A pyridone compound, its biosynthetic method and application

Abstract

This application relates to the field of biosynthesis technology, and specifically discloses a pyridone compound, its biosynthesis method and application. Based on synthetic biology-related technologies, this application modifies non-ribosomal peptide synthetase, heterologously expresses non-ribosomal peptide synthetase mutants in a variety of hosts, and for the first time discovers and obtains a pyridone compound, and determines its chemical formula as C5H4N2O3, named 5-amino-pyridine-2,3,6-trione or 5-hydroxy-3-imino-pyridine-2,6-dione. This novel pyridone compound can be used as a raw material or intermediate in the development and preparation of novel antibiotics, drugs and dyes.

Description

Technical Field

[0001] This application relates to the field of biosynthesis technology, and particularly relates to a pyridinone compound, its biosynthesis method and application. Background Art

[0002] Pyridinone compounds are an important class of organic compounds. Their molecular structure contains a relatively unique six-membered aromatic ring (pyridine ring) and a ketone group, and the pyridine ring contains both a carbonyl group and an N heteroatom. According to the relative position of the nitrogen atom and the carbonyl group, they are divided into 2-pyridinone, 3-pyridinone and 4-pyridinone. Currently, the chemically synthesized pyridinone compounds are mainly mono-carbonyl pyridinones, which can be used as key raw materials or intermediates in the synthesis of organic compounds and have a wide range of applications in the fields of preparing antibiotics, drugs and dyes.

[0003] 2-Pyridinone and 4-pyridinone widely exist in various natural products, and it is reported that their derivatives have biological activities such as antibacterial, anti-inflammatory, anti-tumor and insecticidal effects. Due to the structural characteristics of pyridinone and related biological effects, this structure has become an important skeleton for the synthesis of antibiotics and drugs.

[0004] Chinese Patent with application number 202180007672.3 discloses a pyridinone compound that can be used as a MAT2A inhibitor; pyridinone itself is easily oxidized by air, but it is relatively stable when there is a substituent at the 3-position. Its derivatives can be used as coupling components for green-yellow to orange azo dyes in the dye industry. They have excellent properties such as strong coupling ability, high extinction coefficient of the synthesized dyes, bright color and high light fastness, and are widely used in the synthesis of disperse and reactive dyes. The discovery of new pyridinone compounds is of great significance for the development of new antibiotics, drugs and dyes.

[0005] The products of nonribosomal peptide synthetase (NRPS) present in microorganisms are the sources of various antibiotics and natural pigments, including minimycin and indigoidine. Minimycin and indigoidine have similar structural monomers. In Chinese Patent CN 110777155 B, a pyridone compound was speculated to be their common precursor, but the clear structural characteristics of this compound were not obtained. Zhang et al. (Zhang Z, Li P, Wang M, et al. (S)‐3‐aminopiperidine‐2, 6‐dione is a biosynthetic intermediate of microbial blue pigment indigoidine[J]. Mlife, 2022, 1(2): 146-155.) proposed that (S)-3-aminopiperidine-2,6-dione is one of the synthetic intermediates of indigoidine, and by constructing nonribosomal peptide synthetase mutants, the synthesis of piperidone compounds using glutamine as a substrate was achieved, but the biosynthesis of pyridone compounds was not involved.

[0006] Currently, the preparation of pyridone compounds still mainly relies on chemical synthesis methods, and no methods for the biosynthesis of pyridone compounds have been reported. Summary of the Invention

[0007] The purpose of this application is to overcome the deficiencies of the above-mentioned prior art and provide a pyridone compound, its biosynthesis method and application.

[0008] To achieve the above purpose, the technical solution adopted in this application is as follows:

[0009] This application provides a pyridone compound, the molecular formula of the pyridone compound is C5H4N2O3, and the relative molecular weight is 140.0;

[0010] The chemical name of the pyridone compound is 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione;

[0011] The chemical formula of the 5-aminopyridine-2,3,6-trione is shown in Formula (I):

[0012]

[0013] Formula (I);

[0014] The chemical formula of the 5-hydroxy-3-iminopyridine-2,6-dione is shown in Formula (II):

[0015]

[0016] Formula (II).

[0017] For the first time, the present application discovered and obtained this pyridone compound, and determined its chemical formula as C5H4N2O3, named 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione. This pyridone compound has the potential to be used as a raw material or intermediate in the preparation of new antibiotics, drugs and dyes.

[0018] The present application also provides a pyridone synthase encoding gene encoding the pyridone compound described above. The pyridone synthase encoding gene includes pyridone synthase encoding gene pds1, pyridone synthase encoding gene pds2 and pyridone synthase encoding gene pds3;

[0019] The pyridone synthase encoding gene pds1, pyridone synthase encoding gene pds2 and pyridone synthase encoding gene pds3 are respectively composed of mutating the basic amino acids at the catalytic sites of the oxidation domains of the cyanobactin synthases bpsA, indC and idgS of the cyanobactin synthase into neutral amino acids.

[0020] During the study of the structure of the existing cyanobactin synthase (a non-ribosomal peptide synthase) in the present application, the inventors found that the catalytic sites of the oxidation domains of the cyanobactin synthases bpsA, indC and idgS of the cyanobactin synthase are basic amino acids. Mutating them into neutral amino acids will destroy the catalytic ability of the oxidation domain of the cyanobactin synthase, change the catalytic activity of the enzyme, and thus inhibit the synthesis of cyanobactin, and will promote the accumulation of its precursor - a pyridone compound, thereby converting the cyanobactin synthase into pyridone synthases Pds1, Pds2 and Pds3, realizing the synthesis of 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione using glutamine as a substrate.

[0021] As a preferred embodiment of the pyridone synthase encoding gene encoding the pyridone compound described in the present application, the catalytic site of the oxidation domain of the cyanobactin synthase includes the 876th site of the oxidation domain of the cyanobactin synthase.

[0022] As a preferred embodiment of the pyridone synthase encoding gene encoding the pyridone compound described in the present application, the basic amino acid includes histidine, and the neutral amino acid includes alanine.

[0023] In the technical solution of the present application, the 876th site of the cyan blue synthase bpsA, the cyan blue synthase indC, and the cyan blue synthase idgS are all located in the catalytic pocket of the oxidation domain of the cyan blue synthase, and they are all basic amino acids. After the basic amino acid histidine at this site is directionally mutated to neutral alanine, the catalytic ability of the oxidation domain will be damaged, thereby inhibiting the synthesis of cyan blue and promoting the accumulation of precursor-pyridone compounds, so that the cyan blue synthase is transformed into a pyridone synthase.

[0024] Based on the mutation of the above amino acid site, the basic amino acid histidine at the 876th site of the cyan blue synthase oxidation domain of the cyan blue synthase bpsA is directionally mutated to neutral alanine, forming a cyan blue synthase mutant BpsA-H876A, named the pyridone synthase encoding gene Pds1 (Pyridinone synthetase 1);

[0025] The basic amino acid histidine at the 876th site of the cyan blue synthase oxidation domain of the cyan blue synthase indC is directionally mutated to neutral alanine, forming a cyan blue synthase mutant IndC-H876A, named the pyridone synthase encoding gene Pds2 (Pyridinone synthetase 2);

[0026] The basic amino acid histidine at the 876th site of the cyan blue synthase oxidation domain of the cyan blue synthase idgS is directionally mutated to neutral alanine, forming a cyan blue synthase mutant IdgS-H876A, named the pyridone synthase encoding gene Pds3 (Pyridinone synthetase 2).

[0027] As a preferred embodiment of the pyridone synthase encoding gene of the pyridone compound described in the present application, the nucleotide sequence of the pyridone synthase encoding gene pds1 is shown in SEQ ID NO: 1; the nucleotide sequence of the pyridone synthase encoding gene pds2 is shown in SEQ ID NO: 2; the nucleotide sequence of the pyridone synthase encoding gene pds3 is shown in SEQ ID NO: 3.

