Pyridone compound as well as biosynthesis method and application thereof

Abstract

The invention relates to the technical field of biosynthesis, and particularly discloses a pyridone compound as well as a biosynthesis method and application thereof. On the basis of related technologies of synthetic biology, non-ribosome peptide synthetase is modified, non-ribosome peptide synthetase mutants are heterologously expressed in various hosts, a pyridone compound is found and obtained for the first time, the chemical molecular formula of the pyridone compound is determined to be C5H4N2O3, and the pyridone compound is named as 5-aminopyridine-2, 3, 6-triketone or 5-hydroxy-3-iminopyridine-2, 3, 6-triketone or 5-hydroxy-3-iminopyridine-2, 3, 6-triketone or 5-hydroxy-3-iminopyridine-2, 3, 6-triketone. The novel pyridone compound can be used as a raw material or an intermediate to be applied to development and preparation of novel antibiotics, drugs and dyes.

Description

Technical Field

[0001] The present application relates to the field of biosynthesis technology, and in particular to a pyridone compound and a biosynthesis method and application thereof. Background Art

[0002] Pyridinone compounds are an important type of organic compound. Their molecular structure contains a relatively unique six-membered aromatic ring (pyridine ring) and a ketone group. The pyridine ring contains both a carbonyl group and a N heteroatom. According to the relative position of the nitrogen atom and the carbonyl group, they are divided into 2-pyridone, 3-pyridone and 4-pyridone. At present, the chemically synthesized pyridone compounds are mainly monocarbonyl pyridone, which can be used as a key raw material or intermediate for the synthesis of organic compounds and are widely used in the preparation of antibiotics, drugs and dyes.

[0003] 2-Pyridone and 4-pyridone are widely present in various natural products, and their derivatives are reported to have antibacterial, anti-inflammatory, anti-tumor and insecticidal biological activities. Due to the structural characteristics of pyridone and its related biological effects, this structure has become an important skeleton for the synthesis of antibiotics and drugs.

[0004] The Chinese patent with patent application number 202180007672.3 discloses a pyridone compound that can be used as a MAT2A inhibitor; pyridone 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 of green-yellow to orange azo dyes in the dye industry. It has strong coupling ability, high extinction coefficient of the synthesized dye, bright color, high light fastness and other excellent characteristics, and is widely used in the synthesis of disperse and reactive dyes. The discovery of new pyridone compounds is of great significance for the development of new antibiotics, drugs and dyes.

[0005] The products of nonribosomal peptide synthetases (NRPS) present in microorganisms are the source of a variety of antibiotics and natural pigments, including Minimycin and Indigoidine. Minimycin and Indigoidine have similar structural monomers. Chinese patent CN 110777155 B speculates that a pyridone compound is their common precursor, but the clear structural characteristics of the compound have not been obtained. Zhang et al. (Zhang Z, Li P, Wang M, et al. (S)-3-aminopiperidine-2, 6-dione is abiosynthetic 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 a nonribosomal peptide synthetase mutant, the synthesis of piperidone compounds using glutamine as a substrate was achieved, but the biosynthesis of pyridone compounds was not involved.

[0006] At present, the preparation of pyridone compounds is still mainly based on chemical synthesis methods, and the method of biosynthesis of pyridone compounds has not been reported. Summary of the invention

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

[0008] To achieve the above purpose, the technical solution adopted by this application is: The present application provides a pyridone compound, the molecular formula of which is C 5 H 4 N 2 O 3 , relative molecular weight is 140.0; The chemical name of the pyridone compound is 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione; The chemical formula of the 5-aminopyridine-2,3,6-trione is shown in formula (I): Formula (I); The chemical formula of the 5-hydroxy-3-iminopyridine-2,6-dione is shown in formula (II): Formula (II).

[0009] This application first discovered and obtained the pyridone compound, and determined its chemical formula to be C 5 H 4 N 2 O 3 , named 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione. The pyridone compound has the potential to be used as a raw material or intermediate in the preparation of new antibiotics, drugs and dyes.

[0010] The present application also provides a pyridone synthase encoding gene encoding the pyridone compound, wherein the pyridone synthase encoding gene includes a pyridone synthase encoding gene pds1, a pyridone synthase encoding gene pds2, and a pyridone synthase encoding gene pds3; The pyridone synthase encoding gene pds1, the pyridone synthase encoding gene pds2 and the pyridone synthase encoding gene pds3 are respectively formed by mutation of the basic amino acids in the catalytic sites of the cyanosynthase oxidation domain of the cyanosynthase encoding gene bpsA, the cyanosynthase encoding gene indC and the cyanosynthase encoding gene idgS into neutral amino acids.

[0011] In the process of studying the structure of the existing indigofera synthase (a non-ribosomal peptide synthetase), the inventors of this application found that the catalytic site of the indigofera synthase oxidation domain (Oxidation domain) of indigofera synthase bpsA, indigofera synthase indC and indigofera synthase idgS is a basic amino acid. Mutating it into a neutral amino acid will destroy the catalytic ability of the indigofera synthase oxidation domain, change the catalytic activity of the enzyme, and thus inhibit the synthesis of indigofera. It will promote the accumulation of its precursor - a pyridone compound, thereby converting the indigofera synthase into pyridone synthase Pds1, Pds2 and Pds3, and realizing the synthesis of 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione using glutamine as a substrate.

[0012] As a preferred embodiment of the pyridone synthase encoding gene encoding the pyridone compounds described in the present application, the catalytic site of the indigo synthase oxidation domain includes the 876th site of the indigo synthase oxidation domain.

[0013] 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.

[0014] In the technical scheme of the present application, the 876th sites of indigo blue synthase bpsA, indigo blue synthase indC and indigo blue synthase idgS are all located in the catalytic pocket of the oxidation domain of indigo blue synthase, and are all basic amino acids. After the basic amino acid histidine at this site is directed to mutate to neutral alanine, the catalytic ability of the oxidation domain will be destroyed, thereby inhibiting the synthesis of indigo blue and promoting the accumulation of precursor-pyridone compounds, thereby converting indigo blue synthase into pyridone synthase.

