A nitrate-reducing pseudomonas capable of bioconverting limonene

Abstract

The present application belongs to the technical field of microbial fermentation, and relates to a nitrite-reducing Pseudomonas capable of biotransforming limonene, which is preserved in China Center for Type Culture Collection, named as Pseudomonas nitritireducens GJ-01, has a preservation number of CCTCC M 20251439, and a preservation date of June 23, 2025; the present application separates and screens a nitrite-reducing Pseudomonas strain capable of biotransforming limonene and having high conversion capacity, provides a new strain resource for microbial conversion of D-limonene, and provides a theoretical basis for biosynthesis and metabolic engineering modification of terpenoids, so that the present application has a wide application prospect in the field of microbial fermentation.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology and relates to a nitrate-reducing Pseudomonas bacterium that can bioconvert limonene. Background Technology

[0002] D-limonene, also known as dipentene, is a natural monocyclic monoterpene compound widely found in the essential oils of citrus fruit peels, accounting for 70%-90% of the volatile oil components. Its molecular structure contains conjugated double bonds, endowing it with unique physicochemical properties and biological activities, such as antioxidant, antibacterial, antitumor, and transdermal drug absorption enhancement functions. In the food, pharmaceutical, and daily chemical industries, D-limonene is not only an important flavoring additive but also a core precursor for the synthesis of high-value-added terpene derivatives such as carvone, perillyl alcohol, and limonene oxide.

[0003] Biotransformation utilizes microbial enzymes to specifically transform inexpensive substrates into high-value-added products. Due to its mild reaction conditions, strong stereoselectivity, and environmental friendliness, it has become a research hotspot for replacing traditional production routes.

[0004] Carvone is a chiral compound, specifically L-carvone, primarily found in spearmint. It possesses a minty, sweet, and slightly cooling aroma and has significant applications in the food, daily chemical, pharmaceutical, and agricultural sectors. Its unique spearmint flavor makes it a common flavoring additive in chewing gum, candy, and toothpaste, effectively improving product flavor. Recent studies have also revealed its pharmacological potential, including antioxidant, antitumor, antibacterial, and sedative effects. Limonene, a byproduct of the citrus processing industry, is abundant and inexpensive. Its similar carbon skeleton structure to carvone makes it an ideal precursor for the biosynthesis of L-carvone.

[0005] Currently, no patents have been published regarding the microbial conversion of limonene to carvone. Therefore, the selection of carvone strains for efficient limonene conversion is of great significance for promoting the high-value utilization of limonene and the commercial application of carvone. Summary of the Invention

[0006] The purpose of this invention is to isolate and screen strains with high transformation capabilities from fresh and decaying citrus peels using D-limonene as a carbon source, and to identify the strains through morphological and molecular biological identification. Gas chromatography-mass spectrometry (GC-MS) was used for qualitative analysis of the transformation products, and the product structure was determined by comparison with standards and spectral analysis. The results can provide new strain resources for the microbial transformation of D-limonene, and also provide a theoretical basis for the biosynthesis and metabolic engineering of terpenoids. In this invention, *Pseudomonas nitritireducens* GJ-01 was isolated from citrus peels and deposited with the China Center for Type Culture Collection.

[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 1-7 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] 1 1,5,8-p-Menthatriene 1,5,8-p-Mentholtriene 21195-59-5 0.34 2 1,3,8-p-Menthatriene 1,3,8-p-Mentholtriene 18368-95-1 0.2 3 Linalool Linalool 78-70-6 5.58 4 trans-p-Mentha-2,8-dien-l-ol trans-p-2,8-1-menthol 7212-40-0 26.89 5 (E)-limonene oxide (S)-(-)-Limonene Oxide 4959-35-7 0.46 6 Citral citral 5392-40-5 5.37 7 p-Mentha-1(7),8-dien-2-ol menthol-1(7), 8-dien-2-ol 35907-10-9 0.72 8 .alpha.-Terpineol α-Terpineol 98-55-5 5.38 9 (-)-trans-Isopiperitenol (-)-trans-isopiperol 74410-00-7 6.72 10 cis-Carveol (1R,5R)-rel-carvacrol 1197-06-4 8.66 11 cis-p-mentha-1(7),8-dien-2-ol cis-p-menth-1(7),8-dien-2-ol 22626-43-3 0.26 12 (-)-Carveol L-Carvault alcohol 99-48-9 2.24 13 (-)-Carvone L-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 bacteria are deposited at the China National Center for Culture Collection and are classified as follows: Pseudomonas nitritireducens GJ-01, with accession number CCTCC M 20251439, accession date 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 h.

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