[0028] The present application also provides a vector or recombinant vector containing the pyridone synthase encoding gene of the pyridone compound.

[0029] The present application also provides a method for constructing a metabolic engineering bacterium for biosynthesizing the pyridone compound, and the method for constructing the metabolic engineering bacterium includes the following steps:

[0030] At least one of the pyridone synthase-encoding genes pds1, pds2, and pds3 is introduced into a host bacterium and constructed with a phosphopantetheinyl transferase-encoding gene to obtain a metabolic engineering bacterium;

[0031] The metabolic engineering bacterium biosynthesizes the pyridone compounds described above;

[0032] The pyridone synthase-encoding genes pds1, pds2, and pds3 are respectively composed of mutating the basic amino acids at the catalytic site of the indigo synthase oxidation domain of the indigo synthase-encoding genes bpsA, indC, and idgS into neutral amino acids.

[0033] The indigo synthase-encoding gene bpsA encodes the indigo synthase bpsA; the indigo synthase-encoding gene indC encodes the indigo synthase indC; the indigo synthase-encoding gene idgS encodes the indigo synthase idgS.

[0034] As a preferred embodiment of the method for constructing the metabolic engineering bacterium described in this application, the phosphopantetheinyl transferase-encoding gene includes at least one of the phosphopantetheinyl transferase-encoding genes EntD, IndB, and Sfp.

[0035] As a preferred embodiment of the method for constructing the metabolic engineering bacterium described in this application, the nucleotide sequence of the phosphopantetheinyl transferase-encoding gene EntD is as shown in SEQ ID NO: 4; the nucleotide sequence of the phosphopantetheinyl transferase-encoding gene IndB is as shown in SEQ ID NO: 5; the nucleotide sequence of the phosphopantetheinyl transferase-encoding gene Sfp is as shown in SEQ ID NO: 6.

[0036] As a preferred embodiment of the method for constructing the metabolic engineering bacterium described in this application, the host bacterium includes at least one of Escherichia coli and its derivative strains, Corynebacterium glutamicum and its derivative strains, Streptomyces and its derivative strains, and Saccharomyces cerevisiae and its derivative strains.

[0037] Preferably, the host bacterium includes Escherichia coli or Corynebacterium glutamicum.

[0038] Preferably, the Escherichia coli includes but is not limited to Escherichia coli BL21(DE3); the Corynebacterium glutamicum includes but is not limited to Corynebacterium glutamicum ATCC13032.

[0039] The host bacteria used in this application include but are not limited to common microorganisms such as Escherichia coli, Corynebacterium glutamicum, Streptomyces, and Saccharomyces.

[0040] Through experiments, it is known that the original Escherichia coli BL21(DE3) and Corynebacterium glutamicum ATCC 13032 cannot utilize glucose, glycerol, glutamic acid, or glutamine to synthesize 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione. It is necessary to introduce relevant enzyme genes into Escherichia coli and Corynebacterium glutamicum to metabolic engineering bacteria to achieve the biosynthesis of 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione.

[0041] The metabolic engineering strains include but are not limited to the metabolic engineering strains with similar pyridone synthesis ability constructed in different host bacteria by combining any one or more of pyridone synthases Pds1, Pds2, and Pds3 with any one or more of phosphopantetheinyl transferases EntD, IndB, and Sfp.

[0042] As a preferred embodiment of the construction method of the metabolic engineering bacteria described in this application, the construction method includes any one of the following:

[0043] Amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds1 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene EntD with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Escherichia coli to obtain the metabolic engineering bacteria;

[0044] Or, amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds2 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene indB with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Escherichia coli to obtain the metabolic engineering bacteria;

[0045] Alternatively, amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds3 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene sfp with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Escherichia coli to obtain the metabolic engineering bacterium;

[0046] Alternatively, amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds1 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene EntD with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Corynebacterium glutamicum to obtain the metabolic engineering bacterium;

[0047] Alternatively, amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds2 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene indB with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Corynebacterium glutamicum to obtain the metabolic engineering bacterium;

[0048] Alternatively, amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds3 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene sfp with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Corynebacterium glutamicum to obtain the metabolic engineering bacterium;

[0049] The recombinant vector I includes, but is not limited to, any one of the recombinant vectors of recombinant vector pCDFDuet-Pds1, recombinant vector pCDFDuet-Pds2, recombinant vector pCDFDuet-Pds3, recombinant vector pXMJ19-Pds1, recombinant vector pXMJ19-Pds2, and recombinant vector pXMJ19-Pds3;

[0050] The recombinant vector II includes, but is not limited to, any one of recombinant vector pCDFDuet-Pds1-entD, recombinant vector pCDFDuet-Pds2-indB, recombinant vector pCDFDuet-Pds3-sfp, recombinant vector pXMJ19-Pds1-entD, recombinant vector pXMJ19-Pds2-indB, and recombinant vector pXMJ19-Pds3-sfp.

[0051] As a preferred embodiment of the method for constructing the metabolic engineering bacterium described in the present application, the PCR amplification is performed using primers with nucleotide sequences shown in SEQ ID NO: 7-20.

[0052] Preferably, the recombinant vector includes at least one of pCDFDuet recombinant vector and pXMJ19 recombinant vector.

[0053] This application also provides a metabolic engineering bacterium obtained by the construction method of the above-mentioned metabolic engineering bacterium.

[0054] In some specific embodiments, the metabolic engineering bacterium includes Escherichia coli (E. coli) recombinant strains HG-N-Pd01, HG-N-Pd02, HG-N-Pd03, and Corynebacterium glutamicum (C. glutamicum) HG-N-Pd04, HG-N-Pd05, HG-N-Pd06.

[0055] In some specific embodiments, the metabolic engineering bacterium is Escherichia coli recombinant strain HG-N-Pd01, and the Escherichia coli recombinant strain HG-N-Pd01 is constructed by introducing the recombinant vector pCDFDuet-Pds1-entD into Escherichia coli BL21(DE3).

[0056] The recombinant vector pCDFDuet-Pds1-entD is obtained by respectively subjecting the pyridone synthase encoding gene pds1 and the phosphopantetheinyl transferase encoding gene entD derived from C. glutamicum to PCR amplification, gene synthesis, and ligation to the recombinant vector pCDFDuet-1.

[0057] The pyridone synthase encoding gene pds1 is the gene sequence of the blue pigment synthase site-directed mutant BpsA-H876A, and is obtained by PCR amplification using site-directed mutagenesis primers.

[0058] The construction method of the recombinant vector pCDFDuet-Pds1-entD is specifically as follows:

[0059] (1) The pyridone synthase encoding gene pds1 is obtained by site-directed mutagenesis PCR amplification of the blue pigment synthase encoding gene bpsA, and the phosphopantetheinyl transferase encoding gene entD is obtained by gene synthesis.

[0060] (2) Use a seamless cloning kit to insert the Pds1 and entD gene fragments into the two multiple cloning sites of the pCDFDuet-1 vector in sequence to obtain the recombinant vector pCDFDuet-Pds1-entD.

[0061] (3)The pCDFDuet-Pds1-entD was transferred into Escherichia coli BL21(DE3) using the chemical transformation method to obtain the recombinant Escherichia coli strain HG-N-Pd01 (i.e., recombinant Escherichia coli BL21 (DE3) / pCDFDuet-bpsA-entD). The recombinant Escherichia coli BL21 (DE3) / pCDFDuet-bpsA-entD is derived from Chinese Patent CN 118184576 A.