[0015] Based on the mutation of the above amino acid sites, the basic amino acid histidine at the 876th site of the blue synthase oxidation domain of the blue synthase bpsA was mutated to neutral alanine, forming the blue synthase mutant BpsA-H876A named pyridinone synthetase encoding gene Pds1 (Pyridinone synthetase 1); The basic amino acid histidine at position 876 of the indigo synthase oxidation domain of the indigo synthase indC was mutated to neutral alanine to form the indigo synthase mutant IndC-H876A, which was named the pyridinone synthetase encoding gene Pds2 (Pyridinone synthetase 2); The basic amino acid histidine at position 876 of the indigo synthase oxidation domain of the indigo synthase idgS was mutated to neutral alanine, and the indigo synthase mutant IdgS-H876A was named the pyridinone synthetase encoding gene Pds3 (Pyridinone synthetase 2).

[0016] As a preferred embodiment of the pyridone synthase encoding gene encoding 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.

[0017] The present application also provides a vector or a recombinant vector containing a pyridone synthase encoding gene of the pyridone compound.

[0018] The present application also provides a method for constructing a metabolic engineering bacterium for biosynthesizing the pyridone compounds, and the method for constructing the metabolic engineering bacterium comprises the following steps: At least one of the pyridone synthase encoding gene pds1, the pyridone synthase encoding gene pds2 and the pyridone synthase encoding gene pds3 and a phosphopantetheinyl transferase encoding gene are introduced into the host bacteria to obtain a metabolically engineered bacterium; The metabolically engineered bacteria biosynthesize the pyridone compound; The pyridone synthase encoding gene pds1, the pyridone synthase encoding gene pds2 and the pyridone synthase encoding gene pds3 are respectively formed by mutation of the basic amino acids in the catalytic sites of the cyanosynthase oxidation domain of the cyanosynthase encoding gene bpsA, the cyanosynthase encoding gene indC and the cyanosynthase encoding gene idgS into neutral amino acids.

[0019] The indigo blue synthase encoding gene bpsA encodes the indigo blue synthase bpsA; the indigo blue synthase encoding gene indC encodes the indigo blue synthase indC; the indigo blue synthase encoding gene idgS encodes the indigo blue synthase idgS.

[0020] As a preferred embodiment of the method for constructing the metabolic engineered bacteria described in the present application, the phosphopantetheine transferase encoding gene includes at least one of the phosphopantetheine transferase encoding genes EntD, IndB and Sfp.

[0021] As a preferred embodiment of the method for constructing the metabolic engineered bacteria described in the present application, 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; the nucleotide sequence of the phosphopantetheine transferase encoding gene Sfp is shown in SEQ ID NO: 6.

[0022] As a preferred embodiment of the method for constructing metabolically engineered bacteria described in the present application, the host bacteria include at least one of Escherichia coli and its derivative strains, Corynebacterium glutamicum and its derivative strains, Streptomyces and its derivative strains, and Saccharomyces and its derivative strains.

[0023] Preferably, the host bacteria include Escherichia coli or Corynebacterium glutamicum.

[0024] 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.

[0025] The host bacteria used in the present application include but are not limited to common microorganisms such as Escherichia coli, Corynebacterium glutamicum, Streptomyces and yeast.

[0026] Experiments have shown that the original Escherichia coli BL21 (DE3) and Corynebacterium glutamicum ATCC 13032 cannot use glucose, glycerol, glutamate 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 from Escherichia coli and Corynebacterium glutamicum into metabolically engineered bacteria to achieve the biosynthesis of 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione.

[0027] The metabolically engineered strains include but are not limited to metabolically engineered strains with similar pyridone synthesis capabilities constructed in different host bacteria by combining any one or more of pyridone synthases Pds1, Pds2 and Pds3 with any one or more of phosphopantetheine transferases EntD, IndB and Sfp.

[0028] As a preferred embodiment of the method for constructing the metabolically engineered bacteria described in the present application, the construction method includes any one of the following: Amplifying the recombinant vector, then connecting the pyridone synthase encoding gene pds1 and the recombinant vector, performing PCR amplification to obtain a recombinant vector I, then connecting the phosphopantetheinyl transferase encoding gene EntD and the recombinant vector I to obtain a recombinant vector II, and introducing the recombinant vector II into Escherichia coli to obtain a metabolic engineering bacterium; Alternatively, the recombinant vector is amplified, and then the pyridone synthase encoding gene pds2 is connected to the recombinant vector, and PCR amplification is performed to obtain a recombinant vector I, and then the phosphopantetheinyl transferase encoding gene indB is connected to the recombinant vector I to obtain a recombinant vector II, and the recombinant vector II is introduced into Escherichia coli to obtain a metabolic engineered bacterium; Alternatively, the recombinant vector is amplified, and then the pyridone synthase encoding gene pds3 is connected to the recombinant vector, and PCR amplification is performed to obtain a recombinant vector I, and then the phosphopantetheinyl transferase encoding gene sfp is connected to the recombinant vector I to obtain a recombinant vector II, and the recombinant vector II is introduced into Escherichia coli to obtain a metabolic engineered bacterium; Alternatively, the recombinant vector is amplified, and then the pyridone synthase encoding gene pds1 is connected to the recombinant vector, and PCR amplification is performed to obtain a recombinant vector I, and then the phosphopantetheine transferase encoding gene EntD is connected to the recombinant vector I to obtain a recombinant vector II, and the recombinant vector II is introduced into Corynebacterium glutamicum to obtain a metabolic engineered bacterium; Alternatively, the recombinant vector is amplified, and then the pyridone synthase encoding gene pds2 is connected to the recombinant vector, and PCR amplification is performed to obtain a recombinant vector I, and then the phosphopantetheine transferase encoding gene indB is connected to the recombinant vector I to obtain a recombinant vector II, and the recombinant vector II is introduced into Corynebacterium glutamicum to obtain a metabolic engineered bacterium; Alternatively, the recombinant vector is amplified, and then the pyridone synthase encoding gene pds3 is connected to the recombinant vector, and PCR amplification is performed to obtain a recombinant vector I, and then the phosphopantetheine transferase encoding gene sfp is connected to the recombinant vector I to obtain a recombinant vector II, and the recombinant vector II is introduced into Corynebacterium glutamicum to obtain a metabolic engineered bacterium; The recombinant vector I includes but is not limited to any one of the recombinant vectors pCDFDuet-Pds1, pCDFDuet-Pds2, pCDFDuet-Pds3, pXMJ19-Pds1, pXMJ19-Pds2 and pXMJ19-Pds3; The recombinant vector II includes but is not limited to any one of the recombinant vector pCDFDuet-Pds1-entD, the recombinant vector pCDFDuet-Pds2-indB, the recombinant vector pCDFDuet-Pds3-sfp, the recombinant vector pXMJ19-Pds1-entD, the recombinant vector pXMJ19-Pds2-indB, and the recombinant vector pXMJ19-Pds3-sfp.

