Burkholderia and bacillus capable of efficiently converting monoterpenoids as well as screening method and application of burkholderia and bacillus

Abstract

The invention relates to burkholderia and bacillus capable of efficiently converting monoterpenoids as well as a screening method and application of the burkholderia and the bacillus, and belongs to the technical field of microorganisms and fermentation. According to the burkholderia and bacillus capable of efficiently converting the monoterpenoids, the burkholderia is preserved in Guangdong Microbial Culture Collection Center on August 7, 2025, and the preservation number is GDMCC 66819; the bacillus is preserved in Guangdong Microbial Culture Collection Center on August 7, 2025, and the preservation number is GDMCC 66818. The burkholderia CJ14 and the bacillus L-2 separated from the tobacco not only can effectively tolerate monoterpenoids such as alpha-pinene, beta-myrcene and limonene, but also can convert the monoterpenoids into high-added-value compounds such as rosin carvol, rosin carvone, alpha-terpilenol, dentinidia dentata dienol and dihydrocarvol; wide application prospects are shown in the fields of foods, daily chemicals, medicines, agriculture, biological control and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms and fermentation technology, and particularly relates to a Burkholderia and Bacillus capable of efficiently converting monoterpene compounds, and a screening method and application thereof. BACKGROUND

[0002] Monoterpenes are a class of terpenes condensed from two isoprene units, have strong volatility and rich aroma, and widely exist in various plant essential oils. Due to their rich pharmacology and biological activity, they have been widely used in food, medicine, cosmetics and agriculture and other fields. For example, linalool is often used as a food preservative to extend the shelf life of food due to its significant antibacterial and antioxidant activity. Carveol compounds (such as carveol, dihydrocarveol, dihydrocarveol) can play an insecticidal role by inhibiting GABA receptors, affecting acetylcholinesterase activity, etc. In addition, α-terpineol has analgesic, gastric mucosa protective, heart protective, nerve protective, antidiarrheal and skin permeation promoting effects, while eucalyptol shows good effect in the treatment of respiratory system diseases, which makes it have broad application prospects in the development of new drugs and the treatment of related diseases. A

[0003] At present, there are mainly three methods for synthesizing monoterpene compounds: one is plant extraction method, but it has problems such as low yield, high cost and complex extraction process, which is difficult to meet the needs of large-scale industrial production; the second is chemical synthesis method, although it can realize structure-controllable synthesis to a certain extent, but the operation process is complicated and the environment is not friendly, which limits its wide application; the third is microbial transformation method. Microbial transformation refers to the process of using microorganisms or enzymes contained therein as biological catalysts to transform cheap and abundant substrates into products with similar structures but higher value and different functional characteristics. It has the advantages of mild reaction conditions, high efficiency, environmental protection and strong stereoselectivity, and has become an important direction for the production of monoterpene compounds. Among them, limonene, α-pinene and β-myrcene compounds are ideal starting materials for microbial transformation. From the chemical structure, they are similar in structure to the aforementioned important monoterpene compounds, and are easy to be directionally transformed by microbial catalytic pathway to generate target products. From the economic point of view, these compounds are widely present in the by-products generated in the process of fruit and plant processing, and are widely sourced and low in cost. Taking the market price as an example, the reference price of limonene in 2015 was $34 per liter, while the market price of carveol, the transformation product, was as high as about $530 per liter, showing the significant potential of microbial transformation in improving the added value of raw materials.

[0004] ​In recent years, a variety of microorganisms have been reported to be able to generate functional compounds with high added value by biotransformation of monoterpene compounds such as limonene, alpha-pinene and beta-myrcene. However, due to the high volatility and low water solubility of monoterpene substrates, and the easy cytotoxicity to microorganisms during the transformation process, the transformation efficiency is low. SUMMARY

[0005] The first object of the present application is to provide a Burkholderia and Bacillus which can efficiently transform monoterpene compounds, which not only can tolerate alpha-pinene, beta-myrcene, limonene and other monoterpene compounds, but also shows good biotransformation ability to alpha-pinene, beta-myrcene and limonene.

[0006] The second object of the present application is to provide a screening method for Burkholderia and Bacillus which can efficiently transform monoterpene compounds.

