Strain for efficiently producing 3-hydroxy-2-picolinic acid, construction method and application

Abstract

The invention discloses a strain capable of efficiently producing 3-hydroxy-2-picolinic acid, a construction method and application, and relates to the technical field of gene engineering. According to the 3-hydroxy-2-picolinic acid heterologous expression strain and the application thereof, homologous L-lysine-2-aminotransferase PyrB, bi-component flavin-dependent monooxygenase PyrC / PyrK and flavin adenine nucleotide-dependent dehydrogenase PyrD are cloned from streptomyces picoliniamycin NRRL B-2517 by virtue of a genetic engineering technology, the homologous L-lysine-2-aminotransferase PyrB, the bi-component flavin-dependent monooxygenase PyrC / PyrK and the flavin adenine nucleotide-dependent dehydrogenase PyrD are combined with a strong promoter, and an RBS sequence is optimized, so that the 3-hydroxy-2-picolinic acid heterologous expression strain is successfully constructed; a core foundation is laid for efficient, green and low-cost microbial fermentation production of the product. According to the microbial fermentation method, self production of 3-hydroxy-2-picolinic acid by cells is achieved through metabolic pathway reconstruction, the whole reaction process is a water-phase reaction and can be carried out at normal temperature and normal pressure, toxic reagents and additional coenzymes do not need to be used, the product can be secreted out of the cells, the process is simple and convenient, and the method is a route with great large-scale production potential.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a strain that efficiently produces 3-hydroxy-2-pyridinecarboxylic acid, its construction method, and its uses. Background Technology

[0002] 3-Hydroxypicolinic acid (3-HPA) is a small heterocyclic compound containing a pyridine ring and a carboxylic acid group, first synthesized and characterized in the 1960s. As a key component of bacterial secondary metabolism, it is widely present in the biosynthetic pathways of antibiotics produced by *Streptomyces*, such as etamycin, virginiamycin, and pyridomycin. Due to its unique light absorption and coordination capabilities, 3-HPA has been developed as the preferred matrix for matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), significantly improving the detection sensitivity and resolution of biomolecules such as oligonucleotides, peptides, and nucleic acids. In addition, 3-hydroxy-2-pyridinecarboxylic acid is the core framework of anti-tuberculosis and antifungal lead compounds, and can efficiently chelate Fe3+ for iron overload treatment and diagnosis. Its derivatives have inhibitory effects on Leishmania, HIV and SARS-CoV-2, and have been proven to be key microbial metabolites with antidepressant activity in traditional Chinese medicine, showing multiple medicinal values ​​of antibacterial, antiviral and neuroprotective effects.

[0003] In the traditional route, the chemical synthesis of 3-hydroxy-2-pyridinecarboxylic acid uses 2-hydroxymethyl-3-hydroxypyridine as a raw material. First, the phenolic hydroxyl group is protected with benzyl, then benzyl alcohol is oxidized to carboxylic acid with KMnO4 to obtain 3-benzyloxypyridine-2-carboxylic acid. Finally, 3-hydroxy-2-pyridinecarboxylic acid is obtained by debenzylating with Pd / C hydrogen (John T. Sheehan The Journal of Organic Chemistry 1966 31 (2), 636-638). The steps are complicated and costly. To address the above issues, researchers have proposed a new three-step synthetic route: starting with 3-hydroxypyridine, it is first oxidized in glacial acetic acid with 30% hydrogen peroxide at 60°C to generate pyridine-N-oxide. Then, a cyano group is introduced by reacting trimethylcyanosilane and dimethylcarbamoyl chloride in dichloromethane at room temperature. Finally, the cyano group is hydrolyzed to a carboxyl group by reflux in an ethanol-20% NaOH system at 80°C. After acidification, a solid precipitates to obtain 3-hydroxy-2-pyridinecarboxylic acid. The overall yield of the three steps is 66% (CN 112110853 A). However, this method still has hidden dangers such as the use of highly toxic cyanide, cumbersome steps, and excessive waste. Compared with chemical synthesis, the enzymatic method theoretically replaces chemical synthesis with room temperature, aqueous phase, and highly selective catalysis, eliminating the need for cyanation and allowing for a one-pot reaction to obtain the product. However, there are currently no publicly reported methods, and enzyme activity, substrate spectra, and process parameters all need to be verified.

