Klebsiella pneumoniae and construction method and application thereof

Abstract

The present application belongs to the field of microbial technology, and discloses a Klebsiella pneumoniae, a construction method and application thereof. The present application realizes directional iterative optimization of strain tolerance, substrate utilization ability and product synthesis ability by gradient adaptive evolution of wild-type Klebsiella pneumoniae ATCC 25955 in high-concentration distiller's grains enzymatic hydrolysate, and finally obtains an evolved strain Klebsiella pneumoniae (Kp-1) which can stably grow in a high-concentration distiller's grains enzymatic hydrolysate environment. Klebsiella pneumoniae The strain can normally grow and metabolize in a fermentation system with a distiller's grains enzymatic hydrolysate concentration as high as 60 g / L, and is applied to a 1,3-propanediol production process with high-concentration distiller's grains enzymatic hydrolysate and glycerol as co-substrates. Compared with the wild-type strain, the yield of 1,3-propanediol is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Klebsiella pneumoniae, its construction method, and its application. Background Technology

[0002] 1,3-Propanediol is an important organic compound that plays a crucial role in multiple fields. In the chemical industry, 1,3-propanediol can be used to synthesize polymer materials such as polyesters and polyurethanes, for example, polypropylene terephthalate (PTT). This material has good elasticity, abrasion resistance, and dyeability, and has broad application prospects in carpets, textiles, and engineering plastics. Furthermore, 1,3-propanediol can also be used as a solvent, antifreeze, and humectant in the pharmaceutical and cosmetic industries to improve product stability and water retention.

[0003] The main methods for producing 1,3-propanediol include chemical synthesis and microbial fermentation. With the increasing emphasis on environmental protection and sustainable development, the production of 1,3-propanediol through bio-fermentation has become an important research direction. Numerous studies have been reported on the production of 1,3-propanediol using microbial fermentation. Commonly used strains include Klebsiella pneumoniae, Clostridium, and Citrobacter, among which Klebsiella pneumoniae (…) is particularly valuable. Klebsiella pneumoniae Klebsiella pneumoniae has been widely studied and applied due to its well-defined pathway for synthesizing 1,3-propanediol from glycerol and its rapid growth rate. However, the production of 1,3-propanediol using wild-type Klebsiella pneumoniae through anaerobic fermentation with glycerol as a substrate faces challenges such as high raw material costs and low substrate conversion rates.

[0004] Baijiu (Chinese liquor) lees are a major byproduct of the baijiu brewing industry, rich in nutrients such as polysaccharides, proteins, amino acids, and trace elements, with an annual output exceeding ten million tons. However, the current utilization rate of lees resources in China is low, with most lees waste being directly discarded or incinerated. This not only results in a serious waste of usable biomass resources but also causes environmental pollution problems such as soil and air pollution. Existing technologies include research on using lees enzymatic hydrolysate to assist fermentation in the production of 1,3-propanediol. Enzymatic hydrolysis breaks down large, unusable molecules in lees into smaller, readily absorbed nutrients such as glucose, xylose, and polypeptides, thereby increasing 1,3-propanediol fermentation yield. However, natural lees enzymatic hydrolysate has a complex composition, containing various antibacterial substances that inhibit the growth and metabolism of Klebsiella pneumoniae. Common wild-type Klebsiella pneumoniae has poor tolerance to this hydrolysate and low substrate utilization efficiency, failing to fully utilize the advantages of lees biomass in assisted fermentation, ultimately resulting in a very limited effect on increasing 1,3-propanediol yield.

[0005] In view of this, this invention is hereby proposed. Summary of the Invention

[0006] Based on the above problems, the purpose of this invention is to provide a strain of Klebsiella pneumoniae, its construction method and application, which has strong resistance to enzymatic hydrolysate of distillers' grains and can effectively promote the production of 1,3-propanediol.

