Screening of 2,3-butanediol-producing enterobacter cloacae and its application

Abstract

The application discloses a 2,3-butanediol-producing Enterobacter hormaechei strain and a breeding method and application thereof, and belongs to the fermentation technical field in bioengineering. The application screens a 2,3-butanediol-producing strain from soil in nature, and identifies the strain as Enterobacter hormaechei through 16S rDNA. The screened Enterobacter hormaechei is fermented in a fermentation system containing SOB, SOC, TB or LB medium to produce 2,3-butanediol, and the 2,3-butanediol yield can reach 65.23 g / L by adding glucose. Moreover, the Enterobacter hormaechei can also utilize cheap corncob hydrolysate, the 2,3-butanediol yield can also reach 42.92 g / L by adding the corncob hydrolysate in the fermentation process. The application enriches the types of 2,3-BD-producing strains, and lays a solid foundation for subsequent research.

Description

Technical Field

[0001] This invention relates to the selection and application of a strain of Enterobacter holmium that produces 2,3-butanediol, belonging to the field of fermentation technology in bioengineering. Background Technology

[0002] 2,3-Butanediol, abbreviated as 2,3-BD, is a chiral compound, a colorless viscous liquid with a melting point of 23-27℃ and a boiling point of 170-182℃. It is hygroscopic, miscible with water, and readily soluble in alcohols and ethers.

[0003] 2,3-BD is an important platform chemical material with applications spanning aerospace, food, pharmaceuticals, chemicals, and fuels. 2,3-BD can be added to butter as a flavoring additive, its dehydration form 1,3-butadiene can be used in rubber synthesis, and esterified 2,3-BD is a precursor for polyimide synthesis. Currently, 2,3-BD can be produced through both chemical synthesis and microbial fermentation. Chemical synthesis primarily involves the hydrolysis of four-carbon hydrocarbons produced during petroleum cracking under high temperature and pressure, which suffers from drawbacks such as complex processes, difficulty in operation, and expensive raw materials. In contrast, the biological method uses renewable resources as fermentation feedstock, offering advantages such as environmental friendliness, low cost, low carbon emissions, and green environmental protection. In recent years, with the increasing scarcity of petroleum resources, rising fossil fuel prices, and environmental degradation, the biological synthesis of 2,3-BD has received growing attention. Currently, the main production strains for 2,3-BD are Klebsiella pneumoniae, but the limited variety of production strains severely restricts the development and utilization of 2,3-BD. Therefore, it is urgent to expand the variety of strains capable of producing 2,3-BD. Summary of the Invention

[0004] This invention screened a microbial strain that produces 2,3-BD using glucose as a substrate. The strain was identified as *Enterobacter holmieae*, which possesses advantages such as the ability to utilize multiple carbon sources and rapid growth. Furthermore, its substrate utilization characteristics are similar to those of *Klebsiella pneumoniae*. However, there have been no related reports to date, indicating significant potential for industrial application. Preliminary studies on its fermentation conditions were conducted, achieving a yield of 65.28 g / L and a yield of 0.41% in a 5 L fermenter. This enriches the variety of 2,3-BD-producing strains and lays a solid foundation for subsequent research.

[0005] The purpose of this invention is to provide a strain capable of producing 2,3-BD through fermentation using various carbon sources (such as glucose, corn cob hydrolysate, and glycerol) as substrates, screened from natural soil. The strain was identified using 16S rRNA and named WM11. Qualitative and quantitative identification of the fermentation products was performed, thus broadening the pathway for the industrialization of 2,3-BD production via microbial fermentation.

[0006] This invention provides a strain of Enterobacter cholerae (C. cholerae). Enterobacter hormaechei It was deposited at the China Center for Type Culture Collection on February 28, 2022, with accession number CCTCC NO: M 2022159.

[0007] The present invention provides a microbial preparation containing the aforementioned Enterobacter cholerae.

[0008] In one embodiment, the amount of *Enterobacter holmieae* in the microbial agent is not less than 1.0 × 10⁻⁶. 6 cfu / mL or 1.0×10 6 cfu / g.

[0009] The present invention provides a culture medium or fermentation broth containing the aforementioned Enterobacter holmieae.

[0010] In one embodiment, the amount of *Enterobacter holmieae* in the culture medium or fermentation broth is not less than 1.0 × 10⁻⁶. 6 cfu / mL.

[0011] This invention provides a method for producing 2,3-butanediol, wherein the method involves fermenting 2,3-butanediol using the aforementioned Enterobacter hominis, or the aforementioned microbial preparation, or the aforementioned culture medium.

