An Acinetobacter strain producing orotic acid, its application and method for producing orotic acid

Abstract

The present invention discloses an Acinetobacter strain producing orotic acid, its application, and a method for producing orotic acid. The Acinetobacter strain producing orotic acid is named Acinetobacter sp WJ01, which was deposited at the General Microbiological Center of the China Center for Type Culture Collection on February 27, 2025, with the deposit number: CGMCC NO: 33667. Compared with the existing engineered bacteria for synthesizing orotic acid, this strain has strong environmental adaptability, can naturally synthesize orotic acid under suitable conditions, has a relatively stable genome, no potential risk of foreign genes, and relatively high safety. During the fermentation process, this Acinetobacter has a high yield and a high sugar-acid conversion rate. Applying the strain of the present invention to the fermentation production of orotic acid can significantly increase the yield of orotic acid and greatly shorten the fermentation period.

Description

Technical Field

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

[0002] Orotic acid, also known as vitamin B 13 6, pyrimidine acid, is a nutrient that can be metabolized together with vitamin B 12 6 to reduce the adverse effects of vitamin B 12 6 deficiency in the body. Orotic acid is widely used in the fields of medicine, cosmetics, and food, and has obvious effects such as inhibiting aging and promoting cell metabolism.

[0003] Currently, the production methods of orotic acid mainly include chemical synthesis methods using sodium bromide, chlorine, and maleic hydantoin as raw materials and biological fermentation methods mainly based on microbial fermentation. Among them, the chemical synthesis method has problems such as high production risks, long production cycles, low reaction efficiency, expensive raw materials, and high production costs, and 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), Zou Shaolan (Chinese invention patent with publication number CN115838645A), etc. respectively used Corynebacterium glutamicum and genetically modified strains of Saccharomyces cerevisiae to ferment and produce orotic acid, and their acid production amounts after 72 h of fermentation were 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 strain stability has not been further verified, and there may be factors such as plasmid loss resulting in poor genetic stability. Therefore, it is urgent to explore strains with stronger genetic stability, higher orotic acid production, and lower transformation costs. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides an Acinetobacter strain that produces orotic acid, its application, and a method for producing orotic acid. The Acinetobacter of the present invention has the ability to produce high levels of orotic acid, and it has strong adaptability, high safety, and good stability. 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 the first aspect, the present invention provides an Acinetobacter strain that produces orotic acid, which is achieved through the following technical solutions.

[0006] An Acinetobacter strain that produces orotic acid, named Acinetobacter sp WJ01, was deposited with the China General Microbiological Culture Collection Center on February 27, 2025, and the deposit number is: CGMCC NO: 33667.

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

[0008] Furthermore, the colony is round and raised, with a smooth surface, regular edges, a grayish-white color, and a moist and opaque 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, and does not produce lipase.

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

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

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

[0014] A method for producing orotic acid using the above strain, comprising the following steps:

[0015] a. Streak the strain on an LB medium plate and incubate it statically at 35 - 37 °C for 24 h;

[0016] b. Select single colonies that are far from the group colonies, have a good growth morphology, and are of appropriate size, transfer them to liquid LB medium, and culture them at 35 - 37 °C, 180 - 200 rpm / min for 24 h to obtain a seed solution;

[0017] c. Inoculate the seed solution into the fermentation medium or the optimized fermentation medium at an inoculation amount of 1%, and perform fermentation culture at 35 - 37 °C, 180 - 200 rpm / min for 72 h; or inoculate the seed solution into a fermenter containing the fermentation medium or the optimized fermentation medium at an inoculation amount of 2%, and perform fermentation culture at 35 - 37 °C, 180 - 200 rpm / min, an aeration rate of 2 L / min, and a tank pressure of 0.04 MPa for 80 h.

[0018] Furthermore, the fermentation medium includes: glucose 100 - 140 g / L, corn steep liquor 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.

[0019] Furthermore, the optimized fermentation medium comprises: glucose 140 g / L, corn steep liquor 5 mL / L, 20×trace elements 50 mL / L, and CaCO3 6%; the 20×trace elements include: (NH4)2SO4 4.95 g / L, KH2PO4 5 g / L, MgSO4 0.05 g / L, and Na2SO4 0.1 g / L.

[0020] This application has the following beneficial effects.

