-N-Idg04, and the preparation method of the cell catalyst comprises the following specific steps: picking a single colony of the recombinant S. cerevisiae HG-N-Idg04 and inoculating it into 5 mL of SC-Leu liquid medium, and culturing at 30°C and 220 rpm for 24 h; centrifuging at 4°C and 1500 g for 5 min to remove the supernatant; resuspending the cell pellet with 30 mL of SC-Leu liquid medium, and adding 10 g / L of galactose; culturing at 30°C and 220 rpm for 24 h; and centrifuging to collect the bacterial cells, which are the cell catalyst.

[0068] In some embodiments, the present application provides a cell catalyst containing S. cerevisiae recombinant strain HG-N-Idg04, and the preparation method of the cell catalyst comprises the following specific steps: picking a single colony of the recombinant S. cerevisiae HG-N-Idg04 and inoculating it into 5 mL of SC-Leu liquid medium, and culturing at 30°C and 220 rpm for 24 h; centrifuging at 4°C and 1500 g for 5 min to remove the supernatant; resuspending the cell pellet with 30 mL of SC-Leu liquid medium, and adding 10 g / L of galactose; culturing at 30°C and 220 rpm for 24 h; and centrifuging to collect the bacterial cells, which are the cell catalyst.

[0069] Compared with the prior art, the present application has the following beneficial effects:

[0070] The application provides a natural blue pigment and a biosynthesis method thereof. The natural blue pigment (N-acetylglauconic acid) obtained by the application has a maximum absorption wavelength of 584 nm due to the protection of the acetyl group, has reduced polarity and water solubility, is not easy to fade, has brighter color, has a very wide application range and an industrial production prospect; the application uses metabolic engineering bacteria to express glauconic acid synthetase and phosphopantetheinyl transferase to catalyze glutamine and N-acetylglutamine to biosynthesize N-acetylglauconic acid, the structure of the substance is inferred, and it is identified as a new type of natural blue pigment. The application can also catalyze glutamine and N-acetylglutamine to synthesize N-acetylglauconic acid in Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae and Streptomyces strains. BRIEF DESCRIPTION OF DRAWINGS

[0071] Fig. 1 is a liquid chromatogram of HG-N-Idg01 catalyzing different substrates;

[0072] Among them, (A) is a liquid chromatogram of a glauconic acid standard;

[0073] (B) is a liquid chromatogram of a product when a BL21 (DE3) strain catalyzes a mixture of glutamine and N-acetylglutamine;

[0074] (C) is a liquid chromatogram of a product when a HG-N-Idg01 strain catalyzes glutamine;

[0075] (D) is a liquid chromatogram of a product when a HG-N-Idg01 strain catalyzes N-acetylglutamine;

[0076] (E) is a liquid chromatogram of a product when a HG-N-Idg01 strain catalyzes a mixture of glutamine and N-acetylglutamine;

[0077] Fig. 2 is a UV-visible absorption spectrum of glauconic acid (A) and N-acetylglauconic acid (B);

[0078] Fig. 3 is an LC-MS detection diagram of N-acetylglauconic acid;

[0079] Fig. 4 is a nuclear magnetic hydrogen spectrum of N-acetylglauconic acid 1 H-NMR diagram;

[0080] Fig. 5 is a nuclear magnetic carbon spectrum of N-acetylglauconic acid 13 C-NMR diagram;

[0081] Fig. 6 is a nuclear magnetic HSQC spectrum of N-acetylglauconic acid;

[0082] Fig. 7 is a HMBC spectrum of N-acetylglauconic acid;

[0083] Fig. 8 is a structure inference diagram of N-acetylglauconic acid;

[0084] Figure 9 is a CPMAS spectrum of N-acetylskyrmionin;

[0085] Figure 10 is a CPMAS spectrum of N-acetylskyrmionin;

[0086] Figure 11 is a biosynthetic pathway of N-acetylskyrmionin in HG-N-Idg01 strain;

[0087] Figure 12 is a liquid chromatogram of catalytic product of HG-N-Idg01 (A) and HG-N-Idg02 (B);

[0088] Figure 13 is a liquid chromatogram of catalytic product of C. glutamicum 13032 (A) and HG-N-Idg03 (B);

[0089] Figure 14 is a liquid chromatogram of catalytic product of S. cerevisiae INVSc1 (A) and HG-N-Idg04 (B);

[0090] Figure 15 is a liquid chromatogram of catalytic product of S. lividans TK24 (A) and HG-N-Idg05 (B);

[0091] Figure 16 is a color comparison of N-acetylskyrmionin (A) and skyrmionin (B) in DMSO solution;

[0092] Figure 17 is a color comparison of N-acetylskyrmionin (A) and skyrmionin (B) in water solution. DETAILED DESCRIPTION

[0093] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described in combination with the drawings and specific examples.

[0094] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0095] The medium components involved in the following examples are as follows:

[0096] LB (Luria-Bertani) liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of NaCl, sterilized at 121°C for 20 min.

[0097] LB (Luria-Bertani) solid medium: 10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of NaCl, 15 g / L of agar powder, sterilized at 121°C for 20 min, and the solid medium is cooled to about 50°C, then the required antibiotics are added and poured onto the plate, and after solidification, it is placed at 4°C for standby.

