Salt-tolerant zhanjiangibacter sp. producing r,r-2,3-butanediol and application thereof

Abstract

This invention belongs to the field of microbial metabolic engineering and synthetic biology, specifically relating to a strain for high-yield... R,R -2,3-Butanediol ( R,R Salt-tolerant Jeju Bacillus DT01 IM3 DLB6 (-2,3-BDO), production strain constructed from this strain and its application in fermentation production. R,R Applications of -2,3-BDO. This invention screens a high-yielding strain through heavy ion beam irradiation mutagenesis. R,R Jeju Bacillus DT01 IM3 DL B6 with -2,3-BDO, preservation number CGMCC No. 37311. This strain exhibits excellent salt tolerance and low endotoxin properties. The production strain constructed from it has extremely high product synthesis capabilities, enabling high-concentration, high-intensity industrial fermentation production.

Description

Technical Field

[0001] This invention belongs to the field of microbial metabolic engineering and synthetic biology, specifically relating to a high-yielding strain. R,R -2,3-Butanediol ( R,R Salt-tolerant Jeju Bacillus DT01 IM3 DLB6 (-2,3-BDO) and its application in fermentation production R,R Applications in -2,3-BDO. Background Technology

[0002] R,R -2,3-BDO is an important platform chemical widely used in the fuel, solvent, polymer and pharmaceutical industries. R,R -2,3-BDO is an isomer with special optical activity and has higher application value.

[0003] Currently, biological production R,R -2,3-BDO primarily relies on model strains such as *Escherichia coli* and *Klebsiella pneumoniae*. However, these production processes face numerous challenges:

[0004] High sterilization costs: Traditional strains require strict aseptic conditions, resulting in huge energy consumption in industrial fermentation.

[0005] Biosafety: Gram-negative engineered bacteria (such as Escherichia coli) release large amounts of endotoxins during fermentation, increasing the difficulty and cost of downstream purification.

[0006] Insufficient robustness of strains: In high-concentration product accumulation and complex industrial fermentation environments, common strains often struggle to maintain high activity and high yield.

[0007] Jejubacterium is a non-model salt-tolerant bacterium isolated from superworms, possessing the potential for open fermentation using inexpensive, non-sterile seawater media, and exhibiting extremely low natural endotoxin background. However, the lack of efficient genetic manipulation tools and high-yield strains limits its industrial application. Therefore, developing a genetically stable, salt-tolerant, low-endotoxin, and high-yield strain is crucial. R,R The novel Jeju bacillus with -2,3-BDO has significant industrial implications. Summary of the Invention

[0008] The purpose of this invention is to provide a high-yielding plant. R,R -2,3-BDO Jejubacterium chassis strain DT01 IM3 DLB6 and its applications. This strain, obtained through physical mutagenesis and high-throughput screening, exhibits excellent salt tolerance and low endotoxin properties. The production strain constructed from it possesses extremely high product synthesis capabilities, enabling high-concentration, high-intensity industrial fermentation production.

[0009] To achieve the above objectives, the technical approach adopted by the present invention is as follows: One of the technical solutions provided by this invention is a strain of Jeju bacteria, specifically... Jejubacter sp. DT01 IM3DLB6; the Jeju bacteria Jejubacter sp. DT01 IM3 DLB6 was deposited on January 6, 2026 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC No. 37311.

[0010] The second technical solution provided by this invention is as described in the first technical solution. Jejubacter The application of sp. DT01 IM3DLB6, especially as a chassis bacteria, and even more particularly as a building block for production. R,R Application of strains of -2,3-BDO in the sclerotium.

[0011] The third technical solution provided by this invention is a strain of Jeju Bacillus engineered strain, wherein the Jeju Bacillus engineered strain is the Jeju Bacillus described in one of the technical solutions. Jejubacter sp . Jejubacter sp. DT01 IM3 DLB6 are chassis bacteria, introduced. R,R -2,3-BDO was used to produce plasmid pWT22; Furthermore, the production plasmid pWT22 has the nucleotide sequence shown in SEQ ID NO.2; Furthermore, adopt Jejubacter sp The Jeju Bacillus engineered strain DT01 IM3 DLB6, constructed from chassis bacteria, exhibits the following properties: (1) Gram-negative, rod-shaped.