[0004] 3-Hydroxy-2-pyridinecarboxylic acid (3-H2-C) is a precursor for the synthesis of various antibiotics, and its in vivo enzymatic synthesis mechanism has been characterized. Researchers used L-lysine as a substrate in vitro, involving L-lysine 2-aminotransferase, a two-component flavin-dependent monooxygenase, and a FAD-dependent dehydrogenase, to generate 3-H2-C via C-3 hydroxylation and tautomerism (Yun X, Zhang Q, Lv M, Deng H, Deng Z, Yu Y. Org Biomol Chem. 2019;17(3):454-460.). First, L-lysine 2-aminotransferase catalyzes the transamination of lysine, followed by spontaneous dehydration and cyclization to form piperideine-2-carboxylic acid (P2C). Subsequently, a two-component flavin-dependent monooxygenase system catalyzes the P2C to complete C-3 hydroxylation, forming 3-hydroxy-3,4-dihydropyridine-2-carboxylate. Finally, flavin adenine dinucleotide (FAD)-dependent dehydrogenase catalyzes the aromatization of 3-hydroxy-3,4-dihydropyridine-2-carboxylate through two consecutive 4-electron oxidation steps to form 3-hydroxy-2-pyridinecarboxylic acid. Figure 1 However, the yield of this in vitro enzyme reaction is low, and it requires the addition of flavin and reducing coenzymes, resulting in high costs and making it impossible to manufacture.

[0005] In summary, the current synthesis methods for 3-hydroxy-2-pyridinecarboxylic acid are mainly chemical synthesis, which generally suffers from problems such as cumbersome steps, high cost, and heavy pollution. However, no efficient and green microbial fermentation synthesis method has been reported to date.

[0006] Therefore, those skilled in the art are dedicated to developing a strain that efficiently produces 3-hydroxy-2-pyridinecarboxylic acid. Summary of the Invention

[0007] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to develop a strain that efficiently produces 3-hydroxy-2-pyridinecarboxylic acid.

[0008] To achieve the above objectives, the present invention provides a strain, a method for constructing, and uses for efficiently producing 3-hydroxy-2-pyridinecarboxylic acid.

[0009] Further, the strain is the correct conjugate obtained by introducing the recombinant plasmid pJTH031 into the recipient Streptomyces via conjugation transfer; the construction method includes the following steps: Step 1: Using the genome of *Streptomyces pyridoxine* B-2517 as a template, PCR amplification was performed using specific primers and high-fidelity DNA polymerase to obtain... pyrB, pyrC, pyrD, pyrK Gene fragments and their corresponding ribosome binding sites (RBS) f Simultaneously, using the pSET152 vector as a template, reverse PCR amplification was performed using specific primers to obtain a sample containing the promoter. stnYp The linear vector pSET152 was validated by electrophoresis, and the target fragment was recovered and purified. The fragment was then cloned using a one-step homologous recombination method. pyrB, pyrC, pyrD, pyrK Genes are cloned sequentially into the promoter stnYp Later, among them pyrB, pyrC, pyrK Connect the corresponding RBS before the gene. fb RBS fc RBS fk The recombinant plasmid pJTH031 was obtained. Step 2: Transform the recombinant plasmid pJTH031 into Escherichia coli ET12567 / pUZ8002, and wash the bacterial cells with LB medium to remove antibiotics; then mix the treated E. coli with Streptomyces spores at a ratio of 1... :1 Mix the ingredients in the specified proportions, spread them on MS medium plates, and incubate at 30°C for 18-20 hours. Then, cover the plates with sterile water containing apramycin and TMP, and continue incubation at 30°C for 3-5 days. Step 3: Select the conjugation transferons grown in Step 2 and transfer them to a solid plate containing apramycin and TMP. Verify the correctness through resistance testing to obtain a heterologous expression strain that efficiently produces 3-hydroxy-2-pyridinecarboxylic acid.

