6-Hydroxynicotinic acid-producing Aeromonas salmonicida HD531 and its application

Abstract

The present invention relates to a strain of Aeromonas salmonicida (Aeromonas salmonicida) HD531 for producing 6-hydroxynicotinic acid through microbial fermentation. The strain belongs to the genus Aeromonas (Aeromomas) of the family Vibrionaceae, and its deposit number at the General Microbiology Center of the China Association for Microbiological Culture Collection is CGMCC No. 7.525. The present invention provides a method for producing 6-hydroxynicotinic acid using nicotinic acid fermentation using the strain. Using a resting cell method, the strain can significantly accumulate 6-hydroxynicotinic acid under aerobic conditions, with a maximum accumulation of 70.16 g / L, at an initial niacin concentration of 10 g / L. The method is suitable for industrial production of 6-hydroxynicotinic acid.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme production by microbial fermentation, and relates to Aeromonas salmonicida HD531 producing 6-hydroxynicotinic acid and application thereof. Background Art

[0002] 6-Hydroxynicotinic acid, also known as 6-hydroxypyridine-3-carboxylic acid, is a high-value fine chemical intermediate. Its English name is 6-hydroxynicotinic acid, abbreviated as 6-HNA. 6-Hydroxynicotinic acid has both an alcoholic and a keto form, is insoluble in acid but soluble in alkaline solutions. It is an important intermediate in the synthesis of pesticides, pharmaceuticals, and other chemicals, and has been widely used in the pesticide, dye, medical cosmetics, and materials industries. In the pesticide sector, 6-Hydroxynicotinic acid can be used to produce pyridine methylamine pesticides, such as imidacloprid and acetamiprid, which have high efficacy and low toxicity against plant fungal diseases. It can also react with chlorine to produce the pharmaceutically active ingredient 5,6-dichloronicotinic acid, which promotes lipase degradation, has fat-reducing properties, and is used in the preparation of weight-loss drugs. In chemistry, 6-Hydroxynicotinic acid can also be used as a substrate in the synthesis of pharmaceutical intermediates. For example, starting from 6-Hydroxynicotinic acid, acetonitrile is used as a solvent to synthesize 2-chloro-5-chloromethylpyridine through a stepwise chemical process. In the field of materials, because 6-hydroxynicotinic acid contains electron-rich nitrogen atoms, it has electrocatalytic chemical properties and can be used as an electrode material. For example, using cyclic voltammetry, 6-hydroxynicotinic acid can be polymerized to the surface of a carbon paste electrode to prepare a 6-hydroxynicotinic acid film-modified electrode.

[0003] Due to its promising development prospects, the production of 6-hydroxynicotinic acid has become a research hotspot both domestically and internationally. However, 6-hydroxynicotinic acid is currently difficult to synthesize chemically. Organic synthesis methods for producing 6-hydroxynicotinic acid primarily use fumaric acid or malic acid as raw materials, requiring the addition of reagents such as concentrated sulfuric acid and concentrated hydrochloric acid during the reaction process, which can easily cause environmental pollution and a corresponding increase in production costs. This method also produces numerous byproducts, is costly, and carries significant pollution risks. Compared to traditional chemical methods, the biocatalytic production of 6-hydroxynicotinic acid from nicotinic acid using nicotinic acid dehydrogenase, a microbial enzyme, offers advantages such as environmental friendliness, low cost, and high yield.

[0004] Nicotine dehydrogenase (NDH) is a hydroxylase that catalyzes niacin and various niacin analogs to varying degrees. Research on NDH began with the isolation and purification of NDH from niacin-metabolizing strains. Most reported NDHs are derived from aerobic Pseudomonas putida. For example, in 2005, Xu et al. isolated a strain, Pseudomonas putida NA-1, that effectively catalyzes niacin to 6-hydroxynicotinic acid. In 2010, Luo et al. isolated NDH from Pseudomonas putida BK-1. In 2017, Chen et al. isolated and identified a NDH-producing strain, Pseudomonas putida H9, from rotten fish, capable of catalyzing niacin to 6-hydroxynicotinic acid. In 2021, Shang et al. obtained a NDH-producing strain, Pseudomonas putida S14. However, most reported nicotinic acid dehydrogenase-producing strains still suffer from low enzyme activity and inefficient nicotinic acid conversion, limiting their large-scale industrial application. Therefore, identifying a highly active nicotinic acid dehydrogenase-producing strain, as well as studying its functional expression and efficient catalytic conversion, is of great practical significance for the efficient synthesis and industrial production of 6-hydroxynicotinic acid. Summary of the Invention

[0005] The present invention aims to provide a novel strain of Aeromonas salmonicida HD531 that produces nicotinic acid dehydrogenase. This strain can be used for microbial fermentation of nicotinic acid to produce 6-hydroxynicotinic acid. The present invention also provides a method for producing 6-hydroxynicotinic acid using this strain. This method uses inexpensive nicotinic acid as a raw material and can efficiently synthesize 6-hydroxynicotinic acid.

