Nitrate reducing pseudomonas capable of biologically converting limonene

Abstract

The invention belongs to the technical field of microbial fermentation, and relates to a nitrate reducing pseudomonas capable of biologically converting limonene, which is preserved in the China Center for Culture Collection, the classification name of which is Pseudomonas nitritiducens GJ-01, the preservation number of which is CCTCC M 20251439, and the preservation date of which is June 23, 2025, and the preservation number of which is CCTCC M 20251439. According to the invention, the nitrate reducing pseudomonas strain with efficient conversion capability and capable of biologically converting limonene is separated and screened, so that a new strain resource is provided for microbial conversion of D-limonene, and a theoretical basis is provided for biosynthesis and metabolic engineering modification of terpenoids; therefore, the method has a wide application prospect in the field of microbial fermentation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and relates to a Pseudomonas nitritireducens capable of biotransformation of limonene. BACKGROUND

[0002] D-limonene, also known as dipentene, is a natural monocyclic monoterpene compound widely existing in the peel oil of citrus fruits, and the content of D-limonene can account for 70-90% of the components of the volatile oil of citrus. D-limonene has unique physical and chemical properties and biological activities due to the conjugated double bond in its molecular structure, such as antioxidant, antibacterial, antitumor and promoting transdermal absorption of drugs. In the fields of food, medicine, daily chemical and the like, D-limonene is not only an important flavor additive, but also a core precursor for synthesizing high-value terpene derivatives such as carvone, linalool and limonene oxide.

[0003] The biotransformation method uses microbial enzymes to catalyze specific transformation of cheap substrates to synthesize high-value products, and has become a research hotspot to replace the traditional production route due to the mild reaction conditions, strong stereoselectivity and environmental friendliness.

[0004] Carvone is a chiral compound, and the levorotatory carvone mainly exists in spearmint and has a mint smell, sweet and cool, and has important application value in the fields of food, daily chemical, medicine and agriculture. The levorotatory carvone has a unique spearmint flavor, is commonly used as a flavor additive in food such as chewing gum and candy, and daily necessities such as toothpaste, and can effectively improve the flavor of products. In recent years, researches have also revealed that the levorotatory carvone has pharmacological potentialities such as antioxidant, antitumor, antibacterial and sedative. Limonene is a by-product of citrus processing industry, and has rich sources and low price. The similar carbon skeleton structure of limonene and carvone makes limonene an ideal precursor for biosynthesis of the levorotatory carvone.

[0005] At present, there is no patent related to microbial transformation of limonene to produce carvone, so efficient transformation of limonene to screen carvone-producing strains is of great significance to promote the high value-added of limonene and the commercial application of carvone. SUMMARY

[0006] The present application aims to use D-limonene as a carbon source to screen strains with high transformation capacity from fresh and rotten citrus peels, and to determine the strains through morphological identification and molecular biological identification. The qualitative analysis of the transformation products is carried out by gas chromatography-mass spectrometry (GC-MS) technology, and the structure of the products is determined by comparison with standard products and spectrum analysis. The research results can provide new strain resources for microbial transformation of D-limonene, and provide a theoretical basis for biosynthesis and metabolic engineering of terpenoids. The Pseudomonas nitritireducens GJ-01 in the present application is isolated from citrus peels and preserved in the China General Microbiological Culture Collection Center.

[0007] To achieve the above objectives, the technical solution of the present invention is: a nitrate-reducing Pseudomonas bacterium capable of biotransforming limonene, which is deposited at the China Center for Culture Collection (CCTCC) of Wuhan University, Wuhan City, Hubei Province, and is classified as Pseudomonas nitritireducens GJ-01, with accession number CCTCC M 20251439 and deposit date of June 23, 2025.

[0008] Preferably, the culture characteristics of the nitrate-reducing Pseudomonas are as follows: on LB plates, the colonies are round, translucent, smooth, dull, moist, with regular edges, and appear rod-shaped under a microscope; they are Gram-negative.

[0009] Preferably, the nitrate-reducing Pseudomonas can convert D-limonene into carvone within 72 hours.

[0010] The present invention also discloses the application of nitrate-reducing Pseudomonas aeruginosa in tobacco processing.

