Application of klebsiella pneumoniae in synthesis of genistein

Abstract

The application belongs to the technical field of microorganisms, and discloses application of Klebsiella pneumoniae in synthesis of genistein. Klebsiella pneumoniae It is disclosed and confirmed that Klebsiella pneumoniae (ATCC 43816) can stably endogenously synthesize genistein. The finding breaks the traditional cognition that the synthesis pathway of genistein is specific to plants, and for the first time, reveals that Klebsiella pneumoniae has this atypical secondary metabolic function, provides indispensable core strain resources and empirical basis for opening up a new microbial fermentation method for producing genistein which does not depend on plant extraction, and also provides a new research perspective for understanding the metabolic potential of bacteria, expands the scientific cognition of the endogenous synthesis ability of microorganisms, and brings new strategies and application prospects for biological manufacturing of genistein.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Klebsiella pneumoniae in the synthesis of genistein. Background Technology

[0002] Genistein, chemically named 4',5,7-trihydroxyisoflavone, has the molecular formula C2. 15 H 10 O5, CAS number 446-72-0, is an important natural isoflavone compound found in legumes. Its structure is similar to estradiol, exhibiting estrogen-like activity, and it has been proven to possess broad biological activities and significant application value. In the pharmaceutical field, genistein flavonoids have shown significant anti-diabetic, anti-inflammatory, antioxidant, and anti-obesity effects. Among these, its anti-tumor activity has been the most extensively studied, inhibiting various tumor cells, including breast cancer, cervical cancer, and prostate cancer. Its mechanisms involve regulating the cell cycle, inducing apoptosis, and inhibiting tumor angiogenesis. In the antibacterial field, genistein flavonoids have also shown inhibitory activity against various pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, in agriculture and animal husbandry, it shows potential as a phytoestrogen and natural additive. Therefore, genistein flavonoids demonstrate broad application prospects in medicine, nutrition and health care, and biomaterials.

[0003] Currently, the commercial production of genistein mainly relies on extraction from plant materials such as soybeans. This approach is limited by factors such as the planting cycle of raw materials, geographical and climatic conditions, and resource supply. Furthermore, traditional extraction processes suffer from problems such as complex procedures, low yields, high costs, and potential organic solvent residues. To overcome these bottlenecks, microbial fermentation, as a green, controllable, and easily scalable biomanufacturing technology, has attracted attention. Some studies have attempted to produce genistein through fermentation using heterologous expression of key plant-derived synthetic enzymes by microorganisms. However, technical bottlenecks remain, including mismatch between the exogenous pathway and the host metabolic network, difficulties in coordinating multi-gene co-expression, and low yields of the final product due to insufficient precursor supply, making it difficult to meet the needs of actual industrialization.

[0004] It is worth emphasizing that, in existing publicly available literature, databases, and microbial resource banks, no bacteria have been experimentally proven to endogenously biosynthesize genistein. Whether in plant metabolism research or microbial synthetic biology, genistein is considered a typical plant-specific product, and its biosynthetic pathway from natural bacteria remains completely unexplored. Therefore, obtaining and identifying a natural microbial strain that can stably produce genistein without exogenous enzyme genes will directly break through the boundaries of current technology, providing a completely new technical route for the microbial production of genistein. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art, and for the first time provides a Klebsiella pneumoniae strain capable of endogenously synthesizing genistein (Klebsiella pneumoniae). Klebsiella pneumoniae This study describes a bacterial strain and provides a production method and compound identification method for producing genistein using this strain, thereby filling the technological gap in the existing technology where bacteria cannot naturally synthesize this compound and providing a completely new production route.

[0006] The first aspect of the present invention aims to provide the use of Klebsiella pneumoniae or an agent containing Klebsiella pneumoniae in the production of genistein or in the preparation of products for the production of genistein.

[0007] A second aspect of the present invention is to provide a method for producing genistein.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the invention provides the use of Klebsiella pneumoniae ATCC 43816 or an agent containing Klebsiella pneumoniae ATCC 43816 in the production of genistein or in the preparation of products for the production of genistein.