[0098] ZYM fermentation medium: 96 mL ZY medium + 2 mL 50x M salts + 2 mL 50x5052 + 200 μL 1 mol / L magnesium sulfate + 100 μL 1000x trace elements, 20 g / L glucose.

[0099] ZY medium: 10 g / L peptone, 5 g / L yeast extract, sterilized at 121 °C for 20 min and stored.

[0100] 50x M salts: 1.25 mol / L Na2HPO4, 1.25 mol / L KH2PO4, 2.5 mol / L NH4Cl and 0.25 mol / L Na2SO4.

[0101] 50x5052: 250 g / L glycerol, 25 g / L glucose; 1 mol / L MgSO4.

[0102] 1000x trace elements: 50 mmol / L FeCl3, 20 mmol / L CaCl2, 10 mmol / L MnCl2, 10 mmol / L ZnSO4, 2 mmol / L each of CoCl2, NiCl2, Na2Mo4, Na2SeO3 and H3BO3.

[0103] BHISG medium: 37 g / L brain heart infusion powder, 10 g / L glucose.

[0104] GAP medium: 70 g / L glucose, 40 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.4 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate, 0.01 g / L manganese sulfate, 4 μg / L biotin, 200 μg / L VB1, 50 g / L calcium carbonate. Adjust pH to 8.0 with KOH and sterilize at 115 °C for 30 min.

[0105] MS medium: 20 g / L mannitol, 20 g / L soybean powder and 15 g / L agar.

[0106] TSB medium: 17 g / L casein peptone, 3 g / L soy peptone, 2.5 g / L glucose, 5 g / L NaCl and 2.5 g / L K2HPO4.

[0107] The primers involved in the following examples are shown in Table 1.

[0108] Table 1

[0109] The strain information involved in the following examples is shown in Table 2.

[0110] Table 2

[0111] The bpsA gene for violacein synthetase mentioned in the present application is derived from S. lavendulae; the indC gene for violacein synthetase is derived from S. chromofuscus ATCC49982; the EntD gene for phosphopantetheinyl transferase is derived from C. glutamicum; and the indB gene for phosphopantetheinyl transferase is derived from S. chromofuscus ATCC49982.

[0112] The nucleotide sequence of the bpsA gene for violacein synthetase is shown as SEQ ID NO: 1, the nucleotide sequence of the indC gene for violacein synthetase is shown as SEQ ID NO: 2, the nucleotide sequence of the EntD gene for phosphopantetheinyl transferase is shown as SEQ ID NO: 3, and the nucleotide sequence of the indB gene for phosphopantetheinyl transferase is shown as SEQ ID NO: 4.

[0113] In the following examples, the sample high performance liquid chromatography detection method is as follows.

[0114] Chromatographic conditions:

[0115] Mobile phase: methanol and pure water gradient elution, gradient table 3 as follows:

[0116] Table 3

[0117] Wavelength 600 nm, flow rate 1.0 ml / min, sample solution: DMSO, sample amount: 10 μL, column temperature 35℃, running time 18 min.

[0118] Chromatographic column: EF-C18M 4.6mm id x 250mm L (SN B06211801).

[0119] Example 1, construction of HG-N-Idg01 strain

[0120] The construction method of the HG-N-Idg01 strain of the present example includes the following steps:

[0121] Step one, construction of pCDF-bpsA-entD plasmid:

[0122] (1) The bpsA gene for violacein synthetase (nucleotide sequence as shown in SEQ ID NO. 1) and the entD gene for 4'-phosphopantetheinyl transferase (nucleotide sequence as shown in SEQ ID NO. 3) are synthesized by Qianke Biotechnology Co., Ltd.

[0123] (2) Using the commercially available plasmid pCDFDuet-1 as a template, PCR amplification was performed using primers pCDF-I-F1 and pCDF-I-R1, and the product was purified to obtain the linearized vector pCDFDuet-1.

[0124] (3) The pCDFDuet-1 linearized vector and the bpsA gene (with homologous arms of the vector added to both ends of the gene during gene synthesis) were ligated using a seamless cloning ligation kit. The ligation product was then transformed into Escherichia coli DH5α using chemical transformation. After revival culture, the product was plated onto LB solid medium plates containing 50 μg / mL streptomycin resistance and incubated at 37°C for about 16 h.

[0125] (4) Use primers pCDF-YZ-F1 and pCDF-YZ-R1 to perform colony PCR verification. Culture the strains that are verified correctly by PCR, extract the recombinant plasmid, and send it to Qingke Biotechnology Co., Ltd. for sequencing. The plasmid that is correctly sequenced is pCDF-bpsA plasmid.

[0126] (5) Using pCDF-bpsA plasmid as a template, PCR amplification was performed using pCDF-I-F2 and pCDF-I-R2, and the product was purified to obtain the pCDF-bpsA linearized vector. The pCDF-bpsA linearized vector and the entD fragment encoding the 4'-phosphopanylthioethylamine transferase gene were ligated using a seamless cloning ligation kit. The ligation product was transformed into Escherichia coli DH5α by chemical transformation. After resuscitation culture, it was plated on LB solid medium plates containing 50 μg / mL streptomycin resistance and incubated at 37°C for about 16 h.