[0012] (2) Extremely high product synthesis capability: In fed-batch fermentation, R,R -2,3-BDO accumulation can reach 186 g / L, with a production intensity of 8.28 g / L / h.

[0013] (3) High salt tolerance: It can grow and maintain metabolic activity in an environment with NaCl concentration as high as 60 g / L, and is suitable for high salt fermentation systems.

[0014] (4) Low endotoxin background: The endotoxin content in the bacterial lysate was only 0.039 EU / mL.

[0015] The fourth technical solution provided by this invention is the application of the Jeju Bacillus engineered strain described in the third technical solution; Furthermore, in production R,RApplications in -2,3-BDO; Furthermore, the Jeju Bacillus engineered strain is used for fermentation production. R,R The method for -2,3-BDO is as follows: The seed culture was inoculated into the fermentation medium in the fermenter at an inoculation rate of 8.0-12.0% (v / v). During fermentation, the pH was kept constant at 6.8-7.0. The process adopted a staged temperature control and aeration strategy: the initial stage temperature was set at 36-37 °C, the aeration rate was 2.5-3 vvm, and the rotation speed was 500-600 rpm to promote cell growth (no adjustment was made throughout the fermentation cycle); when the cells grew to the mid-logarithmic growth stage (9 h), the temperature was reduced to 29-31 °C, and the aeration rate was reduced to 1.5-2 vvm; after entering the late logarithmic growth stage (12 h), the aeration rate was further reduced to 0.5-1 vvm. During fermentation, glucose is fed in according to the glucose consumption rate, and as fermentation progresses until the OD600 reaches the stationary phase, the supply is gradually reduced to maintain the cells in a semi-starved state, thereby guiding carbon metabolism to R,R-2,3-butanediol synthesis. Furthermore, the OD600 reached a stable state after approximately 18 hours of fermentation. Furthermore, the glucose concentration in the fermenter is controlled at 5-10 g / L to maintain the cells in a semi-starved state; The fermentation medium contains: 2.8-3.2 g / L KH2PO4, 5.8-6.2 g / L Na2HPO4, 0.8-1.2 g / L NH4Cl, 9.9-10.1 mg / L Vitamin B1, 0.09-0.11 mM CaCl2, 0.9-1.2 mM MgSO4, 4.0-10 g / L yeast extract, and 30.0-40.0 g / L glucose. The prepared medium is placed in a fermenter and sterilized by autoclaving at 121°C for 20 minutes. Furthermore, the fermentation medium also contains no more than 90 g / L of NaCl, preferably 60-90 g / L, and more preferably 60 g / L; at this concentration, the growth of Escherichia coli and other miscellaneous bacteria can be completely inhibited, and there is no need to sterilize the medium.

[0016] Beneficial effects: This invention obtains a high-yielding strain through heavy ion beam irradiation mutagenesis screening. R,R Jeju bacillus basalis with -2,3-BDO Jejubacter sp. DT01 IM3 DLB6, introduced into this chassis bacteria R,R The strain obtained via the -2,3-BDO production route achieved 186 g / L in a 3L fermenter. R,RThe production rate of -2,3-BDO reached 8.28 g / L / h, which is one of the highest levels achieved by non-model microorganisms in the production of this product. 100% of the theoretical yield was achieved in shake-flask fermentation; on an industrial fermenter scale, the yield of glucose reached 0.40 g / g (80.0% of the theoretical value), achieving highly efficient carbon-atom-economical conversion.

[0017] As a chassis cell, DT01 IM3 DLB6 has a naturally low endotoxin content (0.039 EU / mL), which provides significant biosafety advantages. It solves the problem of endotoxin contamination in traditional Gram-negative bacterial fermentation products, greatly reduces subsequent purification costs, and is particularly suitable for applications in the pharmaceutical and high-end daily chemical industries.