[0009] In some embodiments of the present invention, the bacterial agent containing Klebsiella pneumoniae uses Klebsiella pneumoniae ATCC 43816 as the main microorganism.

[0010] In some embodiments of the present invention, the microbial agent is a liquid microbial agent or a solid microbial agent.

[0011] In some embodiments of the present invention, the bacterial agent contains live cells of Klebsiella pneumoniae ATCC 43816, freeze-dried bacterial cells, and immobilized cells.

[0012] This invention marks the first identification of a Klebsiella pneumoniae strain ATCC43816 capable of endogenously synthesizing genistein, filling a gap in the natural production of genistein by microorganisms. Compared to production methods relying on plant extraction or heterologous pathways, the Klebsiella pneumoniae ATCC 43816 provided by this invention can directly generate the target product through its own metabolism without the need for exogenous genes, simplifying the production process and making the system more stable.

[0013] A second aspect of the present invention provides a method for producing genistein, comprising the following steps: fermentation using Klebsiella pneumoniae ATCC 43816 as the generating strain in the first aspect of the present invention.

[0014] In some embodiments of the present invention, the fermentation includes the following steps: activating Klebsiella pneumoniae ATCC43816, inoculating it into a fermentation medium, performing multi-stage seed culture, and then inoculating it into a fermentation medium for fermentation culture.

[0015] In some embodiments of the present invention, the fermentation medium includes at least one of a carbon source, a nitrogen source, and an inorganic salt.

[0016] In some embodiments of the present invention, the carbon source includes at least one selected from glucose, sucrose, maltose, molasses, fructose, rhamnose, arabinose, and sorbitol.

[0017] In some embodiments of the present invention, the nitrogen source includes at least one selected from yeast extract, peptone, yeast extract, glutamic acid, ammonium chloride, ammonia, ammonium sulfate, potassium nitrate, and ammonium nitrate.

[0018] In some embodiments of the present invention, the fermentation medium is selected from tryptone soybean broth (TSB), 7H9 medium, LB medium, and M9 medium.

[0019] In some embodiments of the present invention, an appropriate amount of glucose, such as 0.1%-0.5% glucose, may be added to the 7H9 culture medium as needed.

[0020] In some embodiments of the present invention, the activation includes streaking Klebsiella pneumoniae ATCC 43816 onto LB solid medium plates and culturing at 35-40°C for 8-15 hours; followed by further culturing at 36-38°C for 8-12 hours.

[0021] In some embodiments of the present invention, the multi-stage seed culture includes at least one stage of seed culture. A suitable multi-stage seed culture can be selected based on actual conditions (such as fermentation volume).

[0022] In some embodiments of the present invention, the seed culture conditions are 35-40°C and 100-300 rpm for 6-15 hours; further, 35-38°C and 150-250 rpm for 8-13 hours; and even further, 36-38°C and 190-210 rpm for 8-12 hours.

[0023] In some embodiments of the present invention, the fermentation culture conditions are 35-40°C, 100-300 rpm for 3-25 hours; further, 35-38°C, 150-250 rpm for 3-20 hours; and even further, 35-40°C, 190-210 rpm for 3-8 hours.

[0024] In some embodiments of the present invention, the method further includes enrichment and purification steps.

[0025] In some embodiments of the present invention, the enrichment includes the following steps: collecting the fermentation supernatant after fermentation, extracting it using methyl ethyl ketone as the extraction solvent, collecting the upper organic phase, concentrating it, and obtaining crude extract of genistein.

[0026] In some embodiments of the present invention, the fermentation broth after fermentation is centrifuged to obtain the fermentation supernatant. The centrifugation conditions are 3-5°C and 9000-15000 rpm for 5-15 minutes.

[0027] In some embodiments of the present invention, the volume ratio of the fermentation supernatant to methyl ethyl ketone is 1:(1-5); further, it is 1:(2-4); and even further, it is 1:(2-3).