[0127] (6) Colony PCR verification was performed using primers pCDF-YZ-F2 and pCDF-YZ-R2. The strains that were verified correctly by PCR were cultured, and the recombinant plasmids were extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The plasmids that were correctly sequenced were pCDF-bpsA-entD (the sequence is shown in SEQ ID NO: 31).

[0128] Step 2: Construction of HG-N-Idg01 strain:

[0129] (1) BL21(DE3) strains were purchased from Qingke Biotechnology Co., Ltd., catalog number TSC-C14.

[0130] (2) The pCDF-bpsA-entD plasmid was transformed into Escherichia coli BL21(DE3) by chemical transformation. After recovery and culture, it was spread on LB solid medium plates containing 50 μg / mL streptomycin resistance and incubated at 37°C for about 16 h.

[0131] (3) LB solid medium plate grown single colonies of recombinant E. coli BL21(DE3) / pCDF-bpsA-entD, named HG-N-Idg01.

[0132] Example 2, preparation of recombinant E. coli cell catalyst

[0133] (1) The above-mentioned recombinant E. coli HG-N-Idg01 monoclonal was inoculated into 5 mL LB liquid medium containing 50 μg / mL of streptomycin resistance, and cultured at 37°C, 220 rpm for 8 h.

[0134] (2) According to the inoculation amount of 1%, it was inoculated into 50 mL ZYM fermentation medium containing 50 μg / mL of streptomycin resistance and 0.1 mM of IPTG, and cultured at 30°C, 220 rpm for 16 h.

[0135] (3) After the culture was completed, the bacteria were collected by centrifugation at 4000 rpm for 10 min, and the obtained bacteria were cell catalysts.

[0136] Example 3, catalytic synthesis of N-acetylglucosamine with different substrates

[0137] The HG-N-Idg01 bacteria obtained in Example 1 were used as cell catalysts to perform the following catalytic experiments, and the results are shown in Figure 1.

[0138] Experiment group one: 2.7 mg of standard sample of glucosamine was taken into a 25 mL volumetric flask, DMSO solution was added and ultrasonicated for 15 min to dissolve completely, and then cooled and diluted to the mark. High performance liquid chromatography (HPLC) detection was performed, and the detection results are shown in Figure 1-A.

[0139] Experiment group two: 50 mL of raw E. coli BL21(DE3) fermentation broth cultured according to the method in Example 2 was centrifuged, and 10 mL of catalytic liquid (glutamine 1 g / L, N-acetylglutamine 1 g / L and 50 mM PB buffer) was used to resuspend the bacteria. The reaction was carried out at 25°C, 220 rpm for 6 h, 100 μL of sample was taken, and high performance liquid chromatography detection was performed. The detection results are shown in Figure 1-B.

[0140] Experiment group three: 50 mL of fermentation broth of HG-N-Idg01 strain cultured according to the method in Example 2 was centrifuged, and 10 mL of catalytic liquid (glutamine 2 g / L and 50 mM PB buffer) was used to resuspend the bacteria. The reaction was carried out at 25°C, 220 rpm for 6 h, 100 μL of sample was taken, and high performance liquid chromatography detection was performed. The detection results are shown in Figure 1-C.

[0141] Experimental group four: centrifugal collection of 50 mL of fermentation broth of HG-N-Idg01 strain cultured according to the method in Example 2, resuspension of the bacterial cells using 10 mL of catalytic liquid (N-acetyl glutamine 2 g / L and 50 mM PB buffer), reaction at 25°C, 220 rpm for 6 h, sampling of 100 uL, high performance liquid chromatography detection, and the detection results are shown in Figure 1-D.

[0142] Experimental group five: centrifugal collection of 50 mL of fermentation broth of HG-N-Idg01 strain cultured according to the method in Example 2, resuspension of the bacterial cells using 10 mL of catalytic liquid (glutamine 1 g / L, N-acetyl glutamine 1 g / L and 50 mM PB buffer), reaction at 25°C, 220 rpm for 6 h, sampling of 100 uL, high performance liquid chromatography detection, and the detection results are shown in Figure 1-E.

[0143] The results are shown in Figure 1. In Figure 1-A, the peak time of the observed blue standard is about 8.8 min. In Figure 1-B, no obvious product peak is observed. In Figure 1-C, there is a peak at about 8.8 min, which is consistent with the peak time of the observed blue standard, and the catalytic product is observed blue. In Figure 1-D, no obvious product peak is observed. In Figure 1-E, there are peaks at about 8.8 min and about 9.9 min, respectively, and it is speculated that the products are observed blue and N-acetyl observed blue, respectively. It is shown that the original strain BL21 (DE3) cannot catalyze glutamine 1 g / L and N-acetyl glutamine 1 g / L to synthesize N-acetyl observed blue, and the recombinant Escherichia coli HG-N-Idg01 realizes the synthesis of N-acetyl observed blue from the mixture of glutamine and N-acetyl glutamine by overexpression of bpsA and entD genes through a plasmid, and neither single substrate glutamine nor N-acetyl glutamine can synthesize N-acetyl observed blue.