[0018] The strain is tolerant of high-salt environments (90 g / L NaCl), supports fermentation under open, low-sterilization conditions, effectively inhibits contamination by other microorganisms, and is suitable for large-scale industrial promotion. Attached Figure Description

[0019] Figure 1 Production of different mutagenic bacteria by shake-flask fermentation R,R -2,3-BDO production graph.

[0020] Figure 2 For the production of DT01 IM3 DLB6 / pWT22 by fermentation in a 3L tank R,R Fermentation process curve of -2,3-BDO.

[0021] Figure 3 The curves show the non-sterile fermentation process of DT01 IM3 DLB6 / pWT22 in a 3L tank at high salt concentration. Detailed Implementation

[0022] The present invention will now be described through specific embodiments. All technical means not specifically described herein are methods well-known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of the present invention.

[0023] The starting strain used in this invention Jejubacter sp.DT01 IM3 is existing technology. This bacterium was deposited on September 15, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCC No. 28463.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] The following embodiments are further illustrations of the present invention and do not constitute a limitation on the substantive content of the present invention.

[0027] The present invention will be further explained and illustrated below through specific embodiments.

[0028] Example 1: Obtaining Jeju Bacillus DT01 IM3 DLB6 1. Construction of heavy ion beam irradiation mutagenesis library: Using Jejubacterium DT01 IM3 as the starting strain, DT01 IM3 was cultured to the logarithmic growth phase (approximately OD200). 600 =0.8), take 2 mL of bacterial culture and put it into a sterile irradiation dish and seal it. Use an energy of 80 MeV / u. 12 C 6 The cells were irradiated with a heavy ion beam at a dose of 150 Gy to induce mutagenesis. The irradiated mutagenesis library was then used to prepare competent cells, which were electroporated to contain... R,R -2,3-Butanediol-specific biosensor (BmoR) N89D The plasmid pWT22-GFP, containing the biosynthetic pathway (its complete nucleotide sequence is shown in SEQ ID NO. 1), was used for initial screening using a 96-well plate. The GFP / OD ratio was then detected. 600 The fluorescence intensity was screened to identify 12 mutant strains with significantly higher fluorescence intensity than the control, and these were further screened by shake-flask fermentation. Finally, a strain was obtained after introducing the plasmid pWT22-GFP. R,R The Chameleon mutant DT01 IM3 DLB6 showed a significant increase in -2,3-BDO production.

[0029] (1) Initial screening (96-well plate screening): 129 single clones were randomly selected from the transformation plate and inoculated into 96-well deep plates containing 1 mL LB medium (containing 100 μg / mL ampicillin) and cultured overnight at 37°C. Subsequently, they were transferred at a 1% inoculum to deep-well plates containing 1 mL M9 fermentation medium (containing 40 g / L glucose, 4 g / L yeast extract and 100 μg / L ampicillin) and cultured at 30°C for 12 hours.

[0030] The intensity of green fluorescence (GFP, excitation light 470 nm / emission light 510 nm) and the cell concentration (OD) were detected using a microplate reader. 600 ). Calculate the normalized relative fluorescence intensity (GFP / OD). 600Compared with the control strain, a total of 12 GFP / OD strains were screened. 600 A mutant strain with a significantly increased value.

[0031] (2) Secondary screening (shaking tube screening): The high fluorescence intensity strains obtained in the initial screening were transferred to test tubes for secondary screening. Two GFP / OD strains were selected. 600 The value was significantly higher than that of the control monoclonal clone.