[0029] As a preferred embodiment of the method for constructing metabolically engineered bacteria described in the present application, the PCR amplification uses primers with nucleotide sequences as shown in SEQ ID NOs: 7 to 20.

[0030] Preferably, the recombinant vector includes at least one of the pCDFDuet recombinant vector and the pXMJ19 recombinant vector.

[0031] The present application also provides metabolically engineered bacteria obtained by the construction method of the above-mentioned metabolically engineered bacteria.

[0032] In some specific embodiments, the metabolically engineered bacteria include recombinant Escherichia coli (E. coli) strains HG-N-Pd01, HG-N-Pd02, HG-N-Pd03, and Corynebacterium glutamicum (C. glutamicum) HG-N-Pd04, HG-N-Pd05, HG-N-Pd06.

[0033] In some specific embodiments, the metabolically engineered bacteria is the recombinant Escherichia coli strain HG-N-Pd01, and the recombinant Escherichia coli strain HG-N-Pd01 is constructed by introducing the recombinant vector pCDFDuet-Pds1-entD into Escherichia coli BL21 (DE3).

[0034] The recombinant vector pCDFDuet-Pds1-entD is obtained by PCR amplification, gene synthesis, and ligation of the pyridone synthase encoding gene pds1 and the phosphopantetheinyl transferase encoding gene entD derived from C. glutamicum to the recombinant vector pCDFDuet-1.

[0035] The pyridone synthase encoding gene pds1 is the gene sequence of the cyanobacterial synthase site-directed mutant BpsA-H876A, and is obtained by PCR amplification using site-directed mutation primers.

[0036] The construction method of the recombinant vector pCDFDuet-Pds1-entD comprises the following specific steps: (1) The pyridone synthase encoding gene pds1 was obtained by site-directed mutagenesis PCR amplification of the cyanobacterial synthase encoding gene bpsA, and the phosphopantetheinyl transferase encoding gene entD was obtained by gene synthesis.

[0037] (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.

[0038] (3) pCDFDuet-Pds1-entD was transferred to Escherichia coli BL21 (DE3) by chemical transformation 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 was derived from the Chinese patent CN 118184576 A.

[0039] In some specific embodiments, the metabolically engineered bacteria is the recombinant Escherichia coli strain HG-N-Pd02, and the recombinant Escherichia coli strain HG-N-Pd02 is constructed by introducing the recombinant vector pCDFDuet-Pds2-indB into Escherichia coli BL21 (DE3).

[0040] The recombinant vector pCDFDuet-Pds2-indB is obtained by PCR amplification, gene synthesis, and ligation of the pyridone synthase encoding gene pds2 and the phosphopantetheinyl transferase encoding gene indB from S. chromofuscus to the recombinant vector pCDFDuet-1.

[0041] The pyridone synthase encoding gene pds2 is the gene sequence of the indigo synthase site-directed mutant IndC-H876A, and is obtained by PCR amplification using site-directed mutation primers.

[0042] In some specific embodiments, the metabolically engineered bacteria is the recombinant Escherichia coli strain HG-N-Pd03, and the recombinant Escherichia coli strain HG-N-Pd03 is constructed by introducing the recombinant vector pCDFDuet-Pds3-sfp into Escherichia coli BL21 (DE3).

[0043] The recombinant vector pCDFDuet-Pds3-sfp is obtained by PCR amplification, gene synthesis, and ligation of the pyridone synthase encoding gene pds3 and the phosphopantetheinyl transferase encoding gene sfp derived from B. subtilis to the recombinant vector pCDFDuet-1.

[0044] The pyridone synthase encoding gene pds3 is the gene sequence of the cyanobacterial synthase site-directed mutant IdgS-H876A, and is obtained by PCR amplification using site-directed mutation primers.

[0045] In some specific embodiments, the metabolically engineered bacteria is the recombinant strain HG-N-Pd04 of Corynebacterium glutamicum (C. glutamicum).

[0046] The recombinant strain HG-N-Pd04 of Corynebacterium glutamicum (C. glutamicum) is constructed by introducing the recombinant vector pXMJ19-Pds1-entD into Corynebacterium glutamicum ATCC13032.

[0047] The recombinant vector pXMJ19-Pds1-entD is obtained by PCR amplification, gene synthesis, and ligation of the pyridone synthase encoding gene pds1 and the phosphopantetheinyl transferase encoding gene entD derived from C. glutamicum to the recombinant vector pXMJ19.

[0048] The construction method of the recombinant vector pXMJ19-Pds1-entD comprises the following specific steps: (1) The pyridone synthase encoding gene pds1 was obtained by site-directed mutagenesis PCR amplification of the cyanobacterial synthase encoding gene bpsA, and the phosphopantetheinyl transferase encoding gene entD was obtained by gene synthesis.

[0049] (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.

[0050] (3) The recombinant vector pXMJ19-Pds1-entD was transferred into Corynebacterium glutamicum ATCC13032 by electroporation to obtain the recombinant strain HG-N-Pd04 of Corynebacterium glutamicum.

[0051] In some specific embodiments, the metabolically engineered bacteria is the recombinant strain of Corynebacterium glutamicum HG-N-Pd05, and the recombinant strain of Corynebacterium glutamicum (C. glutamicum) HG-N-Pd05 is constructed by introducing the recombinant vector pXMJ19-Pds2-indB into Corynebacterium glutamicum ATCC 13032.

[0052] The recombinant vector pXMJ19-Pds2-indB is obtained by PCR amplification, gene synthesis, and ligation to the recombinant vector pXMJ19 of the pyridone synthase encoding gene pds2 and the phosphopantetheinyl transferase encoding gene indB derived from S. chromofuscus.

[0053] The pyridone synthase encoding gene pds2 is obtained by site-directed mutagenesis PCR amplification of the indigo synthase encoding gene indC.

[0054] In some specific embodiments, the metabolically engineered bacteria is the recombinant strain of Corynebacterium glutamicum HG-N-Pd06, and the recombinant strain of Corynebacterium glutamicum (C. glutamicum) HG-N-Pd06 is constructed by introducing the recombinant vector pXMJ19-Pds3-sfp into Corynebacterium glutamicum ATCC 13032.

[0055] The recombinant vector pXMJ19-Pds3-sfp is obtained by PCR amplification, gene synthesis, and ligation of the pyridone synthase encoding gene pds3 and the phosphopantetheinyl transferase encoding gene sfp from B. subtilis to the recombinant vector pXMJ19.