[0010] Furthermore, when the recipient Streptomyces is Streptomyces whiteifolia J1074, the heterologous expression strain is AH031; when the recipient Streptomyces is Streptomyces pulvinatus TK24, the heterologous expression strain is LH031.

[0011] Furthermore, in step two, the Streptomyces spores are obtained by aseptically dispersing in water, filtering, resuspending in TES buffer, heat shock in a 50°C water bath for 10 min, pre-germinating in a spore pre-germination medium containing 0.01M calcium chloride at 30°C for about 2 h, and centrifuging and resuspending.

[0012] Furthermore, the construction method includes heterologous expression strains. pyrB, pyrC, pyrD, pyrK Methods for detecting gene transcription levels.

[0013] Furthermore, in the heterologous expression strain pyrB, pyrC, pyrD, pyrKThe method for detecting gene transcription levels was as follows: Heterologous expression strains AH031 and LH031 were inoculated into appropriate amounts of YEME medium and cultured at 30℃ and 220 rpm for 24 h with shaking. 500 μL of bacterial culture was collected by centrifugation, and the cells were washed three times with 10.3% sucrose solution to extract total RNA. Genomic DNA was removed from the total RNA using reverse transcriptase, and cDNA was obtained by reverse transcription. Using the prepared cDNA as a template, real-time PCR was performed using a real-time PCR enzyme. The Ct values ​​of the target genes pyrB, pyrC, pyrD, and pyrK were obtained. The housekeeping genes of *Streptomyces* were then analyzed. hrdB The reference gene was normalized using 2... -△△Ct The method calculates the relative changes in transcriptional levels of each gene.

[0014] Furthermore, quantitative PCR was performed using an ABI 7500 Fast real-time PCR instrument. The reaction program was set as follows: pre-denaturation 95℃, 1 min; 40 cycles: 95℃, 10 s; 60℃, 30 s; melting curve analysis: 95℃, 15 s; 60℃, 1 min; 95℃, 15 s; 60℃, 15 s.

[0015] Furthermore, the construction method includes a fermentation method for heterologous expression strains.

[0016] Furthermore, the fermentation method involves inoculating spores of the heterologous expression strain into 20 ml of YEME medium and culturing at 30°C and 220 rpm for 1 day to obtain a seed culture. Then, 1.5 ml of the seed culture is transferred to 30 ml of new YEME medium and fermented at 30°C and 220 rpm for 3 days to obtain the fermentation broth.

[0017] Furthermore, the HPLC detection method for the 3-hydroxy-2-pyridinecarboxylic acid product in the fermentation broth involves centrifuging the fermentation broth at 10,000 rpm for 20 min to obtain the supernatant. 3 mL of the supernatant is then placed in a pre-cooled freeze dryer to remove moisture, followed by soaking in methanol and sonication for 10 min. The extract is filtered through a 0.22 μm organic phase filter membrane and concentrated to 200 μL using a rotary evaporator. The 305 nm UV absorption is detected using an Agilent 1260 high-performance liquid chromatography system with a 5 μm particle size, 250 mm × 4.6 mm C18 reversed-phase column.

[0018] Furthermore, for HPLC detection, mobile phase A was a 1‰ formic acid aqueous solution, and mobile phase B was 100% acetonitrile; the sample loading volume was 5 μL, and the flow rate was 0.6 mL / min. The specific program was: 0-3 min, 5% acetonitrile; 3-22 min, 5-80% acetonitrile; 22-27 min, 80%-100% acetonitrile; 27-30 min, 100%-5% methanol.

[0019] Furthermore, the described construction method produces a strain that efficiently generates 3-hydroxy-2-pyridinecarboxylic acid.