[0012] In one implementation, OD 600 A 3-5 g / L *Enterobacter holmella* culture was inoculated into a fermentation system containing 4-5 g / L glucose and fermented for 24-72 h at 35-40 °C, 220-250 r / min, and an aeration rate of 1-2 vvm.

[0013] In one implementation, OD 600 A 3-5% concentration of Enterobacter holmium was inoculated into a culture system containing 8-10 g / L glucose and fermented for 10-28 h at 28-35℃, 350-400 r / min, and an aeration rate of 1-2 vvm. In one implementation, OD 600Inoculate a culture of 3-5% Enterobacter holmium into a culture system containing 8-10 g / L glucose. Ferment at 28-35℃, 350-400 r / min, and an aeration rate of 1-2 vvm for 7-9 h. Start adding glucose at a rate of 2-3 g / L / h. Start adding glucose at a rate of 4-5 g / L / h at 23-25 ​​h. Start adding glucose at a rate of 5-6 g / L / h at 36 h. Add 1 L of concentrated nitrogen source at 40-42 h.

[0014] In one implementation, OD 600 A 3-5% concentration of Enterobacter holmium was inoculated into a culture system containing 8-10 g / L glucose and cultured at 28-35°C. The fermentation speed was 300-350 r / min for the first 24 h, and 350-400 r / min after 24 h. During the first 8-24 h of fermentation, corn cob hydrolysate containing 200-250 g / L glucose was added at a flow rate of 20-22 mL / h. After 24 h, corn cob hydrolysate containing 450-500 g / L glucose was added at a flow rate of 18-20 mL / h. At 40-42 h, a concentrated acid nitrogen source containing peptone and yeast powder was added. The aeration rate was controlled at 1-2 vvm during fermentation.

[0015] In one embodiment, the fermentation system contains SOB, SOC, TB, or LB culture medium.

[0016] Preferably, TB medium is used as the fermentation medium.

[0017] In one embodiment, the peptone in the concentrated nitrogen source is added at 6 g per liter of fermentation system, and the yeast powder is added at 12 g per liter of fermentation system.

[0018] This invention provides the application of the aforementioned Enterobacter holmie in the preparation of 2,3-butanediol, 2,3-butanediol derivatives, and products containing 2,3-butanediol and its derivatives.

[0019] This invention provides the application of the aforementioned microbial preparation in the preparation of 2,3-butanediol, 2,3-butanediol derivatives and products containing 2,3-butanediol and its derivatives.

[0020] This invention provides the application of the culture medium or fermentation broth in the preparation of 2,3-butanediol, 2,3-butanediol derivatives and products containing 2,3-butanediol and its derivatives.

[0021] The beneficial effects of this invention are as follows: Using strains screened from natural soil as test strains, the starting strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium and cultured at 37°C and 250 r / min for 12 h. After two subcultures, the strain was inoculated into fermentation medium at a 2% inoculum and fermented at 37°C and 250 r / min for 48 h. The fermentation broth was collected, centrifuged, and the supernatant was analyzed. 2 ml of the supernatant was centrifuged through an aqueous membrane, and the 2,3-BD content in the fermentation broth was determined by high-performance liquid chromatography (HPLC). This invention enriches the variety of 2,3-BD producing strains and lays a solid foundation for subsequent industrial research on the production of 2,3-BD through microbial fermentation.

[0022] The relatively inexpensive substrate glucose or corn cob hydrolysate can be converted into 2,3-BD with higher added value. At the same time, the microbial method for producing 2,3-BD has advantages over the chemical method, such as mild reaction conditions, high raw material utilization, high product purity, simple process, and easy control. It is also beneficial to environmental protection and easy to promote and apply.

[0023] Preservation of biological materials The Enterobacter cholerae provided in this invention is classified and named as follows: Enterobacter hormaechei WM11 was deposited at the China Center for Type Culture Collection (CCTCC) on February 28, 2022, with accession number CCTCC NO: M 2022159, and the deposit address is Wuhan University, Wuhan, China. Attached Figure Description

[0024] Figure 1 This is a screening diagram of the 2,3-BD strain produced in this invention.

[0025] Figure 2 Phylogenetic tree of 16S rRNA sequence of strain WM11.

[0026] Figure 3 GC-MS spectra of 2,3-BD standard and fermentation broth sample.

[0027] Figure 4 HPLC chromatograms of 2,3-BD standard and fermentation broth sample.