[0144] Figure 2 is the wavelength corresponding to the maximum absorption peak of experimental group five, in which the absorption peak of observed blue at 8.8 min corresponds to a wavelength of 600 nm, as shown in Figure 2-A; the absorption peak of N-acetyl observed blue at 9.9 min corresponds to a wavelength of 584 nm, as shown in Figure 2-B.

[0145] Example 4, structure identification of N-acetyl observed blue sample:

[0146] The catalytic reaction liquid of experimental group five in Example 3 was collected, centrifuged at 10000 g for 5 min, and after removing the supernatant, the precipitated cells were resuspended with 10 mL of dimethylformamide (DMF), and the cells were broken by ultrasonic to extract N-acetyl observed blue into the solvent. The supernatant was obtained by centrifugation again, the organic solvent was evaporated under vacuum, and then the dark solid was washed with 10 mL of pure water, methanol, ethyl acetate and hexane twice, respectively, and finally vacuum freeze-dried to obtain 0.14 g of N-acetyl observed blue powder. The above sample was sent to the Analysis and Test Center of Nanjing Normal University for LC-MS detection and NMR detection.

[0147] Figure 3 shows the LC-MS detection pattern; Figure 4 shows the N-acetylated blue color. 1 ¹H-NMR spectrum; Figure 5 shows N-acetylated blue. 13 C-NMR spectrum; Figures 6-7 are the NMR HSQC and HMBC spectra of N-acetylglucosamine; Figure 8 is the inferred structure of N-acetylglucosamine; Figure 9 is the NMR CPMAS spectrum of glimbrolizumab; Figure 10 is the NMR CPMAS spectrum of N-acetylglucosamine.

[0148] Table 4 shows 1 H-NMR (Figure 4) and 13 Correspondence between C-NMR data (Figure 5) and element positions in the inferred structure (Figure 8).

[0149] Table 4. Analysis of NMR Data

[0150] The relative molecular mass of the blue acetyl group was 248.19, and the relative molecular mass of the acetyl group was 43. The LC-MS detection results (Figure 3) showed that the relative molecular mass of the sample was 289.9, which was consistent with the theoretical value of N-acetylated blue acetyl.

[0151] like 1 The 1H-NMR spectrum (Figure 4) shows a peak with a δ value of approximately 2.50 ppm, corresponding to the solvent DMSO, and a peak with a δ value of 3.36 ppm, corresponding to water. Using the isolated peak at 11.71 ppm as the integration standard, the integral is 1. Looking at the HSQC spectrum (Figure 6), there is no point on the horizontal axis corresponding to 11.71 ppm, suggesting hydrogen on nitrogen; similarly, 11.43 ppm, 7.74 ppm, and 9.34 ppm also suggest hydrogen on nitrogen; the peaks at 8.35 ppm and 9.62 ppm are singlets, corresponding to carbon atoms at 100-160 ppm in the HSQC spectrum (Figure 6), suggesting alkene carbons; the 2.10 ppm peak is an s peak with an integral of 3, suggesting the presence of a methyl group. The 7.74 ppm peak has an integral of 2, suggesting a primary amine; the peaks at 11.71 ppm, 11.43 ppm, and 9.34 ppm have integrals of 1, suggesting a secondary amine. 1 H-NMR analysis revealed that the compound contains one primary amine, three secondary amines, two double bonds, and one methyl group.

[0152] Combined 13C-NMR spectrum (Figure 5) analysis, the δ value is 24.54 ppm is the methyl signal; the δ value is 160 ppm-180 ppm is the carbonyl signal, so there are δ value of 169.50 ppm, 165.44 ppm, 165.06 ppm, 160.70 ppm, 160.38 ppm five carbon base; generally 100-150 ppm is SP2 hybrid carbon atom, 122.88 ppm and 106.31 ppm peak is and 1 H-NMR spectrum corresponding to the carbon, suggesting that the olefin carbon, the remaining 143.01 ppm, 134.87 ppm, 125.72 ppm, 118.39 ppm are unsaturated carbon. In summary, and combined with the blue structure, it is speculated that the structure of N-acetyl blue is shown in Figure 8. 1 H-NMR spectrum and 13 The assignment of C-NMR spectrum signal is shown in Table 4.

[0153] By calculation, the molecular formula of the new substance is C 12 H 10 N4O5, by comparing the nuclear magnetic CPMAS spectrum of blue and N-acetyl blue (Figure 9 and Figure 10), δ = 172.23 ppm and δ = 24.90 ppm respectively appear the characteristic peaks of -C = O and -CH3 on the acetyl group, so it is named as N-acetyl blue. This structure has not been reported in the literature, it is a new substance. This new substance presents a deep blue color in solution, the maximum light absorption value is 584 nm, it is inferred to be a new type of blue pigment.

[0154] The biosynthetic pathway of N-acetyl blue of HG-N-Idg01 strain is shown in Figure 11.

[0155] Example 5, construction of HG-N-Idg02 strain

[0156] In order to compare the biological activities of blue synthetase and phosphopantetheinyl transferase from different species, the present application constructs an Escherichia coli recombinant strain HG-N-Idg02 which overexpresses the blue synthetase encoding gene (indC) and the phosphopantetheinyl transferase encoding gene (indB) from S. chromofuscus ATCC49982.