[0028] In some embodiments of the present invention, the number of extractions may be adjusted according to actual conditions, such as 2-5 extractions.

[0029] In some embodiments of the present invention, the concentration includes gentle concentration using a rotary evaporator under a water bath at 35-45°C until the volatile extraction solvent is completely removed, and excess water is removed by a cryogenic pump.

[0030] In some embodiments of the present invention, the purification includes the following steps: treating the crude extract of genistein using semi-preparative liquid chromatography to obtain purified genistein.

[0031] The beneficial effects of this invention are: This invention discloses and confirms that *Klebsiella pneumoniae* ATCC 43816 can stably and endogenously synthesize genistein. This discovery breaks through the traditional understanding that "genistein synthesis is unique to plants," revealing for the first time this atypical secondary metabolic function possessed by *Klebsiella pneumoniae*. It provides an indispensable core strain resource and empirical basis for developing a novel microbial fermentation method for producing genistein that does not rely on plant extraction. Simultaneously, it offers a new research perspective on the metabolic potential of bacteria, expands the scientific understanding of the endogenous synthetic capabilities of microorganisms, and brings new strategies and application prospects for the biomanufacturing of genistein.

[0032] This invention provides a method for producing genistein, which can achieve effective accumulation of genistein under conventional conditions. This method is characterized by convenient operation, short cycle, and good scalability, providing a new technical route for large-scale production. Attached Figure Description

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1The process flow diagram of the method for producing and identifying genistein from Klebsiella pneumoniae (ATCC 43816) provided by the present invention.

[0034] Figure 2 This is a photograph of the extract (paste) rich in genistein obtained by fermentation and separation of Klebsiella pneumoniae according to the present invention.

[0035] Figure 3 Comparative high-performance liquid chromatography (HPLC) chromatograms of the extract (sample solution) rich in genistein obtained from the production of Klebsiella pneumoniae of the present invention and the genistein standard (control) (B).

[0036] Figure 4 For the present invention to Figure 3 Mass spectrometry analysis of the chromatographic peak (sample solution) of suspected genistein.

[0037] Figure 5 For the present invention to Figure 3 Hydrogen-1 nuclear magnetic resonance spectrum analysis of the chromatographic peak (sample solution) of suspected genistein.

[0038] Figure 6 For the present invention to Figure 3 Carbon-13 nuclear magnetic resonance spectrum analysis of the chromatographic peak (sample solution) of suspected genistein. Detailed Implementation

[0039] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0040] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0041] TSB medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd., catalog number 024048; Middlebrook 7H9 broth medium (7H9 medium) was purchased from BD Biosciences Inc., catalog number 271310; M9 medium was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number PM0570-5L; yeast extract and tryptone were purchased from ThermoFisher Scientific, catalog numbers LP0021 and LP0042B respectively; sodium chloride was purchased from Genview, catalog number 7647-14-5.

[0042] This invention systematically confirms the genistein synthesis capacity of the strain by constructing a complete "fermentation-separation-identification" system. First, the strain is fermented in a standardized culture system to accumulate the target metabolite. Then, the target component is efficiently separated from the complex fermentation system using solvent extraction and other methods. Finally, analytical techniques such as chromatography-mass spectrometry are used to accurately compare the obtained component with standards, thereby confirming the genistein at the chemical structure level.

[0043] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0044] Example 1: Fermentation Production and Separation of Dysplasia Flavonoids This embodiment provides a method for producing genistein based on the fermentation of Klebsiella pneumoniae (ATCC strain 43816). Figure 1 ), as detailed below: (1) Preparation of seed liquid Klebsiella pneumoniae (ATCC 43816 strain) was streaked onto LB agar plates and incubated overnight at 37°C. Subsequently, a single, well-formed colony was picked and inoculated into a test tube containing 1 mL of liquid culture medium. The tube was then incubated overnight at 37°C and 200 rpm with shaking to allow for regeneration. This was used as the seed culture.