[0007] The third object of the present application is to provide an application of Burkholderia and Bacillus which can efficiently transform monoterpene compounds.

[0008] In order to achieve the above objects, the technical scheme adopted by the present application is:

[0009] The Burkholderia and Bacillus which can efficiently transform monoterpene compounds, the Burkholderia was preserved in the Guangdong Microbial Culture Collection Center on August 7, 2025, and the preservation number is GDMCC 66819.

[0010] The Burkholderia and Bacillus which can efficiently transform monoterpene compounds, the Bacillus was preserved in the Guangdong Microbial Culture Collection Center on August 7, 2025, and the preservation number is GDMCC 66818.

[0011] Further, the 16S rDNA sequence of the Burkholderia is shown in SEQ ID NO. 1.

[0012] Further, the 16S rDNA sequence of the Bacillus is shown in SEQ ID NO. 2.

[0013] The application of the Burkholderia and Bacillus which can efficiently transform monoterpene compounds in transforming alpha-pinene, beta-myrcene and limonene.

[0014] Further, the step of the Burkholderia in transforming alpha-pinene, beta-myrcene and limonene is: inoculating the Burkholderia in the culture medium and culturing to OD 600 0.8-1.0, and then adding 0.1% of alpha-pinene, beta-myrcene and limonene respectively for fermentation for 24-72h.

[0015] Further, the step of transforming the alpha-pinene, beta-myrcene and limonene by the Bacillus is: inoculating the Burkholderia in the culture medium and culturing to OD 600 0.8-1.0, and then adding 0.1% of the alpha-pinene, beta-myrcene and limonene respectively and fermenting for 24-72h.

[0016] The screening method of the Burkholderia and the Bacillus capable of efficiently transforming monoterpenes, the screening method of the Burkholderia comprising the following steps: taking tobacco samples as the bacterial source, gradient culturing in liquid culture medium with mixed substrates from low concentration to high concentration, screening out the well-growing bacterial strains, and obtaining after screening with the mixed substrates as the sole carbon source; the mixed substrates are mixed substrates of alpha-pinene, beta-myrcene and limonene; the volume ratio of the alpha-pinene, beta-myrcene and limonene is 1:1:1.

[0017] The screening method of the Burkholderia and the Bacillus capable of efficiently transforming monoterpenes, the screening method of the Bacillus comprising the following steps: taking tobacco samples as the bacterial source, gradient culturing in liquid culture medium with mixed substrates from low concentration to high concentration, screening out the well-growing bacterial strains, and obtaining after screening with the mixed substrates as the sole carbon source; the mixed substrates are mixed substrates of alpha-pinene, beta-myrcene and limonene; the volume ratio of the alpha-pinene, beta-myrcene and limonene is 1:1:1.

[0018] The beneficial effects of the present application are:

[0019] The Burkholderia CJ14 and the Bacillus L-2 separated from tobacco in the present application not only can effectively resist alpha-pinene, beta-myrcene, limonene and other monoterpenes, but also show good biological transformation capacity to these compounds, and can transform them into high-value-added compounds such as pinocamphone, pinocarvone, alpha-terpineol, ipsdienol and dihydrocarveol, and show broad application prospects in the fields of food, daily chemicals, medicine, agriculture and biological control.

[0020] The existing ipsdienol is synthesized by chemical synthesis method, and has problems of high energy consumption and serious environmental pollution, the Bacillus L-2 in the present application can transform beta-myrcene into ipsdienol by biological transformation method, the reaction condition is mild, the environment is friendly, and the product has strong specificity, so that the Bacillus L-2 is a green synthesis path with application prospect. The Bacillus L-2 in the present application can also transform alpha-pinene into pinocamphone and pinocarvone by taking alpha-pinene as the substrate. The Burkholderia CJ14 and the Bacillus L-2 in the present application can directly transform limonene into dihydrocarveol by taking limonene as the substrate.

