Salt-tolerant Jezhou bacillus capable of tolerating higher alcohol and application thereof

Abstract

The invention relates to a high-alcohol-tolerant salt-tolerant microorganism Jezhou bacillus and application thereof, and belongs to the technical field of microorganisms. The preservation number of the Jejubacter sp. DT01IM3 is CGMCC (China General Microbiological Culture Collection Center) No.28463, and the preservation number of the Jejubacter sp. The Jezhou bacillus DT01IM3 can normally grow under the conditions of relatively high salt concentration (0-150g / L) and relatively high higher alcohol concentration (0-8000mg / L), and the problem of tolerance of microorganisms to higher alcohols is solved, so that a foundation is laid for producing higher alcohols by the microorganisms.

Description

Technical field:

[0001] The invention relates to a salt-tolerant microorganism Jeju bacillus tolerant of higher alcohols and application thereof, belonging to the technical field of microorganisms. Background technology:

[0002] Higher alcohols, also known as higher fatty alcohols, refer to mixtures of monohydric alcohols containing more than three carbon atoms. 6 ~C 10 Alcohols in the range are called plasticizer alcohols, and C 12 The above alcohols are called detergent alcohols. Higher alcohols include but are not limited to n-propanol, isopropanol, n-butanol, isobutanol, n-pentanol, isopentanol, etc. These higher alcohols affect the growth, development and metabolism of microorganisms through various pathways such as enzyme activity, cell membrane permeability, photosynthesis, respiration and ion absorption, and have toxic effects on microorganisms.

[0003] Higher alcohols are hydrophobic and can be mixed with gasoline at will; using microorganisms to ferment renewable materials to produce higher alcohol fuels to replace mineral fuels is clean and environmentally friendly, and is a major development trend. However, when using microorganisms for industrial production, the ability of microorganisms to produce higher alcohols is usually affected by the tolerance of microorganisms to higher alcohols.

[0004] Currently, the production of higher alcohols by microorganisms is a common method in metabolic engineering. Therefore, it is very necessary to improve the tolerance of microorganisms to higher alcohols, which is also an important way to solve the problem of limiting the ability of microorganisms to produce higher alcohols. Summary of the invention:

[0005] The object of the present invention is to provide a salt-tolerant Jeju bacterium strain having the property of tolerating higher alcohols, and to use the same for the production of higher alcohols such as isobutanol.

[0006] One of the technical solutions provided by the present invention is a strain of Jejubacter sp., specifically Jejubacter sp. DT01 IM3. The strain still grows well in 8 g / L isobutanol and has the characteristic of tolerating higher alcohols.

[0007] The Jejubacter sp. DT01 IM3 strain was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC) on September 15, 2023, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, and the deposit number is CGMCC No. 28463.

[0008] The Jejubacter sp. DT01 IM3 strain has the following physicochemical properties:

[0009] (1) Gram-negative bacteria, after being cultured in LB solid medium at 37°C for 12 hours, the colonies are round, convex, transparent, smooth, and white. Their morphological characteristics are rod-shaped and have flagella, with a length of 0.8-1.5μm and a diameter of 0.4-0.8μm;

[0010] (2) It can grow in a medium with a salt concentration of 0-150 g / L (preferably 0-90 g / L), and the salt includes but is not limited to NaCl, KCl, MgCl 2 MgSO 4 , CaCl 2 、NaHCO 3 NH 4 Cl et al.

[0011] (3) It can grow in a medium with a pH value of 3 to 11 (preferably 4 to 10);

[0012] (4) It can grow at a temperature of 5 to 55°C (preferably 16 to 40°C);

[0013] (5) Positive motility test, negative oxidase test, positive catalase test, positive nitrate reduction test, weakly positive glycerol test, positive arabinose test, positive xylose test, positive mannose test, positive rhamnose test, positive maltose test, positive trehalose test, positive mannitol test, positive arabitol test, positive potassium gluconate test, negative cellobiose test, negative lactose test, negative starch hydrolysis test, negative inulin hydrolysis test, negative fucose test, negative raffinose test;

[0014] (6) Isobutanol tolerance: Able to tolerate more than 8 g / L of isobutanol;

[0015] (7) n-Butanol tolerance: Able to tolerate n-butanol above 8 g / L;

[0016] (8) Isoamyl alcohol tolerance: Able to tolerate isoamyl alcohol above 4 g / L.

[0017] The second technical solution provided by the present invention is the application of Jejubacter sp. DT01 IM3;

[0018] Further, the application in the production of higher alcohols;

[0019] Furthermore, the invention relates to an application in non-sterile fermentation of a high-salt medium, in particular, an application in the production of higher alcohols by non-sterile fermentation of a high-salt medium; since the DT01 IM3 strain can grow in a medium with a salt concentration of 0-150 g / L, the high-salt medium can be directly fermented without sterilization; the salt concentration in the high-salt medium is not higher than 120 g / L;

[0020] Furthermore, it is used as a base bacteria for constructing a higher alcohol production strain;

[0021] Furthermore, the higher alcohol includes but is not limited to: n-propanol, isopropanol, n-butanol, isobutanol, n-pentanol, isopentanol and the like.