[0006] The salmonicidal Aeromonas HD531 that produces 6-hydroxynicotinic acid, whose classification name is Aeromonas salmonicida, was deposited at the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on July 10, 2023, with the deposit number CGMCC NO.7.525.

[0007] The strain HD531 belongs to the genus Aeromonas of the family Vibrionaceae and the genus Aeromonas salmonicida.

[0008] The method for growing cells and producing 6-hydroxynicotinic acid using the strain comprises the following steps: streaking the strain onto a solid culture medium, culturing at 30°C and 200 rpm for 24 hours, picking a single bacterium onto a liquid seed culture medium, culturing at 30°C and 200 rpm for 24 hours, inoculating the liquid fermentation culture medium at an inoculum rate of 1 to 5%, fermenting and culturing at 30°C under aerobic conditions for 24 hours, and detecting the concentration of the product 6-hydroxynicotinic acid by high performance liquid chromatography (HPLC).

[0009] The method for producing 6-hydroxynicotinic acid by resting cells using the strain comprises the following steps: firstly spreading and streaking the strain liquid onto a solid plate culture medium, and culturing at 30°C for 24 to 36 hours under aerobic conditions; then picking the cultured single colony of the strain onto a primary seed culture medium, and aerobically culturing at 30°C for 12 to 24 hours; then inoculating the cultured primary seeds into a secondary seed culture medium at an inoculation rate of 1 to 10%, and aerobically culturing at 30°C for 8 to 12 hours; after 8 to 12 hours, inoculating the cultured secondary seeds into a liquid fermentation culture medium at an inoculation rate of 1 to 10%, and fermenting and culturing at 30°C for 10 to 12 hours under aerobic conditions; and after the fermentation is completed, collecting the fermentation liquid, collecting the bacterial cells by centrifugation, adding 1% nicotinic acid conversion liquid, concentrating the bacterial cells by 20 times, and performing biocatalytic conversion for 60 to 80 hours.

[0010] The solid culture medium comprises: 17 g / L tryptone, 3 g / L soy peptone, 2.5 g / L glucose, 2.5 g / L dipotassium hydrogen phosphate, 10 g / L sodium chloride, 20 g / L agar powder, with the balance being water, and a pH of 7.0. The liquid seed culture medium comprises: 17 g / L tryptone, 3 g / L soy peptone, 2.5 g / L glucose, 2.5 g / L dipotassium hydrogen phosphate, 10 g / L sodium chloride, 2 g / L nicotinic acid, with the balance being water, and a pH of 7.0. The liquid fermentation culture medium comprises: 10-20 g / L nicotinic acid, 8-10 g / L yeast extract, 8-10 g / L peptone, 0.5-1 g / L KH2PO4, 2-4 g / L K2HPO4·3H2O, with the balance being water, and a pH of 7.0.

[0011] The compositions of the primary seed culture medium and the secondary seed culture medium are both: 1-2 g / L of nicotinic acid, 2-5 g / L of beef extract, 8-10 g / L of peptone, 0.5-1 g / L of NaCl, and the balance is water, with a pH of 7.0; the composition of the liquid fermentation culture medium is the same as that of the growing cell method.

[0012] Specifically, the niacin conversion solution is a mixture of niacin and a buffer solution at different concentrations. For example, 100 ml of phosphate buffer plus 1 g of niacin produces a 10 g / L niacin conversion solution, and 100 ml of phosphate buffer plus 2 g of niacin produces a 20 g / L niacin conversion solution. The buffer solution is made of NaH2PO4 and Na2HPO4 and has a pH of 7.0. The carbon and nitrogen sources are preferably yeast extract, beef extract, or peptone.

[0013] The pH of the culture medium can be adjusted by conventional methods in the art, such as adding inorganic acids and bases such as hydrochloric acid and sodium hydroxide. The pH of the buffer solution is adjusted using its component reagents, namely NaH2PO4 and Na2HPO4. The culture medium is sterilized at 115-121°C for 20 minutes.