[0028] Figure 5 This is a diagram showing the results of shake-flask fermentation.

[0029] Figure 6 The results are from batch loading into the tank.

[0030] Figure 7 Results of fed-batch fermentation of glucose.

[0031] Figure 8 Results of batch fermentation of corn cob hydrolysate. Detailed Implementation

[0032] Solid culture medium for plates and slant agar: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 2% agar powder, natural pH.

[0033] SOC medium: disodium hydrogen phosphate 6.78 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, ammonium chloride 1 g / L, magnesium sulfate 2 mmol / L, calcium chloride 0.1 mmol / L, pH natural; SOB medium: peptone 20 g / L, yeast extract 5 g / L, magnesium chloride hexahydrate 2.03 g / L, sodium chloride 0.5 g / L, potassium chloride 0.186 g / L, pH natural; TB medium: peptone 12 g / L, yeast extract 24 g / L, potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate monohydrate 16.43 g / L, pH natural; LB medium (seed enrichment medium): peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH natural.

[0034] HPLC detection conditions: Agilent liquid chromatograph, Aminex HPX-87H (300x7.8mm) column, column temperature: 35℃; mobile phase: 5 mM sulfuric acid; flow rate: 0.6 ml / min; injection volume: 10 μL; detector: differential refractive index detector.

[0035] GC-MS identification: The fermentation supernatant was extracted with ethyl acetate, and the remaining water in the extract was removed by blotting with anhydrous sodium sulfate. The extract was then filtered and purged with nitrogen. After drying, a silanizing reagent was added, and the extract was purged with nitrogen again. The solution was redissolved in n-hexane for GC-MS identification. GC-MS detection conditions: SP-5 column, TSQ-8000 detector, injection temperature 290℃, injection volume 1 μL, high-purity helium carrier gas, carrier gas flow rate 1.2 mL / min, detector temperature 280℃; TraceMS mass spectrometry conditions: EI + bombardment source, full scan mode, scan quality range of 50 550 amu, emission current of 200 μA, electron energy of 70 eV, and mass spectrometry detection library is NIST library.

[0036] Example 1: Screening and Identification of Strains Initial screening: Collected soil samples were cultured in seed enrichment medium for 12 h. Different dilutions of bacterial suspension were spread onto plates and incubated at 37℃ until single colonies appeared. A large number of single colonies were picked and fermented in SOB medium at 37℃ and 250 r / min for 48 h. After centrifugation, the supernatant was placed in an ELISA plate for acetoin colorimetric reaction to detect the presence of acetoin (an essential precursor for the synthesis of 2,3-BD; its presence indicates a positive colorimetric reaction). Figure 1 As shown in a. The red-colored positive strains were preserved for further screening and later use.

[0037] Secondary screening: Strains were picked from the slant and inoculated into enrichment medium, and cultured until the logarithmic OD phase. 600 The inoculum was 3-5, and 2% was inoculated into SOB fermentation medium containing 4 g / L glucose. The culture was carried out with shaking at 250 r / min for 48 h. The fermentation broth was collected, centrifuged at 12000 r / min for 5 min, and the supernatant was collected for identification by gas chromatography-mass spectrometry (GC-MS) and detection by high performance liquid chromatography (HPLC). (1) GC-MS identification: The supernatant was identified by GC-MS. The GC-MS spectra of the 2,3-BD standard and the fermentation broth sample are shown below. Figure 3 c~ Figure 3 d (2,3-BD standard) Figure 3 a~ Figure 3 b (fermentation sample) is shown.

[0038] (2) HPLC detection: The yield of the supernatant was detected by HPLC, and the results are as follows. Figure 1 As shown in b, strain WM11 had the highest yield, with a 2,3-BD content of 1.12 g / L in the fermentation broth and a molar yield of 0.28. HPLC chromatograms of the 2,3-BD standard and fermentation broth samples are shown below. Figure 4 As shown, a is the fermentation broth sample and b is the standard sample.

[0039] The 16S rRNA sequencing of strain WM11, which had the highest yield, was performed by Tianlin Biotechnology Co., Ltd. The results were compared with existing sequences in the NCBI database, and a phylogenetic tree was constructed using MEGA 7.0. The 16S rRNA sequence of the strain is shown in SEQ ID NO:1, and the phylogenetic tree of the 16S rRNA sequence is shown below. Figure 2 As shown.