[0062] In some specific embodiments, the metabolic engineering bacterium is the recombinant Escherichia coli strain HG-N-Pd02, which is constructed by introducing the recombinant vector pCDFDuet-Pds2-indB into Escherichia coli BL21(DE3).

[0063] The recombinant vector pCDFDuet-Pds2-indB is obtained by respectively subjecting the pyridone synthase encoding gene pds2 and the phosphopantetheinyl transferase encoding gene indB derived from S. chromofuscus to PCR amplification, gene synthesis, and ligation to the recombinant vector pCDFDuet-1.

[0064] The pyridone synthase encoding gene pds2 is the gene sequence of the site-directed mutant IndC-H876A of the indigo synthase, and is obtained by PCR amplification using site-directed mutagenesis primers.

[0065] In some specific embodiments, the metabolic engineering bacterium is the recombinant Escherichia coli strain HG-N-Pd03, which is constructed by introducing the recombinant vector pCDFDuet-Pds3-sfp into Escherichia coli BL21(DE3).

[0066] The recombinant vector pCDFDuet-Pds3-sfp is obtained by respectively subjecting the pyridone synthase encoding gene pds3 and the phosphopantetheinyl transferase encoding gene sfp derived from B. subtilis to PCR amplification, gene synthesis, and ligation to the recombinant vector pCDFDuet-1.

[0067] The pyridone synthase encoding gene pds3 is the gene sequence of the site-directed mutant IdgS-H876A of the indigo synthase, and is obtained by PCR amplification using site-directed mutagenesis primers.

[0068] In some specific embodiments, the metabolic engineering bacterium is the recombinant strain HG-N-Pd04 of Corynebacterium glutamicum (C. glutamicum).

[0069] Among them, the recombinant strain HG-N-Pd04 of Corynebacterium glutamicum is constructed by introducing the recombinant vector pXMJ19-Pds1-entD into Corynebacterium glutamicum ATCC13032.

[0070] The recombinant vector pXMJ19-Pds1-entD is obtained by respectively subjecting the pyridone synthase-encoding gene pds1 and the phosphopantetheinyl transferase-encoding gene entD derived from C. glutamicum to PCR amplification, gene synthesis, and ligation to the recombinant vector pXMJ19.

[0071] The construction method of the recombinant vector pXMJ19-Pds1-entD is as follows:

[0072] (1) The pyridone synthase-encoding gene pds1 is obtained by site-directed mutagenesis PCR amplification of the blue pigment synthase-encoding gene bpsA, and the phosphopantetheinyl transferase-encoding gene entD is obtained by gene synthesis.

[0073] (2) Use a seamless cloning kit to insert the Pds1 and entD gene fragments into the multiple cloning site of the pXMJ19 vector to obtain the recombinant vector pXMJ19-Pds1-entD.

[0074] (3) Use electroporation to transfer the recombinant vector pXMJ19-Pds1-entD into Corynebacterium glutamicum ATCC13032 to obtain the recombinant strain HG-N-Pd04 of Corynebacterium glutamicum.

[0075] In some specific embodiments, the metabolic engineering bacterium is the recombinant strain HG-N-Pd05 of Corynebacterium glutamicum, and the recombinant strain HG-N-Pd05 of Corynebacterium glutamicum is constructed by introducing the recombinant vector pXMJ19-Pds2-indB into Corynebacterium glutamicum ATCC 13032.

[0076] The recombinant vector pXMJ19-Pds2-indB is obtained by respectively subjecting the pyridone synthase-encoding gene pds2 and the phosphopantetheinyl transferase-encoding gene indB derived from S. chromofuscus to PCR amplification, gene synthesis, and ligation to the recombinant vector pXMJ19.

[0077] The pyridone synthase-encoding gene pds2 is obtained by site-directed mutagenesis PCR amplification of the blue pigment synthase-encoding gene indC.

[0078] In some specific embodiments, the metabolic engineering bacterium is the recombinant strain HG-N-Pd06 of Corynebacterium glutamicum, and the recombinant strain HG-N-Pd06 of Corynebacterium glutamicum (C. glutamicum) is constructed by introducing the recombinant vector pXMJ19-Pds3-sfp into Corynebacterium glutamicum ATCC 13032.

[0079] The recombinant vector pXMJ19-Pds3-sfp is obtained by respectively subjecting the pyridone synthase encoding gene pds3 and the phosphopantetheinyl transferase encoding gene sfp derived from B. subtilis to PCR amplification, gene synthesis, and ligation to the recombinant vector pXMJ19.

[0080] The pyridone synthase encoding gene pds3 is obtained by site-directed mutagenesis PCR amplification of the indigoidine synthase encoding gene idgS.

[0081] The recombinant strains HG-N-Pd01, HG-N-Pd02, HG-N-Pd03 of Escherichia coli (E. coli), and the recombinant strains HG-N-Pd04, HG-N-Pd05, HG-N-Pd06 of Corynebacterium glutamicum (C. glutamicum) constructed in this application efficiently express pyridone synthase and phosphopantetheinyl transferase through the vector, and all achieve the synthesis of 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione.

[0082] This application also provides the use of the above-mentioned metabolic engineering bacterium in the biosynthesis of pyridone compounds.

[0083] This application also provides a method for the biosynthesis of the pyridone compounds, which uses the above-mentioned metabolic engineering bacterium to express pyridone synthase to catalyze the biosynthesis of natural pyridones from glutamine.

[0084] This application also provides a fermentation broth or extract of the above-mentioned metabolic engineering bacterium, and the fermentation broth or extract includes the above-mentioned pyridone compounds.

[0085] This application also provides a culture medium for fermenting the above-mentioned pyridone compounds, and the culture medium includes the following components in mass concentration:

[0086] Glucose 30 - 50 g / L, glycerol 5 - 10 g / L, molasses 5 - 10 g / L, corn steep liquor 5 - 10 g / L, dipotassium hydrogen phosphate dodecahydrate 2 - 5 g / L, potassium dihydrogen phosphate 2 - 5 g / L, ammonium sulfate 20 - 30 g / L, magnesium sulfate heptahydrate 0.5 - 1.5 g / L, ferrous sulfate heptahydrate 0.05 - 0.1 g / L, and the solvent is water.

[0087] Preferably, the initial pH of the culture medium is 6.5.

[0088] Preferably, the culture medium comprises components with the following mass concentrations: 40 g / L of glucose, 7 g / L of glycerol, 7 g / L of molasses, 8 g / L of dry corn steep liquor, 3 g / L of dipotassium hydrogen phosphate dodecahydrate, 3.5 g / L of potassium dihydrogen phosphate, 25 g / L of ammonium sulfate, 0.8 g / L of magnesium sulfate heptahydrate, 0.07 g / L of ferrous sulfate heptahydrate, and the solvent is water.

[0089] The metabolic engineering bacterium of the present application can be used for fermentation production, and pyridone compounds can be fermentatively synthesized in the culture medium.

[0090] Glucose and glycerol are used as carbon sources in the culture medium. The mixed carbon sources improve the efficiency of the tricarboxylic acid cycle of the strain and enhance the synthesis of pyridone precursors glutamate and glutamine; corn steep liquor and molasses provide the necessary organic nitrogen sources for the growth of the strain, while supplementing some trace elements and growth factors, ensuring the normal growth of the engineered bacterium in the early stage; dipotassium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate act as a buffer pair to maintain the stability of the fermentation pH; ferrous sulfate heptahydrate inhibits the activity of glutamine degrading enzyme and increases the accumulation of pyridone precursors; magnesium sulfate heptahydrate provides cofactors for key enzymes and maintains their catalytic activity.