[0056] The pyridone synthase encoding gene pds3 is obtained by site-directed mutagenesis PCR amplification of the indigo synthase encoding gene idgS.

[0057] The Escherichia coli (E. coli) recombinant strains HG-N-Pd01, HG-N-Pd02, HG-N-Pd03 and the C. glutamicum (C. glutamicum) recombinant strains HG-N-Pd04, HG-N-Pd05, and HG-N-Pd06 constructed in the present application efficiently express pyridone synthase and 4'-phosphopantetheinyl transferase through vectors, and all achieve the synthesis of 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione.

[0058] The present application also provides the use of the above metabolically engineered bacteria in the biosynthesis of pyridone compounds.

[0059] The present application also provides a biosynthetic method for the pyridinone compounds, wherein the biosynthetic method utilizes the metabolically engineered bacteria to express pyridinone synthase to catalyze glutamine biosynthesis of natural pyridinone compounds.

[0060] The present application also provides a fermentation broth or extract of the metabolically engineered bacteria, wherein the fermentation broth or extract comprises the pyridone compound.

[0061] The present application also provides a culture medium for fermenting the pyridone compound, wherein the culture medium comprises the following components in mass concentrations: Glucose 30-50 g / L, glycerol 5-10 g / L, molasses 5-10 g / L, corn syrup 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, the solvent is water.

[0062] Preferably, the initial pH of the culture medium is 6.5.

[0063] Preferably, the culture medium comprises the following components in mass concentrations: 40 g / L glucose, 7 g / L glycerol, 7 g / L molasses, 8 g / L corn steep liquor powder, 3 g / L dipotassium hydrogen phosphate dodecahydrate, 3.5 g / L potassium dihydrogen phosphate, 25 g / L ammonium sulfate, 0.8 g / L magnesium sulfate heptahydrate, 0.07 g / L ferrous sulfate heptahydrate, and the solvent is water.

[0064] The metabolically engineered bacteria of the present application can be used for fermentation production, and can ferment and synthesize pyridone compounds in the culture medium.

[0065] Glucose and glycerol are used as carbon sources in the culture medium. The mixed carbon source improves the tricarboxylic acid cycle efficiency of the strain and enhances the synthesis of glutamate and glutamine, the precursors of pyridone; corn syrup and molasses provide the necessary organic nitrogen source for the growth of the strain, while supplementing some trace elements and growth factors to ensure the normal growth of the engineered bacteria in the early stage; dipotassium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate are used as buffers to maintain the stability of the fermentation pH; ferrous sulfate heptahydrate inhibits the activity of glutamine degrading enzymes and increases the accumulation of pyridone precursors; magnesium sulfate heptahydrate provides key enzyme cofactors to maintain their catalytic activity.

[0066] In some specific embodiments, the fermentation broth containing pyridone compounds provided in the present application is obtained by fermentation of the recombinant strain HG-N-Pd01 of Escherichia coli (E. coli), and the specific steps of the preparation method of the fermentation broth are as follows: pick a single colony of the recombinant strain HG-N-Pd01 and transfer it to 5 mL LB medium, the medium contains 50 μg / mL streptomycin, culture at 37°C, 220rpm for 16 hours, inoculate it into 50 mL Pds fermentation medium with a 1% inoculum amount, add a final concentration of 50 μg / mL streptomycin and a final concentration of 0.1 mM inducer IPTG, continue to culture for 48 hours, and obtain a fermentation broth containing pyridone compounds.

[0067] In some specific embodiments, the fermentation broth containing pyridone compounds provided in the present application is obtained by fermentation of the recombinant strain HG-N-Pd04 of Corynebacterium glutamicum (C. glutamicum), and the specific steps of the preparation method of the fermentation broth are as follows: pick a single colony of the recombinant strain HG-N-Pd04 and transfer it to 5 mL BHISG medium, the medium contains 15 μg / mL chloramphenicol and 50 μg / L biotin, culture at 30°C, 220rpm for 16 hours, inoculate into 50 mL Pds fermentation medium with a 1% inoculum amount, add 15 μg / mL chloramphenicol and 50 μg / L biotin to the medium, culture at 32°C, 220rpm, add IPTG with a final concentration of 1 mM after 6 hours, and continue to culture for 48 hours to obtain a fermentation broth containing pyridone compounds.

[0068] The present application also provides the use of the above-mentioned pyridone compounds in the preparation of new antibiotics, drugs or dyes.

[0069] The present application utilizes genetic engineering technology to transform the metabolic pathways of Escherichia coli and Corynebacterium glutamicum, respectively, and express non-ribosomal peptide synthetase, thereby realizing fermentation synthesis of pyridone compounds or their enol tautomers using glucose as a substrate. The pyridone compounds are isolated and obtained for the first time from fermentation broths of Escherichia coli and Corynebacterium glutamicum, and their molecular structures are inferred by mass spectrometry, nuclear magnetic resonance and other analytical methods.