[0007] To achieve the above objectives, the first technical solution of the present invention provides a strain of Klebsiella pneumoniae (… Klebsiella pneumoniae The strain Kp-1 is deposited at the China Center for Type Culture Collection (CCTCC) on April 30, 2026, with accession number CCTCC NO: M 2026879.

[0008] The second technical solution adopted in this invention provides the above-mentioned Klebsiella pneumoniae ( Klebsiella pneumoniae The Kp-1 strain was constructed by using an adaptive evolution method to continuously passage and screen wild-type Klebsiella pneumoniae under stress of gradually increasing concentration of distillers' grains hydrolysate.

[0009] Preferably, the wild-type Klebsiella pneumoniae is Klebsiella pneumoniae ATCC 25955.

[0010] Preferably, the preparation method of the fermented lees hydrolysate is as follows: the dried fermented lees are crushed, mixed with buffer solution, and then microwaved. After that, buffer solution is added again, mixed evenly, and then enzymatically hydrolyzed with laccase and compound hydrolase in sequence. Finally, the pH is adjusted to 6-7.

[0011] Preferably, the complex hydrolase includes cellulase and amylase.

[0012] Preferably, the concentration of the enzymatic hydrolysate of the distiller's grains gradually increases from low to high within the range of 10 g / L to 60 g / L.

[0013] The third technical solution adopted in this invention provides the above-mentioned Klebsiella pneumoniae ( Klebsiella pneumoniae The application of Kp-1 in the production of 1,3-propanediol involves inoculating fermentation medium containing distillers' grains hydrolysate and glycerol with Klebsiella pneumoniae. Klebsiella pneumoniae Kp-1 seed liquid was fermented.

[0014] Preferably, in the fermentation medium, the concentration of the distillers' grains hydrolysate is 40-60 g / L, and the concentration of glycerol is 40-70 g / L.

[0015] Preferably, the Klebsiella pneumoniae ( Klebsiella pneumoniae The inoculation amount of Kp-1 seed solution is 1.5-2%.

[0016] Preferably, the fermentation temperature is 30-37 ℃ and the time is 36-48 h.

[0017] The beneficial effects of this invention are as follows: This invention achieves targeted iterative optimization of strain tolerance, substrate utilization, and product synthesis capabilities by performing gradient adaptive evolution on wild-type Klebsiella pneumoniae ATCC 25955 in high-concentration distillers' grains enzymatic hydrolysate, ultimately obtaining an evolved strain of Klebsiella pneumoniae capable of stable growth in a high-concentration distillers' grains enzymatic hydrolysate environment. Klebsiella pneumoniae Kp-1. This strain can grow and metabolize normally in fermentation systems with a distillers' grains hydrolysate concentration as high as 60 g / L, effectively overcoming the technical bottleneck of traditional Klebsiella pneumoniae strains' poor tolerance to distillers' grains substrate and limited distillers' grains concentration in the initial fermentation system.

[0018] This invention utilizes Klebsiella pneumoniae ( Klebsiella pneumoniae Kp-1, using high-concentration distillers' grains hydrolysate and glycerol as co-substrate, produced 1,3-propanediol through fermentation, and the yield of 1,3-propanediol was significantly increased compared to the wild-type strain. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description of the invention is provided in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are merely exemplary and not intended to limit the scope of the invention. Furthermore, in the following description, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0020] This invention provides a strain of Klebsiella pneumoniae ( Klebsiella pneumoniae The strain Kp-1 is deposited at the China Center for Type Culture Collection (CCTCC) on April 30, 2026, with accession number CCTCC NO: M2026879.

[0021] The embodiments of the present invention also provide the above-mentioned Klebsiella pneumoniae ( Klebsiella pneumoniae The Kp-1 strain was constructed by using an adaptive evolution method to continuously passage and screen wild-type Klebsiella pneumoniae under stress of gradually increasing concentration of distillers' grains hydrolysate.

