Acinetobacter strain for producing orotic acid, application of acinetobacter strain and method for producing orotic acid

Abstract

The invention discloses an acinetobacter strain for producing orotic acid, application of the acinetobacter strain and a method for producing orotic acid. The acinetobacter strain for producing the orotic acid is named as Acinetobacter sp WJ01, the strain is preserved in the China General Microbiological Culture Collection Center on February 27, 2025, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO: 33667. Compared with the existing engineering bacteria for synthesizing the orotic acid, the strain has the advantages that the environmental adaptability is higher, the orotic acid can be naturally synthesized under proper conditions, the genome is relatively stable, the potential risk of exogenous genes does not exist, and the safety is relatively high. In the fermentation process, the acinetobacter yield is high, and the sugar-acid conversion rate is high. When the strain is applied to the fermentation production of the orotic acid, the yield of the orotic acid can be obviously improved, and the fermentation period is greatly shortened.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a strain of Acinetobacter that produces orotic acid ( Acinetobacter sp WJ01) and use and method for producing orotic acid. Background Art

[0002] Orotic acid, also known as vitamin B 13 , pyrimidine acid, is a nutrient that can be combined with vitamin B 12 Metabolize together, thereby reducing the body's lack of vitamin B 12 Orotic acid is widely used in medicine, cosmetics and food, and has obvious effects of inhibiting aging and promoting cell metabolism.

[0003] At present, the main production methods of orotic acid are chemical synthesis using sodium bromide, chlorine and maleurea as raw materials and biological fermentation based on microbial fermentation. Among them, the chemical synthesis method has problems such as high production risk, long production cycle, low reaction efficiency, expensive raw materials and high production cost, which is not suitable for industrial production. In the reported studies, Zhang Yiping (Production of orotic acid by microbial fermentation. Journal of Wuxi University of Light Industry. Vol. 22, No. 3, May 2023) and Zou Shaolan (Chinese invention patent with publication number CN115838645A) respectively used genetically modified strains of Corynebacterium glutamicum and Saccharomyces cerevisiae to ferment and produce orotic acid, and the acid production after 72 h of fermentation was 8.6 g / L and 3.75 g / L, respectively. However, since most of them are model strains, they are not suitable for industrial production, and the stability of the strains has not been further verified, there may be factors such as plasmid loss leading to poor genetic stability. Therefore, it is urgent to explore strains with stronger genetic stability, higher orotic acid production and lower transformation cost. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a strain of acinetobacter that produces orotic acid, an application thereof, and a method for producing orotic acid. The acinetobacter of the present invention has the ability to produce high orotic acid, and has strong adaptability, high safety, good stability, and the strain is cheap and easy to obtain, and has low requirements for culture conditions. The acinetobacter can be used as an excellent strain for producing organic acids, and is the best choice for producing orotic acid.

[0005] In a first aspect, the present invention provides a strain of Acinetobacter that produces orotic acid, which is achieved through the following technical scheme.

[0006] A strain of Acinetobacter that produces orotic acid, named Acinetobacter sp WJ01, the strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 27, 2025, with the deposit number: CGMCC NO: 33667.

[0007] Further, the strain is an obligate aerobe, a Gram-negative bacterium, the culture temperature is 25-37°C, and the culture pH is 4.5-9.0. Preferably, the culture temperature is 35-37°C, and the culture pH is 6.0-8.0.

[0008] Furthermore, the colonies are round protrusions with smooth surfaces and neat edges, and the colonies are off-white in color and moist and opaque in texture.

[0009] Furthermore, the carbon sources utilized by the strain include fructose, maltose, sucrose, and glycerol.

[0010] Furthermore, the strain produces oxidase, catalase, cellulase, and amylase, but does not produce lipase.

[0011] In a second aspect, the present invention provides a use of a strain of Acinetobacter that produces orotic acid, which is achieved through the following technical solution.

[0012] An application of the above strain in producing orotic acid.

[0013] In a third aspect, the present invention provides a method for producing orotic acid, which is achieved through the following technical scheme.

[0014] A method for producing orotic acid using the above strain comprises the following steps: a. Streak the strain on LB medium plate and incubate at 35-37°C for 24h; b. Select a single colony that is away from the colony group, has good growth morphology, and is of moderate size, transfer it to liquid LB medium, and culture it at 35-37°C and 180-200 rpm / min for 24 hours to obtain seed solution; c. Inoculate the seed liquid into the fermentation medium or optimized fermentation medium at an inoculation rate of 1%, and ferment at 35-37°C, 180-200 rpm / min for 72 h; or inoculate the seed liquid into a fermentation tank containing the fermentation medium or optimized fermentation medium at an inoculation rate of 2%, and ferment for 80 h at 35-37°C, 180-200 rpm / min, ventilation volume of 2L / min, and tank pressure of 0.04MPa.