[0070] Compared with the prior art, this application has the following beneficial effects: The present application provides a pyridone compound and a biosynthesis method and application thereof. The present application is based on synthetic biology related technologies, and by modifying non-ribosomal peptide synthetase (blue synthetase), heterologously expressing non-ribosomal peptide synthetase mutants in various hosts, the pyridone compound is discovered and obtained for the first time, and its chemical formula is determined to be C 5 H 4 N 2 O 3 , named 5-aminopyridine-2,3,6-trione or 5-hydroxy-3-iminopyridine-2,6-dione. This new pyridone compound can be used as a raw material or intermediate in the development and preparation of new antibiotics, drugs and dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 This is the liquid phase detection diagram of the original E. coli BL21 fermentation product; Figure 2 This is a liquid phase detection diagram of the fermentation product of recombinant Escherichia coli BL21 (DE3) / pCDFDuet-bpsA-entD; Figure 3 This is the liquid phase detection diagram of the fermentation product of recombinant Escherichia coli HG-N-Pd01; Figure 4 This is a liquid phase detection diagram of the fermentation product of recombinant Escherichia coli HG-N-Pd01 supplemented with glutamine; Figure 5 It is the UV-visible absorption spectrum of pyridone compounds; Figure 6 It is a high-resolution mass spectrometry spectrum of pyridone compounds; Figure 7 H NMR spectrum of pyridone compounds 1 H-NMR spectrum; Figure 8 The NMR carbon spectrum of pyridone compounds 13 C-NMR spectrum; Fig. 9 This is the NMR HSQC spectrum of pyridone compounds; Fig.10 It is the NMR HMBC spectrum of pyridone compounds; Fig.11 It is the structural analysis and appearance diagram of pyridone compounds; Wherein, A is the inferred structure diagram of pyridone compounds; B is a liquid phase detection diagram of tautomers of pyridone compounds; C is the UV-visible absorption spectrum of pyridone compounds; Fig.12 The figure is a speculated diagram of the biosynthetic pathway of pyridone compounds; Fig.13 This is the liquid phase detection diagram of the fermentation synthesis of pyridone compounds by the recombinant strain HG-N-Pd01; Fig.14 This is a liquid phase detection diagram of the fermentation synthesis of pyridone compounds by the recombinant strain HG-N-Pd02; Fig.15 This is a liquid phase detection diagram of the fermentation synthesis of pyridone compounds by the recombinant strain HG-N-Pd03; Fig.16 This is a liquid phase detection diagram of the fermentation synthesis of pyridone compounds by the recombinant strain HG-N-Pd04; Fig.17 This is a liquid phase detection diagram of the fermentation synthesis of pyridone compounds by the recombinant strain HG-N-Pd05; Fig.18 This is a liquid phase detection diagram of the fermentation synthesis of pyridone compounds by the recombinant strain HG-N-Pd06; Fig.19 This is a liquid phase detection diagram of the fermentation product of the mutant bpsA-H876D engineered bacteria; Fig. 20 This is a liquid phase detection diagram of the fermentation product of the mutant indC-H876D engineered bacteria; Fig.21 This is a liquid phase detection diagram of the fermentation product of the mutant idgS-H876D engineered bacteria. DETAILED DESCRIPTION

[0072] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0073] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0074] The culture medium components involved in the following examples are as follows: LB (Luria-Bertani) liquid medium: molasses 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, sterilized at 121°C for 20 min.

[0075] LB (Luria-Bertani) solid medium: 10 g / L molasses, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar powder, sterilize at 121℃ for 20 min, cool the solid medium to about 50℃, add the required antibiotics, pour into plates, and place at 4℃ for use after solidification.

[0076] BHISG medium: 37 g / L brain heart extract powder, 10 g / L glucose, sterilized at 115℃ for 30 min; 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.

[0077] The primers involved in the following examples are shown in Table 1.

[0078] Table 1 Primer information The strains involved in the following examples are shown in Table 2.

[0079] Table 2 Metabolic engineering strain information The phosphopantetheine transferase encoding gene entD mentioned in the present application 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.

[0080] 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 phosphopantetheinyl transferase encoding gene EntD is shown in SEQ ID NO: 4, the nucleotide sequence of the phosphopantetheinyl transferase encoding gene indB is shown in SEQ ID NO: 5, and the nucleotide sequence of the phosphopantetheinyl transferase encoding gene sfp is shown in SEQ ID NO: 6.

[0081] In the following examples, the high performance liquid chromatography detection method of the samples is as follows.

[0082] HPLC conditions: Mobile phase: methanol and pure water gradient elution, gradient table 3 is as follows: Table 3 Liquid phase gradient elution table The wavelength was 400 nm, the flow rate was 1.0 mL / min, the sample preparation solution was DMSO, the injection volume was 10 μL, the column temperature was 35 °C, and the running time was 18 min.

[0083] Chromatographic column model: GalasilEF-C18M 4.6mm id×250mm L (SN B06211801).

[0084] Example 1. Construction of metabolically engineered strain HG-N-Pd01 The method for constructing the metabolically engineered strain HG-N-Pd01 of this embodiment comprises the following steps: Step 1: Construction of pyridone synthase Pds1: (1) Using primer point mutagenesis technology, the cyanobacterial synthase encoding gene bpsA was used as a template and site-directed mutagenesis was performed to bpsA-H876A by PCR to obtain the mutant encoding gene sequence. The sequences of the mutation primers used are shown in SEQ ID NO: 7 and SEQ ID NO: 8 in Table 1.

[0085] (2) The PCR product was tested by gene sequencing to confirm that the mutant sequence had no mismatch or other abnormalities and then stored for future use. The gene obtained after mutation was named pyridone synthase gene pds1, and its nucleotide sequence was shown in SEQ ID NO: 1. Hereinafter, the mutant was referred to as Pds1.

[0086] Step 2: Construction of pCDFDuet-Pds1-entD vector: (1) The pyridone synthase encoding gene pds1 (nucleotide sequence as shown in SEQ ID NO: 1) was obtained by site-directed mutagenesis PCR amplification of the cyanobacterial synthase encoding gene bpsA as described in step 1, and the 4'-phosphopantetheinyl transferase encoding gene entD (nucleotide sequence as shown in SEQ ID NO: 4) was synthesized by Qingke Biotechnology Co., Ltd.

[0087] (2) Using the commercial vector pCDFDuet-1 purchased on the market as a template, primers pCDF-PF (SEQ ID NO: 13) and pCDF-PR (SEQ ID NO: 14) were used for PCR amplification, and the product was purified to obtain the pCDFDuet-1 linearized vector.

[0088] (3) The pCDFDuet-1 linearized vector and the Pds1 gene (homologous arms of the fragments have been added at both ends of pCDFDuet-1) were connected using a seamless cloning ligation kit, and the ligation product was transferred into Escherichia coli DH5α by chemical transformation. After recovery and cultivation, it was spread on an LB solid culture medium plate containing 50 μg / mL streptomycin resistance and cultured in a 37°C incubator for about 16 h.

[0089] (4) Use primers pCDF-YZ-F (SEQ ID NO: 15) and pCDF-YZ-R (SEQ ID NO: 16) to perform colony PCR verification. Cultivate the strain that is verified correctly by PCR, extract the recombinant vector, and send it to Qingke Biotechnology Co., Ltd. for sequencing. The vector that is sequenced correctly is the pCDFDuet-Pds1 vector.

[0090] (5) Using pCDFDuet-Pds1 vector as template, pCDF-IF (SEQ ID NO: 17) and pCDF-IR (SEQ ID NO: 18) were used for PCR amplification, and the product was purified to obtain the pCDFDuet-Pds1 linearized vector. The pCDFDuet-Pds1 linearized vector and the entD fragment of the 4'-phosphopantetheine transferase encoding gene were ligated using a seamless cloning ligation kit, and the ligation product was transferred into Escherichia coli DH5α by chemical transformation. After recovery culture, it was spread on an LB solid culture medium plate containing 50 μg / mL streptomycin resistance and cultured in a 37°C incubator for about 16 hours.