[0091] In some specific embodiments, the fermentation broth containing pyridone compounds provided by the present application is obtained by fermenting the recombinant strain HG-N-Pd01 of Escherichia coli (E. coli). The specific steps for preparing the fermentation broth are as follows: Pick a single colony of the recombinant strain HG-N-Pd01 and transfer it to 5 mL of LB medium containing 50 μg / mL of streptomycin. Culture at 37 °C and 220 rpm for 16 h, and then inoculate it into 50 mL of Pds fermentation medium at an inoculation amount of 1%. Add streptomycin with a final concentration of 50 μg / mL and an inducer IPTG with a final concentration of 0.1 mM, and continue to culture for 48 h to obtain the fermentation broth containing pyridone compounds.

[0092] In some specific embodiments, the fermentation broth containing pyridone compounds provided by the present application is obtained by fermenting the recombinant strain HG-N-Pd04 of Corynebacterium glutamicum (C. glutamicum). The specific steps for preparing the fermentation broth are as follows: Pick a single colony of the recombinant strain HG-N-Pd04 and transfer it to 5 mL of BHISG medium containing 15 μg / mL chloramphenicol and 50 μg / L biotin. Incubate at 30 °C and 220 rpm for 16 h, then inoculate it into 50 mL of Pds fermentation medium at an inoculation amount of 1%, with a final concentration of 15 μg / mL chloramphenicol and 50 μg / L biotin added to the medium. Incubate at 32 °C and 220 rpm. After 6 h, add IPTG with a final concentration of 1 mM and continue to incubate until 48 h to obtain the fermentation broth containing pyridone compounds.

[0093] The present application also provides the use of the above pyridone compounds in the preparation of novel antibiotics, drugs or dyes.

[0094] The present application uses genetic engineering techniques to respectively modify the metabolic pathways of Escherichia coli and Corynebacterium glutamicum and express non-ribosomal peptide synthetases, thereby achieving the fermentation synthesis of pyridone compounds or their enol tautomers using glucose as a substrate. The pyridone compounds were first isolated from the fermentation broths of Escherichia coli and Corynebacterium glutamicum, and their molecular structures were deduced by analytical means such as mass spectrometry and nuclear magnetic resonance.

[0095] Compared with the prior art, the present application has the following beneficial effects:

[0096] The present application provides a pyridone compound, its biosynthesis method and application. Based on synthetic biology-related technologies, the present application modifies non-ribosomal peptide synthetase (guanamine synthetase), heterologously expresses non-ribosomal peptide synthetase mutants in multiple hosts, discovers and obtains the pyridone compound for the first time, and determines its chemical formula as C5H4N2O3, named 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione. This novel pyridone compound can be used as a raw material or intermediate in the development and preparation of novel antibiotics, drugs and dyes. Description of the Drawings

[0097] Figure 1 It is the liquid phase detection chart of the fermentation product of the original Escherichia coli BL21;

[0098] Figure 2 It is the liquid phase detection chart of the fermentation product of the recombinant Escherichia coli BL21 (DE3) / pCDFDuet-bpsA-entD;

[0099] Figure 3 It is the liquid phase detection chart of the fermentation product of the recombinant Escherichia coli HG-N-Pd01;

[0100] Figure 4 Liquid chromatography detection chart of the fermentation product of recombinant Escherichia coli HG-N-Pd01 supplemented with glutamine;

[0101] Figure 5 Ultraviolet-visible absorption spectrum of pyridone compounds;

[0102] Figure 6 High-resolution mass spectrometry chart of pyridone compounds;

[0103] Figure 7 1H-NMR of pyridone compounds 1 H-NMR chart;

[0104] Figure 8 13C-NMR of pyridone compounds 13 C-NMR chart;

[0105] Figure 9 HSQC spectrum of pyridone compounds;

[0106] Figure 10 HMBC spectrum of pyridone compounds;

[0107] Figure 11 Structure analysis and appearance diagram of pyridone compounds;

[0108] Among them, A is the structural speculation diagram of pyridone compounds;

[0109] B is the liquid chromatography detection chart of the tautomeric form of pyridone compounds;

[0110] C is the ultraviolet-visible absorption spectrum of pyridone compounds;

[0111] Figure 12 Biosynthesis pathway speculation diagram of pyridone compounds;

[0112] Figure 13 Liquid chromatography detection chart of the fermentation synthesis of pyridone compounds by recombinant strain HG-N-Pd01;

[0113] Figure 14 Liquid chromatography detection chart of the fermentation synthesis of pyridone compounds by recombinant strain HG-N-Pd02;

[0114] Figure 15 Liquid chromatography detection chart of the fermentation synthesis of pyridone compounds by recombinant strain HG-N-Pd03;

[0115] Figure 16 Liquid chromatography detection chart of the fermentation synthesis of pyridone compounds by recombinant strain HG-N-Pd04;

[0116] Figure 17 It is the liquid-phase detection diagram of the fermentation synthesis of pyridone compounds by the recombinant strain HG-N-Pd05;

[0117] Figure 18 It is the liquid-phase detection diagram of the fermentation synthesis of pyridone compounds by the recombinant strain HG-N-Pd06;

[0118] Figure 19 It is the liquid-phase detection diagram of the fermentation broth product of the mutant bpsA-H876D engineering bacteria;

[0119] Figure 20 It is the liquid-phase detection diagram of the fermentation broth product of the mutant indC-H876D engineering bacteria;

[0120] Figure 21 It is the liquid-phase detection diagram of the fermentation broth product of the mutant idgS-H876D engineering bacteria. Detailed implementation manners

[0121] To better illustrate the purpose, technical solutions and advantages of this application, the following will further illustrate this application in combination with the accompanying drawings and specific embodiments.

[0122] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels, and the component raw materials used in each parallel experiment are the same.

[0123] The components of the culture media involved in the following examples are as follows:

[0124] LB (Luria-Bertani) liquid culture medium: molasses 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, sterilized at 121 °C for 20 min.

[0125] LB (Luria-Bertani) solid culture medium: molasses 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, 15 g / L of agar powder, sterilized at 121 °C for 20 min. The solid culture medium is cooled to about 50 °C, and the required antibiotics are added for pouring plates. After solidification, it is placed at 4 °C for standby.

[0126] BHISG culture medium: 37 g / L of brain heart infusion powder, 10 g / L of glucose, sterilized at 115 °C for 30 min;

[0127] Pds fermentation medium: glucose 40 g / L, glycerol 7 g / L, molasses 7 g / L, corn steep liquor 8 g / L, dipotassium hydrogen phosphate dodecahydrate 3 g / L, potassium dihydrogen phosphate 3.5 g / L, ammonium sulfate 25 g / L, magnesium sulfate heptahydrate 0.8 g / L, ferrous sulfate heptahydrate 0.07 g / L, sterilized at 115 °C for 30 min.

[0128] The primers involved in the following examples are shown in Table 1.

[0129] Table 1 Primer information table

[0130]

[0131] The strains involved in the following examples are shown in Table 2.

[0132] Table 2 Metabolic engineering strain information table

[0133]

[0134] In this application, the phosphopantetheine transferase encoding gene entD is derived from C. glutamicum, the phosphopantetheine transferase encoding gene indB is derived from S. chromofuscus, and the phosphopantetheine transferase encoding gene sfp is derived from B. subtilis.

[0135] The nucleotide sequence of the pyridone synthase encoding gene pds1 is shown in SEQ ID NO: 1, the nucleotide sequence of the pyridone synthase encoding gene pds2 is shown in SEQ ID NO: 2, and the nucleotide sequence of the pyridone synthase encoding gene pds3 is shown in SEQ ID NO: 3; the nucleotide sequence of the phosphopantetheine transferase encoding gene EntD is shown in SEQ ID NO: 4, the nucleotide sequence of the phosphopantetheine transferase encoding gene indB is shown in SEQ ID NO: 5, and the nucleotide sequence of the phosphopantetheine transferase encoding gene sfp is shown in SEQ ID NO: 6.