[0011] Preferably, the applications include: aroma optimization and improvement, harm reduction function development, and processing technology optimization in tobacco processing. Specifically, this includes:

[0012] Targeted synthesis and enhancement of aroma components. Through the biotransformation of limonene by nitrate-reducing Pseudomonas, the transformed product has more complex aroma characteristics and can be used as a natural tobacco flavoring to enhance the smoothness and harmony of tobacco, suitable for cigarette flavoring.

[0013] Harm reduction function development. Limonene itself has antioxidant and anti-inflammatory properties, and its microbial transformation products may further enhance this function, which can be used to reduce free radicals in smoke or neutralize harmful substances (such as polycyclic aromatic hydrocarbons), thereby reducing the health risks of tobacco products.

[0014] Optimize tobacco fermentation and improve quality. Microbial transformation can be combined with tobacco fermentation processes to regulate the composition of volatile substances in tobacco leaves, accelerate the fermentation process and improve aroma purity, or to further shorten aging time and improve raw material utilization.

[0015] The beneficial effects of this invention are:

[0016] This invention isolates and screens nitrate-reducing Pseudomonas strains with high conversion efficiency for biotransformation of limonene from fresh and rotten citrus peels, providing new strain resources for the microbial transformation of D-limonene. At the same time, it provides a theoretical basis for the biosynthesis and metabolic engineering of terpenoids. Therefore, this invention has broad application prospects in the field of microbial fermentation. Attached Figure Description

[0017] Figure 1 This diagram shows the number and types of nitrate-reducing Pseudomonas strains that can bioconvert limonene according to the present invention.

[0018] Figure 2 Radar diagram of olfactory and palpable flavor of Pseudomonas nitritireducens GJ-01 after biotransformation according to the present invention;

[0019] Figure 3 This is a colony and cell morphology diagram of Pseudomonas nitritireducens GJ-01 of the present invention;

[0020] Figure 4 The 16S rDNA gene sequence of Pseudomonas nitritireducens GJ-01 is the present invention.

[0021] Figure 5 This is a phylogenetic tree diagram of the 16S rDNA gene sequence of Pseudomonas nitritireducens GJ-01 of the present invention;

[0022] Figure 6 This is a GC-MS chromatogram of the conversion of limonene in Pseudomonas nitritireducens GJ-01 according to the present invention;

[0023] Figure 7 This is a chemical structure diagram of the D-limonene metabolite biotransformed by Pseudomonas nitritireducens GJ-01 according to the present invention. Detailed Implementation

[0024] The related technologies of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] like Figures 1 to 7 As shown, this embodiment uses fresh and rotten citrus peels, branches, leaves, and nearby soil collected from the Citrus Research Institute of Southwest University in Chongqing as raw materials for screening limonene-resistant strains. A strain with limonene as a carbon source was obtained. Gram staining microscopy and 16S rDNA sequencing confirmed that this strain is *Pseudomonas nitritireducens* (see attached image). Figure 1 and attached Figure 2The strain, classified and named *Pseudomonas nitritireducens* GJ-01, was deposited on June 23, 2025, at the China Center for Culture Collections (CCTCC) of Wuhan University, Wuhan, Hubei Province, with accession number CCTCC M 20251439. On LB agar plates, the colonies are round, translucent, smooth, dull, moist, with regular edges, and appear rod-shaped under a microscope. It is Gram-negative. Using LB medium supplemented with 0.5% limonene, the culture was carried out in shake flasks at 30°C and 220 rpm for 24 h and 72 h. After extraction of the supernatant with n-hexane, the transformation products were analyzed by GC-MS. The results showed that *Pseudomonas nitritireducens* GJ-01 can convert D-limonene to carvone after 72 h.

[0026] Example

[0027] 1. Initial screening of strains:

[0028] 1.1 Sample Collection and Processing: Fresh and rotten citrus peels, branches, leaves, and nearby soil were collected from the Citrus Research Institute of Southwest University in Chongqing. The collected samples were sealed in sample bags. 10g of each sample was placed in 150mL of physiological saline and incubated in a constant-temperature shaker at 30℃ and 180r / min for 20min.