[0091] (6) Use primers pCDF-YZ-F (SEQ ID NO: 15) and pCDF-YZ-R (SEQ ID NO: 16) to perform colony PCR verification. Cultivate the strain that is correct by PCR verification, extract the recombinant vector, and send it to Qingke Biotechnology Co., Ltd. for sequencing. The vector that is correctly sequenced is the pCDFDuet-Pds1-entD vector.

[0092] Step 3: Construction of HG-N-Idg01 strain: (1) The BL21(DE3) competent strain was purchased from Qingke Biotechnology Co., Ltd. with the catalog number TSC-C14.

[0093] (2) The pCDFDuet-Pds1-entD vector was transferred into Escherichia coli BL21(DE3) by chemical transformation. After recovery and culture, it was spread on LB solid culture medium plates containing 50 μg / mL streptomycin resistance and cultured in a 37°C incubator for about 16 h.

[0094] (3) The single colony grown on the LB solid medium plate is the recombinant Escherichia coli BL21 (DE3) / pCDFDuet-Pds1-entD, named HG-N-Pd01.

[0095] Example 2: Preparation of recombinant Escherichia coli fermentation broth containing pyridone compounds (1) Seed solution preparation: Pick a single colony of the recombinant strain HG-N-Pd01 and transfer it to 5 mL LB medium containing 50 μg / mL streptomycin. Cultivate at 37°C and 220 rpm for 16 h to obtain seed solution. Preparation of fermentation broth: The seed liquid was inoculated into 50 mL of Pds fermentation medium at a 1% inoculum, and streptomycin with a final concentration of 50 μg / mL and an inducer IPTG with a final concentration of 0.1 mM were added. After continuing to culture for 48 hours, a fermentation broth containing pyridone compounds was obtained.

[0096] Example 3: Fermentation products of different recombinant Escherichia coli strains Experimental group 1, using the original E. coli BL21 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 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 1 shown.

[0097] Experimental group 2, using recombinant Escherichia coli BL21 (DE3) / pCDFDuet-bpsA-entD 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 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 2 shown.

[0098] Experimental group 3, using recombinant E. coli 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 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 3 shown.

[0099] Experimental group 4: Using recombinant E. coli HG-N-Pd01 as the fermentation strain, 50 mL of fermentation broth was prepared by the method of Example 2, and glutamine was added at a final concentration of 1 g / L after 24 hours of fermentation. After 48 hours of fermentation, 100 μL of fermentation broth was taken, 900 μL of DMSO was added, mixed and shaken for 10 minutes, centrifuged at 12000 rpm for 5 minutes, and 10 μL of supernatant was taken and tested by high performance liquid chromatography. The test results are as follows: Figure 4 shown.

[0100] The experimental results are as follows Figures 1 to 4 As shown, Figure 1 and Figure 2 No peak was observed around 5.7min. Figure 3 and Figure 4 A peak was observed at a liquid phase retention time of 5.7 min. Figure 5 As shown, the maximum absorption peak of the compound is 396nm. The comparison of the results 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 the compound by the method of Example 2, while the recombinant Escherichia coli BL21 (DE3) / pCDFDuet-Pds1-entD can synthesize the compound under the same conditions. The comparison of the results of experimental group 3 and experimental group 4 shows that the peak area of ​​the compound increases significantly after adding glutamine, and it is speculated that glutamine may be a precursor for the synthesis of the compound.

[0101] Example 4: Structural Identification of Unknown New Compound Samples 40 mL of the fermentation broth of Experimental Group 4 in Example 3 was collected and centrifuged at 12000 g for 5 min. After removing the supernatant, the precipitated cells were resuspended with 10 mL of dimethyl sulfoxide (DMSO), and the cells were broken by ultrasound to extract the pyridone compounds into the solvent. The supernatant was obtained by centrifugation again, the organic solvent was evaporated in vacuum, and then the solid was washed twice with 10 mL of pure water, methanol, ethyl acetate and hexane, respectively, and finally vacuum freeze-dried to obtain 0.04 g of yellow powder. The above samples were sent to the Analysis and Testing Center of Nanjing Normal University for high-resolution mass spectrometry and NMR detection.

[0102] Figure 6 This is a high-resolution mass spectrometry detection diagram of the pyridone compound; Figure 7 The pyridone compound 1 H-NMR spectrum; Figure 8 The pyridone compound 13 C-NMR spectrum; Fig. 9 and Fig.10 They are respectively the NMR HSQC spectrum and HMBC spectrum of the pyridone compound; Fig.11 This is a speculated diagram of the structure of the pyridone compound.

[0103] Table 4 shows 1 H-NMR( Figure 7 )and 13 C-NMR( Figure 8 ) data and inferred structure ( Fig.11 ) corresponds to the position of the elements in .

[0104] Table 4 NMR data analysis According to the high-resolution mass spectrometry ( Figure 6 ), the relative molecular weight of the compound was determined to be 140.0.

[0105] like Figure 7 As shown in the 1H-NMR spectrum, the peak at δ value of about 2.57 ppm is DMSO solvent, and the peak at δ value of about 3.52 ppm is H 2 O peak. The peak at 11.88 ppm was used as the integration standard, and the product was 1. From the HSQC spectrum ( Fig. 9 ), there is no point corresponding to 11.88 ppm on the horizontal axis, and it is speculated that hydrogen is connected to nitrogen here; similarly, the peaks at 7.61 ppm and 8.24 ppm are also not reflected in the HSQC spectrum, and it is speculated that the hydrogen at these two places is also connected to nitrogen; the peak at 5.72 pmm is a single peak, which is not reflected in the HSQC spectrum ( Fig. 9 ) has carbons corresponding to 100-110 ppm, which are presumed to be olefinic carbons; the integrals of the peaks at 11.88 ppm, 8.24 ppm, and 7.61 ppm are 1, indicating secondary amines. 1H-NMR can infer that the compound has three secondary amines and one double bond.

[0106] like Figure 8 As shown, 13 The peak with a δ value of about 40.6 ppm in the C-NMR spectrum is DMSO solvent, 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, which indicates that the compound contains 5 carbon groups; the δ value of 150 ppm-170 ppm is the carbonyl signal, so the two peaks with δ values ​​of 161.38 ppm and 159.03 ppm are inferred to be carbonyl carbon; generally, 100-150 ppm is SP2 hybridized carbon atom, and the peak at 102.24 ppm is the carbon corresponding to the 1H-NMR spectrum, indicating olefinic carbon.