[0022] It should be noted that while the distillers' grains hydrolysate contains small-molecule nutrients such as glucose, xylose, and polypeptides that can be utilized by microorganisms and help increase the fermentation yield of 1,3-propanediol, it also contains various substances that inhibit microbial growth, such as furfural, phenolic compounds, and organic acids. Under high concentrations, these inhibitors can significantly suppress the growth and metabolism of wild-type Klebsiella pneumoniae. Therefore, current technologies for producing 1,3-propanediol using distillers' grains hydrolysate generally employ a fed-batch fermentation process to mitigate the inhibitory effects of high-concentration substrates. The concentration of distillers' grains hydrolysate in the initial fermentation system is typically controlled at 4-10 g / L, resulting in low substrate utilization and fermentation efficiency. To address the aforementioned issues, this embodiment employs directed adaptive evolution of wild-type Klebsiella pneumoniae. The strain is continuously passaged in a gradient stress environment with the concentration of the distillers' grains hydrolysate gradually increasing from 10 g / L to 60 g / L. This process continuously screens for bacterial populations with strong tolerance and high growth and metabolic activity, while gradually eliminating strains with weak tolerance and low metabolic efficiency. This achieves directed iterative optimization of the strain's tolerance, substrate utilization, and product synthesis capabilities, ultimately yielding an evolved strain capable of stable growth and high production of 1,3-propanediol in a high-concentration distillers' grains hydrolysate environment.

[0023] In this embodiment, Klebsiella pneumoniae ATCC 25955 was selected as the wild-type original strain. This strain was purchased from the American Type Culture Collection. Its glycerol metabolic pathway is complete and stable, providing a reliable basis for subsequent adaptive evolution and industrial fermentation applications.

[0024] In this embodiment, the preparation method of the fermented lees hydrolysate is as follows: the dried fermented lees are crushed, mixed with buffer solution, and then microwaved. After that, buffer solution is added again, mixed evenly, and then enzymatically hydrolyzed with laccase and compound hydrolase in sequence. Finally, the pH is adjusted to 6-7.

[0025] In a specific implementation, the complex hydrolytic enzyme includes cellulase and amylase. To fully release the small-molecule nutrients in the distiller's grains that can be utilized by microorganisms, the dosage of the above components and processing parameters can be routinely adjusted as needed. For example, in the preparation of the distiller's grains enzymatic hydrolysate, the buffer solution is an acetate buffer solution with a pH of 4.5-5.5. The first addition is 5-8 mL / g of dry distiller's grains, and the second addition is 2-7.5 mL / g of dry distiller's grains. The microwave treatment power is 300-500W, and the time is 5-10 min. The amount of laccase added is 0.5-2 mg / g of dry distiller's grains. After adding laccase, enzymatic hydrolysis is carried out at 40-50 °C for 3-5 h. Then, the complex hydrolytic enzyme is added, and enzymatic hydrolysis is carried out at 45-55 °C for 24 h. The complex hydrolytic enzyme includes cellulase and amylase, and the addition amounts are 15-20 mg / g of dry distiller's grains and 5-10 mg / g, respectively.

[0026] The embodiments of the present invention also provide the above-mentioned Klebsiella pneumoniae ( Klebsiella pneumoniae The application of Kp-1 in the production of 1,3-propanediol involves inoculating fermentation medium containing distillers' grains hydrolysate and glycerol with Klebsiella pneumoniae. Klebsiella pneumoniae Kp-1 seed liquid was fermented.

[0027] In the fermentation medium of this embodiment, the concentration of the distillers' grains hydrolysate is 40-60 g / L, and the concentration of glycerol is 40-70 g / L. The high-concentration distillers' grains hydrolysate and glycerol are used together as fermentation substrates. It is understood that, in addition to the distillers' grains hydrolysate and glycerol, the fermentation medium also includes the following components: yeast extract 7 g / L, K2HPO4·3H2O 7 g / L, KH2PO4 2 g / L, (NH4)2SO4 1.25 g / L, MgSO4·7H2O 0.1 g / L, and trace element solution 1.0 mL / L; the trace element solution contains: CaCl2·2H2O 3.2 mg / L, ZnCl2 3.8 mg / L, FeCl3·6H2O 30 mg / L, MnCl2·4H2O 11.4 mg / L, CuCl2·2H2O 0.96 mg / L, CoCl2·6H2O 2.64 mg / L, H3BO3 0.35 mg / L, and Na2MoO4·2H2O 24.5 μg / L.