[0021] (1) The present invention solves the problems in the existing process of producing orotic acid by microbial fermentation, including low yield, many by-products, and high cost of subsequent orotic acid purification process. The Acinetobacter of the present invention hardly produces by-products during the shake-flask fermentation process, and the orotic acid yield is as high as 74.95 g / L;

[0022] (2) The present invention solves the problem in the existing process of producing orotic acid by microbial fermentation that a plasmid needs to be introduced to modify the strain metabolic pathway, resulting in genetic instability of the strain. The Acinetobacter strain of the present invention is a natural strain with strong environmental adaptability and can stably grow in the natural environment. It can form biofilms on various surfaces, which not only enhances its environmental adaptability but also improves its tolerance to antibiotics;

[0023] (3) Through the optimization of microbial fermentation, the efficiency of producing orotic acid by the Acinetobacter strain of the present invention is significantly improved. The yield in the shake-flask fermentation for 72 hours is increased from 36.87 g / L to 74.95 g / L, and the yield in a 5 L fermenter for 80 h reaches 112.46 g / L, providing an excellent strain for the preparation of orotic acid by the microbial fermentation method. Description of the Drawings

[0024] Figure 1 is the electrophoresis diagram of the PCR product of the present invention (where M: marker; 1, 2, 3, 4: PCR products);

[0025] Figure 2 is the phylogenetic tree of Acinetobacter of the present invention;

[0026] Figure 3 is the optimization result diagram of seeds at different culture temperatures of the present invention;

[0027] Figure 4 is the optimization result diagram of seeds at different culture pH values of the present invention;

[0028] Figure 5 is the standard curve diagram of orotic acid in the present invention;

[0029] Figure 6 is the HPLC verification diagram of the standard product and the sample of the present invention;

[0030] Figure 7 It is the optimized fermentation result diagram of different carbon sources in the present invention;

[0031] Figure 8 It is the optimized fermentation result diagram of different nitrogen sources in the present invention;

[0032] Figure 9 It is the optimized fermentation result diagram of different metal ions in the present invention;

[0033] Figure 10 It is the optimized fermentation result diagram of different calcium carbonate concentrations in the present invention;

[0034] Figure 11 It is the fermentation result diagram in the bioreactor of the present invention. Detailed implementation manners

[0035] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods used in the present invention are conventional methods, and the experimental equipment, materials, reagents, etc. used can be purchased from relevant material sales companies.

[0036] An Acinetobacter sp. strain ( Acinetobacter sp WJ01) producing orotic acid provided by the present invention was screened from the surrounding sea area of Binhai New Area, Tianjin, and was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 27, 2025, with the deposit number: CGMCC NO: 33667; after optimization by the microbial fermentation method, the orotic acid yield is as high as 74.95 g / L.

[0037] I. Isolation and screening of the strain

[0038] (1) Take 1 ml of seawater from the surrounding sea area of Binhai New Area, Tianjin and add it to 50 mL of sterilized LB liquid medium, and shake and culture at 35 - 37 °C and 180 - 200 rpm / min for 24 h;

[0039] (2) Take 1 mL of the enriched bacterial suspension for enrichment culture, dilute the enriched bacterial liquid by 10 -5 , take 100 μL of the diluted solution and spread it on the LB solid medium, and culture at 35 - 37 °C for 24 h;

[0040] (3) Pick a single colony and perform three-zone streaking on the LB activation medium plate with an inoculation loop, and place the plate at 35 - 37 °C for 24 h;

[0041] (4) Select single colonies with good growth morphology and appropriate size, transfer them to a sterile liquid LB medium, and culture at 35 - 37 °C and 180 - 200 rpm / min for 24 h;

[0042] (5) Take the fresh bacterial liquid and perform PCR amplification using 16S primers. After electrophoresis detection of the PCR products, determine the 16r DNA sequence.

[0043] II. Identification of Strains

[0044] 1. Identification of Physiological and Biochemical Characteristics

[0045] The colonies of the strain on the LB (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.0) solid medium are round and raised, with a smooth surface, neat edges, grayish-white, moist and opaque texture. After Gram staining, it is negative. The carbon sources that can be utilized are fructose, maltose, sucrose, and glycerol. It can produce oxidase, catalase, cellulase, and amylase, but does not produce lipase.