[0107] In summary, combined with the blue structure, it is estimated 1 H-NMR spectrum and 13 The assignment of C-NMR spectrum signals is shown in Table 4, and the molecular formula of the compound is determined to be C 5 H 4 N 2 O 3 , the structure is as follows Fig.11 ( Fig.11 This structure has not been reported in the literature and is a new pyridone compound named 5-hydroxy-3-iminopyridine-2,6-dione.

[0108] Since the compound has the structural characteristics of the enol isomer, there is keto-enol tautomerism, such as Fig.11 ( Fig.11 As shown in B), the liquid phase detection of the separated product found an obvious peak at 6.3min. Based on the study of 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 6.3min in the liquid phase was named 5-aminopyridine-2,3,6-trione, and its structure and liquid phase detection are shown in Fig.11 ( Fig.11 As shown in Figure C, the maximum absorption peak is 394nm. The existing separation process can only separate the enol structure of the compound at 5.7min, and the keto structure at 6.3min has not yet been obtained as a pure product, so NMR and other tests have not been performed.

[0109] It is currently speculated that there may be two reasons why 5-aminopyridine-2,3,6-trione could not be successfully separated and purified: (1) 5-aminopyridine-2,3,6-trione with a keto structure is more likely to react with other substances during the fermentation process, participating in the synthesis of metabolites of the engineered strain and failing to accumulate. Fig.13 , Fig.14 , Fig.15 and Fig.18 As shown in the figure, 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 the loss is greater during the separation and purification process. The current separation and purification methods are unable to distinguish between keto-enol tautomers.

[0110] 5-Hydroxy-3-iminopyridine-2,6-dione appears yellow in DMSO solution. Based on the physical and chemical properties of the compound, it is speculated that the pyridone compound can be used as a new type of yellow pigment or drug intermediate, and has great application potential in the fields of printing and dyeing and medicine.

[0111] The biosynthetic pathway of pyridone in metabolically engineered strain HG-N-Pd01 is as follows Fig.12 shown.

[0112] Example 5. Construction of metabolically engineered strain HG-N-Pd2 The difference between this embodiment and embodiment 1 is that: 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 mutation 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.

[0113] 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).

[0114] Example 6. Construction of metabolically engineered strain HG-N-Pd03 The difference between this embodiment and embodiment 1 is that: 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.

[0115] 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).

[0116] Example 7. Construction of metabolically engineered strain HG-N-Pd04 The host of the metabolic engineering strain described in this example is Corynebacterium glutamicum ATCC 12032, and the experimental steps are as follows: Step 1: The method for obtaining the pyridone synthase gene pds1 in this example is the same as step 1 in Example 1.

[0117] Step 2. In this embodiment, the commercial vector pXMJ19 purchased from the market is used as a template, and primers pXMJ19-PF (SEQ ID NO: 19) and pXMJ19-PR (SEQ ID NO: 20) are used for PCR amplification to obtain a pXMJ19 linearized vector for the construction of the recombinant vector pXMJ19-Pds1-entD; primers pXMJ19-YZ-F (SEQ ID NO: 21) and pXMJ19-YZ-R (SEQ ID NO: 22) are used for colony PCR verification, and the strain verified by PCR is cultured, the recombinant vector is extracted, and sent to Qingke Biotechnology Co., Ltd. for sequencing. The vector with correct sequencing is the pXMJ19-Pds1-entD vector.

[0118] Step 3, prepare the electroporation competent state of Corynebacterium glutamicum ATCC 13032, pick a single colony of ATCC13032 in 5mL of antibiotic-free BHISG medium, and culture it overnight at 30℃ and 220rpm; take 15 OD bacterial solution and inoculate it in 50 mL of BHISG GT medium (0.1% Tween 80 and 50μg / L biotin), culture it at 30℃ and 220rpm until OD600 is about 1.0, then take it out and place it on ice, and ice bath it for 20min; transfer the bacterial solution to a 50ml centrifuge tube, centrifuge it at 4℃ and 2600×g for 10 min, discard the supernatant, add 50 mL of 10% glycerol (precooled) to resuspend the bacteria, centrifuge it at 4℃ and 2600×g for 10min, discard the supernatant, and use a gun to suck away the residual bacterial solution; repeat this step once; resuspend the bacteria with 100 μL of 10% glycerol (precooled), transfer it to a 1.5 mL EP tube, and set aside.

[0119] Step 4: Construction of recombinant strain HG-N-Pd04: (1) Take 200 ng of pXMJ19-Pds1-entD vector and add it to the electroporation competent cells prepared in step 3. Mix gently and add it to the electroporation cup and place it on ice for 5-10 minutes. (2) The electroporation condition was 1.8 KV. After electroporation, BHISG medium preheated at 46°C was quickly added. After heat shock in a 46°C water bath for 6 minutes, the transformation solution was placed in a 30°C shaker and incubated for 2 hours. (3) After incubation, take 1 mL of the transformation solution and centrifuge at 4000 rpm. Discard most of the supernatant and resuspend the remaining part. Spread it on a chloramphenicol-resistant BHISG plate and incubate it in a 30°C incubator for 48 h.

[0120] (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.

[0121] Example 8: Preparation of recombinant Corynebacterium glutamicum fermentation broth containing pyridone compounds (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. (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.

[0122] Example 9. Construction of metabolically engineered strain HG-N-Pd05 The difference between this embodiment and embodiment 7 is that: 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 mutation 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.

[0123] 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).

[0124] Example 10. Construction of metabolically engineered strain HG-N-Pd06 The difference between this embodiment and embodiment 7 is that: 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.

[0125] Step 2: construct the recombinant vector pXMJ19-Pds3-sfp. The pyridone synthase encoding gene used is pds3 (nucleotide sequence is shown in SEQ ID NO: 3).

[0126] Example 11: Fermentation synthesis of pyridone compounds by different recombinant strains Experimental group 1: The recombinant strain HG-N-Pd01 was used as the fermentation strain, and 50 mL of fermentation broth was prepared by the method of Example 2. 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: Fig.13 shown.

[0127] 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 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: Fig.14 shown.

[0128] 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 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: Fig.15 shown.

[0129] 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 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: Fig.16 shown.

[0130] Experimental group 5: The recombinant strain HG-N-Pd05 was used as the fermentation strain, and 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: Fig.17 shown.

[0131] 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: Fig.18 shown.

[0132] The experimental results are as follows Figures 13 to 18As 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.