[0015] Furthermore, the fermentation medium includes: glucose 100-140 g / L, corn syrup 5-6 mL / L, 20× trace elements 50 mL / L, CaCO 3 6-8%; 20× trace elements include: (NH 4 ) 2 SO 4 4.8-4.95 g / L, KH 2 PO 44.5-5 g / L, MgSO 4 0.05-0.1g / L.

[0016] Furthermore, the optimized fermentation medium included: glucose 140 g / L, corn syrup 5 mL / L, 20× trace elements 50 mL / L, CaCO 3 6%; 20× trace elements include: (NH 4 ) 2 SO 4 4.95 g / L, KH 2 PO 4 5 g / L, MgSO 4 0.05g / L, Na 2 SO 4 0.1g / L.

[0017] This application has the following beneficial effects.

[0018] (1) The present invention solves the problem of low yield and high by-products in the existing process of producing orotic acid by microbial fermentation, which results in increased costs for the subsequent orotic acid purification process. The Acinetobacter of the present invention produces almost no by-products during the shake flask fermentation process, and the orotic acid yield is as high as 74.95 g / L; (2) The present invention solves the problem that in the existing process of producing orotic acid by microbial fermentation, plasmids need to be introduced to transform the metabolic pathway of the strain, resulting in genetic instability of the strain. The Acinetobacter strain of the present invention is a natural strain with strong environmental adaptability and can grow stably in the natural environment. It can form biofilms on various surfaces, which not only enhances its adaptability to the environment, but also improves its tolerance to antibiotics; (3) Through the optimization of microbial fermentation, the efficiency of the present invention's Acinetobacter strain in producing orotic acid was significantly improved. The yield after 72 hours of shake flask fermentation increased from 36.87 g / L to 74.95 g / L, and the yield after 80 hours in a 5L fermenter reached 112.46 g / L, providing an excellent strain for the preparation of orotic acid by microbial fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the electrophoresis diagram of the PCR product of the present invention (wherein, M: marker; 1, 2, 3, 4: PCR products); Figure 2 is the evolutionary tree of Acinetobacter of the present invention; Figure 3 This is a diagram of seed optimization results at different culture temperatures of the present invention; Figure 4 This is a diagram of the optimization results of seeds at different culture pH values ​​of the present invention; Figure 5 is a standard curve diagram of orotic acid in the present invention; Figure 6 HPLC verification diagram of the standard product and the sample of the present invention; Figure 7 This is a diagram of the optimization results of fermentation with different carbon sources in the present invention; Figure 8 This is a diagram showing the optimization results of fermentation with different nitrogen sources in the present invention; Fig. 9 This is a diagram of the fermentation optimization results of different metal ions in the present invention; Fig.10 This is a diagram of fermentation optimization results at different calcium carbonate concentrations in the present invention; Fig.11 This is a diagram of the fermentation results in the upper tank of the present invention. DETAILED DESCRIPTION

[0020] The invention is further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental method used in the present invention is a conventional method, and the experimental equipment, materials, reagents, etc. used can be purchased from relevant material sales companies.

[0021] The present invention provides a strain of Acinetobacter that produces orotic acid ( Acinetobacter sp WJ01) was screened from the sea areas around Tianjin Binhai New Area and was deposited in the General Microbiology Center of China Microorganism Culture Collection on February 27, 2025, with the deposit number: CGMCC NO: 33667; after optimization by microbial fermentation method, the orotic acid production reached 74.95 g / L.

[0022] 1. Isolation and screening of strains (1) Take 1 ml of seawater from the surrounding sea areas of Tianjin Binhai New Area and add it to 50 mL of sterilized LB liquid culture medium. Incubate at 35-37 °C and 180-200 rpm / min for 24 h. (2) Take 1 mL of bacterial suspension for enrichment culture and dilute the enriched bacterial suspension by 10 -5 , spread 100 μL of the dilution on LB solid medium and culture at 35-37 ℃ for 24 h; (3) Pick a single colony and use an inoculation loop to make three zones on an LB activated medium plate. Incubate the plate at 35-37°C for 24 h. (4) Select a single colony with good growth morphology and appropriate size, transfer it to sterile liquid LB medium, and culture it at 35-37°C and 180-200 rpm / min for 24 h; (5) Take fresh bacterial culture medium and use 16S primers for PCR amplification. After electrophoresis detection of PCR products, determine the 16S DNA sequence.