[0021] The Burkholderia CJ14 and Bacillus L-2 of the present application not only can tolerate monoterpene, but also can efficiently convert the monoterpene into high value-added compounds, which enriches the microbial resource library that can be used for bioconversion. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Growth curve of Burkholderia CJ14 and Bacillus L-2 of Example 1 and Example 2 in M9 medium;

[0023] Figure 2 Colony chart of Burkholderia CJ14 of Example 1, wherein A is a colony morphology chart, B is a scanning electron microscope chart, and C is a scanning electron microscope chart;

[0024] Figure 3 Colony chart of Bacillus L-2 of Example 2, wherein A is a colony morphology chart, B is a scanning electron microscope chart, and C is a scanning electron microscope chart;

[0025] Figure 4 PCR agarose gel electrophoresis chart of Burkholderia CJ14 and Bacillus L-2 of Example 1 and Example 2;

[0026] Figure 5 Phylogenetic tree chart of Burkholderia CJ14 of Example 1;

[0027] Figure 6 Phylogenetic tree chart of Bacillus L-2 of Example 2;

[0028] Figure 7 Growth curve of Burkholderia CJ14 and Bacillus L-2 of Example 1 and Example 2;

[0029] Figure 8 Chart of Burkholderia CJ14 and Bacillus L-2 of Example 1 and Example 2 under different environmental influences, wherein A is the influence of temperature on Burkholderia CJ14 and Bacillus L-2, B is the influence of rotation speed on Burkholderia CJ14 and Bacillus L-2, and C is the influence of pH on Burkholderia CJ14 and Bacillus L-2;

[0030] Figure 9 Hemolytic test chart of Burkholderia CJ14 and Bacillus L-2 of Example 1 and Example 2. DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with the embodiments of the present application and the drawings.

[0032] Tobacco material: tobacco samples Kunming CJ34 and Zimbabwe L10 / C, provided by China Tobacco Industry Co., Ltd.

[0033] YM liquid medium includes glucose at a final concentration of 10 g / L, peptone at a final concentration of 5 g / L, yeast extract powder at a final concentration of 3 g / L, and malt extract at a final concentration of 3 g / L. YM solid medium is YM liquid medium with agar at a final concentration of 20 g / L; M9 medium includes Na2HPO4 at a final concentration of 6 g / L, NaCl at a final concentration of 0.5 g / L, KH2PO4 at a final concentration of 3 g / L, NH4Cl at a final concentration of 1 g / L, and MgSO4 at a final concentration of 2 mmol / L after sterilization.

[0034] The mixed substrate is a mixture of a-pinene, β-myrcene, and limonene, and the volume ratio of a-pinene, β-myrcene, and limonene is 1:1:1. The mixed substrate is sterilized by filtering through a 0.22 μm filter membrane.

[0035] Example 1

[0036] The screening and identification of Burkholderia CJ14 in Example 1 include the following steps:

[0037] 1) 0.1% mixed substrate conversion screening: 1 g of Kunming CJ34 tobacco sample was taken in 9 mL of sterile physiological saline, and incubated at 30°C with shaking at 150 rpm for 30 min to obtain a bacterial suspension. 0.5 mL of the bacterial suspension was inoculated into 50 mL of YM liquid medium, and 0.1% (v / v) of the mixed substrate was added. After incubation at 30°C with shaking at 150 rpm for 48 h, 1 mL of the culture was taken and diluted to 10 -7 , and spread on YM solid plates. After incubation at 30°C until the colonies were fully grown, single colonies with different shapes, colors, sizes, and morphologies were selected for streak culture. All the isolated single colonies were picked from the plates with obvious and well-grown colonies, and a total of 50 strains were obtained.

[0038] 2) 2% mixed substrate conversion screening: the strains after streak culture were transferred to YM liquid medium for activation culture. 1 mL of the activated bacterial solution was inoculated into 100 mL of YM liquid medium, and 2% (v / v) of the mixed substrate was added. After incubation at 30°C with shaking at 150 rpm for 48 h, 1 mL of the culture was taken and diluted to 10 -7 , and spread on YM solid plates. After incubation at 30°C until the colonies were fully grown, 6 strains were able to grow normally and well in the medium containing 2% mixed substrate.