[0022] Beneficial effects:

[0023] The present invention obtains a strain DT01 IM3 capable of tolerating higher alcohols through multiple rounds of domestication based on the salt-tolerant Jeju bacillus DT01. The strain can tolerate more than 8 g / L of isobutanol, more than 8 g / L of n-butanol, and more than 4 g / L of isopentanol, solving the problem in the prior art that microorganisms are intolerant to higher alcohols and thus limit the production of higher alcohols. In addition, since DT01 IM3 also has the ability to tolerate high salt, it can be applied to non-sterile fermentation in high-salt culture medium. Experiments have shown that after DT01 and DT01 IM3 are used as base bacteria to construct higher alcohol production strains and fermentation production, DT01 IM3 can improve the yield of higher alcohols compared to DT01 as a base bacteria, and on the other hand, it also proves the feasibility of non-sterile fermentation production of host DT01 IM3 in a high-salt environment. Description of the drawings:

[0024] Figure 1 The colony morphology of Jejubacter sp. DT01 strain on LB solid medium;

[0025] Figure 2 The bacterial morphology of Jejubacter sp. DT01 strain observed by transmission electron microscopy;

[0026] Figure 3 The growth status of Jejubacter sp. DT01 strain under different NaCl concentration conditions;

[0027] Figure 4 The growth status of Jejubacter sp. DT01 strain under different pH conditions;

[0028] Figure 5 The growth status of Jejubacter sp. DT01 strain under different temperature conditions;

[0029] Figure 6 Phylogenetic tree of the DT01 strain constructed based on the 16S rDNA sequence;

[0030] Figure 7 The strain was domesticated and grown on a solid LB plate containing 8 g / L isobutanol;

[0031] Figure 8 The growth curves of Jejubacter sp. DT01 and DT01 IM3 in 8 g / L isobutanol;

[0032] Fig. 9 The growth curves of Jejubacter sp. DT01 and DT01 IM3 in 8 g / L n-butanol;

[0033] Fig.10 The growth curves of Jejubacter sp. DT01 and DT01 IM3 in 4 g / L isoamyl alcohol;

[0034] Fig.11 The map of the isobutanol production plasmid pDT192 of Jejubacter sp. DT01 and DT01 IM3;

[0035] Fig.12 This is a comparison chart of the production of isobutanol by Jejubacter sp. DT01 and DT01 IM3 using glucose in high-salt non-sterile fermentation medium.

[0036] Fig.13 This is a comparison chart of the production of isobutanol by Jejubacter sp. DT01 and DT01 IM3 using glucose in high-salt sterilized fermentation medium. Specific implementation method:

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] The following examples are provided to further illustrate the present invention and are not intended to limit the present invention.

[0040] The original strain Jejubacter sp. DT01 used in the present invention was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration (CGMCC for short) on March 9, 2023, with the deposit number CGMCC No. 26777.

[0041] The strain Jejubacter sp. DT01 IM3 involved in the present invention was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC for short) on September 15, 2023, with the deposit number CGMCC No. 28463.

[0042] The present invention is further explained below by means of specific examples.

[0043] Example 1: Isolation, identification and preservation of DT01 strain

[0044] 1. Isolation of DT01 strain

[0045] (1) The test saline-alkali soil sample was diluted with 0.9% physiological saline at a ratio of 1:1000 and inoculated into brain heart infusion broth (BHI) medium. The culture was carried out in an aerobic constant temperature shaker at 30°C and a speed of 220 rpm for 3 days to obtain a mixed bacterial solution.

[0046] The brain heart infusion broth (BHI) medium is composed of: calf brain infusion powder 12.5 g / L, beef heart infusion powder 5 g / L, peptone 10 g / L, glucose 2 g / L, sodium chloride 5 g / L, disodium hydrogen phosphate 2.5 g / L, and the rest is water, with a pH value of about 6.8;

[0047] (2) The mixed bacterial solution was diluted and spread on LB solid medium. After culturing at 30°C for 24 h, single clones were streaked on LB solid medium. Single colonies were randomly selected and streaked repeatedly to finally obtain colonies with a single morphology.

[0048] The LB solid culture medium is composed of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 10-20 g / L agar, and the rest is water;

[0049] (3) culturing the microorganisms separated in step (2) in LB medium containing different concentrations of sodium chloride;

[0050] The LB medium consists of: 10 g / L tryptone, 5 g / L yeast extract, and X g / L sodium chloride;

[0051] The sodium chloride concentration X in the above LB medium was set to different concentration gradients, and the specific concentration gradient was designed to be 10 g / L, 30 g / L, 50 g / L, 70 g / L, 90 g / L, and 110 g / L, and the salt-tolerant strains were screened step by step.