[0136] In the following examples, the high-performance liquid chromatography detection method for samples is as described below.

[0137] High-performance liquid chromatography conditions:

[0138] Mobile phase: gradient elution of methanol and pure water, and the gradient table is as follows in Table 3:

[0139] Table 3 Liquid phase gradient elution table

[0140]

[0141] Wavelength: 400 nm, flow rate: 1.0 mL / min, sample preparation solution: DMSO, injection volume: 10 μL, column temperature: 35 °C, running time: 18 min.

[0142] Column model: Galasil EF-C18M 4.6 mm id × 250 mm L (SN B06211801).

[0143] Example 1. Construction of metabolic engineering strain HG-N-Pd01

[0144] The construction method of the metabolic engineering strain HG-N-Pd01 in this example includes the following steps:

[0145] Step 1: Construction of pyridone synthase Pds1:

[0146] (1) Using primer site-directed mutagenesis technology, with the cyan blue synthase-encoding gene bpsA as a template, PCR site-directed mutagenesis was performed to bpsA-H876A to obtain the mutant-encoding gene sequence. The mutant primer sequences used are shown as SEQ ID NO: 7 and SEQ ID NO: 8 in Table 1.

[0147] (2) The PCR product was sequenced to determine that there were no abnormalities such as mismatches in the mutant sequence, and then it was stored for later use. The gene obtained after mutation was named pyridone synthase gene pds1, and its nucleotide sequence is shown as SEQ ID NO: 1. Hereinafter, this mutant will be abbreviated as Pds1.

[0148] Step 2: Construction of pCDFDuet-Pds1-entD vector:

[0149] (1) The pyridone synthase-encoding gene pds1 (nucleotide sequence shown as SEQ ID NO: 1), as described in Step 1, was obtained by PCR amplification with site-directed mutagenesis of the cyan blue synthase-encoding gene bpsA. The 4'-phosphopantetheine transferase-encoding gene entD (nucleotide sequence shown as SEQ ID NO: 4) was synthesized by Tsingke Biotechnology Co., Ltd.

[0150] (2) Using the commercially available vector pCDFDuet-1 purchased on the market as a template, primers pCDF-P-F (SEQ ID NO: 13) and pCDF-P-R (SEQ ID NO: 14) were used for PCR amplification, and the product was purified to obtain the pCDFDuet-1 linearized vector.

[0151] (3) Linearize the pCDFDuet-1 vector and the Pds1 gene (homologous arms of the added fragments have been added to both ends of pCDFDuet-1), and ligate them using a seamless cloning and ligation kit. Transform the ligation product into Escherichia coli DH5α using the chemical transformation method. After resuscitation and culture, spread it onto an LB solid medium plate containing 50 μg / mL streptomycin resistance, and culture it in a 37°C incubator for about 16 h.

[0152] (4) Use primers pCDF-YZ-F (SEQ ID NO: 15) and pCDF-YZ-R (SEQ ID NO: 16) for colony PCR verification. Culture the strains with correct PCR verification, extract the recombinant vector, and send it to Tsingke Biotechnology Co., Ltd. for sequencing. The strain with correct sequencing is the pCDFDuet-Pds1 vector.

[0153] (5) Using the pCDFDuet-Pds1 vector as a template, use pCDF-I-F (SEQ ID NO: 17) and pCDF-I-R (SEQ ID NO: 18) for PCR amplification, and purify the product to obtain the linearized pCDFDuet-Pds1 vector. Use a seamless cloning and ligation kit to ligate the linearized pCDFDuet-Pds1 vector and the fragment of the 4'-phosphopantetheine transferase-encoding gene entD. Transform the ligation product into Escherichia coli DH5α using the chemical transformation method. After resuscitation and culture, spread it onto an LB solid medium plate containing 50 μg / mL streptomycin resistance, and culture it in a 37°C incubator for about 16 h.

[0154] (6) Use primers pCDF-YZ-F (SEQ ID NO: 15) and pCDF-YZ-R (SEQ ID NO: 16) for colony PCR verification. Culture the strains with correct PCR verification, extract the recombinant vector, and send it to Tsingke Biotechnology Co., Ltd. for sequencing. The strain with correct sequencing is the pCDFDuet-Pds1-entD vector.

[0155] Step 3: Construction of HG-N-Idg01 strain:

[0156] (1) The competent cells of BL21(DE3) strain were purchased from Tsingke Biotechnology Co., Ltd., and the product number is TSC-C14.

[0157] (2) Use the chemical transformation method to transform the pCDFDuet-Pds1-entD vector into Escherichia coli BL21(DE3). After resuscitation and culture, spread it onto an LB solid medium plate containing 50 μg / mL streptomycin resistance, and culture it in a 37°C incubator for about 16 h.

[0158] (3)The single colonies grown on the LB solid medium plate are the recombinant Escherichia coli BL21 (DE3) / pCDFDuet-Pds1-entD, named HG-N-Pd01.

[0159] Example 2. Preparation of the fermentation broth of recombinant Escherichia coli containing pyridone compounds

[0160] (1)Seed broth preparation: Pick a single colony of the recombinant strain HG-N-Pd01 and transfer it to 5 mL of LB medium containing 50 μg / mL streptomycin. Incubate at 37 °C and 220 rpm for 16 h to obtain the seed broth.

[0161] Fermentation broth preparation: Inoculate the seed broth at an inoculation amount of 1% into 50 mL of Pds fermentation medium, add streptomycin with a final concentration of 50 μg / mL and an inducer IPTG with a final concentration of 0.1 mM, and continue to culture for 48 h to obtain the fermentation broth containing pyridone compounds.

[0162] Example 3. Fermentation products of different recombinant Escherichia coli strains

[0163] Experimental group 1: Use the original Escherichia coli BL21 as the fermentation strain, and prepare 50 mL of fermentation broth by the method of Example 2. After fermentation for 48 h, take 100 μL of the fermentation broth, add 900 μL of DMSO, mix well and shake for 10 min, centrifuge at 12000 rpm for 5 min, take 10 μL of the supernatant, and detect it by high performance liquid chromatography. The detection results are as Figure 1 shown.

[0164] Experimental group 2: Use the recombinant Escherichia coli BL21 (DE3) / pCDFDuet-bpsA-entD as the fermentation strain, and prepare 50 mL of fermentation broth by the method of Example 2. After fermentation for 48 h, take 100 μL of the fermentation broth, add 900 μL of DMSO, mix well and shake for 10 min, centrifuge at 12000 rpm for 5 min, take 10 μL of the supernatant, and detect it by high performance liquid chromatography. The detection results are as Figure 2 shown.

[0165] Experimental group 3: Use the recombinant Escherichia coli HG-N-Pd01 as the fermentation strain, and prepare 50 mL of fermentation broth by the method of Example 2. After fermentation for 48 h, take 100 μL of the fermentation broth, add 900 μL of DMSO, mix well and shake for 10 min, centrifuge at 12000 rpm for 5 min, take 10 μL of the supernatant, and detect it by high performance liquid chromatography. The detection results are as Figure 3 shown.

[0166] Experimental group 4: Using recombinant Escherichia coli HG-N-Pd01 as the fermentation strain, 50 mL of fermentation broth was prepared by the method of Example 2, and glutamine with a final concentration of 1 g / L was added at 24 h of fermentation. After 48 h of fermentation, 100 μL of the fermentation broth was taken, 900 μL of DMSO was added, and after mixing, it was shaken for 10 min. After centrifugation at 12000 rpm for 5 min, 10 μL of the supernatant was taken and detected by high performance liquid chromatography. The detection results are as Figure 4 shown.