[0028] In this embodiment, Klebsiella pneumoniae ( Klebsiella pneumoniae The inoculum size of Kp-1 seed culture is 1.5-2%, and the fermentation temperature after inoculation is 30-37 ℃, with a fermentation time of 36-48 h. The seed culture is prepared by inoculating Klebsiella pneumoniae (Kp-1) with... Klebsiella pneumoniae Kp-1 was inoculated into seed culture medium and cultured at 37 ℃ for 8-12 h to obtain seed culture. The seed culture medium used consisted of: 20 g / L glycerol, 7 g / L yeast extract, 7 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 1.25 g / L (NH4)2SO4, 0.1 g / L MgSO4·7H2O, and 1.0 mL / L trace element solution; the trace element solution had the same composition as the trace element solution in the fermentation culture medium described above.

[0029] The following are several specific examples of Klebsiella pneumoniae (Klebsiella pneumoniae) Klebsiella pneumoniae This paper provides a detailed explanation of Kp-1, its construction method, and its applications.

[0030] The purchase information for some of the raw materials in this example is as follows: Cellulase was purchased from Novozymes (China) Investment Co., Ltd. (Beijing); The amylase was purchased from Shandong Longkete Enzyme Preparation Co., Ltd. Klebsiella pneumoniae ATCC 25955 was purchased from the American Center for Type Culture Collection.

[0031] The culture medium and its formulation used in the examples are as follows: Seed culture medium: glycerol 20 g / L, yeast extract 7 g / L, K2HPO4·3H2O 7 g / L, KH2PO4 2 g / L, (NH4)2SO4 1.25 g / L, MgSO4·7H2O 0.1 g / L, trace element solution 1.0 mL / L (solution composition: CaCl2·2H2O 3.2 mg / L, ZnCl2 3.8 mg / L, FeCl3·6H2O 30 mg / L, MnCl2·4H2O 11.4 mg / L, CuCl2·2H2O 0.96 mg / L, CoCl2·6H2O 2.64 mg / L, H3BO3 0.35 mg / L, Na2MoO4·2H2O 24.5 μg / L).

[0032] Fermentation medium: 40-60 g / L of distiller's grains hydrolysate, 40-70 g / L of glycerol, 7 g / L of yeast extract, 7 g / L of K2HPO4·3H2O, 2 g / L of KH2PO4, 1.25 g / L of (NH4)2SO4, 0.1 g / L of MgSO4·7H2O, and 1.0 mL / L of trace element solution (same composition as the trace element solution in the above fermentation medium).

[0033] The preparation method of the distillers' grains enzymatic hydrolysate used in the above culture medium is as follows: The dried distiller's grains were crushed and mixed with 5 mL / g of dry distiller's grains in a pH 4.5 acetate buffer solution. After mixing thoroughly, the mixture was microwaved at 300 W for 5 min. Then, 2 mL / g of dry distiller's grains in a pH 4.5 acetate buffer solution was added and mixed thoroughly. Laccase was added at 0.5 mg / g of dry distiller's grains and the mixture was hydrolyzed at 40 °C for 3 h. Then, a complex hydrolytic enzyme (including cellulase and amylase, added at 15 mg / g and 5 mg / g of dry distiller's grains, respectively) was added and the mixture was hydrolyzed at 45 °C for 24 h to obtain the distiller's grains hydrolysate.