[0052] A strain that grew well under 90 g / L salt ions was selected and named strain DT01.

[0053] 2. Identification of DT01 strain

[0054] 1. Morphological identification of DT01 strain

[0055] Morphological identification of DT01 strain: After culturing in LB solid medium at 37°C for 12 hours, the colonies were round, convex, transparent, smooth, and white ( Figure 1 ). Using an optical microscope to observe the morphology of the bacteria, at 100 times magnification, the bacteria are rod-shaped. At the same time, using a transmission electron microscope, it can be observed that the bacteria are rod-shaped and have flagella, with a length of 0.8-1.5μm and a diameter of 0.4-0.8μm ( Figure 2 ). DT01 strain is a Gram-negative bacterium.

[0056] 2. Physiological and biochemical identification of DT01 strain

[0057] The physiological and biochemical characteristics of strain DT01 are shown in Table 1.

[0058] Table 1 Physiological and biochemical characteristics of strain DT01

[0059]

[0060]

[0061] Note: +: positive reaction; -: negative reaction; W: weak positive reaction

[0062] 3. Performance testing of DT01 strain

[0063] The DT01 strain was tested for performance. A single clone was obtained by streaking an LB plate from the frozen stock of DT01. A single clone was selected and placed in LB medium and cultured at 37°C with a shaker at 220 rpm for 12 hours as seed liquid. The seed liquid was then inoculated into LB liquid medium at a volume ratio of 1% (volume ratio), and cultured under different conditions for 48 hours, and its OD was measured. 600 , as follows:

[0064] (1) Different concentrations of NaCl (specifically: 0, 10, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165 g / L) were added to LB medium to conduct a NaCl concentration gradient tolerance experiment. After culturing at 37°C and pH 7 for 48 h, the OD 600 Situation Figure 3 The results showed that the DT01 strain could grow in a medium with a salt concentration of 0 to 150 g / L.

[0065] (2) The pH value of the LB medium was adjusted to 2-12 (specifically: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) to conduct a pH gradient experiment. After culturing for 48 h at 37°C and a NaCl concentration of 10 g / L, the OD 600 Situation Figure 4 The results showed that the DT01 strain could grow in a medium with a pH value of 3 to 11.

[0066] (3) Adjust different culture temperatures (specifically: 5, 10, 15, 25, 30, 37, 40, 45, 50, 55, 60°C) to conduct temperature gradient experiments. After culturing for 48 h at a pH of 7 and a NaCl concentration of 10 g / L, the OD 600 Situation Figure 5 The results showed that the DT01 strain could grow at a culture temperature of 5 to 55°C.

[0067] The above experiments were repeated 3 times.

[0068] 4. Molecular identification of DT01 strain

[0069] The activated DT01 strain was inoculated into LB medium containing 10 g / L sodium chloride and cultured at 30°C for 24 h. 2 mL of fresh culture solution was centrifuged at 4°C and 12000 rpm for 3 min, the bacteria were collected in a 2 mL centrifuge tube, and genomic DNA was extracted using a bacterial genomic DNA extraction kit.

[0070] The 16S rDNA sequence of the strain was detected. The primers used to amplify the 16S rDNA sequence were universal primers: 16F (5'-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG-3') and 16R (5'-GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT-3').

[0071] The specific sequence of 16S rDNA of DT01 strain is as follows (shown in SEQ ID NO.1):

[0072] attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggcagcggaa ggaagcttgc