[0014] The beneficial effects achieved by the present invention are:

[0015] The salmonicidal Aeromonas HD531 capable of producing nicotinic acid dehydrogenase can significantly accumulate 60-70 g / L of 6-hydroxynicotinic acid under aerobic conditions. The method is green, friendly and sustainable, has low cost and high yield, can realize industrial production, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0017] Figure 1 This is a photo of the colony of Aeromonas salmonicida HD531 on a plate (×10);

[0018] Figure 2 This is a scanning electron micrograph of Aeromonas salmonicida HD531 (×10000);

[0019] Figure 3 Phylogenetic tree constructed for Aeromonas salmonicida HD531 based on BLAST results. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0021] Example

[0022] Initial screening

[0023] Soil samples were collected from different areas of Kaifeng City, Henan Province. The soil was added with 100 ml of PBS solution and shaken at 30°C and 200 rpm for 12 h. The supernatant was taken and diluted into a series of concentration gradients: 10-1 , 10 -2 , 10 -3 Spread 0.2 ml of each gradient onto a plate containing 5 g / L niacin, 5 g / L yeast extract, 20 g / L agar, 1.0 g / L NaCl, and the balance water (pH 7.0). Incubate at 30°C under aerobic conditions for 48–72 h.

[0024] After the colonies grow, place the plates under ultraviolet light for observation, pick the brightly colored colonies and inoculate them into the seed culture medium for enrichment culture (the brighter the color of the colonies, the higher the enzyme activity). Each colored strain is inoculated into one shake flask. The seed culture medium composition is 10 g / L peptone, 5.0 g / L beef extract, 1.0 g / L NaCl, and the balance is water. The pH is 7.0.

[0025] 2. Rescreening

[0026] The seed culture was incubated at 30°C under aerobic conditions for 24 hours and then transferred to a fermentation medium composed of 10 g / L yeast extract, 10 g / L peptone, 2.7 g / L niacin, 1.0 g / L KH2PO4, 3.93 g / L K2HPO4·3H2O, and the balance water at a pH of 7.0. The culture was continued at 30°C for 24 hours.

[0027] 1.5 ml of fermentation broth was centrifuged at 12,000 rpm for 5 minutes. The supernatant was collected, diluted to an appropriate ratio, and the 6-hydroxynicotinic acid production and residual nicotinic acid content were determined by high-performance liquid chromatography (HPLC). HPLC conditions were as follows: CST Daiso C18 column (5 μm, 4.6 × 250 mm), mobile phase: methanol:water (pH = 3.0) = 20:80 (v / v), detector: RI detector, UV detector (detection wavelength 260 nm), column temperature 30°C, flow rate 0.4 ml / min, wavelength 260 nm, injection volume 20 μl. The following table lists the 6-hydroxynicotinic acid production of some of the tested chromogenic strains. Strain #36 is the newly screened strain Aeromonas salmonicida.

[0028] Table 1 6-hydroxynicotinic acid production by some test strains

[0029]

[0030]

[0031] Identification of Aeromonas salmonicida HD531:

[0032] A novel strain, Aeromonas salmonicida HD531, was screened for its ability to effectively accumulate 6-hydroxynicotinic acid. This strain is a Gram-negative bacterium with straight, rod-shaped cells that are rounded at the ends, to cocciform, ranging in diameter from 1.0 to 4.4 microns, occasionally forming filaments up to 8 microns long. It occurs singly, in pairs, or in chains. It motility is by polar flagella, usually single; some are non-motile. It is unknown whether it has a dormant period. It is a chemoorganic bacterium with respiratory and fermentative metabolism, breaking down carbohydrates to produce either acid or acid and gas (CO2 and H2).

[0033] Physiological and biochemical characterization was performed according to the Bergey's Manual of Bacterial Identification (8th Edition) (results are shown in Table 2). The strain was identified as belonging to the genus Aeromonas based on Gram staining, growth in a nutrient solution containing 7.5% NaCl, butanediol dehydrogenase, gas production from glycerol, gas production from glucose, lysine decarboxylase reactions, and decomposition of galactose, mannitol, and arabinose.