[0032] (3) Shake-flask fermentation verification: The mutant strains DT01 IM3 DLB3 and DT01 IM3 DLB6 containing plasmid pWT22-GFP obtained from the secondary screening were selected and fermented with the control strain DT01 IM3 at 30°C and 220 rpm for 48 h in shake flasks containing 20 mL of M9 fermentation medium (containing 40 g / L glucose, 4 g / L yeast extract and 100 μg / L ampicillin). Finally, the DT01 IM3 DLB6 strain containing plasmid pWT22-GFP was fermented in shake flasks for 24 h. R,R The yield of 2,3-butanediol was 20.3 g / L, which was 20.8% higher than that of strain DT01 IM3 containing plasmid pWT22-GFP (e.g., Figure 1 (As shown).

[0033] The DT01 IM3 DLB6 strain with the pWT22-GFP plasmid lost was preserved for further application as a chassis bacterium.

[0034] The chassis strain DT01 IM3 DLB6 obtained after final screening was named Jeju Bacillus. Jejubacter sp. DT01IM3 DLB6 was deposited on January 6, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37311.

[0035] Example 2: High-density fermentation of DT01 IM3 DLB6 / pWT22 strain in a 3L fermenter 1. Construction of production strains (1) Preparation of competent cells of DT01 IM3 DLB6: First, a single colony of the chassis strain DT01 IM3 DLB6 was inoculated into LB liquid medium and cultured overnight at 37°C with shaking at 220 rpm. Then, it was transferred to 2 mL of fresh LB medium at a ratio of 1:100 (v / v) and cultured at 37°C with shaking at 220 rpm until OD. 600The bacterial culture reached the logarithmic mid-growth phase of 0.5. Immediately afterwards, the culture was cooled in an ice bath for 30 min, and the cells were collected by centrifugation at 4°C and 4500 rpm for 3 min. After discarding the supernatant, the cells were resuspended in 2 mL of 10% (v / v) glycerol and centrifuged again at 4°C and 4500 rpm for 3 min (this step was repeated two to three times). After the final discarding of the supernatant, the cells were resuspended in 100 μL of 10% (v / v) glycerol and stored at -80°C for later use.

[0036] (2) Production plasmid: pWT22 plasmid. The pWT22 plasmid is a recombinant plasmid using pUC19 as a vector plasmid to express acetyllactate synthase (AlsS), acetyllactate decarboxylase (AlsD), and (secondary) alcohol dehydrogenase (sADH). The nucleotide sequence of the pWT22 plasmid is shown in SEQ ID NO.2. The difference between the pWT22 plasmid and the pWT22-GFP plasmid used for screening high-yield strains is that the pWT22 plasmid does not contain a biosensor and a GFP reporter protein; it only contains... R,R -2,3-Butanediol biosynthetic pathway.

[0037] The pWT22 plasmid was electroporated into the above competent cells to obtain... R,R -2,3-Butanediol producing strain DT01IM3 DLB6 / pWT22.

[0038] The specific electroporation transformation steps are as follows: 100 μL of frozen competent cells were slowly thawed on ice, and 1000 ng of pWT22 plasmid DNA was added for electroporation transformation (voltage 2.6 kV, capacitance 25 μF, resistance 200 Ω). Immediately after electroporation, the cells were resuspended in 900 μL of fresh LB medium and cultured at 37℃ and 220 rpm for 2.5 h with shaking. Then, the cells were collected by centrifugation at room temperature and 4500 rpm for 3 min, and resuspended in 100 μL of fresh LB medium before being plated onto plates containing Amp... R Incubate overnight at 37°C on resistant LB solid culture dishes. Randomly selected single colonies were then further sequenced for verification. R,R -2,3-Butanediol producing strain DT01 IM3 DLB6 / pWT22.

[0039] 2. Fermentation System: A 3L fermenter was used, with a liquid volume of 1.5L. The fermentation medium contained 3.0 g / L KH₂PO₄, 6.0 g / L Na₂HPO₄, 1.0 g / L NH₄Cl, 10.0 mg / L Vitamin B1, 0.1 mM CaCl₂, 1.0 mM MgSO₄, 4.0 g / L yeast extract, and 40.0 g / L glucose. The prepared medium was poured into the 3L fermenter and autoclaved at 121℃ for 20 minutes.