[0157] The construction process is as follows:

[0158] Step one, pCDF-indB-indC plasmid construction:

[0159] (1) The indC gene encoding the violacein synthase (nucleotide sequence as shown in SEQ ID NO. 3) and the indB gene encoding the 4'-phosphopantetheinyl transferase (nucleotide sequence as shown in SEQ ID NO. 4) were synthesized by Genescript Biotech Co., Ltd.

[0160] (2) The pCDF-indB-indC plasmid was constructed according to the method of Example 1.

[0161] Step two, construction of HG-N-Idg02 strain:

[0162] The pCDF-indB-indC constructed in step one was transformed into BL21 (DE3) by chemical transformation method to obtain recombinant Escherichia coli BL21 (DE3) / pCDF-indB-indC, named HG-N-Idg02.

[0163] Example 6, comparison of catalytic ability of violacein synthases from different sources

[0164] The cell catalysts of HG-N-Idg01 and HG-N-Idg02 were prepared using the culture method in Example 2, and 10 mL of catalytic liquid (1 g / L glutamine + 1 g / L N-acetyl glutamine + 50 mM PB) was used to resuspend the bacterial cells, which were placed at 20°C, 220 rpm for 6 h, and 100 uL of each was taken for HPLC detection. The test results are shown in Figure 12, and two peaks appeared at 8.8 min and 9.9 min in the HG-N-Idg01 and HG-N-Idg02 catalytic reaction liquid, and the peak area of the catalytic products of HG-N-Idg01 and N-acetyl violacein was greater than that of HG-N-Idg02. The results show that indB and indC can catalyze glutamine and N-acetyl glutamine to generate violacein and N-acetyl violacein, but the enzyme activity is lower than that of bpsA and entD.

[0165] The present application uses violacein synthases and 4'-phosphopantetheinyl transferases from different sources to realize the biosynthesis of N-acetyl violacein in Escherichia coli.

[0166] Example 7, construction of recombinant Corynebacterium glutamicum HG-N-Idg03

[0167] The present example provides a method for constructing recombinant Corynebacterium glutamicum HG-N-Idg03, comprising the following steps:

[0168] Step one, construction of pXMJ19-bpsA-entD plasmid:

[0169] (1) Using the pCDF-bpsA-entD plasmid constructed in Example 1 as a template, primers pXMJ19-bpsA-F and pXMJ19-bpsA-R were used to perform PCR amplification to obtain a bpsA gene fragment; primers pXMJ19-entD-F and pXMJ19-entD-R were used to perform PCR amplification to obtain an entD gene fragment;

[0170] (2) Using the commercially available plasmid pXMJ19 as a template, primers pXMJ19-I-F and pXMJ19-I-R were used to perform PCR amplification to obtain a pXMJ19 linearized vector.

[0171] (3) The seamless cloning kit of Baobio was used for ligation, and the ligation product was transformed into E. coli DH5α. After recovery and culture, it was plated on LB solid medium containing 15 μg / mL chloramphenicol resistance and cultured in a 37°C incubator for about 16 h.

[0172] (4) Primers pXMJ19-YZ-F and pXMJ19-YZ-R were used for colony PCR verification, and the strain with correct PCR verification was cultured to extract the recombinant plasmid, which was sent to the Qikang Biotechnology Company for sequencing. The correct sequencing was the pXMJ19-bpsA-entD plasmid.

[0173] Step two, preparation of ATCC13032 electrotransformation competent cells:

[0174] (1) A single colony of ATCC13032 was picked into 5 mL of antibiotic-free BHISG medium and cultured overnight at 30°C and 220 rpm;

[0175] (2) OD 600 was measured, and 15 OD of bacterial solution was inoculated into 50 mL of BHISGGT medium (0.1% Tween 80 and 50 μg / L biotin) and cultured at 30°C and 220 rpm until the OD 600 was about 1.0, and then it was removed and placed on ice for 20 min;

[0176] (3) The bacterial solution was transferred to a 50 ml centrifuge tube, centrifuged at 4°C and 2600 x g for 10 min, the supernatant was discarded, and the residual bacterial solution was removed with a gun;

[0177] (4) 50 mL of 10% glycerol (pre-cooled) was added to resuspend the bacterial cells, centrifuged at 4°C and 2600 x g for 10 min, the supernatant was discarded, and the residual bacterial solution was removed with a gun; this step was repeated once;

[0178] (5) 100 μL of 10% glycerol (pre-cooled) was used to resuspend the bacterial cells, which were transferred to a 1.5 mL EP tube and used as needed.

[0179] Step three, construction of recombinant strain HG-N-Idg03:

[0180] (1) Take 200 ng pXMJ19-bpsA-entD plasmid and add to the electrotransformation competent cells prepared in step two, mix gently and uniformly, then add to the electrotransformation cup, ice bath for 5-10 min;

[0181] (2) After 1.8KV electric shock for 2-3 times, quickly add 46℃ preheated BHISG medium, heat shock in 46℃ water bath for 6 min, then place the heat shocked transformation liquid in 30℃ incubator for 2 h;

[0182] (3) After incubation, centrifuge 1 mL transformation liquid at 4000 rpm, discard most of the supernatant, resuspend the remaining part, then spread on chloramphenicol resistant BHISG plate, and place in 30℃ incubator for 48 h.