[0040] Example 2: Fermentation production of 2,3-butanediol in different culture media SOB, SOC, TB and LB media were selected for the study, with an initial sugar concentration of 4 g / L.

[0041] Develop to the logarithmic OD phase 600Seed culture of 3-5 was added at a 2% (v / v) inoculation rate to 50 mL / 250 mL shake flask fermentation medium containing glucose. Fermentation conditions were: temperature 37℃, rotation speed 250 r / min, aeration rate 1 vvm, and fermentation time 72 h. The fermentation broth was collected, centrifuged, and the supernatant was analyzed. The highest 2,3-BD content was found in the TB medium, reaching 1.23 g / L at 48 h, with a yield of 0.3%. The results are as follows: Figure 5 As shown.

[0042] Example 3: 5L batch loading test Develop to the logarithmic OD phase 600 Seed culture of 3-5 was inoculated at a rate of 5% (v / v) into a 2L fermenter containing 10 g / L glucose. The culture medium was TB medium. Fermentation conditions were: temperature 30℃, rotation speed 400 rpm, aeration rate 1 vvm, and fermentation time 28 h. The fermentation broth was collected, and the results are as follows: Figure 6 As shown, the 2,3-BD content of the supernatant obtained after centrifugation was measured to be 0.8 g / L.

[0043] Example 4: Experiment on batch feeding of glucose Develop to the logarithmic OD phase 600 Seed culture of 3-5 was inoculated at a 5% (v / v) inoculation rate into a 2 L fermenter containing 10 g / L glucose. Fermentation conditions were: temperature 30℃, rotation speed 300 r / min for the first 24 hours, 400 r / min for the next 48 hours, and aeration rate of 1 vvm. After 8 hours of fermentation, 500 g / L glucose was added at a rate of 2.5 g / L / h. At 24 hours, the glucose flow rate was increased to 4 g / L / h, and at 36 hours to 5 g / L / h. At 42 hours, 200 mL of concentrated nitrogen source (containing 12 g peptone and 24 g yeast extract) was added. The fermentation broth was collected after 72 hours of fermentation, and the results are as follows: Figure 7 As shown, the 2,3-BD content of the supernatant obtained after centrifugation was 65.23 g / L.

[0044] Example 5: Experiment on batch feeding of corn cob hydrolysate into the tank The seed culture described in Example 4 was inoculated at a 5% (v / v) inoculation rate into a 5 L fermenter containing 10 g / L glucose and a volume of 2 L. Fermentation conditions were: temperature 30°C, rotation speed 300 r / min for the first 24 hours and 400 r / min for the next 48 hours, and aeration rate of 1 vvm. During fermentation for 8-24 hours, corn cob hydrolysate containing 228 g / L glucose (provided by Yigao Environmental Investment Group) was added at a rate of 22 mL / h. After 24 hours, corn cob hydrolysate containing 464 g / L glucose was added at a rate of 20 mL / h. At 42 hours, 1 L of concentrated nitrogen source was added. The fermentation broth was collected after 72 hours of fermentation, and the results are as follows: Figure 8 As shown, the 2,3-BD content of the supernatant obtained after centrifugation was 42.92 g / L.

[0045] Example 6: Preparation of microbial inoculant containing strain WM11 200 μL of WM11 strain was inoculated into 20 mL of LB liquid medium and activated at 37°C for 2 to 3 generations until the WM11 strain reached 10⁻⁶. 8 When the viable cell count is above CFU / mL, centrifuge at 5000-10000 rpm for 10-20 min, remove the supernatant, and add buffer and lyophilization protectant sequentially under aseptic conditions until the cell concentration is not lower than 10⁻⁶. 6 Solid bacterial agent was obtained by vacuum freeze-drying at cfu / mL.