[0040] Fermentation control: Seed culture was expanded to OD in two stages using LB liquid medium. 600 Once the pH reaches 0.8, the inoculum is introduced into the fermenter at a rate of 10.0% (v / v). The pH is maintained constant between 6.8 and 7.0 during fermentation. A phased temperature and aeration strategy is employed: initially, the temperature is set at 37 °C, the aeration rate is 3 vvm, and the rotation speed is 500-600 rpm to promote cell growth; when the cells reach mid-logarithmic growth (approximately 9 h, biomass ≈ 4 g / L), the temperature is reduced to 30 °C, and the aeration rate is reduced to 2 vvm; after entering late-logarithmic growth (approximately 12 h, biomass ≈ 17 g / L), the aeration rate is further reduced to 1 vvm. This gradual reduction in aeration aims to prevent excessive carbon source from being diverted to the tricarboxylic acid cycle for growth, thus inhibiting the production of the main product.

[0041] Residual sugar was sampled and tested every 4 hours during fermentation. A feed-in program was implemented based on the glucose consumption rate (calculating the glucose consumed every 4 hours to predict the amount likely to be consumed in the next 4 hours, and using this amount to calculate and set the automatic glucose feed rate per minute). As fermentation progressed until OD600 reached a stationary phase (approximately 12 hours), the feed rate was gradually reduced to maintain the cells in a semi-starved state (maintaining a glucose concentration of 5-10 g / L in the fermenter), thereby guiding the carbon metabolism flow towards... R,R Synthesis of 2,3-butanediol.

[0042] Fermentation process curve as shown Figure 2 As shown.

[0043] Final yield: At the end of 48 hours of fermentation, calculate the product in the fermenter. R,R The yield of 2,3-butanediol reached as high as 186 g / L.

[0044] Efficiency indicators: the efficiency of the entire fermentation process R,R The production intensity of 2,3-butanediol is 8.28 g / L / h, and the yield of glucose is 0.40 g / g.

[0045] Example 3: Fermentation performance of DT01 IM3 DLB6 / pWT22 under high salt conditions High salt test: 60 g / L NaCl was added to the fermentation medium in Example 2 to simulate a high salt environment. The medium was not sterilized.

[0046] The fermentation process was controlled in the same way as in Example 2.

[0047] Results: The fermentation process curve is as follows: Figure 3 As shown, strains DT01IM3 DLB6 / pWT22 and *Dendrocalamus DT01IM3 DLB6* can adapt to high-salt environments and carry out [activities]. R,R 2,3-Butanediol production was carried out in a 3L fermenter under high-salt (60 g / L NaCl) non-sterile conditions. R,R The yield of 2,3-butanediol can still reach 175 g / L.

[0048] The low-salt sterilized fermentation (186 g / L) in Example 2 and the high-salt non-sterile fermentation (175 g / L) in Example 3 R,R The yield difference of 2,3-butanediol was only 5.9%, which is well within the margin of error, proving that Jejubacterium DT01 IM3 DLB6 can undergo non-sterile fermentation and achieve the same yield as sterile fermentation. R,R The yield of 2,3-butanediol was also increased. This demonstrates the robustness of this strain under high-salinity processing conditions.

[0049] Example 4: Detection of Endotoxin Content Endotoxin levels were determined in the bacterial lysate of DT01 IM3 DLB6 chassis bacteria to assess its safety as a biomanufacturing chassis.

[0050] Methods: The standard Limulus Amebocyte Lysate (LAL) assay was used. The specific steps are as follows: (1) Sample preparation: The DT01 IM3 DLB6 strain was inoculated into LB liquid medium and cultured until the logarithmic growth phase (approximately 4 h, OD) was reached. 600 =0.8). Collect bacterial cells and wash them three times with pyrogen-free water to completely eliminate interference from culture medium components in the detection.

[0051] (2) Cell lysis: The washed bacterial precipitate was resuspended in pyrogen-free water and ultrasonically disrupted and lysed under ice bath conditions.