[0020] Furthermore, the use of the strain that efficiently produces 3-hydroxy-2-pyridinecarboxylic acid is for its use in the synthesis of 3-hydroxy-2-pyridinecarboxylic acid.

[0021] In a preferred embodiment 1 of the present invention, the construction process of the heterologous expression plasmid vector is described in detail.

[0022] In another preferred embodiment 2 of the present invention, the construction process of the heterologous expression strain is described in detail.

[0023] In another preferred embodiment 3 of the present invention, the process of detecting the transcriptional levels of pyrB, pyrC, pyrD, and pyrK genes in heterologous expression strains is described in detail.

[0024] In another preferred embodiment 4 of the present invention, the shake-flask small-scale culture and fermentation process of the heterologous expression strain of 3-hydroxy-2-pyridinecarboxylic acid is described in detail.

[0025] In another preferred embodiment 5 of the present invention, the detection process of 3-hydroxy-2-pyridinecarboxylic acid product in the fermentation broth of heterologous expression strains is described in detail.

[0026] Technical effects: This invention utilizes genetic engineering technology to clone homologous L-lysine-2-aminotransferase PyrB, two-component flavin-dependent monooxygenases PyrC / PyrK, and flavin-adenine nucleotide-dependent dehydrogenase PyrD from *Streptomyces pyridinium* NRRL B-2517. By combining these with a strong promoter and optimizing the RBS sequence, a heterologous expression strain of 3-hydroxy-2-pyridinecarboxylic acid was successfully constructed, laying a core foundation for the efficient, green, and low-cost microbial fermentation production of this product.

[0027] This invention employs a microbial fermentation method to achieve cellular "self-production" of 3-hydroxy-2-pyridinecarboxylic acid through metabolic pathway reconstruction. The entire reaction is carried out in an aqueous phase at room temperature and pressure, requiring no toxic reagents or additional coenzymes. Furthermore, the product can be secreted extracellularly, making the process simple and a route with significant potential for large-scale production. Simultaneously, the heterologous expression strain constructed in this invention can utilize inexpensive glucose as a raw material to synthesize the target product, further reducing production costs and demonstrating clear prospects for industrial application.

[0028] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the biosynthetic pathway of 3-hydroxy-2-pyridinecarboxylic acid; Figure 2 This is a schematic diagram of the genome assembly of a heterologous expression plasmid in a preferred embodiment 1 of the present invention; the diagram shows the genome... pyrB, pyrC, pyrD, pyrK Overexpression with the same operator as the strong promoter stnYp, and pyrB, pyrC, pyrK Add ribosome binding sites (RBS) before each of the three genes. fb 、 RBS fc 、 RBS fk ; Figure 3 This is a preferred embodiment 3 of the present invention, using a heterologous expression strain. pyrB, pyrC, pyrD, pyrK Gene transcription level analysis diagram: Calculation of gene transcription levels in heterologous expression strains AH031 and LH031 using quantitative real-time PCR. pyrB, pyrC, pyrD, pyrK The relative expression level of gene mRNA, in order to pyrB, pyrC, pyrD, pyrK The original strain of Streptomyces pyridycin, from which the gene originated. Streptomyces pyridomyceticus B-2517 was used as a control group, and the results showed that AH031 pyrB, pyrC, pyrD, pyrK The mRNA expression levels of the gene were upregulated by 27.1, 7.6, 7.8, and 13.2 times, respectively, compared to B2517. In LH031... pyrB, pyrC, pyrD, pyrK The mRNA expression levels of the gene were upregulated by 27.8, 7.7, 14.1, and 17.9 times, respectively, compared to B2517. p≤0.05; p≤0.01; (p≤0.001); Figure 4 This is an HPLC analysis chromatogram of the fermentation products of two heterologous expression strains in a preferred embodiment 5 of the present invention. The left image shows the heterologous expression strain AH031 (S. albus J1074-pJTH031), the right figure shows the heterologous expression strain LH031 (J1074-pJTH031), S. lividans TK24-pJTH031). Detailed Implementation