[0023] 2. Identification of strains 1. Identification of physiological and biochemical characteristics The colonies of the strain on LB (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.0) solid culture medium are round protrusions with a smooth surface, neat edges, grayish white color, moist and opaque texture, and Gram staining is negative. It can utilize fructose, maltose, sucrose, and glycerol as carbon sources and can produce oxidase, catalase, cellulase, and amylase, but no lipase.

[0024] 2. 16S rDNA sequence identification Take 1 μL of bacterial solution as template and amplify the 16S rDNA gene of the strain using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO.2) and 1492R (5'-GGTTACCTTGTTACGACTT-3') (SEQ ID NO.3). Take 2 μL of PCR product and verify it by 1% agarose gel electrophoresis ( Figure 1 ), and the products were sent to Suzhou Jinweizhi Company.

[0025] The determined 16S rDNA gene sequence is shown below (SEQ ID NO. 1).

[0026] Based on the above identification results, and after sequence comparison, it was determined that strain WJ01 belonged to the genus Acinetobacter ( Acinetobacter sp. ). Strain WJ01 was identified as Acinetobacter by comparison with NCBI data, and the query coverage was 99%. Its evolutionary tree is shown in Figure 2 shown.

[0027] 3. Seed culture optimization Using the Acinetobacter strain WJ01 as the starting strain, the three-zone streak method was used to streak onto LB solid culture medium, and then placed in incubators at 20°C, 25°C, 30°C, 37°C, and 40°C for 24 hours; single colonies that were away from the colonies, had good growth morphology, and were of moderate size were selected and transferred to sterile liquid LB culture medium with pH values ​​of 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, and 9.0, respectively, and cultured at 35-37°C and 180-200 rpm / min for 24 hours. After 24 hours of seed culture, sterile sampling was performed and the OD was measured after dilution 10 times. 600 , determine the specific bacterial concentration in the seed solution with different pH values. Figure 3 , Figure 4 , the seed culture temperature range is 25-37℃, the optimal culture temperature is 37℃; the seed culture pH range is 4.5-9.0, the optimal culture pH is 8.0. The following experiments are all fermented and cultured under the optimal temperature and optimal pH conditions.

[0028] 4. Determination of orotic acid standard curve and calculation method of sugar-acid conversion rate in fermentation tank Dilute the orotic acid standard with 5mM dilute sulfuric acid as the mobile phase to 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L, 70g / L, 80g / L, and 90g / L standard solutions. Use concentration as the abscissa and peak area as the ordinate to establish a regression curve between orotic acid concentration and detection peak area. Figure 5 As shown ( Figure 5 The red line in the middle represents the regression curve of orotic acid concentration and detection peak area obtained by fitting the data points. In the concentration range of 10~90g / L, the linear relationship is excellent. The regression equation is: y=498.35x+277.4, R 2 =0.99.

[0029] The calculation formula for the sugar-acid conversion rate in the fermentation tank is: Sugar-acid conversion rate (g / g) = residual sugar (g / L) ÷ orotic acid production (g / L) Among them, the residual sugar content is measured using a biosensor.

[0030] 5. Fermentation and cultivation of orotic acid The cultured seed liquid was added to the fermentation medium at a 1% inoculum rate and fermented for 72 h at 37 °C and 200 rpm / min. The fermentation medium included 140 g / L glucose, 5 mL / L corn syrup, 50 mL / L 20× trace elements, and CaCO 3 8%; 20× trace elements include: (NH 4 ) 2 SO 4 4.95 g / L, KH 2 PO 4 5 g / L, MgSO 4 0.05g / L. After the fermentation broth was acidified and analyzed by HPLC, it was found that the peak was consistent with that of the orotic acid standard product. Figure 6 The peak area of ​​orotic acid was recorded as y, and the orotic acid concentration x was calculated by substituting it into the above standard curve. The final orotic acid content was 36.87 g / L.

[0031] The method for acidifying the fermentation broth is as follows: centrifuge 2 mL of the fermentation broth at 8000 r / min for 15 min, take 100 uL of the supernatant and add 1900 uL of 5 mmol / L sulfuric acid solution, vortex mix, filter with a 0.22 μm filter membrane into a liquid phase vial, and perform HPLC analysis.

[0032] HPLC detection conditions: organic acid column, mobile phase 5 mmol / L sulfuric acid solution, flow rate 0.6 mL / min, column temperature 60°C, injection volume 10 μL, UV detection wavelength 218 nm.