[0029] 1.2 Initial Screening of Limonene-Resistant Strains: 0.3 mL of the above culture medium was transferred to 30 mL of YM medium (1% glucose, 0.5% peptone, 0.3% malt extract, 0.3% yeast extract) and 30 μL (0.1% v / v) of D-limonene. After one week of incubation at 30℃ and 150 rpm on a shaker, the culture was serially diluted, and 0.2 mL was evenly spread onto YM solid medium plates. The growth status of the strains was observed during one week of incubation at 30℃. The grown strains were purified by streak plating, and this process was repeated three times. Gram staining was then performed, and the strains were observed under a microscope to confirm their isolation. (See attached image) Figure 1 As shown, a total of 52 bacterial strains were isolated in the initial screening stage, mainly including Bacillus, Pseudomonas, and Bacillus.

[0030] 2. Screening of limonene-resistant strains: The isolated strains were re-inoculated into 30 mL of YM medium and 600 μL (2%, v / v) of D-limonene. The cultures were incubated at 30 °C and 150 r / min for 48 h. 100 μL of the culture was then spread onto YM solid medium and incubated at 30 °C for 48 h. Growth was evaluated afterward. All strains with satisfactory growth (>30 CFU) were considered resistant to 2% (v / v) limonene.

[0031] 3. Sensory analysis of the transformation products of the strains: The screened strains were activated overnight and inoculated with 1% (v / v) in LB medium (1% tryptone, 1% NaCl, and 0.5% yeast extract). After cultivation at 30℃ and 220 rpm to the logarithmic phase, 0.5% (v / v) D-limonene was added for 24 h and 72 h of cultivation, respectively. After the biotransformation experiment, the fermentation broth of each strain was centrifuged at 8000 rpm for 10 min. The supernatant was extracted with n-hexane, and the upper organic phase was volatilized at room temperature for several hours to obtain the sample. The sample was sent to Shaanxi Tobacco Industry Co., Ltd. for sensory evaluation. The results are attached. Figure 2 As shown, the transformation product of strain Pseudomonas nitritireducens GJ-01 was found to have a resinous, sour, sweet, spicy, fruity and fresh aroma, with the resinous and fruity aromas being more prominent.

[0032] 4. Identification of strains:

[0033] 4.1 Preliminary identification of morphological characteristics: Colony morphology, color, size, and Gram staining results were observed. On LB agar plates, *Pseudomonas nitritireducens* GJ-01 colonies were round, translucent, smooth, dull, moist, with regular edges. Under a microscope, they appeared rod-shaped and Gram-negative. (See attached image for details.) Figure 3 As shown.

[0034] 4.2 Molecular Biological Identification: Using the strain's genome as a template, the 16S sequence of ribosomal DNA was amplified using universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-TACGGCTACCTTGTTACGACT-3). After verifying the PCR product by agarose gel electrophoresis, a PCR fragment of approximately 1500 bp was obtained. The PCR product was recovered using a DNA purification and recovery kit and sequenced (performed by Shenzhen BGI Genomics Co., Ltd.). The sequencing results were submitted to the NCBI website for BLAST search. The results showed that the strain belonged to the genus Pseudomonas and showed the highest homology (100%) with Pseudomonas nitritireducens, and was named Pseudomonas nitritireducens GJ-01. Multiple sequence matching was performed using MEGA 6.06 software to construct a phylogenetic tree, as shown in the attached figure. Figure 5 As shown.

[0035] 5. Biotransformation of 0.5% limonene (v / v) by Bacillus cereus GJ-02:

[0036] 5.1 Sample preparation: The method was the same as in Example 2.2. The sample was filtered through a 0.22 μm filter membrane and then added to a sample vial. Analysis was performed using an Agilent 8890-5977B gas chromatograph-mass spectrometer.

[0037] 5.2 Gas Chromatography Conditions: HP-5MS UI column (30m × 250μm × 0.25μm), high-purity helium as carrier gas, constant flow rate of 1.0mL / min, injection volume of 0.3μL, split injection of 10:1, injection port temperature of 250℃. Temperature program: initial column temperature 80℃, hold time 1min, then increase to 170℃ at a rate of 5℃ / min, hold time 3min.

[0038] 5.3 Mass spectrometry conditions: Electron impact ionization (EI) source, transfer line temperature 280℃, ion source temperature 230℃, quadrupole temperature 150℃, electron energy 70eV. Full scan mode (SCAN) was used, with a mass scan range of m / z 30-500 and a scan frequency of 3.2 scan / s.