[0046] SEQ ID NO:1 Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims. SEQUENCE LISTING <110> Jiangnan University <120> Breeding and Application of a 2,3-Butanediol-Producing Enterobacter holmieae <130> BAA220219A <160> 1 <170> PatentIn version 3.3 <210> 1 <211> 1449 <212> DNA <213> Enterobacter hormaechei <400> 1 gtcgagggca gaaggactag acatgcaagt cgaacggtaa caggaagcag cttgctgctt 60 tgctgacgag tggcggacgg gtgagtaatg tctgggaaac tgcctgatgg agggggataa 120 ctactggaaa cggtagctaa taccgcataa cgtcgcaaga ccaaagaggg ggaccttcgg 180 gcctcttgcc atcggatgtg cccagatggg attagctagt aggtggggta acggctcacc 240 taggcgacga tccctagctg gtctgagagg atgaccagcc acactggaac tgagacacgg 300 tccagactcc tacgggaggc agcagtgggg aatattgcac aatgggcgca agcctgatgc 360 agccatgccg cgtgtatgaa gaaggccttc gggttgtaaa gtactttcag cggggaggaa 420 ggcgataagg ttaataacct tgtcgattga cgttacccgc agaagaagca ccggctaact 480 ccgtgccagc agccgcggta atacggaggg tgcaagcgtt aatcggaatt actgggcgta 540 aagcgcacgc aggcggtctg tcaagtcgga tgtgaaatcc ccgggctcaa cctgggaact 600 gcattcgaaa ctggcaggct agagtcttgt agaggggggt agaattccag gtgtagcggt 660 gaaatgcgta gagatctgga ggaataccgg tggcgaaggc ggccccctgg acaaagactg 720 acgctcaggt gcgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgccg 780 taaacgatgt cgacttggag gttgtgccct tgaggcgtgg cttccggagc taacgcgtta 840 agtcgaccgc ctggggagta cggccgcaag gttaaaactc aaatgaattg acgggggccc 900 gcacaagcgg tggagcatgt ggtttaattc gatgcaacgc gaagaacctt acctactctt 960 gacatccaga gaacttagca gagatgcttt ggtgccttcg ggaactctga gacaggtgct 1020 gcatggctgt cgtcagctcg tgttgtgaaa tgttgggtta agtcccgcaa cgagcgcaac 1080 ccttatcctt tgttgccagc ggttaggccg ggaactcaaa ggagactgcc agtgataaac 1140 tggaggaagg tggggatgac gtcaagtcat catggccctt acgagtaggg ctacacacgt 1200 gctacaatgg cgcatacaaa gagaagcgac ctcgcgagag caagcggacc tcataaagtg 1260 cgtcgtagtc cggattggag tctgcaactc gactccatga agtcggaatc gctagtaatc 1320 gtggatcaga atgccacggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1380 atgggagtgg gttgcaaaag aagtaggtag cttaaccttc gggagggcgc taccactttg 1440 gattcccgg 1449

Claims

1. A strain of Enterobacter cholerae ( Enterobacter hormaechei It was deposited at the China Center for Type Culture Collection on February 28, 2022, with accession number CCTCC NO: M 2022159.

2. A microbial preparation containing the Enterobacter cholerae described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The amount of *Enterobacter holmieae* in the microbial preparation is not less than 1.0 × 10⁻⁶. 6 CFU / mL or 1.0×10 6 CFU / g.

4. A method for producing 2,3-butanediol, characterized in that, 2,3-Butanediol is produced by fermentation using the Enterobacter holmium of claim 1 or the microbial preparation of claim 2 or 3.

5. The method according to claim 4, characterized in that, OD 600 A culture of 3–5 *Enterobacter holmella* was inoculated into a culture system containing 8–10 g / L glucose and fermented for 10–28 h at 28–35 °C, 350–400 r / min, and an aeration rate of 1–2 vvm.

6. The method according to claim 4, characterized in that, OD 600 Inoculate a culture of 3-5% Enterobacter holmium into a culture system containing 8-10 g / L glucose. Ferment at 28-35°C, 350-400 rpm, and an aeration rate of 1-2 vvm for 7-9 hours. Start adding glucose at a rate of 2-3 g / L / h. Change the glucose flow rate to 4-5 g / L / h at 23-25 ​​hours, and to 5-6 g / L / h at 36 hours. Add 1 L of concentrated nitrogen source at 40-42 hours.

7. The method according to claim 4, characterized in that, OD 600 A culture of 3–5% *Enterobacter holmella* was inoculated into a culture system containing 8–10 g / L glucose and cultured at 28–35°C. The fermentation speed was 300–350 r / min for the first 24 hours, and 350–400 r / min after 24 hours. During the first 8–24 hours of fermentation, corn cob hydrolysate containing 200–250 g / L glucose was added at a flow rate of 20–22 mL / h. After 24 hours, corn cob hydrolysate containing 450–500 g / L glucose was added at a flow rate of 18–20 mL / h. A concentrated nitrogen source containing peptone and yeast powder was added at 40–42 hours. The aeration rate was controlled at 1–2 vvm during fermentation.

8. The use of the Enterobacter holmieus of claim 1, or the microbial preparation of claim 2 or 3, in the preparation of 2,3-butanediol.

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