[0052] (3) Obtaining the supernatant: Centrifuge the lysis buffer at 12,000 rpm for 10 minutes and collect the clear supernatant as the sample to be tested.

[0053] (4) Colorimetric reaction: In a pyrogen-free 96-well microplate, strictly follow the instructions of the kit to mix the supernatant to be tested with LAL reagent and colorimetric substrate, and incubate under constant temperature to carry out the colorimetric reaction.

[0054] (5) Detection and calculation: The absorbance of each well was measured at a wavelength of 405 nm using an ELISA reader. At the same time, a standard curve was plotted using the Control Standard Endotoxin (CSE), and the endotoxin concentration (EU / mL) in the sample was calculated based on the standard curve.

[0055] Results: The endotoxin content of DT01 IM3 DLB6 was extremely low, only 0.039 EU / mL fermentation broth.

[0056] Conclusion: Compared with traditional hosts such as Escherichia coli, the endotoxin level of DT01 IM3 DLB6 is almost negligible, which greatly simplifies the downstream processing steps of the product and ensures the safety of the final product.

[0057] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A strain of Jeju bacteria, characterized in that, The Jeju bacteria specifically refers to Jejubacter sp. DT01 IM3 DLB6, accession number CGMCC No. 37311.

2. The Jeju bacteria as described in claim 1 Jejubacter sp Application of DT01 IM3 DLB6 as a chassis bacteria.

3. The application as described in claim 2, characterized in that, In the production R,R Application of 2,3-butanediol in stellate bacteria.

4. A strain of Jeju Bacillus engineered bacteria, characterized in that, The Jeju bacteria is the Jeju bacteria described in claim 1. Jejubacter sp DT01, IM3, and DLB6 are chassis bacteria; import them. R,R 2,3-Butanediol was used to produce plasmid pWT22; The production plasmid pWT22 has the nucleotide sequence shown in SEQ ID NO.

2.

5. The Jeju Bacillus engineered strain according to claim 4 in production R,R Applications in 2,3-butanediol.

6. The application as described in claim 5, characterized in that, Fermentation production R,R The method for producing 2,3-butanediol is as follows: The seed culture was inoculated into the fermentation medium at an inoculum rate of 8.0-12.0%. During fermentation, the pH was maintained at a constant 6.8-7.

0. A phased temperature and aeration strategy was employed: initially, the temperature was set at 36-37 °C, the aeration rate at 2.5-3 vvm, and the rotation speed at 500-600 rpm; when the cells reached mid-logarithmic growth, the temperature was lowered to 29-31 °C, and the aeration rate was reduced to 1.5-2 vvm; after entering late logarithmic growth, the aeration rate was further reduced to 0.5-1 vvm. During fermentation, glucose is fed in according to the glucose consumption rate, and as fermentation progresses until the cell OD600 reaches the stationary phase, the supply is gradually reduced to maintain the cells in a semi-starved state, thereby guiding carbon metabolism to R,R-2,3-butanediol synthesis.

7. The application as described in claim 6, characterized in that, During fermentation, the amount of glucose consumed at certain time intervals is used as the glucose feed rate for the next time interval. Maintain the glucose concentration in the fermenter at 5-10 g / L to keep the cells in a semi-starved state.

8. The application as described in claim 6, characterized in that, The fermentation medium contains the following components: 2.8-3.2 g / L KH2PO4, 5.8-6.2 g / L Na2HPO4, 0.8-1.2 g / L NH4Cl, 9.9-10.1 mg / L Vitamin B1, 0.09-0.11 mM CaCl2, 0.9-1.2 mM MgSO4, 4.0-10 g / L yeast extract, and 30.0-40.0 g / L glucose.

9. The application as described in claim 7, characterized in that, The fermentation medium also contains no more than 90 g / L of NaCl; preferably, the fermentation medium also contains 60-90 g / L of NaCl.

10. The application as described in claim 9, characterized in that, The fermentation medium does not require sterilization and can be used directly for fermentation production.

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