[0183] (4) The grown single colony is the recombinant coryneform bacterium HG-N-Idg03.

[0184] Example 8, recombinant coryneform bacterium HG-N-Idg03 catalyzes synthesis of N-acetylglutamic acid

[0185] Pick the recombinant coryneform bacterium strain HG-N-Idg03 single colony and transfer to 5 mL BHISG medium containing 15 μg / mL chloramphenicol and 50 μg / L biotin, incubate at 30℃, 220 rpm for 16 h, inoculate into 50 mL GAP medium with 1% inoculation amount, add 15 μg / mL chloramphenicol and 50 μg / L biotin to the medium to a final concentration, incubate at 30℃, 220 rpm for 3 h, then add 1 mM IPTG to a final concentration, continue to incubate for 24 h, centrifuge to collect the bacterial cells as the cell catalyst.

[0186] Resuspend the bacterial cells with 10 mL catalytic reaction liquid (glutamine 1 g / L, N-acetylglutamine 1 g / L, 50 mM PB), incubate at 25℃, 220 rpm for 6 h, take 100 uL sample, and detect by HPLC. Use the original strain coryneform bacterium ATCC13032 as the control group.

[0187] The test results are shown in Figure 13, Figure 13-A is the test results of ATCC13032 strain, no obvious peak is found at 8.8 min and 9.9 min; Figure 13-B is the test results of HG-N-Idg03 strain, there is a peak at 8.8 min and 9.9 min, which is consistent with the peak time of indigo and N-acetyl indigo. It is shown that the original strain ATCC13032 cannot catalyze the synthesis of N-acetyl indigo from glutamine and N-acetyl glutamine, and the recombinant corynebacterium glutamicum strain HG-N-Idg03 realizes the catalysis of the synthesis of N-acetyl indigo from glutamine and N-acetyl glutamine by overexpressing the bpsA and entD genes through the plasmid.

[0188] Example 9, construction of recombinant saccharomyces cerevisiae HG-N-Idg04

[0189] The present embodiment provides a method for constructing recombinant saccharomyces cerevisiae HG-N-Idg04, comprising the following steps:

[0190] Step one, construction of pRS425-bpsA-entD plasmid:

[0191] (1) The bpsA gene encoding indigo synthase is synthesized by GenScript, and the TEF1 promoter and CYC1 terminator sequences are introduced during synthesis. The TEF1 promoter-bpsA-CYC1 terminator expression frame nucleotide sequence is shown in SEQ ID NO: 32.

[0192] (2) The entD gene encoding 4'-phosphopantetheinyl transferase is synthesized by GenScript, and the TEF1 promoter and ADH1 terminator sequences are introduced during synthesis. The TEF1 promoter-entD-ADH1 terminator expression frame nucleotide sequence is shown in SEQ ID NO: 33.

[0193] (3) The pRS425 plasmid purchased in the market is used as a template, and primers pRS425-I-F and pRS425-I-R are used for PCR amplification to obtain a pRS425 linearized vector.

[0194] (4) The pRS425 linearized vector, TEF1 promoter-bpsA-CYC1 terminator fragment, and TEF1 promoter-entD-ADH1 terminator are connected using a seamless cloning kit. The ligation product is transformed into E. coli DH5a using chemical transformation method. After recovery and culture, it is spread on LB solid medium containing 100 μg / mL ampicillin resistance, and cultured in a 37°C incubator for about 16 h.

[0195] (5) Using primers pRS425-YZ-F and pRS425-YZ-R for colony PCR verification, the correct strain was cultured, the recombinant plasmid was extracted, and sent to Genescript Biotech Co., Ltd. for sequencing. The correct sequencing is pRS425-bpsA-entD plasmid.

[0196] Step two, HG-N-Idg04 strain construction:

[0197] The recombinant plasmid pRS425-bpsA-entD was transformed into Saccharomyces cerevisiae INVSc1 by lithium acetate transformation method. After recovery and culture, it was coated on SC-Leu selective medium and cultured at 30°C for 48h. The single colony that grew was the recombinant Saccharomyces cerevisiae INVSc1 / pRS425-bpsA-entD, named as recombinant Saccharomyces cerevisiae HG-N-Idg04.

[0198] Example 10, recombinant Saccharomyces cerevisiae HG-N-Idg04 catalyzing the synthesis of N-acetyl-oxynanduline

[0199] The recombinant Saccharomyces cerevisiae HG-N-Idg04 single colony was inoculated into 5mL of SC-Leu liquid medium and cultured at 30°C, 220rpm for 24h. Centrifuged at 4°C, 1500g for 5min, and the supernatant was removed. Resuspend the cell pellet with 50mL of SC-Leu liquid medium, add 10g / L of galactose, and culture at 30°C, 220rpm for 24h. Centrifuge to collect the bacteria, which is the HG-N-Idg04 cell catalyst. Resuspend the bacteria with 10mL of catalytic liquid (glutamine 1g / L, N-acetyl glutamine 1g / L, 50mM PB), and react at 25°C, 220rpm for 6h. Take 100uL of sample for HPLC detection. The control group is the original strain INVSc1.