[0039] 3) Conversion screening with a single carbon source: the strains after streak culture were again transferred to YM liquid medium, and incubated at 30°C with shaking at 150 rpm for 12 h. The bacterial cells were collected by centrifugation (5500 x g, 15 min) and washed with 0.9% physiological saline for three times to remove the culture medium. The bacterial cells were resuspended in M9 medium, and the initial OD 600The concentration was 0.2, and 1% (v / v) of the mixed substrate was added. The mixture was incubated with shaking at 30°C and 150 rpm. OD was measured at regular intervals. 600 To determine whether the microorganism is growing, and based on the measurement results, a growth curve of the strain is plotted to preliminarily screen strains capable of transforming α-pinene, β-myrcene, or limonene. The growth curves of the strains in M9 medium are shown below. Figure 1 As shown, OD 600 The increase in the value indicates that the strain can use the mixed substrate as the sole carbon source for growth, suggesting that the strain can not only effectively tolerate these monoterpenoids, but may also have α-pinene, β-myrcene or limonene degradation pathways.

[0040] 4) Secondary screening of strains: The strains obtained in step 3) were inoculated into YM liquid medium and cultured at 30°C and 150 rpm for OD250. 600 Fermentation was carried out by adding 0.1% (v / v) of α-pinene, β-myrcene, and limonene to a temperature of 0.8-1.0. The aroma components of the products from different fermentation times (24h, 48h, 72h) were analyzed using SPME-GC-MS to screen for strains capable of transforming monoterpenoids. The results are shown in Table 1. This strain exhibits a certain transformation ability for all three substrates mentioned above, converting them into other terpenoid aroma components.

[0041] 5) Analysis of aroma components in fermentation products: The aroma components were searched and analyzed using computer spectral libraries (NIST05 / WILEY7.0), and the retention index (RI) of the compounds was calculated and verified with the RI values ​​in the literature to identify the types of aroma substances. The internal standard method was used for quantitative analysis.

[0042] 6) Morphological observation: After streaking the activated strain on YM solid medium plates at 30℃ for 48 hours, the colony morphology characteristics were observed. Figure 2 As can be seen from A, the strain is round, milky yellow, opaque, with regular edges, a raised center, and a smooth, moist surface. Its cell structure was observed using a scanning electron microscope. Figure 2 B and Figure 2 As can be seen from C, the bacterial cells of this strain are rod-shaped and oval.

[0043] 7) Molecular biology identification: Genomic DNA of the sample was extracted using Dzup Genomic DNA Quick Extraction Kit, PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), the amplification product was sent to Shengqun Bioengineering (Shanghai) Co., Ltd. for sequencing, the sequencing results were subjected to BLAST comparison on NCBI, and a phylogenetic tree was constructed using MEGA 11 software to identify the strain. The results showed that after 16S rDNA primer amplification, a PCR fragment of about 1439 bp was obtained. The sequencing results were subjected to comparison analysis and phylogenetic tree construction on the NCBI website, and the results showed that the strain clustered with Burkholderia sp. TNe-862 in the same branch. Figure 5 Therefore, the strain was identified as Burkholderia sp. by morphological and molecular biology methods, and was named Burkholderia sp. CJ14.

[0044] The 16S rDNA sequence of Burkholderia sp. CJ14 is shown as SEQ ID NO. 1:

[0045]

[0046] Example 2

[0047] Screening and identification of Bacillus sp. L-2 in Example 1 includes the following steps:

[0048] 1) 0.1% mixed substrate conversion screening: 1 g of Zimbabwe L10 / C tobacco sample was taken in 9 mL of sterile physiological saline, and the bacterial suspension was obtained by incubation at 30°C with 150 rpm shaking for 30 min. 0.5 mL of the bacterial suspension was inoculated into 50 mL of YM liquid medium, and 0.1% (v / v) of mixed substrate was added. After incubation at 30°C with 150 rpm shaking for 48 h, it was taken out for concentration gradient dilution to 10 -7 , and spread on YM solid plates for incubation at 30°C until the colonies were fully grown. Single colonies with different shapes, colors, sizes and morphologies were selected for streak culture, and all isolated single colonies were picked from the plates with obvious and well-grown colonies.

[0049] 2) 2% mixed substrate conversion screening: the strains after streak culture were transferred to YM liquid medium for activation culture. 1 mL of the activated bacterial liquid was inoculated into 100 mL of YM liquid medium, and 2% (v / v) of mixed substrate was added. After incubation at 30°C with 150 rpm shaking for 48 h, it was taken out for concentration gradient dilution to 10 -7 , and spread on YM solid plates for incubation at 30°C until the colonies were fully grown.