[0167] The experimental results are as Figures 1 - 4 shown, Figure 1 and Figure 2 no peak was observed near 5.7 min, Figure 3 and Figure 4 a peak was observed at the liquid phase retention time of 5.7 min. As Figure 5 shown, the maximum absorption peak of this compound is 396 nm. The result comparison of experimental group 1, experimental group 2, experimental group 3 and experimental group 4 shows that the original strain BL21(DE3) and the control recombinant Escherichia coli BL21 (DE3) / pCDFDuet-bpsA-entD cannot synthesize this compound by the method of Example 2, while the recombinant Escherichia coli BL21 (DE3) / pCDFDuet-Pds1-entD can synthesize this compound under the same conditions. The result comparison of experimental group 3 and experimental group 4 shows that the peak area of this compound increases significantly after adding glutamine, and it is speculated that glutamine may be the precursor for synthesizing this compound.

[0168] Example 4, Identification of the structure of the unknown new compound sample

[0169] Collect 40 mL of the fermentation broth of experimental group 4 in Example 3, centrifuge at 12000 g for 5 min, remove the supernatant, resuspend the precipitated cells with 10 mL of dimethyl sulfoxide (DMSO), and break the cells by ultrasonic treatment to extract the pyridone compound into the solvent. Centrifuge again to obtain the supernatant, evaporate the organic solvent under vacuum, then wash the solid twice with 10 mL of pure water, methanol, ethyl acetate and hexane respectively, and finally freeze-dry under vacuum to obtain 0.04 g of yellow powder. The above sample was sent to the Analysis and Testing Center of Nanjing Normal University for high resolution mass spectrometry detection and NMR detection.

[0170] Figure 6 is the high resolution mass spectrometry detection chart of this pyridone compound; Figure 7 is the 1 H-NMR chart of this pyridone compound; Figure 8 is the 13 C-NMR chart of this pyridone compound; Figure 9 and Figure 10 are the NMR HSQC spectrum and HMBC spectrum of this pyridone compound respectively;Figure 11 This is a speculative diagram of the structure of the pyridone compound.

[0171] Table 4 shows 1 1H-NMR ( Figure 7 ) and 13 13C-NMR ( Figure 8 ) data corresponding to the elemental positions in the speculative structure ( Figure 11 ).

[0172] Table 4 NMR data analysis

[0173]

[0174] According to the high-resolution mass spectrometry ( Figure 6 ), the relative molecular weight of this compound is determined to be 140.0.

[0175] As Figure 7 shown, the peak at about 2.57 ppm in the 1H-NMR spectrum is the DMSO solvent peak, and the peak at about 3.52 ppm is the H2O peak. Taking the peak at 11.88 ppm as the integration standard, the integration is 1. From the HSQC spectrum ( Figure 9 ), there is no corresponding point on the abscissa for 11.88 ppm, so it is speculated that the hydrogen here is connected to nitrogen; similarly, the peaks at 7.61 ppm and 8.24 ppm are also not shown in the HSQC spectrum, so it is speculated that the hydrogens at these two positions are also connected to nitrogen; the peak at 5.72 ppm is a singlet, and there is a corresponding carbon at 100 - 110 ppm in the HSQC spectrum ( Figure 9 ), so it is speculated to be an olefinic carbon; the integrals of the peaks at 11.88 ppm, 8.24 ppm, and 7.61 ppm are 1, indicating a secondary amine. It can be inferred from 1H-NMR that this compound has three secondary amines and one double bond.

[0176] As Figure 8 shown, 13 in the 13C-NMR spectrum, the peak at about 40.6 ppm is the DMSO solvent peak, and there are five peaks at δ values of 173.12 ppm, 161.38 ppm, 159.03 ppm, 150.57 ppm, and 102.24 ppm. It is speculated that this compound contains 5 carbonyl groups; the δ values of 150 ppm - 170 ppm are carbonyl signals, so it is speculated that the two peaks at δ values of 161.38 ppm and 159.03 ppm are carbonyl carbons; generally, the SP2 hybridized carbon atoms are at 100 - 150 ppm, and the peak at 102.24 ppm is the carbon corresponding to the 1H-NMR spectrum, indicating an olefinic carbon.

[0177] In summary, combined with the cyan blue structural formula, it is calculated that 1 1H-NMR spectrum and13 The attribution of the signals in the 13C-NMR spectrum is shown in Table 4. It is determined that the molecular formula of this compound is C5H4N2O3, and the structure is as Figure 11 shown in Figure 11 A) below. This structure has not been reported in the literature and is a novel pyridone compound named 5-hydroxy-3-iminopyridine-2,6-dione.

[0178] Due to the structural characteristics of the enol isomer of this compound, keto-enol tautomerism exists, as shown in Figure 11 shown in Figure 11 B) below. It was found that a significant peak appeared at 6.3 min in the liquid-phase detection of the separated product. Based on the research on the tautomerism of pyridone compounds, it is speculated that it may be a tautomer of 5-hydroxy-3-iminopyridine-2,6-dione. Therefore, the compound at 6.3 min in this liquid phase is named 5-aminopyridine-2,3,6-trione, and its structure and liquid-phase detection are shown in Figure 11 shown in Figure 11 C) below, with a maximum absorption peak at 394 nm. The existing separation process can only separate the enol structure of this compound at 5.7 min, and the keto structure at 6.3 min has not been obtained in pure form, so nuclear magnetic resonance and other detections have not been carried out.

[0179] Currently, it is speculated that there may be two reasons why 5-aminopyridine-2,3,6-trione cannot be successfully separated and purified: (1) The keto form of 5-aminopyridine-2,3,6-trione is more likely to react with other substances during the fermentation process and participates in the synthesis of the metabolites of the engineering strain, and thus cannot accumulate. Therefore, as shown in Figure 13 , Figure 14 , Figure 15 , Figure 18 shown, the peak of 5-aminopyridine-2,3,6-trione is not obvious in the liquid-phase detection of the fermentation broth sample; (2) The keto structure is less stable and has a lower content than the enol structure, and there is a large loss during the separation and purification process. The current separation and purification methods are still unable to distinguish between keto-enol tautomers.

[0180] 5-Hydroxy-3-iminopyridine-2,6-dione appears yellow in DMSO solution. Based on the physical and chemical properties of this compound, it is speculated that this pyridone compound can be used as a novel yellow pigment or pharmaceutical intermediate and has great application potential in the printing and dyeing and pharmaceutical fields.

[0181] The biosynthetic pathway of pyridone in the metabolic engineering strain HG-N-Pd01 is as shown in Figure 12 below.

[0182] Example 5. Construction of the metabolic engineering strain HG-N-Pd2

[0183] The difference between this embodiment and embodiment 1 is that:

[0184] Step 1: In this example, the indC gene was used as a template, and site-directed mutation was performed to indC-H876A by PCR to obtain the gene sequence encoding the mutant. The sequences of the mutant primers used are shown in SEQ ID NO: 9 and SEQ ID NO: 10 in Table 1. The gene obtained after mutation was named pyridone synthase gene pds2, and its nucleotide sequence is shown in SEQ ID NO: 2. Hereinafter, the mutant is referred to as Pds2.

[0185] Step 2: construct the recombinant vector pCDFDuet-Pds2-entD. The pyridone synthase encoding gene used is pds2 (the nucleotide sequence is shown in SEQ ID NO: 2).

[0186] Example 6. Construction of metabolically engineered strain HG-N-Pd03

[0187] The difference between this embodiment and embodiment 1 is that:

[0188] Step 1: In this example, the idgS gene was used as a template, and site-directed mutation was performed to idgS-H876A by PCR to obtain the gene sequence encoding the mutant. The sequences of the mutation primers used are shown in SEQ ID NO: 11 and SEQ ID NO: 12 in Table 1. The gene obtained after mutation was named pyridone synthase gene pds3, and its nucleotide sequence is shown in SEQ ID NO: 3. Hereinafter, the mutant is referred to as Pds3.