[0039] 5.4 Qualitative Analysis: GC-MS was used for analysis and identification. The results were retrieved and analyzed using computer-aided spectroscopy (NIST 23.L) and standard reference materials. Table 1 shows the volatile compound analysis of D-limonene in the 72-hour biotransformation of *Pseudomonas nitritireducens* GJ-01:

[0040] Table 1

[0041] No. English Name Compound Name CAS No. Percentage Content (%) 1 1,5,8-p-Menthatriene 1,5,8-p-Menthatriene 21195-59-5 0.34 2 1,3,8-p-Menthatriene 1,3,8-p-Menthatriene 18368-95-1 0.2 3 Linalool Linalool 78-70-6 5.58 4 trans-p-Mentha-2,8-dien-l-ol trans-p-Mentha-2,8-dien-l-ol 7212-40-0 26.89 5 (E)-limonene oxide (E)-limonene oxide 4959-35-7 0.46 6 Citral Citral 5392-40-5 5.37 7 p-Mentha-1(7),8-dien-2-ol p-Mentha-1(7),8-dien-2-ol 35907-10-9 0.72 8 .alpha.-Terpineol .alpha.-Terpineol 98-55-5 5.38 9 (-)-trans-Isopiperitenol (-)-trans-Isopiperitenol 74410-00-7 6.72 10 cis-Carveol cis-Carveol 1197-06-4 8.66 11 cis-p-mentha-1(7),8-dien-2-ol cis-p-mentha-1(7),8-dien-2-ol 22626-43-3 0.26 12 (-)-Carveol (-)-Carveol 99-48-9 2.24 13 (-)-Carvone (-)-Carvone 6485-40-1 28.89

[0042] The total ion chromatograms of the biotransformation of strain Pseudomonas nitritireducens GJ-01 at 24 h and 72 h were detected by GC-MS and are shown in the attached figures. Figure 6 As shown. Where δ is the relative intensity, t R The retention times of each compound peak are shown. A comparison with the NIST23.L database and identification of chemical components using a reference quality spectrum revealed that peak 1 is D-limonene (+)-limonene; peak 2 is L-carvone (-)-carvone. Based on a search of the NIST23.L database and the mechanism of biotransformation of limonene, the chemical structure of the D-limonene metabolite from the strain Pseudomonas nitritireducens GJ-01 was deduced, as shown in the attached figure. Figure 7As shown, *Pseudomonas nitritireducens* GJ-01 bioconverts D-limonene to L-carvone, which is then further converted to L-carvone. GC-MS analysis of the volatile compounds generated during the D-limonene bioconversion of *Pseudomonas nitritireducens* GJ-01 was performed. Table 1 shows that after 72 hours of bioconversion, 13 volatile compounds were identified, mainly including alkenes, alcohols, and ketones. *Pseudomonas nitritireducens* GJ-01 bioconverted 28.89% of L-carvone.

[0043] In summary, this invention isolates and screens nitrate-reducing Pseudomonas strains with high conversion efficiency capable of biotransforming limonene, providing new strain resources for the microbial transformation of D-limonene. It also provides a theoretical basis for the biosynthesis and metabolic engineering of terpenoids, and can be used for aroma optimization, harm reduction development, and processing technology optimization in tobacco processing.

[0044] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A nitrate-reducing Pseudomonas bacterium capable of biotransforming limonene, characterized in that, The nitrate-reducing Pseudomonas is deposited at the China Center for Culture Collection, classified as Pseudomonas nitritireducens GJ-01, with accession number CCTCC M20251439, and deposited on June 23, 2025.

2. The nitrate-reducing Pseudomonas bacillus capable of biotransforming limonene according to claim 1, characterized in that, The culture characteristics of the nitrate-reducing Pseudomonas are as follows: on LB plates, the colonies are round, translucent, smooth, dull, moist, with regular edges, and appear rod-shaped under a microscope; they are Gram-negative.

3. The nitrate-reducing Pseudomonas bacillus capable of biotransforming limonene according to claim 1, characterized in that, The nitrate-reducing Pseudomonas can convert D-limonene into carvone within 72 hours.

4. The use of nitrate-reducing Pseudomonas as described in any one of claims 1 to 3 in tobacco processing.

5. The application of a nitrate-reducing Pseudomonas bacterium capable of bioconverting limonene according to claim 4 in tobacco processing, characterized in that, The applications include: aroma optimization and improvement, harm reduction development, and processing technology optimization in tobacco processing.

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