[0034] The method for determining the concentration of 1,3-propanediol involved in the examples is as follows: High-performance liquid chromatography (HPLC) was used for detection. The HPLC system was an Agilent 1260 Infinity II, the detector was a differential detector, the column was an Aminex HPX-87H (Bio-Rad), the injection volume was 20 μL, the column temperature was 40 ℃, the mobile phase was 5 mM H₂SO₄ solution, and the flow rate was 0.6 mL / min.

[0035] Construction of standard curves: Prepare 1,3-propanediol standard samples with concentrations of 0.01 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, and 5 g / L respectively, and perform high performance liquid chromatography detection together. Plot concentration-peak area standard curves based on the detection results.

[0036] Sample preparation: Take 1 mL of fermentation broth, centrifuge at 10000 rpm for 3 min, take the supernatant and dilute it with ultrapure water to an appropriate ratio, then filter the supernatant into a sample vial using a 0.22 μm nylon aqueous filter cartridge for high performance liquid chromatography detection, and substitute the detection results into the above standard curve to calculate the concentration of 1,3-propanediol.

[0037] Example 1: Adaptive Evolution of Klebsiella pneumoniae ATCC 25955 Single colonies of wild-type Klebsiella pneumoniae ATCC 25955 were picked and inoculated into seed culture medium, and cultured at 37 ℃ and 150 rpm for 8-12 h to obtain Klebsiella pneumoniae ATCC 25955 seed culture.

[0038] At an inoculation rate of 2%, Klebsiella pneumoniae ATCC 25955 seed culture was inoculated into seed culture medium containing 10 g / L distillers' grains hydrolysate, and cultured at 37℃ and 150 rpm for 12-24 h. When OD... 600 A concentration of 1.5 or higher indicates that the strain has adapted to that concentration. Each concentration level should be passaged for at least five consecutive generations until the strain reaches a stable OD value at the fixed concentration. 600 Once the concentration reaches a stable value of 1.8 or higher, take the culture medium and inoculate it at a rate of 2% into a seed culture medium with a higher concentration of distiller's grains hydrolysate for further cultivation.

[0039] The concentrations of the distillers' grains enzymatic hydrolysate were set sequentially as follows: 10 g / L, 20 g / L, 30 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, and 60 g / L. When the strain could grow normally in seed culture medium containing 60 g / L distillers' grains enzymatic hydrolysate (OD within 24 h...), the growth rate was determined. 600 (≥1.8) is considered to be the completion of evolution.

[0040] Example 2 Klebsiella pneumoniae ( Klebsiella pneumoniae Isolation, screening and identification of Kp-1 The culture medium obtained from the final evolution in Example 1 was serially diluted with physiological saline (10). -4 10 -5 10 -6 100 μL of each dilution was spread onto a solid seed culture medium containing 60 g / L distiller's grains hydrolysate (15 g / L agar powder was added to the seed culture medium), and anaerobically cultured at 37℃ for 48 h. Fifteen single colonies with large diameters, plump morphology, and neat edges were selected and inoculated into seed culture medium containing 60 g / L distiller's grains hydrolysate, and cultured at 37℃ for 12 h. The 1,3-propanediol concentration in the culture medium of each strain was measured. The five strains with the highest 1,3-propanediol concentrations were screened for secondary screening, and finally, the strain with the highest 1,3-propanediol yield was obtained.

[0041] The selected strain was purified and cultured, and named Klebsiella pneumoniae Kp-1. It was deposited at the China Center for Type Culture Collection (CCTCC) on April 30, 2026, with accession number CCTCC NO: M 2026879, and classified as follows: Klebsiella pneumoniae The strain was sequenced, and its 16S rDNA nucleotide sequence is shown in SEQ ID NO: 1.

[0042] Example 3 Klebsiella pneumoniae ( Klebsiella pneumoniae Application of Kp-1 in the production of 1,3-propanediol Experimental group 1: Klebsiella pneumoniae ( Klebsiella pneumoniae Kp-1 was inoculated into the seed culture medium and cultured at 37℃ for 12 h to obtain the seed culture. 1.5% of the seed culture was then inoculated into a fermentation medium containing 40 g / L distiller's grains hydrolysate and 40 g / L glycerol, and fermented at 37℃ for 36 h. After fermentation, 1 mL of the fermentation broth was taken to determine the concentration of 1,3-propanediol, which was 25.55 g / L.