[0189] Step 2: In this embodiment, the pyridone synthase encoding gene used in constructing the recombinant vector pCDFDuet-Pds2-entD is pds3 (the nucleotide sequence is shown in SEQ ID NO: 4).

[0190] Example 7. Construction of metabolically engineered strain HG-N-Pd04

[0191] The host of the metabolic engineering strain described in this example is Corynebacterium glutamicum ATCC 12032, and the experimental steps are as follows:

[0192] Step 1: The method for obtaining the pyridone synthase gene pds1 in this example is the same as step 1 in Example 1.

[0193] Step 2: Using the commercially available vector pXMJ19 purchased from the market as a template, PCR amplification was performed using primers pXMJ19-P-F (SEQ ID NO: 19) and pXMJ19-P-R (SEQ ID NO: 20) to obtain the linearized pXMJ19 vector, which was used for the construction of the recombinant vector pXMJ19-Pds1-entD; colony PCR verification was performed using primers pXMJ19-YZ-F (SEQ ID NO: 21) and pXMJ19-YZ-R (SEQ ID NO: 22). The strains with correct PCR verification were cultured, and the recombinant vector was extracted and sent to Tsingke Biotechnology Co., Ltd. for sequencing. The vector with correct sequencing was the pXMJ19-Pds1-entD vector.

[0194] Step 3: Preparation of electrocompetent cells of Corynebacterium glutamicum ATCC 13032. Pick a single colony of ATCC13032 into 5 mL of antibiotic-free BHISG medium and culture overnight at 30 °C and 220 rpm; take 15 OD of the bacterial solution and inoculate it into 50 mL of BHISG GT medium (0.1% Tween 80 and 50 μg / L biotin), culture at 30 °C and 220 rpm until the OD600 is about 1.0, then take it out and place it on ice for 20 min; transfer the bacterial solution to a 50 ml centrifuge tube, centrifuge at 4 °C and 2600×g for 10 min, discard the supernatant, add 50 mL of 10% glycerol (pre-cooled) to resuspend the cells, centrifuge at 4 °C and 2600×g for 10 min, discard the supernatant, and aspirate the residual bacterial solution with a pipette; repeat this step once; resuspend the cells with 100 μL of 10% glycerol (pre-cooled) and transfer them to a 1.5 mL EP tube for use.

[0195] Step 4: Construction of the recombinant strain HG-N-Pd04:

[0196] (1) Add 200 ng of the pXMJ19-Pds1-entD vector to the electrocompetent cells prepared in Step 3, gently mix evenly, then add it to the electroporation cuvette, and incubate on ice for 5 - 10 min;

[0197] (2) The electroporation conditions were 1.8 KV. After electroporation, quickly add the pre-warmed BHISG medium at 46 °C, heat shock in a 46 °C water bath for 6 minutes, and then incubate the transformation solution in a 30 °C shaker for 2 hours;

[0198] (3) After incubation, take 1 mL of the transformation solution and centrifuge at 4000 rpm, discard most of the supernatant, resuspend the remaining part, and spread it on a chloramphenicol-resistant BHISG plate, and culture it in a 30 °C incubator for 48 h.

[0199] (4) Single clones on the plate were picked for colony PCR verification, and sequencing confirmed that the correct strain was the recombinant strain of Corynebacterium glutamicum HG-N-Pd04.

[0200] Example 8: Preparation of recombinant Corynebacterium glutamicum fermentation broth containing pyridone compounds

[0201] (1) Pick a single colony of the recombinant strain HG-N-Pd04 and transfer it to 5 mL of BHISG medium containing 15 μg / mL chloramphenicol and 50 μg / L biotin. Cultivate at 30°C and 220 rpm for 16 h.

[0202] (2) Inoculate 50 mL of Pds fermentation medium at a 1% inoculum size, add chloramphenicol at a final concentration of 15 μg / mL and biotin at 50 μg / L to the medium, culture at 32°C, 220 rpm, and add IPTG at a final concentration of 1 mM after 6 h of culture. Continue culturing for 48 h to obtain a fermentation broth containing pyridone compounds.

[0203] Example 9. Construction of metabolically engineered strain HG-N-Pd05

[0204] The difference between this embodiment and embodiment 7 is that:

[0205] Step 1: In this example, the indC gene was used as a template, and site-directed mutation was performed to indC-H876A by PCR to obtain the gene sequence encoding the mutant. The sequences of the mutant primers used are shown in SEQ ID NO: 9 and SEQ ID NO: 10 in Table 1. The gene obtained after mutation was named pyridone synthase gene pds2, and its nucleotide sequence is shown in SEQ ID NO: 2. Hereinafter, the mutant is referred to as Pds2.

[0206] Step 2: construct the recombinant vector pCDFDuet-Pds2-entD. The pyridone synthase encoding gene used is pds2 (the nucleotide sequence is shown in SEQ ID NO: 2).

[0207] Example 10. Construction of metabolically engineered strain HG-N-Pd06

[0208] The difference between this embodiment and embodiment 7 is that:

[0209] Step 1: In this example, the idgS gene was used as a template, and site-directed mutation was performed to idgS-H876A by PCR to obtain the gene sequence encoding the mutant. The sequences of the mutation primers used are shown in SEQ ID NO: 11 and SEQ ID NO: 12 in Table 1. The gene obtained after mutation was named pyridone synthase gene pds3, and its nucleotide sequence is shown in SEQ ID NO: 3. Hereinafter, the mutant is referred to as Pds3.

[0210] In Step 2, the pyridone synthase encoding gene used to construct the recombinant vector pXMJ19-Pds3-sfp is pds3 (the nucleotide sequence is as shown in SEQ ID NO: 3).

[0211] Example 11: Fermentation of Different Recombinant Strains to Synthesize Pyridone Compounds

[0212] Experimental Group 1: Using the recombinant strain HG-N-Pd01 as the fermentation strain, 50 mL of fermentation broth was prepared by the method of Example 2. After 48 h of fermentation, 100 μL of the fermentation broth was taken, 900 μL of DMSO was added, and after mixing, it was shaken for 10 min. After centrifugation at 12,000 rpm for 5 min, 10 μL of the supernatant was taken and detected by high-performance liquid chromatography. The detection results are as Figure 13 shown.

[0213] Experimental Group 2: Using the recombinant strain HG-N-Pd02 as the fermentation strain, 50 mL of fermentation broth was prepared by the method of Example 2. After 48 h of fermentation, 100 μL of the fermentation broth was taken, 900 μL of DMSO was added, and after mixing, it was shaken for 10 min. After centrifugation at 12,000 rpm for 5 min, 10 μL of the supernatant was taken and detected by high-performance liquid chromatography. The detection results are as Figure 14 shown.

[0214] Experimental Group 3: Using the recombinant strain HG-N-Pd03 as the fermentation strain, 50 mL of fermentation broth was prepared by the method of Example 2. After 48 h of fermentation, 100 μL of the fermentation broth was taken, 900 μL of DMSO was added, and after mixing, it was shaken for 10 min. After centrifugation at 12,000 rpm for 5 min, 10 μL of the supernatant was taken and detected by high-performance liquid chromatography. The detection results are as Figure 15 shown.

[0215] Experimental Group 4: Using the recombinant strain HG-N-Pd04 as the fermentation strain, 50 mL of fermentation broth was prepared by the method of Example 8. After 48 h of fermentation, 100 μL of the fermentation broth was taken, 900 μL of DMSO was added, and after mixing, it was shaken for 10 min. After centrifugation at 12,000 rpm for 5 min, 10 μL of the supernatant was taken and detected by high-performance liquid chromatography. The detection results are as Figure 16 shown.