[0200] The test results are shown in Figure 14, Figure 14-A is the test result of INVSc1 strain, no obvious peak is found at 8.8min and 9.9min; Figure 14-B is the test result of HG-N-Idg04 strain, there is a peak at 8.8min and 9.9min, which is consistent with the peak time of oxynanduline and N-acetyl-oxynanduline. It shows that the original strain INVSc1 cannot catalyze glutamine and N-acetyl glutamine to synthesize N-acetyl-oxynanduline, and the recombinant Saccharomyces cerevisiae HG-N-Idg04 can catalyze glutamine and N-acetyl glutamine to synthesize N-acetyl-oxynanduline by overexpressing bpsA and entD genes through plasmid.

[0201] Example 11, construction of recombinant Streptomyces lividans HG-N-Idg05

[0202] The embodiment provides a construction method of a recombinant Streptomyces lividans HG-N-Idg05, comprising the following steps.

[0203] Step one, construction of a hygromycin B resistant plasmid pKC-hyg:

[0204] (1) Taking a commercially available plasmid pKC1139 as a template, PCR amplification is carried out by using primers pKCH-hyg-I-F and pKCH-hyg-I-R, so that a pKC1139 linearized vector is obtained.

[0205] (2) A hygromycin B resistance gene (hyg r ) is synthesized by a company, and the nucleotide sequence is shown in SEQ ID NO: 34.

[0206] (3) The pKC1139 linearized vector and the hygromycin B resistance gene fragment are connected by using a seamless cloning kit of a company, and the connection product is transformed into E. coli DH5α; after recovery culture, coating is carried out on an LB solid culture medium plate containing 100 μg / mL of hygromycin B resistance, and culture is carried out in a 37°C incubator for about 16 hours.

[0207] (4) Colony PCR verification is carried out by using primers pKC-hyg-YZ-F and pKC-hyg-YZ-R, and a strain that is correctly verified by PCR is cultured, a recombinant plasmid is extracted, and the sequencing is carried out by a company, so that the correct sequencing is the hygromycin B resistant plasmid pKC-hyg.

[0208] Step two, construction of a pKCH-PkasO-bpsA-entD plasmid:

[0209] (1) Taking the pKC-hyg plasmid constructed in step one as a template, PCR amplification is carried out by using primers pKCH-I-F and pKCH-I-R, so that a pKCH-hyg linearized vector is obtained.

[0210] (2) A PkasO-bpsA-entD expression frame gene sequence is synthesized by a company, and the nucleotide sequence is shown in SEQ ID NO: 35.

[0211] (3) The pKCH-hyg linearized vector and the PkasO-bpsA-entD are connected by using a seamless cloning kit of a company, and the connection product is transformed into E. coli DH5α; after recovery culture, coating is carried out on an LB solid culture medium plate containing 100 μg / mL of hygromycin B resistance, and culture is carried out in a 37°C incubator for about 16 hours.

[0212] (4) Using primers pKCH-YZ-F and pKCH-YZ-R for colony PCR verification, the correct strain for PCR verification was cultured, the recombinant plasmid was extracted, and was sent to the Bio-Company for sequencing. The correct sequencing was pKCH-PkasO-bpsA-entD plasmid.

[0213] Step three, recombinant strain HG-N-Idg05 construction:

[0214] Streptomyces lividans TK24 and E. coli ET12567 / pUZ8002 were purchased from Bio-Company. The recombinant plasmid pKCH-PkasO-bpsA-entD was transformed into Streptomyces lividans TK24 by conjugation transfer, and then HG-N-Idg05 was constructed.

[0215] The specific experimental process is as follows:

[0216] (1) The plasmid pKCH-PkasO-bpsA-entD used for conjugation transfer was transformed into E. coli ET12567 / pUZ8002;

[0217] (2) The E. coli ET12567 / pUZ8002 containing the plasmid was used for two-parent conjugation transfer with Streptomyces lividans TK24;

[0218] (3) After 18h incubation at 28℃, hygromycin B and nalydic acid were used for covering;

[0219] (4) After 3-5 days of incubation at 28℃, the conjugation transfer was picked up and the genomic DNA was extracted;

[0220] (5) Using primers pKCH-YZ-F and pKCH-YZ-R for PCR verification, the correct recombinant Streptomyces lividans HG-N-Idg05: TK24 / pKCH-PkasO-bpsA-entD was obtained.

[0221] Example 12, recombinant Streptomyces lividans HG-N-Idg05 catalyzing synthesis of N-acetylglucosamine

[0222] The recombinant Streptomyces lividans HG-N-Idg05 grown on MS medium for 4-5 days was picked up with a sterile gun head and inoculated into a primary shake flask containing 10 mL of TSB medium. After 48 h of culture at 28°C, 1 mL was transferred into a secondary shake flask containing 50 mL of TSB medium, and cultured at 28°C for 72 h. The bacterial cells were collected by centrifugation, which were the HG-N-Idg06 cell catalyst. The bacterial cells were resuspended in 10 mL of catalytic liquid (glutamine 1 g / L, N-acetylglutamine 1 g / L, 50 mM PB) and reacted at 25°C and 220 rpm for 6 h. 100 uL of the sample was taken for HPLC detection. The control group was the original strain Streptomyces lividans TK24.