[0050] 3) Conversion screening with unique carbon source: the strains after streak culture were again transferred to YM liquid medium, and incubated at 30°C with 150 rpm shaking for 12 h. The bacterial cells were collected by centrifugation (5500 x g, 15 min) and washed with 0.9% physiological saline for three times to remove the culture medium. The bacterial cells were resuspended in M9 medium, and the initial OD 600 was controlled at 0.2. 1% (v / v) of mixed substrate was added, and the culture was incubated at 30°C with 150 rpm shaking. The OD 600 was measured at certain time intervals to determine whether the microorganism grew, and the growth curve of the strain was plotted according to the measurement results. The strains capable of converting α-pinene, β-myrcene or limonene were preliminarily screened. The growth curve of the strain in M9 medium is shown in Figure 1 , and the increase of OD 600 value indicates that the strain can grow with mixed substrate as the only carbon source, which shows that the strain not only can effectively tolerate these monoterpene compounds, but also may have α-pinene, β-myrcene or limonene degradation pathway.

[0051] 4) Strain re-screening: the strains preliminarily screened in step 3) were inoculated into YM liquid medium, and incubated at 30°C with 150 rpm shaking until the OD 600To 0.8-1.0, respectively, 0.1% (v / v) of a-pinene, β-myrcene and limonene were added for fermentation. The SPME-GC-MS technique was used to analyze the aroma components of the products at different fermentation times (24h, 48h, 72h), and the strain capable of converting monoterpene compounds was screened. The results are shown in Table 1. The strain has certain conversion capacity for the above three substrates, and can convert them into other terpene flavor components.

[0052] 5) Analysis of the aroma components of the fermentation products: The computer library (NIST05 / WILEY7.0) was used for retrieval and analysis, and the retention index (Retention Index, RI) and literature RI value were used for verification to confirm the type of aroma substances. The internal standard method was used for quantitative analysis.

[0053] 6) Morphological observation of Bacillus L-2: After activation, the strain was streaked on YM solid medium plate and cultured at 30°C for 48h, and the colony morphological characteristics were observed. From Figure 3 A, it can be seen that the colony of the strain is irregular round, the edge is leaf-shaped diffusion, and the surface is wrinkled. The bacterial structure was observed by scanning electron microscope, from Figure 3 B and Figure 3 C, it can be seen that the bacterial body of the strain is rod-shaped.

[0054] 7) Molecular biology identification: The Dzup genomic DNA rapid extraction kit was used to extract the sample genomic DNA, and the universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used for PCR amplification. The amplification product was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing, and the sequencing results were subjected to BLAST comparison on NCBI, and the MEGA 11 software was used to construct a phylogenetic tree to identify the strain. The strain obtained a PCR fragment of about 1490bp after 16S rDNA primer amplification. Figure 4 The results showed that the strain was clustered in the same branch with Bacillus siamensis KCTC13613, Bacillus subtilis IAM 12118 and Bacillus cabrialesii TE3. Figure 6 Therefore, the strain is Bacillus sp., named Bacillus L-2.

[0055] The 16S rDNA sequence of Bacillus L-2 is shown as SEQ ID NO. 2:

[0056]

[0057] Table 1. Transformation products of different substrates by strains of Example 1 and Example 2 at different transformation times

[0058]

[0059]

[0060] Note: "-" indicates that there is no RI value of the compound in the database or the compound is not detected. The RI value in the literature is derived from the database of NIST Chemistry WebBook website (https: / / webbook.nist.gov).

[0061] As can be seen from Table 1, the Burkholderia CJ14 shows certain biological transformation ability on monoterpene compounds such as α-pinene, β-myrcene and limonene. Among them, the main products of CJ14 biological transformation of α-pinene are (-)-trans-sabinyl alcohol, sabinaketone and α-terpineol. CJ14 can directly transform limonene to dihydrocarveol. The Bacillus L-2 screened from tobacco samples not only has high biological safety, but also shows good transformation ability on α-pinene, β-myrcene and limonene, and has potential industrial application prospect. The products of L-2 biological transformation of α-pinene include limonene, β-myrcene, (-)-trans-sabinyl alcohol and sabinaketone. The biological transformation product of L-2 on β-myrcene is ipsdienol.