[0216] Experimental Group 5: Using the recombinant strain HG-N-Pd05 as the fermentation strain, 50 mL of fermentation broth was prepared by the method of Example 8. After 48 h of fermentation, 100 μL of the fermentation broth was taken, 900 μL of DMSO was added, and after mixing, it was shaken for 10 min. After centrifugation at 12,000 rpm for 5 min, 10 μL of the supernatant was taken and detected by high-performance liquid chromatography. The detection results are as Figure 17 shown.

[0217] Experimental group 6: Using the recombinant strain HG-N-Pd06 as the fermentation strain, 50 mL of fermentation broth was prepared by the method of Example 8. After 48 h of fermentation, 100 μL of fermentation broth was taken, 900 μL of DMSO was added, mixed and shaken for 10 min, centrifuged at 12000 rpm for 5 min, and 10 μL of supernatant was taken and tested by high performance liquid chromatography. The test results are as follows: Figure 18 shown.

[0218] The experimental results are as follows Figures 13 - 18 As shown, the compound of formula I was detected in the fermentation broth of the six recombinant strains, indicating that the three pyridone synthases Pds1, Pds2 and Pds3 can synthesize 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione in the hosts of Escherichia coli and Corynebacterium glutamicum, and the six metabolically engineered strains all have the ability to ferment and produce the compound, laying a foundation for the large-scale production of the pyridone compound.

[0219] Comparative Example 1

[0220] This comparative example is different from Example 1 in that:

[0221] Step 1: In this comparative example, the bpsA gene was used as a template, and site-directed mutation was performed to idgS-H876D by PCR, and the histidine at position 876 was mutated to the acidic amino acid aspartic acid to obtain the gene sequence encoding the mutant. Subsequently, the Escherichia coli engineering strain was constructed in the same manner as in Example 1, and the fermentation liquid of the Escherichia coli engineering strain was obtained in the same manner as in Example 2, and liquid phase detection was performed. The test results are shown in FIG. Figure 19 As shown, no pyridone compounds were observed.

[0222] Comparative Example 2

[0223] This comparative example is different from Example 5 in that:

[0224] Step 1: In this comparative example, the indC gene was used as a template, and site-directed mutation was performed to indC-H876D by PCR, and the histidine at position 876 was mutated to the acidic amino acid aspartic acid to obtain the gene sequence encoding the mutant. Subsequently, the Escherichia coli engineering strain was constructed in the same manner as in Example 1, and the fermentation liquid of the Escherichia coli engineering strain was obtained in the same manner as in Example 2, and liquid phase detection was performed. The detection results are shown in FIG. Figure 20 As shown, no pyridone compound peak was observed.

[0225] Comparative Example 3

[0226] This comparative example is different from Example 6 in that:

[0227] Step 1: This comparative example uses the idgS gene as a template, and uses PCR to site-directed mutagenesis to idgS-H876D, mutating the histidine at position 876 to the acidic amino acid aspartic acid to obtain the gene sequence encoding the mutant. Subsequently, the E. coli engineering strain was constructed in the same manner as in Example 1, and the fermentation broth of the E. coli engineering strain was obtained in the same manner as in Example 2, and liquid phase detection was performed. The test results are shown in FIG. Figure 21 As shown, no pyridone compound peak was observed.

[0228] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A pyridone synthase-encoding gene encoding a pyridone compound, characterized in that, The pyridone synthase-encoding genes include pyridone synthase-encoding gene pds1, pyridone synthase-encoding gene pds2, and pyridone synthase-encoding gene pds3; The pyridone synthase-encoding gene pds1, pyridone synthase-encoding gene pds2, and pyridone synthase-encoding gene pds3 are respectively constituted by mutating the basic amino acids at the catalytic site of the blue pigment synthase oxidation domain of the blue pigment synthase-encoding gene bpsA, blue pigment synthase-encoding gene indC, and blue pigment synthase-encoding gene idgS into neutral amino acids; The catalytic site of the blue pigment synthase oxidation domain includes the 876th site of the blue pigment synthase oxidation domain; The basic amino acid includes histidine, and the neutral amino acid includes alanine; The nucleotide sequence of the pyridone synthase-encoding gene pds1 is as shown in SEQ ID NO: 1; the nucleotide sequence of the pyridone synthase-encoding gene pds2 is as shown in SEQ ID NO: 2; the nucleotide sequence of the pyridone synthase-encoding gene pds3 is as shown in SEQ ID NO: 3; The structural formula of the pyridone compound is as shown in formula (I) or formula (II); Formula (I); Formula (II).

2. A vector or recombinant vector containing the pyridone synthase-encoding gene of the pyridone compound as described in claim 1.

3. A method for constructing a metabolically engineered bacterium, characterized in that, The method for constructing the metabolic engineering bacterium comprises the following steps: Introducing at least one of the pyridone synthase-encoding gene pds1, pyridone synthase-encoding gene pds2, and pyridone synthase-encoding gene pds3 as described in claim 1 and the phosphopantetheinyl transferase-encoding gene into a host bacterium to construct a metabolic engineering bacterium; The metabolic engineering bacterium biosynthesizes the pyridone compound as described in claim 1; The phosphopantetheinyl transferase-encoding gene includes at least one of the phosphopantetheinyl transferase-encoding genes EntD, IndB, and Sfp; The nucleotide sequence of the phosphopantetheinyl transferase-encoding gene EntD is as shown in SEQ ID NO: 4; the nucleotide sequence of the phosphopantetheinyl transferase-encoding gene IndB is as shown in SEQ ID NO: 5; the nucleotide sequence of the phosphopantetheinyl transferase-encoding gene Sfp is as shown in SEQ ID NO: 6; The host bacterium includes at least one of Escherichia coli and its derivative strains, Corynebacterium glutamicum and its derivative strains.

4. The method for constructing a metabolically engineered bacterium according to claim 3, wherein The construction method includes any one of the following: Amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds1 and the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene EntD and recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Escherichia coli to obtain a metabolic engineering bacterium; Alternatively, amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds2 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene indB with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Escherichia coli to obtain a metabolic engineering bacterium; Alternatively, amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds3 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene sfp with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Escherichia coli to obtain a metabolic engineering bacterium; Alternatively, amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds1 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene EntD with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Corynebacterium glutamicum to obtain a metabolic engineering bacterium; Alternatively, amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds2 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene indB with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Corynebacterium glutamicum to obtain a metabolic engineering bacterium; Alternatively, amplify the recombinant vector, then ligate the pyridone synthase-encoding gene pds3 with the recombinant vector, perform PCR amplification to obtain recombinant vector I, then ligate the phosphopantetheinyl transferase-encoding gene sfp with recombinant vector I to obtain recombinant vector II, and introduce the recombinant vector II into Corynebacterium glutamicum to obtain a metabolic engineering bacterium.

5. The method for constructing the metabolically engineered bacterium according to claim 4, wherein The PCR amplification is carried out using primers with nucleotide sequences as shown in SEQ ID NO: 7-20.

6. A metabolic engineering bacterium obtained by the method for constructing a metabolic engineering bacterium according to any one of claims 3-5.

7. Use of the metabolic engineering bacterium according to claim 6 in the biosynthesis of pyridone compounds.

8. A method for the biosynthesis of a pyridone compound as described in claim 1, characterized in that, The biosynthesis method uses the metabolic engineering bacterium according to claim 6 to express pyridone synthase and catalyze the biosynthesis of pyridone compounds from glutamine.

9. A fermentation broth or extract containing the metabolically engineered bacterium as described in claim 6, characterized in that, The fermentation broth or extract comprises the pyridone compounds according to claim 1.

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