[0223] The test results are shown in FIG. 15. FIG. 15-A is the test results of Streptomyces lividans TK24, and no obvious peaks are observed at 8.8 min and 9.9 min. FIG. 15-B is the test results of the HG-N-Idg05 strain, and there is a peak at about 8.8 min and 9.9 min, which is consistent with the peak time of observarine and N-acetyl observarine. It is indicated that the original strain TK24 cannot catalyze the synthesis of N-acetyl observarine from glutamine and N-acetylglutamine, and the recombinant Streptomyces lividans HG-N-Idg05 realizes the catalysis of N-acetyl observarine from glutamine and N-acetylglutamine by overexpressing the bpsA and entD genes through a plasmid.

[0224] The application realizes the catalysis of N-acetyl observarine from glutamine and N-acetylglutamine in Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae and Streptomyces.

[0225] Comparative Example 1, color comparison of observarine and N-acetyl observarine DMSO solution

[0226] 1 mg of observarine standard was weighed and resuspended in 10 mL of DMSO, and 1 mg of purified N-acetyl observarine in Example 4 was weighed and resuspended in 10 mL of DMSO. The color difference between the two groups was observed, and the results are shown in FIG. 16. FIG. 16-A is the N-acetyl observarine DMSO solution, which is bright blue. FIG. 16-B is the observarine DMSO solution, which is blue. The N-acetyl observarine DMSO solution is brighter and more vibrant than the observarine DMSO solution.

[0227] Comparative Example 2, color comparison of observarine and N-acetyl observarine aqueous solution

[0228] 1 mg of observarine standard was weighed and resuspended in 10 mL of DMSO, and 1 mg of purified N-acetyl observarine in Example 4 was weighed and resuspended in 10 mL of DMSO. The color difference between the two groups was observed, and the results are shown in FIG. 16. FIG. 16-A is the N-acetyl observarine DMSO solution, which is bright blue. FIG. 16-B is the observarine DMSO solution, which is blue. The N-acetyl observarine DMSO solution is brighter and more vibrant than the observarine DMSO solution.

[0229] Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A natural blue pigment, characterized in that, The chemical name of the natural blue pigment is N-acetyl-indigoidine, the molecular formula is C 12 H 10 N4O5, the chemical structure is shown as formula I; 2. The method for biosynthesis of natural blue pigments according to claim 1, characterized in that, The biosynthesis method utilizes a metabolic engineering bacterium to express an orceline synthase and a phosphopantetheinyl transferase to catalyze biosynthesis of natural blue pigment from glutamine and N-acetylglutamine.

3. The method for biosynthesis of natural blue pigments according to claim 2, characterized in that, The orceline synthase-encoding gene comprises an orceline synthase-encoding gene bpsA or an orceline synthase-encoding gene indC. The phosphopantetheinyl transferase-encoding gene comprises a phosphopantetheinyl transferase-encoding gene EntD or a phosphopantetheinyl transferase-encoding gene indB.

4. The method for biosynthesis of natural blue pigments according to claim 2, characterized in that, The metabolic engineering bacterium is: The metabolic engineering bacterium is constructed by introducing the orceline synthase-encoding gene bpsA and the phosphopantetheinyl transferase-encoding gene EntD into a host bacterium. Alternatively, The metabolic engineering bacterium is constructed by introducing the orceline synthase-encoding gene indC and the phosphopantetheinyl transferase-encoding gene indB into a host bacterium.

5. The method of biosynthesis of natural blue pigments according to claim 4, characterized in that, The construction method of the metabolic engineering bacterium comprises the following steps: The orceline synthase-encoding gene bpsA and the phosphopantetheinyl transferase-encoding gene EntD are respectively amplified by PCR and connected to a plasmid to obtain the metabolic engineering bacterium. Alternatively, the orceline synthase-encoding gene indC and the phosphopantetheinyl transferase-encoding gene indB are respectively amplified by PCR and connected to a plasmid to obtain the metabolic engineering bacterium.

6. The method of biosynthesis of natural blue pigments according to claim 5, characterized in that, The plasmid comprises at least one of pCDFDuet, pXMJ19, pRS425, and pKC1139 plasmids.

7. The method of biosynthesis of natural blue pigments according to claim 5, characterized in that, The PCR amplification is performed using primers with sequences shown in SEQ ID NOs: 5-30.

8. The method for biosynthesis of natural blue pigments according to claim 4, characterized in that, The host bacterium comprises at least one of Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Streptomyces lividans.

9. The method of biosynthesis of natural blue pigments according to claim 4, characterized in that, The biosynthesis method comprises: centrifuging to collect bacterial cells of the metabolic engineering bacterium induced for culture, resuspending the bacterial cells in a catalytic liquid containing a phosphate buffer, glutamine, and N-acetylglutamine, obtaining a conversion liquid after catalysis, and centrifuging to collect natural blue pigment in the conversion liquid.

10. The method of biosynthesis of natural blue pigments according to claim 9, characterized in that, The concentration of glutamine in the catalytic liquid is 0.1-10 g / L, and the concentration of N-acetylglutamine is 0.1-10 g / L.

11. A cell catalyst, characterized by, The cell catalyst contains the metabolic engineering bacterium according to claim 4 or 5.

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