[0062] Growth characteristics and safety of Burkholderia CJ14 and Bacillus L-2

[0063] 1. Growth curve: Burkholderia CJ14 and Bacillus L-2 were inoculated into 100 mL of YM liquid medium at 1% (v / v), and cultured at 30°C and 150 rpm. Samples were taken every 2h, and the blank control was YM liquid medium. The absorbance value of the bacterial solution at 600 nm was measured, and the time was taken as the abscissa and the OD 600 value as the ordinate to draw the growth curve. As shown in Figure 7 Burkholderia CJ-14 was in the lag phase at 0-4h, and rapidly grew at 6-48h, which was in the logarithmic growth phase. Bacillus L-2 was in the lag phase at 0-6h, rapidly grew at 6-16h, which was in the logarithmic growth phase, and entered the stationary phase after 16h.

[0064] 2. Effects of different environmental factors on bacterial growth: Burkholderia CJ14 and Bacillus L-2 were inoculated into 100 mL of YM liquid medium at a 1% (v / v) inoculum and cultured with shaking for 24 h. The effects of different temperatures (20, 25, 30, 35, 40℃), rotation speeds (100, 150, 200, 250, 300 rpm), and pH (3, 5, 7, 9, 11) on bacterial growth were investigated. Figure 8 As shown, strains CJ14 and L-2 can grow in the temperature range of 20–40℃, with an optimal growth temperature of 30℃ for both. The growth of the strains decreased when the temperature was higher or lower than 30℃. Particularly for Bacillus L-2, after culturing at 20℃ for 24 hours, the OD of the fermentation broth... 600 The value was only 0.369, significantly lower than its growth at other temperatures (P<0.05). Figure 8 A). Low temperatures can reduce cell membrane fluidity, decrease the activity of ATP synthase, and reduce proton-driven force, leading to a decrease in the rate of nutrient uptake (such as glucose), thereby inhibiting the overall metabolism and growth of the strain.

[0065] The effect of rotation speed on the growth of strains CJ14 and L-2 is as follows: Figure 8 As shown in Figure B, both strains CJ14 and L-2 can grow within a rotation speed range of 100-300 rpm. The growth rate of Burkholderia CJ-14 increases with increasing rotation speed; when the rotation speed reaches 300 rpm, the OD of its fermentation broth... 600 The value reached 7.992. Increased rotation speed helps enhance oxygen transfer rate and nutrient diffusion, thus favoring the growth of aerobic Burkholderia. For Bacillus L-2, growth was low below 150 rpm; maximum growth was achieved at 150 rpm, with an OD value of 7.992. 600 The value is 3.744; when the speed is between 200-300 rpm, the OD... 600 The values ​​tended to stabilize, and the differences were not significant (P>0.05).

[0066] The growth of strains CJ14 and L-2 at different pH values ​​are as follows: Figure 8 As shown in Figure C, strains CJ14 and L-2 could not grow at pH 3; however, they could grow normally within a pH range of 5–11. Low pH environments lead to protonation of the polar heads of membrane lipids, a sharp decrease in membrane fluidity, the formation of leakage channels, resulting in intracellular ion leakage and damage to cell structural integrity. The optimal growth pH for Burkholderia CJ-14 is 9, and for Bacillus L-2 it is 5. Growth of these strains is inhibited when the pH is higher or lower than their optimal values.