[0034] At the same time, Shanghai Sangon Biotech Co., Ltd. was commissioned to perform 16S RNA full sequence sequencing on the strain. BLAST was used to search the 16S RNA gene sequences of related strains in GenBank on the NCBI website (see Table 3), and MEGA 11.0 software was used to construct a phylogenetic tree analysis (see Figure 3 Based on 16S RNA sequence analysis and phylogenetic tree construction, it was concluded that HD531 was closely related to Aeromonas salmonicida strain ODK2, Aeromonas salmonicida strain YTU1, and Aeromonas salmonicida strain B52. Strain HD531 can be identified as Aeromonas salmonicida.

[0035] The above identification results show that strain HD531 belongs to Aeromonas salmonicida of the genus Aeromonas of the family Vibrionaceae. The deposit number of this strain at the General Microbiology Center of the China Culture Collection Administration is 7.525.

[0036] Table 2 Comparison table of physiological and biochemical characteristics identification results

[0037]

[0038] Table 3 Comparison of 16S RNA sequence homology (or similarity)

[0039]

[0040]

[0041] Production of 6-hydroxynicotinic acid using Aeromonas salmonicida fermentation:

[0042] The method for producing 6-hydroxynicotinic acid by growing cells and fermenting nicotinic acid using the strain is specifically as follows: the strain culture liquid is first spread and streaked onto a tryptone soy broth solid medium (TSA) (the tryptone soy broth solid medium is composed of: tryptone 17g / L, soy peptone 3g / L, glucose 2.5g / L, dipotassium hydrogen phosphate 2.5g / L, sodium chloride 10g / L, agar powder 20g / L, the balance is water, pH 7.0), and cultured at 30°C for 24h under aerobic conditions; then a single colony of the cultured strain is picked onto a tryptone soy broth solid medium. A tryptone soy broth liquid medium (TSB) (the composition of the tryptone soy broth liquid medium is: 17 g / L tryptone, 3 g / L soy peptone, 2.5 g / L glucose, 2.5 g / L dipotassium hydrogen phosphate, 10 g / L sodium chloride, 2 g / L nicotinic acid, the balance being water, and the pH being 7.0) is cultured aerobically at 30°C for 24 hours; then the cultured seed liquid is inoculated into a fermentation medium (the composition of the fermentation medium is: 10 g / L nicotinic acid, 10 g / L yeast extract, 10 g / L peptone, 1 g / L KH2PO4, 3.93 g / L K2HPO4·3H2O, the balance being water, and the pH being 7.0) at a 2% inoculum rate, and cultured aerobically at 30°C for 24 hours.

[0043] The yield of 6-hydroxynicotinic acid was 3.71 g / L as determined by high performance liquid chromatography.

[0044] The method for producing 6-hydroxynicotinic acid by catalyzing nicotinic acid through resting cell fermentation using the strain comprises the following steps: firstly spreading and streaking the strain liquid onto a TSA flat solid culture medium, and culturing at 30°C for 24-36 hours under aerobic conditions; then picking the cultured single colony of the strain onto a primary seed culture medium, and aerobically culturing at 30°C for 12-24 hours; then inoculating the cultured primary seeds into a secondary seed culture medium at an inoculation rate of 1-10%, and aerobically culturing at 30°C for 8-12 hours; after 8-12 hours, inoculating the cultured secondary seeds into a 20L fermentation tank containing 10L liquid fermentation culture medium at an inoculation rate of 1-10%, and fermenting and culturing at 30°C for 10-12 hours under aerobic conditions; and after the fermentation is completed, collecting the fermentation liquid, collecting the bacterial cells by centrifugation, adding 1% nicotinic acid conversion liquid, concentrating the bacterial cells by 20 times, and performing biocatalytic conversion for 60-80 hours.

[0045] The composition of the nicotinic acid conversion solution is: 100 ml of 20 mM buffer, 10 g / L of nicotinic acid, and the pH is adjusted to 7.0. During the conversion process, nicotinic acid is added to maintain the substrate concentration at about 10 g / L.

[0046] The 6-hydroxynicotinic acid content in the conversion solution was measured at 0, 4, 6, 12, 16, 20, 24, 28, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, and 76 hours of conversion. The experimental results are shown in Table 4. The product yield began to decrease at 76 hours of conversion, and the conversion was terminated. The 6-hydroxynicotinic acid content reached a maximum of 70.16 g / L.

[0047] Table 4 6-hydroxynicotinic acid production by resting cell fermentation of strain HD531

[0048]

[0049]

[0050] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Aeromonas salmonicida HD531, which produces 6-hydroxynicotinic acid, is classified as Aeromonas salmonicida , was deposited at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on July 10, 2023, with the deposit number CGMCC NO. 7.