[0030] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0031] Example 1: Construction of heterologous expression plasmid vectors

[0032] Using the genome of *Streptomyces pyridomyceticus* B-2517 as a template, high-fidelity DNA polymerase was used to amplify the pyrB, pyrC, pyrD, and pyrK gene fragments containing the corresponding ribosome binding site RBSf using primers designed in Table 1 (the nucleotide sequences of the primers are shown in SEQ ID NO:13-20). Specifically, the nucleotide sequence of the pyrB gene is shown in SEQ ID NO:5, the amino acid sequence of the L-lysine-2-aminotransferase it encodes is shown in SEQ ID NO:6, and the ribosome binding site RBSf it carries... b The nucleotide sequence of the gene is shown in SEQ ID NO:2; the nucleotide sequence of the pyrC gene is shown in SEQ ID NO:7, and the amino acid sequence of the bicomponent flavin-dependent monooxygenase (PyrC subunit) it encodes is shown in SEQ ID NO:8, carrying the ribosome binding site RBSf. c The nucleotide sequence of the pyrK gene is shown in SEQ ID NO:3; the nucleotide sequence of the pyrK gene is shown in SEQ ID NO:9; the amino acid sequence of the bicomponent flavin-dependent monooxygenase (PyrK subunit) it encodes is shown in SEQ ID NO:10; and the ribosome binding site RBSf it carries... k The nucleotide sequence of the pyrD gene is shown in SEQ ID NO:4; the nucleotide sequence of the pyrD gene is shown in SEQ ID NO:11; and the amino acid sequence of the FAD-dependent dehydrogenase it encodes is shown in SEQ ID NO:12.

[0033] Simultaneously, using the pSET152 vector as a template (the pSET152 vector used was a derivative vector pre-integrated with the stnYp promoter), reverse PCR amplification was performed using the vector-specific reverse primers pSET-stnYp-Spe1-R and RpSET-stnYp-F listed in Table 1 to obtain the linear vector pSET152 containing the stnYp promoter (the nucleotide sequence of the stnYp promoter is shown in SEQ ID NO:1). After electrophoresis verification and gel recovery, purified pyrB, pyrC, pyrD, and pyrK gene fragments (with RBSf) and the linear vector pSET152 containing the stnYp promoter were obtained. Homologous recombination was used for one-step cloning to clone the pyrB, pyrC, pyrD, and pyrK genes (where pyrB, pyrC, and pyrK genes are each preceded by their corresponding RBSf). fb RBS fc RBS fk ) cloned sequentially in the promoter stnYp Subsequently, the recombinant plasmid pJTH031 was obtained. The vector was validated by sequencing.

[0034] Table 1 Primers used in Example 1

[0035] Example 2: Construction of Heterologous Expression Strains

[0036] The recombinant plasmid pJTH031 was transformed into *E. coli* ET12567 / pUZ8002, and then 5 mL of *E. coli* ET12567 / pUZ8002 containing the target plasmid was cultured overnight (LB medium, 50 µg / mL apramycin, 50 µg / mL kanamycin, and 25 µg / mL chloramphenicol). The culture was then transferred to 50 mL of LB medium at a 1% inoculum and incubated at 37°C until the OD600 reached 0.4-0.6. The cells were then washed with LB medium to thoroughly remove antibiotics and set aside. Meanwhile, *Streptomyces* spores, serving as the recipient, underwent heat shock and pre-germination treatment. *Streptomyces albopictus* (white *Streptomyces*) were collected from sporulated plates. Streptomyces albus J1074 and Streptomyces cerevisiae ( Streptomyces lividansTK24 spores. Resuspend Streptomyces spores in a centrifuge tube containing 10 mL of sterile water and shake thoroughly for at least 5 min to disperse the spore clusters and spores adhering to the mycelium. Collect the dispersed spore suspension through a sterile spore filter, wash once with water, and resuspend in 5 mL of TES buffer (0.05 M, pH 8.0). Heat shock at 50°C for 10 min, cool to room temperature, and add an equal volume of spore pre-germination medium (containing 0.01 M calcium chloride). Pre-germinate at 30°C for approximately 2 h. Centrifuge to collect spores and resuspend in an appropriate amount of LB spores. Shake to disperse the spores on a mixer, using a 1:1 ratio for Escherichia coli and Streptomyces. 9 10 8 The mixture was prepared in the specified proportions, spread onto MS agar plates, dried, and incubated at 30°C for 18-20 hours. Afterward, it was covered with 1 ml of sterile water containing appropriate amounts of apramycin and TMP (to inhibit the growth of E. coli), and incubated at 30°C for another 3-5 days. Conjugation transferons were observed to grow. These were then transferred to MS agar plates containing 50 µg / mL apramycin and 50 µg / mL TMP. The correct conjugation was verified by antibiotic resistance testing. The correct conjugants were strains AH031 (host J1074) and LH031 (host J1074), which were used for subsequent operations.