[0067] 3. Hemolysis experiment: After Burkholderia CJ14 and Bacillus L-2 were activated in YM liquid medium, 1 μL of bacterial solution was taken and streaked on 5% sheep blood agar medium, and cultured at 30°C for 24 h. The results showed that Burkholderia CJ14 and Bacillus L-2 did not show hemolysis on 5% sheep blood agar medium, as shown in Figure 2. It was indicated that Burkholderia CJ14 and Bacillus L-2 were safe strains. Figure 9

[0068] 4. Antibiotic sensitivity experiment: After Burkholderia CJ14 and Bacillus L-2 were activated in YM liquid medium, 100 μL of bacterial solution was uniformly coated on the surface of YM solid medium, and antibiotic-containing paper discs were placed on the medium with sterile forceps, and cultured at 30°C for 24 h. The diameter of the inhibition zone was measured. The results are shown in Table 2. It was indicated that strain L-2 was sensitive to penicillin, amoxicillin, cefotaxime, streptomycin, kanamycin, norfloxacin, ciprofloxacin, vancomycin, erythromycin, and rifampicin; moderately sensitive to gentamicin; and resistant to lincomycin. Strain CJ-14 showed resistance to all the above antibiotics.

[0069] Table 2 Antibiotic sensitivity experiment

[0070]

[0071] Note: S, sensitive; I, moderately sensitive; R, resistant.

[0072] Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the present disclosure. The present disclosure is intended to cover any variations, uses, or adaptations of the present disclosure following the general principles thereof and including modifications and equivalents of the present disclosure. The specification and examples are to be regarded as illustrative only, and the true scope and spirit of the present disclosure are indicated by the claims.

[0073] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. The patent protection scope of the present application is subject to the claims, and any equivalent structural changes made according to the content of the specification of the present application shall be included in the protection scope of the present application.​

Claims

1. Burkholderia and Bacillus which can efficiently convert monoterpenoids, characterized in that, The Burkholderia is preserved in Guangdong Microbial Culture Collection Center on August 7, 2025, and the preservation number is GDMCC 66819.

2. Burkholderia and Bacillus strains that can efficiently convert monoterpenoids, characterized in that, The Bacillus is preserved in Guangdong Microbial Culture Collection Center on August 7, 2025, and the preservation number is GDMCC 66818.

3. The Burkholderia and Bacillus strains capable of efficiently converting monoterpenes according to claim 1, characterized in that, The 16S rDNA sequence of the Burkholderia is shown as SEQ ID NO.

1.

4. The Burkholderia and Bacillus strains capable of efficiently converting monoterpenes according to claim 2, characterized in that, The 16S rDNA sequence of the Bacillus is shown as SEQ ID NO.

2.

5. Application of the Burkholderia and Bacillus capable of efficiently converting monoterpene compounds in converting α-pinene, β-myrcene and limonene.

6. The use of the Burkholderia and Bacillus which can efficiently convert monoterpenes according to claim 5, characterized in that, The step of converting alpha-pinene, beta-myrcene and limonene by the Burkholderia bacteria is: inoculating the Burkholderia bacteria in a culture medium to OD 600 0.8-1.0, followed by adding 0.1% of alpha-pinene, beta-myrcene and limonene respectively and fermenting for 24-72h.

7. The use of the Burkholderia and Bacillus which can efficiently convert monoterpenes according to claim 5, characterized in that, The steps for the conversion of α-pinene, β-myrcene and limonene by the Bacillus sp. are: inoculation of the Bacillus sp. in a culture medium to an OD 600 of 0.8-1.0, followed by the addition of 0.1% of α-pinene, β-myrcene and limonene, respectively, and fermentation for 24-72 h.

8. The screening method for Burkholderia and Bacillus that can efficiently transform monoterpenoid compounds as described in claim 1, characterized in that, The screening method of the Burkholderia comprises the following steps: taking tobacco samples as the source of bacteria, gradient culturing in liquid culture medium with mixed substrates from low concentration to high concentration, screening out well-growing strains, and obtaining after screening with the mixed substrates as the only carbon source; the mixed substrates are mixed substrates of α-pinene, β-myrcene and limonene; the volume ratio of α-pinene, β-myrcene and limonene is 1:1:

1.

9. The screening method for Burkholderia and Bacillus that can efficiently transform monoterpenoid compounds as described in claim 2, characterized in that, The screening method of the Bacillus comprises the following steps: taking tobacco samples as the source of bacteria, gradient culturing in liquid culture medium with mixed substrates from low concentration to high concentration, screening out well-growing strains, and obtaining after screening with the mixed substrates as the only carbon source; the mixed substrates are mixed substrates of α-pinene, β-myrcene and limonene; the volume ratio of α-pinene, β-myrcene and limonene is 1:1:1.

Images