[0046] 2. Identification of 16S rDNA Sequence

[0047] Take 1 μL of the bacterial liquid as a template and amplify the 16S rDNA gene of the strain using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO.2) and 1492R (5'-GGTTACCTTGTTACGACTT-3') (SEQ ID NO.3). Take 2 μL of the PCR product and verify it by 1% agarose gel electrophoresis ( Figure 1 ) and then send the product to Genewiz (Suzhou) Inc.

[0048] The determined 16S rDNA gene sequence is as follows (SEQ ID NO.1).

[0049]

[0050] Based on the above identification results and after sequence alignment, it was determined that strain WJ01 belongs to the genus Acinetobacter ( Acinetobacter sp. ). Strain WJ01 was aligned with NCBI data and determined to be Acinetobacter, with a query coverage of 99%. Its phylogenetic tree is as Figure 2 shown.

[0051] III. Optimization of Seed Culture

[0052] Using Acinetobacter strain WJ01 as the starting strain, the three-zone streaking method was used to streak it onto LB solid medium, and it was placed in incubators at 20 °C, 25 °C, 30 °C, 37 °C, and 40 °C for 24 h; single colonies far from the colony group, with good growth morphology and moderate size were selected and transferred to sterile liquid LB media with pH values of 3.5, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, and 9.0, and shaken at 35 - 37 °C and 180 - 200 rpm / min for 24 h. After 24 h of seed culture, aseptic sampling was carried out, and after dilution by 10 times, OD 600 was measured to determine the specific cell concentration in the seed solution at different pH values. The experimental results are as Figure 3 , Figure 4 shown. The seed culture temperature range is 25 - 37 °C, and the optimal culture temperature is 37 °C; the seed culture pH range is 4.5 - 9.0, and the optimal culture pH is 8.0. All the following experiments were carried out on the strain under the optimal temperature and optimal pH conditions for fermentation culture.

[0053] IV. Determination of the Standard Curve of Orotic Acid and Calculation Method of the Sugar Acid Conversion Rate in the Fermenter

[0054] The orotic acid standard was diluted with mobile phase 5 mM dilute sulfuric acid to standard solutions of 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, and 90 g / L respectively. With the concentration as the abscissa and the peak area as the ordinate, a regression curve of orotic acid concentration and detected peak area was established as Figure 5 shown ( Figure 5 the red line in it represents the regression curve of orotic acid concentration and detected peak area obtained by fitting through the data points). In the concentration range of 10 - 90 g / L, the linear relationship is excellent, and the regression equation is: y = 498.35x + 277.4, R 2 = 0.99.

[0055] The calculation formula for the sugar acid conversion rate in the fermenter is:

[0056] Sugar acid conversion rate (g / g) = Residual sugar amount (g / L) ÷ Orotic acid yield (g / L)

[0057] Among them, the residual sugar content was measured using a biosensor.

[0058] V. Fermentation culture of orotic acid

[0059] The cultured seed liquid was added to the fermentation medium at an inoculation amount of 1%, and fermentation culture was carried out at 37 °C and 200 rpm / min for 72 h. The fermentation medium included 140 g / L of glucose, 5 mL / L of corn steep liquor, 50 mL / L of 20×trace elements, and 8% of CaCO3; the 20×trace elements included: 4.95 g / L of (NH4)2SO4, 5 g / L of KH2PO4, and 0.05 g / L of MgSO4. After the fermentation broth was acidified and analyzed by HPLC, it was detected that the peak was consistent with the orotic acid standard product, as Figure 6 shown. 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. Finally, the content of orotic acid was 36.87 g / L.

[0060] The method for acidifying the fermentation broth was as follows: 2 mL of the fermentation broth was centrifuged at 8000 r / min for 15 min, 100 μL of the supernatant was taken and added to 1900 μL of 5 mmol / L sulfuric acid solution. After vortex mixing, it was filtered through a 0.22 μm filter membrane into a liquid phase vial for HPLC analysis.

[0061] HPLC detection conditions: organic acid chromatographic column, mobile phase 5 mmol / L sulfuric acid solution, flow rate 0.6 mL / min, column temperature 60 °C, injection volume 10 μL, ultraviolet detection wavelength 218 nm.