[0037] Example 3: In heterologous expression strains pyrB, pyrC, pyrD, pyrK Gene transcription level detection

[0038] Heterologous expression strains AH031 and LH031 were inoculated into appropriate amounts of YEME medium (3 g / L yeast extract, 3 g / L malt extract, 5 g / L tryptone, 10 g / L glucose, pH 7.2) and cultured at 30°C and 220 rpm with shaking for 24 h. 500 μL of the bacterial culture was collected, centrifuged at 5000 rpm for 10 min, and the cells were washed three times with 10.3% sucrose solution. Total RNA was extracted using an RNA extraction kit (Beijing TransGen Biotech Co., Ltd.) and extracted using reverse transcriptase (ReverTraceAce). TM Genomic DNA was removed from total RNA using qPCR RT Master (TOYOBO), and cDNA was obtained by reverse transcription. Using the primers in Table 2 (primer nucleotide sequences shown in SEQ ID NO:21-32), and with the prepared cDNA as a template, real-time PCR was performed using ArtiCan. ATMQuantitative real-time PCR was performed using SYBR qPCR Mix (Tsingke). An ABI 7500Fast real-time PCR instrument was used, with the following reaction program: Step 1: Pre-denaturation: 95°C for 1 min, 95°C for 10 s, 60°C for 30 s, then return to Step 2 for 40 cycles: 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, 60°C for 15 s. The Ct values ​​of the target genes (pyrB, pyrC, pyrD, pyrK) were obtained. (Streptomyces housekeeping genes were used.) hrdB Genes were used as internal references for normalization. Two [types of genes were used]. -△△Ct The method calculates the relative changes in transcriptional levels of each gene.

[0039] The results showed that the strain was similar to the original strain of Streptomyces pyridoxine from which the gene originated. Streptomyces pyridomyceticus Compared to B-2517, heterologous expression strains AH031 and LH031... pyrB Gene expression levels were upregulated by 27.1 and 27.8 times, respectively. pyrC Gene expression levels were upregulated by 7.6 and 7.7 times, respectively. pyrD Gene expression levels were upregulated by 7.8 and 14.1 times, respectively. pyrK Gene expression levels were upregulated by 13.2 and 17.9 times, respectively. Figure 3 ).

[0040] Table 2 Primers used in Example 3

[0041] Example 4: Shake-flask small-scale fermentation of heterologous expression strain of 3-hydroxy-2-pyridinecarboxylic acid

[0042] 3-hydroxy-2-pyridinecarboxylic acid heterologous expression strains AH031 and LH031 and wild-type Streptomyces albopictus ( Streptomyces albus J1074 and Streptomyces cerevisiae ( Streptomyces lividans Spores of strain TK24 were inoculated into 20 ml of yeast extract-maltose (YEME) medium (3 g yeast extract, 5 g tryptone, 3 g malt extract, 10 g glucose, 103 g sucrose, dissolved in ultrapure water and brought to a final volume of 1000 mL, autoclaved at 115℃ for 30 min, and 2 mL of sterilized 2.5 M MgCl2·6H2O was added per 1000 mL before use) and cultured at 30℃ and 220 rpm for 1 day to obtain seed culture. Then, 1.5 ml of seed culture was transferred to 30 ml of new YEME medium and fermented at 30℃ and 220 rpm for 3 days.