[0033] 6. Fermentation medium optimization The cultured seed liquid was added to the culture medium with different carbon sources, nitrogen sources, metal ions, and calcium carbonate concentrations at an inoculum rate of 1% for cultivation. The selected carbon sources for fermentation medium optimization were glucose, maltose, fructose, soluble starch, sodium citrate, lactose, and sucrose; the selected nitrogen sources for fermentation medium optimization were corn steep liquor, yeast powder (FM888, FM305, FM508, FM503), soybean cake powder, peanut cake powder, cottonseed cake powder, and tryptone; the selected metal ions for fermentation medium optimization were FeSO 4 ,CuSO 4 , KH 2 PO 4 、Na 2 SO 4 、MnSO 4 MgSO 4 etc.; The selected calcium carbonate concentration (wt%) for fermentation medium optimization is: 2, 4, 6, 8. The experimental results are as follows Figure 7-10As shown, the optimal carbon source for the fermentation medium is glucose; the optimal nitrogen source for the fermentation medium is corn steep liquor; the optimal metal ion for the fermentation medium is Na 2 SO 4 The optimal calcium carbonate concentration of the fermentation medium is 6%. The optimized fermentation medium composition is: glucose 140g / L, corn syrup 5 mL / L, 20× trace elements 50mL / L, CaCO 3 6%; 20× trace elements include: (NH 4 ) 2 SO 4 4.95 g / L, KH 2 PO 4 5 g / L, MgSO 4 0.05g / L, Na 2 SO 4 0.1g / L.

[0034] Using the optimized fermentation medium, after 72 hours of shake flask fermentation, the content of orotic acid produced by the strain WJ01 of the present invention can reach 74.95 g / L, which is 103.28% higher than the content of orotic acid produced before fermentation optimization.

[0035] The seed liquid was inoculated into a 5L fermentation tank at a 2% inoculation rate, and the optimized fermentation medium was added into the fermentation tank. The fermentation culture was carried out for 80 hours at 37°C, 200 rpm / min, aeration volume of 2L / min, and tank pressure of 0.04MPa. After 80 hours of fermentation in the 5L tank, the whey acid produced by the strain WJ01 of the present invention can reach 112.46g / L ( Fig.11 ), the sugar-acid conversion rate is 0.76g / g.

[0036] The embodiments of this specific implementation method are all preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Acinetobacter that produces orotic acid, named Acinetobacter sp WJ01, the strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 27, 2025, with the deposit number: CGMCC NO: 33667.

2. The strain according to claim 1, characterized in that: The strain is an obligate aerobic, Gram-negative bacterium, the culture temperature is 25-37° C., and the culture pH is 4.5-9.

0.

3. The strain according to claim 1, characterized in that: The colonies are round protrusions with a smooth surface and neat edges. The colonies are grayish white in color and have a moist and opaque texture.

4. The strain according to claim 1, characterized in that: The carbon sources utilized by the strain include fructose, maltose, sucrose and glycerol.

5. The strain according to claim 1, characterized in that: The strain produces oxidase, catalase, cellulase, and amylase, but no lipase.

6. Use of the strain according to any one of claims 1 to 5 in producing orotic acid.

7. A method for producing orotic acid using the strain described in any one of claims 1 to 5, characterized in that: The following steps are involved: a. Streak the strain on LB medium plate and incubate at 35-37°C for 24h; b. Select a single colony that is away from the colony group, has good growth morphology, and is of moderate size, transfer it to liquid LB medium, and culture it at 35-37°C and 180-200 rpm / min for 24 hours to obtain seed solution; c. Inoculate the seed liquid into the fermentation medium or optimized fermentation medium at an inoculation rate of 1%, and ferment at 35-37°C, 180-200 rpm / min for 72 h; or inoculate the seed liquid into a fermentation tank containing the fermentation medium or optimized fermentation medium at an inoculation rate of 2%, and ferment for 80 h at 35-37°C, 180-200 rpm / min, ventilation volume of 2L / min, and tank pressure of 0.04MPa.

8. The method according to claim 7, characterized in that: The fermentation medium includes: glucose 100-140 g / L, corn syrup 5-6 mL / L, 20× trace elements 50 mL / L, CaCO3 6-8%; 20× trace elements include: (NH4)2SO4 4.8-4.95 g / L, KH2PO4 4.5-5 g / L, MgSO4 0.05-0.1 g / L.

9. The method according to claim 7, characterized in that: The optimized fermentation medium included: glucose 140 g / L, corn syrup 5 mL / L, 20× trace elements 50 mL / L, CaCO3 6%; 20× trace elements included: (NH4)2SO4 4.95 g / L, KH2PO4 5 g / L, MgSO4 0.05g / L, Na2SO40.1g / L.

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