[0062] VI. Optimization of fermentation medium

[0063] The cultured seed liquid was added to the media with different carbon sources, nitrogen sources, metal ions, and calcium carbonate concentrations at an inoculation amount of 1% for culture. The carbon sources selected for optimizing the fermentation medium were: glucose, maltose, fructose, soluble starch, sodium citrate, lactose, sucrose; the nitrogen sources selected for optimizing the fermentation medium were: corn steep liquor, yeast powder (FM888, FM305, FM508, FM503), soybean cake powder, peanut cake powder, cottonseed cake powder, tryptone; the metal ions selected for optimizing the fermentation medium were: FeSO4, CuSO4, KH2PO4, Na2SO4, MnSO4, MgSO4, etc.; the calcium carbonate concentrations (wt%) selected for optimizing the fermentation medium were: 2, 4, 6, 8. The experimental results are as Figures 7 - 10As shown in the figure, the optimal carbon source of the fermentation medium is glucose; the optimal nitrogen source of the fermentation medium is corn steep liquor; the optimal metal ion of the fermentation medium is Na2SO4; the optimal calcium carbonate concentration of the fermentation medium is 6%. The composition of the optimized fermentation medium is as follows: glucose 140 g / L, corn steep liquor 5 mL / L, 20× trace elements 50 mL / L, CaCO3 6%; 20× trace elements include: (NH4)2SO4 4.95 g / L, KH2PO4 5 g / L, MgSO4 0.05 g / L, Na2SO4 0.1 g / L.

[0064] Using the optimized fermentation medium and through 72-hour 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.

[0065] Inoculate the seed liquid into a 5 L fermentation tank at an inoculation amount of 2%, add the optimized fermentation medium to the fermentation tank, and carry out fermentation culture for 80 h under the conditions of 37 °C, 200 rpm / min, aeration rate of 2 L / min, and tank pressure of 0.04 MPa. After 80-hour fermentation in a 5 L fermentation tank, the orotic acid produced by the strain WJ01 of the present invention can reach 112.46 g / L ( Figure 11 ), and the sugar-acid conversion rate is 0.76 g / g.

[0066] The examples of this specific implementation mode are all preferred examples of the present invention, and do not limit the protection scope of the present invention accordingly. Therefore, all equivalent changes made according to the structure, shape, and principle of the present invention shall be covered within the protection scope of the present invention.

Claims

1. An Acinetobacter sp. WJ01 producing orotic acid, characterized in that: Acinetobacter The Acinetobacter was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 27, 2025, with the deposit number: CGMCC NO: 33667. ​ 2. Use of the Acinetobacter described in claim 1 in the production of orotic acid.

3. A method for producing orotic acid using Acinetobacter as described in claim 1, characterized in that: It includes the following steps: a. Streak the strain on an LB medium plate and statically culture it at 35 - 37 °C for 24 h; b. Select a single colony that is far from the group colonies, has a good growth morphology, and is of moderate size, transfer it to a liquid LB medium, and culture it at 35 - 37 °C, 180 - 200 rpm / min for 24 h to obtain a seed solution; c. Inoculate the seed solution into the fermentation medium or the optimized fermentation medium at an inoculation amount of 1%, and carry out fermentation culture at 35 - 37 °C, 180 - 200 rpm / min for 72 h; or inoculate the seed solution into a fermenter containing the fermentation medium or the optimized fermentation medium at an inoculation amount of 2%, and carry out fermentation culture at 35 - 37 °C, 180 - 200 rpm / min, aeration rate of 2 L / min, and tank pressure of 0.04 MPa for 80 h.

4. The method according to claim 3, wherein: The fermentation medium includes: 100 - 140 g / L of glucose, 5 - 6 mL / L of corn steep liquor, 50 mL / L of 20× trace elements, 6 - 8% of CaCO3; 20× trace elements include: 4.8 - 4.95 g / L of (NH4)2SO4, 4.5 - 5 g / L of KH2PO4, 0.05 - 0.1 g / L of MgSO4.

5. The method according to claim 3, wherein: The optimized fermentation medium includes: 140 g / L of glucose, 5 mL / L of corn steep liquor, 50 mL / L of 20× trace elements, 6% of CaCO3; 20× trace elements include: 4.95 g / L of (NH4)2SO4, 5 g / L of KH2PO4, 0.05 g / L of MgSO4, 0.1 g / L of Na2SO4.

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