525.

2. Use of the 6-hydroxynicotinic acid-producing Aeromonas salmonicida HD531 according to claim 1 in producing 6-hydroxynicotinic acid.

3. The method for producing 6-hydroxynicotinic acid by liquid fermentation of Aeromonas salmonicida HD531 according to claim 1, comprising the following steps: The strain was streaked onto solid culture medium and cultured at 30°C, 200 rpm for 24 h. A single bacterium was picked and transferred to liquid seed culture medium and cultured at 30°C, 200 rpm for 24 h. The inoculum was then inoculated into liquid fermentation culture medium at a rate of 1-5%. The culture was fermented at 30°C under aerobic conditions for 24 h. The concentration of the product 6-hydroxynicotinic acid was detected by high-performance liquid chromatography (HPLC).

4. The method according to claim 3, wherein The solid culture medium is composed of: 17 g / L tryptone, 3 g / L soy peptone, 2.5 g / L glucose, 2.5 g / L dipotassium hydrogen phosphate, 10 g / L sodium chloride, 20 g / L agar powder, and the balance is water, with a pH of 7.0; The liquid seed culture medium comprises: 17 g / L tryptone, 3 g / L soy peptone, 2.5 g / L glucose, 2.5 g / L dipotassium hydrogen phosphate, 10 g / L sodium chloride, 2 g / L nicotinic acid, the balance being water, and a pH of 7.0; the liquid fermentation culture medium comprises: 10-20 g / L nicotinic acid, 8-10 g / L yeast extract, 8-10 g / L peptone, 0.5-1 g / L KH2PO4, 2-4 g / L K2HPO4·3H2O, the balance being water, and a pH of 7.

0.

5. The method for producing 6-hydroxynicotinic acid in resting cells using the 6-hydroxynicotinic acid-producing Aeromonas salmonicida HD531 of claim 1, comprising the following steps: S1. First, spread the bacterial liquid of the strain onto a solid plate medium and culture it at 30°C under aerobic conditions for 24-36 hours. Then, pick a single colony of the cultured strain onto a primary seed medium and culture it aerobically at 30°C for 12-24 hours. Then, inoculate the cultured primary seeds into a secondary seed medium at a rate of 1-10%, culture it aerobically at 30°C for 8-12 hours, and after 8-12 hours, inoculate the cultured secondary seeds into a liquid fermentation medium at a rate of 1-10%. S2. Ferment the 6-hydroxynicotinic acid-producing strain HD531 under aerobic conditions for 10 to 12 hours, and then collect the cells by centrifugation of the fermentation broth; S3. Add 1% niacin conversion solution at 1 / 20 the volume of the original fermentation broth to the collected cells to concentrate the cells and then perform the conversion. Add the substrate niacin every 4 hours to stabilize the niacin concentration in the system at 10 g / L. S4. After a certain period of conversion, a conversion solution containing 6-hydroxynicotinic acid is obtained.

6. The method according to claim 5, wherein The composition of the 1% niacin conversion solution is: 100 ml of 20 mM buffer, 10 g / L of niacin, and the pH is adjusted to 7.

0.

7. The method according to claim 5, wherein The solid culture medium is composed of: 17 g / L tryptone, 3 g / L soy peptone, 2.5 g / L glucose, 2.5 g / L dipotassium hydrogen phosphate, 10 g / L sodium chloride, 20 g / L agar powder, and the balance is water, with a pH of 7.0; The liquid seed culture medium comprises: 17 g / L tryptone, 3 g / L soy peptone, 2.5 g / L glucose, 2.5 g / L dipotassium hydrogen phosphate, 10 g / L sodium chloride, 2 g / L nicotinic acid, the balance being water, and a pH of 7.0; the liquid fermentation culture medium comprises: 10-20 g / L nicotinic acid, 8-10 g / L yeast extract, 8-10 g / L peptone, 0.5-1 g / L KH2PO4, 2-4 g / L K2HPO4·3H2O, the balance being water, and a pH of 7.

0.

8. The method according to claim 5, wherein The compositions of the primary seed culture medium and the secondary seed culture medium are: niacin 1-2 g / L, beef extract 2-5 g / L, peptone 8-10 g / L, NaCl 0.5-1 g / L, the balance being water, and the pH is 7.0.

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