[0133] Comparative Example 1 This comparative example is different from Example 1 in that: 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. Fig.19 As shown, no pyridone compounds were observed.

[0134] Comparative Example 2 This comparative example is different from Example 5 in that: 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. Fig. 20 As shown, no pyridone compounds were observed.

[0135] Comparative Example 3 This comparative example is different from Example 6 in that: 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. Fig.21 As shown, no pyridone compounds were observed.

[0136] 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 compound, characterized in that The structural formula of the pyridone compound is shown in formula (I) or formula (II); Formula (I); Formula (II).

2. A pyridone synthase encoding gene encoding the pyridone compound according to claim 1, characterized in that: The pyridone synthase encoding gene includes a pyridone synthase encoding gene pds1, a pyridone synthase encoding gene pds2 and a pyridone synthase encoding gene pds3; The pyridone synthase encoding gene pds1, the pyridone synthase encoding gene pds2 and the pyridone synthase encoding gene pds3 are respectively formed by mutation of the basic amino acid at the catalytic site of the cyanosynthase oxidation domain of the cyanosynthase encoding gene bpsA, the cyanosynthase encoding gene indC and the cyanosynthase encoding gene idgS to a neutral amino acid; The catalytic site of the cyanobacterium synthase oxidation domain includes the 876th site of the cyanobacterium synthase oxidation domain.

3. The pyridone synthase encoding gene of the pyridone compound according to claim 2, characterized in that: The basic amino acid includes histidine, and the neutral amino acid includes alanine.

4. The pyridone synthase encoding gene of the pyridone compound according to claim 2, characterized in that: 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.

5. A vector or recombinant vector containing a pyridone synthase encoding gene of the pyridone compound according to any one of claims 2 to 4.

6. A method for constructing a metabolically engineered bacterium for biosynthesizing the pyridone compound according to claim 1, characterized in that: The method for constructing the metabolic engineering bacteria comprises the following steps: Introducing at least one of the pyridone synthase encoding gene pds1, the pyridone synthase encoding gene pds2 and the pyridone synthase encoding gene pds3 as described in any one of claims 2 to 4 and a phosphopantetheinyl transferase encoding gene into a host bacterium to obtain a metabolically engineered bacterium; The metabolically engineered bacteria biosynthesize the pyridone compound as claimed in claim 1.

7. The method for constructing metabolically engineered bacteria according to claim 6, characterized in that: The phosphopantetheinyl transferase encoding gene includes at least one of the phosphopantetheinyl transferase encoding genes EntD, IndB and Sfp.

8. The method for constructing metabolically engineered bacteria according to claim 6, characterized in that: The nucleotide sequence of the phosphopantetheinyl transferase encoding gene EntD is shown in SEQ ID NO: 4; the nucleotide sequence of the phosphopantetheinyl transferase encoding gene IndB is shown in SEQ ID NO: 5; and the nucleotide sequence of the phosphopantetheinyl transferase encoding gene Sfp is shown in SEQ ID NO:

6.

9. The method for constructing metabolically engineered bacteria according to claim 6, characterized in that: The host bacteria include at least one of Escherichia coli and its derivative strains, Corynebacterium glutamicum and its derivative strains, Streptomyces and its derivative strains, and Saccharomyces and its derivative strains.

10. The method for constructing metabolically engineered bacteria according to claim 7, characterized in that: The construction method includes any one of the following: Amplifying the recombinant vector, then connecting the pyridone synthase encoding gene pds1 and the recombinant vector, performing PCR amplification to obtain a recombinant vector I, then connecting the phosphopantetheinyl transferase encoding gene EntD and the recombinant vector I to obtain a recombinant vector II, and introducing the recombinant vector II into Escherichia coli to obtain a metabolic engineering bacterium; Alternatively, the recombinant vector is amplified, and then the pyridone synthase encoding gene pds2 is connected to the recombinant vector, and PCR amplification is performed to obtain a recombinant vector I, and then the phosphopantetheinyl transferase encoding gene indB is connected to the recombinant vector I to obtain a recombinant vector II, and the recombinant vector II is introduced into Escherichia coli to obtain a metabolic engineered bacterium; Alternatively, the recombinant vector is amplified, and then the pyridone synthase encoding gene pds3 is connected to the recombinant vector, and PCR amplification is performed to obtain a recombinant vector I, and then the phosphopantetheinyl transferase encoding gene sfp is connected to the recombinant vector I to obtain a recombinant vector II, and the recombinant vector II is introduced into Escherichia coli to obtain a metabolic engineered bacterium; Alternatively, the recombinant vector is amplified, and then the pyridone synthase encoding gene pds1 is connected to the recombinant vector, and PCR amplification is performed to obtain a recombinant vector I, and then the phosphopantetheine transferase encoding gene EntD is connected to the recombinant vector I to obtain a recombinant vector II, and the recombinant vector II is introduced into Corynebacterium glutamicum to obtain a metabolic engineered bacterium; Alternatively, the recombinant vector is amplified, and then the pyridone synthase encoding gene pds2 is connected to the recombinant vector, and PCR amplification is performed to obtain a recombinant vector I, and then the phosphopantetheine transferase encoding gene indB is connected to the recombinant vector I to obtain a recombinant vector II, and the recombinant vector II is introduced into Corynebacterium glutamicum to obtain a metabolic engineered bacterium; Alternatively, the recombinant vector is amplified, and then the pyridone synthase encoding gene pds3 is connected to the recombinant vector, and PCR amplification is performed to obtain a recombinant vector I, and then the phosphopantetheine transferase encoding gene sfp is connected to the recombinant vector I to obtain a recombinant vector II, and the recombinant vector II is introduced into Corynebacterium glutamicum to obtain a metabolic engineered bacterium.

11. The method for constructing metabolically engineered bacteria according to claim 10, characterized in that: The PCR amplification uses primers with nucleotide sequences as shown in SEQ ID NOs: 7-20.

12. The metabolically engineered bacteria obtained by the method for constructing metabolically engineered bacteria according to any one of claims 6 to 11.

13. Use of the metabolically engineered bacteria according to claim 12 in the biosynthesis of pyridone compounds.

14. A biosynthetic method for pyridone compounds as claimed in claim 1, characterized in that: The biosynthesis method utilizes the metabolically engineered bacteria as claimed in claim 12 to express pyridone synthase to catalyze the biosynthesis of pyridone compounds from glutamine.

15. A fermentation broth or extract containing the metabolically engineered bacteria according to claim 12, characterized in that: The fermentation broth or extract comprises the pyridone compound as claimed in claim 1.

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