[0043] Example 5: Detection of 3-hydroxy-2-pyridinecarboxylic acid product (HPLC)

[0044] The fermentation broths obtained in Example 4 were centrifuged at 10,000 rpm for 20 min to obtain the supernatant. 3 mL of the supernatant was placed in a pre-cooled freeze dryer to remove moisture, and then soaked in an appropriate amount of methanol and sonicated for 10 min. The extract was filtered through a 0.22 μm organic phase filter membrane and concentrated to 200 μL by rotary evaporator. The 305 nm UV absorption was detected by high performance liquid chromatography (HPLC, Agilent 1260) with a reversed-phase C18 column (250 × 4.6 mm, SuperLu C18(2) 5 μm). Mobile phase A was 1 / 1000 formic acid water, and mobile phase B was 100% acetonitrile. The sample loading volume was 5 μL, and the flow rate was 0.6 mL / min. The specific program was as follows: 0-3 min, 5% acetonitrile; 3-22 min, 5-80% acetonitrile; 22-27 min, 80%-100% acetonitrile; 27-30 min, 100%-5% methanol.

[0045] The results showed that: in the HPLC chromatogram ( Figure 4 On the fermentation broth, no characteristic peak of 3-hydroxy-2-pyridinecarboxylic acid was detected in the wild-type strains corresponding to strains AH031 and LH031, while 3-hydroxypyridinecarboxylic acid was detected in the fermentation broth of both heterologous expression strains AH031 and LH031 at a retention time of 9.7 min. The calculated yields of 3-hydroxy-2-pyridinecarboxylic acid in strains AH031 and LH031 were 9.89 mg / L and 2.78 mg / L, respectively.

[0046] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for constructing a strain efficiently producing 3-hydroxy-2-pyridinecarboxylic acid, characterized by, The strain is a correct conjugant obtained by introducing the recombinant plasmid pJTH031 into the recipient Streptomyces by conjugation; the construction method comprises the following steps: Step one, with pyridine streptomyces B-2517 genome as template, using specific primers, PCR amplification with high fidelity DNA polymerase, get pyrB, pyrC, pyrD, pyrK Gene fragments and the corresponding ribosome binding sites RBS of each gene f , and at the same time, pSET152 vector as template, and then using specific primers for reverse PCR amplification, get linear vector pSET152 containing promoter stnYp , electrophoresis verification, recovery and purification of the target fragment, by homologous recombination one step cloning method to pyrB, pyrC, pyrD, pyrK Gene cloned in the promoter stnYp , respectively, wherein pyrB, pyrC, pyrK The gene is connected with the corresponding RBS fb , RBS fc , RBS fk , obtain recombinant plasmid pJTH031; Step two, transform the recombinant plasmid pJTH031 into E. coli ET12567 / pUZ8002, after culture, wash the bacteria with LB medium to remove antibiotics; mix the treated E. coli with Streptomyces spores at a ratio of 1 :1 , spread on MS medium plates, and after 18-20 h of culture at 30°C, cover the plates with sterile water containing apramycin and TMP, and continue to culture at 30°C for 3-5 days; Step three, picking up the conjugation sub in step two, transferring to a solid plate containing apramycin and TMP, and obtaining the heterologous expression strain for producing 3-hydroxy-2-pyridine carboxylic acid by resistance verification.

2. The method for constructing a high 3-hydroxy-2-pyridinecarboxylic acid-producing strain according to claim 1, wherein When the recipient Streptomyces is Streptomyces albus J1074, the heterologous expression strain is AH031; when the recipient Streptomyces is Streptomyces variabilis TK24, the heterologous expression strain is LH031.