[0045] (2) Fermentation culture At an inoculum ratio of 1:1000 (volume ratio), transfer the seed culture to a 1-liter Erlenmeyer flask containing 100 mL of fresh liquid culture medium. Continue fermentation under the same conditions with shaking until the logarithmic phase (5 hours) or plateau phase (16 hours). After fermentation, centrifuge the fermentation broth at 4°C and 10,000 rpm for 10 minutes to completely separate the cells, and collect the clear supernatant.

[0046] The liquid culture media mentioned in steps (2) and (3) are tryptone soybean broth (TSB), 7H9 medium (containing 0% or 0.2% glucose by volume), LB medium, and M9 medium.

[0047] (3) Preparation of fermented extract (crude extract of genistein) Add 2 volumes of methyl ethyl ketone (butanone) as the extraction solvent to the supernatant obtained in step (2). Shake thoroughly in a separatory funnel, let stand to allow separation, collect the upper organic phase, and discard the lower aqueous phase. Repeat this extraction process three times to ensure that the target product is fully extracted. Combine all organic phases and gently concentrate them using a rotary evaporator at 40°C until the volatile extraction solvent is completely removed. After repeated extraction and rotary evaporation 2-3 times, use a 4 mL sample bottle to hold the concentrated extract. After removing excess water from the extract using a cryogenic pump, a brownish-yellow, viscous crude extract (extract) with a characteristic odor is finally obtained (its physical form is as shown in the image). Figure 2 (As shown). This extract is the preliminary extraction product (crude extract of genistein). After weighing and recording, it was stored at -80℃ for subsequent analysis.

[0048] Among various fermentation media, TSB as the liquid medium yielded the highest extract yield, at 17 mg / 100 mL. The extract yields from the other liquid media are shown in Table 1. When further separation was performed using either the logarithmic or plateau phase fermentation broth, the extract yield from the logarithmic phase fermentation broth was superior to that from the plateau phase fermentation broth.

[0049] Table 1. Yields of extracts obtained from various fermentation media

[0050] Further chromatographic and mass spectrometric analyses were performed on the extract obtained from the TSB fermentation to confirm whether the component in the extract was genistein, as detailed below: First, the above extract was dissolved in an appropriate amount of chromatographically pure methanol and filtered through a microporous membrane with a pore size of 0.22 micrometers to prepare a test solution. Simultaneously, genistein standards were accurately weighed and prepared into a standard solution of known concentration using the same amount of methanol as a control. Analysis was performed using an Agilent 1260 high-performance liquid chromatography system. The chromatographic column used was a Phenomenex Prodigy ODS (2) column (4.6 mm × 150 mm, 5 μm). Gradient elution was performed at a column temperature of 30℃ using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase, with a flow rate set at 1.0 mL per minute. The test solution and standard solution were detected separately at a wavelength of 260 nm.

[0051] High performance liquid chromatography analysis results are as follows Figure 3 As shown, in the chromatogram of the test solution, a significant chromatographic peak appears at a retention time of approximately 15 minutes. Figure 3 (A), the retention time of this peak is related to that of the genistein standard solution ( Figure 3The main chromatographic peaks (retention time 15 minutes) of the extract (B) were completely identical, providing preliminary and strong evidence that the extract contains genistein.

[0052] Secondly, the components corresponding to the aforementioned chromatographic peaks were subjected to semi-preparative separation and structural identification. Semi-preparative high-performance liquid chromatography (SHPLC) was performed using a Hitachi Primaide system (equipped with a 1110 isocratic pump and a 1430 DAD detector). A C18 semi-preparative column (10 mm × 250 mm, 5 μm) was used, and the fractions corresponding to the target peaks were collected and concentrated under reduced pressure to obtain purified samples. Further analysis of the purified samples was performed by high-resolution electrospray ionization mass spectrometry (HRESIMS), and the data were obtained on a Bruker MaXis 4G UHR59 TOFMS mass spectrometer.