[0043] Experimental group 2: Klebsiella pneumoniae ( Klebsiella pneumoniae Kp-1 was inoculated into the seed culture medium and cultured at 37℃ for 12 h to obtain the seed culture. 1.8% of the seed culture was then inoculated into a fermentation medium containing 50 g / L distiller's grains hydrolysate and 70 g / L glycerol, and fermented at 35℃ for 42 h. After fermentation, 1 mL of the fermentation broth was taken to determine the concentration of 1,3-propanediol, which was 41.26 g / L.

[0044] Experimental group 3: Klebsiella pneumoniae ( Klebsiella pneumoniae Kp-1 was inoculated into the seed culture medium and cultured at 37℃ for 12 h to obtain the seed culture. 2% (by weight) of the seed culture was then inoculated into a fermentation medium containing 60 g / L distiller's grains hydrolysate and 55 g / L glycerol, and fermented at 30℃ for 48 h. After fermentation, 1 mL of the fermentation broth was taken to determine the concentration of 1,3-propanediol, which was 32.58 g / L.

[0045] Control group: Compared with experimental group 1, the only difference is that Klebsiella pneumoniae (Klebsiella pneumoniae) was used. Klebsiella pneumoniae Kp-1 was replaced with wild-type Klebsiella pneumoniae ATCC 25955. After fermentation, 1 mL of fermentation broth was taken to test the concentration of 1,3-propanediol, which was 10.64 g / L.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Klebsiella pneumoniae ( Klebsiella pneumoniae Kp-1, characterized in that, This strain is deposited at the China Center for Type Culture Collection (CCTCC) on April 30, 2026, with accession number CCTCC NO: M 2026879.

2. Klebsiella pneumoniae as described in claim 1 ( Klebsiella pneumoniae The method for constructing Kp-1 is characterized by, Wild-type Klebsiella pneumoniae was obtained by continuous subculturing under gradually increasing concentration of distillers' grains enzymatic hydrolysate using an adaptive evolution method.

3. The construction method as described in claim 2, characterized in that, The wild-type Klebsiella pneumoniae is Klebsiella pneumoniae ATCC 25955.

4. The construction method as described in claim 2, characterized in that, The preparation method of the fermented lees hydrolysate is as follows: the dried fermented lees are crushed, mixed with buffer solution, and then microwaved. After that, buffer solution is added again, mixed evenly, and then enzymatically hydrolyzed with laccase and compound hydrolase in sequence. Finally, the pH is adjusted to 6-7.

5. The construction method as described in claim 4, characterized in that, The complex hydrolase includes cellulase and amylase.

6. The construction method as described in claim 2, characterized in that, The concentration of the enzymatic hydrolysate from the distiller's grains gradually increases from low to high within the range of 10 g / L to 60 g / L.

7. Klebsiella pneumoniae as described in claim 1 ( Klebsiella pneumoniae The application of Kp-1 in the production of 1,3-propanediol is characterized by, Klebsiella pneumoniae was inoculated into a fermentation medium containing distillers' grains hydrolysate and glycerol. Klebsiella pneumoniae Kp-1 seed liquid was fermented.

8. The application as described in claim 7, characterized in that, In the fermentation medium, the concentration of the distillers' grains hydrolysate is 40-60 g / L, and the concentration of glycerol is 40-70 g / L.

9. The application as described in claim 7, characterized in that, The Klebsiella pneumoniae ( Klebsiella pneumoniae The inoculation amount of Kp-1 seed solution is 1.5-2%.

10. The application as described in claim 7, characterized in that, The fermentation temperature is 30-37 ℃ and the time is 36-48 h.