3. The method for constructing a high 3-hydroxy-2-pyridinecarboxylic acid-producing strain according to claim 1, wherein In step two, the spores of Streptomyces are spores of Streptomyces albus J1074 or Streptomyces variabilis TK24, which are dispersed with sterile water, filtered, resuspended with TES buffer, heat shocked at 50℃ for 10 min, pre-germinated in spore pre-germination medium containing 0.01M calcium chloride at 30℃ for about 2h, and resuspended by centrifugation.

4. The method for constructing a high 3-hydroxy-2-pyridinecarboxylic acid-producing strain according to claim 1, wherein The construction method includes heterologous expression of a strain in pyrB, pyrC, pyrD, pyrK A method for detecting the transcription level of a gene.

5. The method for constructing a strain highly producing 3-hydroxy-2-pyridinecarboxylic acid according to claim 4, wherein in the heterologous expression strain pyrB, pyrC, pyrD, pyrK The method for detecting the transcription level of the gene is as follows: the heterologous expression strains AH031 and LH031 are respectively inoculated into appropriate YEME medium, and cultured at 30°C with 220 rpm for 24 h; 500 μL of bacterial solution is taken, centrifuged, and the bacterial body is collected; the bacterial body is washed with 10.3% sucrose solution for three times; total RNA is extracted; reverse transcriptase is used to remove genomic DNA in the total RNA, and cDNA is obtained by reverse transcription; the prepared cDNA is used as a template, and fluorescent quantitative PCR is carried out using a fluorescent quantitative PCR enzyme; the reaction program is set as follows: pre-denaturation at 95°C for 1 min; 40 cycles of 95°C for 10 s, 60°C for 30 s; melting curve analysis: 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, 60°C for 15 s; and the Ct value of the target genes pyrB, pyrC, pyrD and pyrK is obtained. Streptomyces housekeeping genes hrdB The transcription level of each gene was calculated by the 2 -△△Ct method.

6. The method for constructing a high 3-hydroxy-2-pyridinecarboxylic acid-producing strain according to claim 1, wherein The construction method comprises a fermentation method of the heterologous expression strain, and the specific method is that spores of the heterologous expression strain are inoculated into 20ml YEME medium and cultured at 30℃ and 220 rpm for 1 day to obtain a seed culture solution, then 1.5ml of the seed culture solution is transferred into 30ml of new YEME medium, and the medium is fermented at 30℃ and 220 rpm for 3 days to obtain a fermentation liquor.

7. The method for constructing a strain highly producing 3-hydroxy-2-pyridinecarboxylic acid according to claim 6, wherein The HPLC detection method of 3-hydroxy-2-pyridine carboxylic acid product in the fermentation liquor is that the fermentation liquor is centrifuged at 10000 rpm for 20 min to obtain a fermentation liquor supernatant, 3 mL of the supernatant is taken and water is removed in a pre-cooled freeze dryer, then methanol is added and soaked for ultrasonic treatment for 10 min, the leaching liquor is filtered through a 0.22μm organic phase filter membrane, concentrated to 200μL by a rotary evaporator, and detected by an Agilent 1260 high performance liquid chromatography system, a particle size of 5μm, a 250 mm×4.6 mm C18 reverse phase chromatographic column, and 305 nm ultraviolet absorption.

8. The method for constructing a strain efficiently producing 3-hydroxy-2-pyridinecarboxylic acid according to claim 7, wherein The mobile phase A is 1‰ formic acid aqueous solution, the mobile phase B is 100% acetonitrile, the sample amount is 5μL, the flow rate is 0.6mL / min, and the specific program is: 0-3 min, 5% acetonitrile; 3-22 min, 5-80% acetonitrile; 22-27 min, 80%-100% acetonitrile; 27-30 min, 100%-5% methanol.

9. The strain for efficiently producing 3-hydroxy-2-pyridine carboxylic acid constructed by the construction method of claim 1.

10. Use of the strain for efficiently producing 3-hydroxy-2-pyridine carboxylic acid of claim 9 in synthesis of 3-hydroxy-2-pyridine carboxylic acid.

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