[0053] Mass spectrometry results are attached. Figure 4 As shown, the quasi-molecular ion peak [MH] of this component - mass-to-charge ratio ( m / z The value was 269.04550, compared to genistein (C). 15 H 10 The theoretical molecular weight of O5 is precisely matched. The characteristic fragment ions produced by its secondary mass spectrometry are highly consistent with the fragmentation mode of the standard under the same conditions.

[0054] Finally, the purified sample was subjected to one-dimensional nuclear magnetic resonance hydrogen spectrum analysis. 1 H NMR spectra were acquired on a Bruker Ascend 700 MHz NMR spectrometer, with chemical shifts (δ) using tetramethylsilane (TMS) as an internal standard.

[0055] The spectrum shows that the characteristic signals of this component at multiple locations are completely consistent with the 1H NMR data of genistein reported in the literature and measured by the standard. Figure 5 Simultaneously, the acquired one-dimensional carbon nuclear magnetic resonance spectrum (NMR spectrum) 13 C NMR clearly showed the signals of all carbon atoms in the sample, and its chemical shift values ​​were completely consistent with the theoretical and standard spectral data of genistein. Figure 6 This provides confirmation at the carbon skeleton level.

[0056] The consistent retention times in high-performance liquid chromatography, the precise match of molecular weight in high-resolution mass spectrometry, and the comprehensive matching of proton and carbon NMR signals constitute a complete and rigorous chain of evidence, conclusively proving that the extract prepared above contains genistein, thus confirming that Klebsiella pneumoniae ATCC 43816 can synthesize genistein.

[0057] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Klebsiella pneumoniae ( Klebsiella pneumoniae The use of ATCC 43816 or an agent containing Klebsiella pneumoniae ATCC43816 in the production of genistein or in the preparation of products for the production of genistein.

2. The application according to claim 1, characterized in that, The bacterial agent can be a liquid bacterial agent or a solid bacterial agent.

3. A method for producing genistein, comprising the following steps: Fermentation was carried out using the Klebsiella pneumoniae ATCC 43816 strain as described in claim 1 or 2.

4. The method according to claim 3, characterized in that, The fermentation includes the following steps: activating Klebsiella pneumoniae ATCC 43816, inoculating it into a fermentation medium, performing multi-stage seed culture, and then transferring it to the fermentation medium for fermentation culture.

5. The method according to claim 4, characterized in that, The fermentation medium includes at least one of a carbon source, a nitrogen source, and an inorganic salt; Preferably, the carbon source includes at least one selected from glucose, sucrose, maltose, molasses, fructose, rhamnose, arabinose, and sorbitol; Preferably, the nitrogen source includes at least one selected from yeast extract, peptone, yeast extract, glutamic acid, ammonium chloride, ammonia, ammonium sulfate, potassium nitrate, and ammonium nitrate. Preferably, the inorganic salt includes at least one selected from ammonium citrate, sodium citrate, ferrous sulfate, manganese sulfate, magnesium sulfate, zinc sulfate, copper sulfate, calcium sulfate, calcium carbonate, potassium dihydrogen phosphate, disodium hydrogen phosphate, calcium chloride, and sodium chloride.

6. The method according to claim 4, characterized in that, The seed culture conditions are 35-40℃ and 100-300rpm for 6-15 hours.

7. The method according to claim 4, characterized in that, The fermentation conditions are 35-40℃ and 100-300 rpm for 3-25 hours.

8. The method according to any one of claims 3-7, characterized in that, The method also includes enrichment and purification steps.

9. The method according to claim 8, characterized in that, The enrichment includes the following steps: collecting the fermentation supernatant after fermentation, extracting it using methyl ethyl ketone as the extraction solvent, collecting the upper organic phase, concentrating it, and obtaining crude extract of genistein.

10. The method according to claim 9, characterized in that, The purification process includes the following steps: treating the crude extract of genistein using semi-preparative liquid chromatography to obtain purified genistein.

Images