[0073] ttccttgccg gcgagcggcg gacgggtgag taatgtctgg ggatctgcct gatggagggg

[0074] gataaccact ggaaacggtg gctaataccg cataatctcg aaagagcaaa gtgggggacc

[0075] ttcgggcctc acgccatcag atgaacccag atgggattag ctagcaggtg aggtaatggc

[0076] ccacctgggc gacgatccct agctggtctg agaggatgac cagccacact ggaactgaga

[0077] cacggtccag actcctacgg gaggcagcag tggggaatat tgcacaatgg gcgcaagcct

[0078] gatgcagcca tgccgcgtgt atgaagaagg ccttcgggtt gtaaagtact ttcagtcagg

[0079] aggaagggtg tgagcttaat acgttcatgc attgacgtta ctgacagaag aagcaccggc

[0080] taactccgtg ccagcagccg cggtaatacg gagggtgcaa gcgttaatcg gaattactgg

[0081] gcgtaaagcg cacgcaggcg gttggttaag tcagatgtga aatccccggg ctcaacccgg

[0082] gaactgcatt tgaaactggc cagctggagt ctcgtagagg gaggtagaat tccaggtgta

[0083] gcggtgaaat gcgtagagat ctggaggaat accggtggcg aaggcggcct cctggacgaa

[0084] gactgacgct caggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca

[0085] cgccgtaaac gatgtcgact tggaggctgt gagcttgact cgtggcttcc ggagctaacg

[0086] cgttaagtcg accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgacggg

[0087] ggcccgcaca agcggtggag catgtggttt aattcgatgc aacgcgaaga accttacctg

[0088] gtcttgacat ccacggaagg tttcagagat gagactgtgc cttcgggaac cgtgagacag

[0089] gtgctgcatg gctgtcgtca gctcgtgttg tgaaatgttg ggttaagtcc cgcaacgagc

[0090] gcaaccctta tcctttgttg ccagcgattc ggtcgggaac tcaaaggaga ctgccggtga

[0091] taaaccggag gaaggtgggg atgacgtcaa gtcatcatgg cccttacgac cagggctaca

[0092] cacgtgctac aatggcgcat acaaagagaa gcgacctcgc gagagcaagc ggacctcata

[0093] aagtgcgtcg tagtccggat tggagtctgc aactcgactc catgaagtcg gaatcgctag

[0094] taatcgtgaa tcagaatgtc acggtgaata cgttcccggg ccttgtacac accgcccgtc

[0095] acaccatggg agtgggttgc aaaagaagta ggtagcttaa ccttcgggag ggcgcttacc

[0096] actttgtgat tcatgactgg ggtg

[0097] The sequence was searched and compared with the taxonomically united 16S rRNA gene database in the EZBioCloud database, and a phylogenetic tree based on the complete 16S rDNA sequence was constructed using the Neighbor-Joining method using Mega11.0 software (see Figure 6 ), and DT01 was identified as belonging to the genus Jejubacter.

[0098] Based on the above identification results, the DT01 strain is a new Jeju bacillus, named Jejubacter sp. DT01. The strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No. 26777.

[0099] Example 2: Screening of DT01 IM3 strain

[0100] (1) The frozen bacterial liquid of the original strain Jeju bacillus DT01 was streaked on an LB plate without antibiotics, cultured in a 37°C incubator for 12 hours, and a single clone was picked and inoculated into a shaking tube containing 5 ml LB and 4 g / L isobutanol, cultured in a 37°C shaker for 24 hours, and the bacterial liquid was transferred to 4 g / L isobutanol for 10 consecutive rounds. The isobutanol concentration was set to 6 g / L, and the bacterial liquid was transferred from LB containing 4 g / L isobutanol to LB containing 6 g / L isobutanol, cultured in a 37°C shaker for 24 hours and transferred for 10 consecutive rounds. The isobutanol concentration was set to 7 g / L, and the bacterial liquid was transferred from LB containing 6 g / L isobutanol to LB containing 7 g / L isobutanol, cultured in a 37°C shaker for 48 hours and transferred for 10 consecutive rounds. The isobutanol concentration was set to 8 g / L, and the bacterial solution was transferred from LB containing 7 g / L isobutanol to LB containing 8 g / L isobutanol. The culture was incubated in a shaker at 37°C for 48 h and the transfer was repeated for 10 rounds.

[0101] The LB antibiotic-free plate medium is composed of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar, and the rest is water;

[0102] The LB medium consists of: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride;

[0103] (2) The final round of enriched bacterial solution obtained in step (1) was subjected to plate colony screening. The mixed bacterial solution was diluted and spread on LB solid medium containing 8 g / L isobutanol. After culturing at 37°C for 24 h, a relatively large single clone (such as Figure 7 ), followed by a growth test.

[0104] (3) The microorganisms separated in step (2) were cultured in LB medium containing 8 g / L isobutanol. The strain with the best growth ability under 8 g / L isobutanol was selected and determined as the most ideal mutant, which was named Jejubacter sp.DT01 IM3 and deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee (CGMCC) with a deposit number of CGMCC No.28463.

[0105] Example 3: Growth test of DT01 IM3 tolerating higher alcohols

[0106] The DT01 strain and DT01 IM3 were tested for their tolerance to isobutanol and other higher alcohols. LB plates were streaked from the frozen bacteria of DT01 and DT01 IM3 to obtain single clones. Single clones were picked and placed in LB medium and cultured at 37°C with a shaker at 220 rpm for 12 hours to serve as seed liquid. The seed liquid was then inoculated into LB liquid medium containing different higher alcohols at an inoculum volume of 1% (volume ratio), and cultured in LB containing isobutanol, n-butanol, and isopentanol for different time periods, and the OD values ​​were measured. 600 , as follows:

[0107] (1) 8 g / L isobutanol was added to the culture medium and cultured at 37°C for 4 h, 8 h, 12 h, 24 h, and 48 h. The OD 600 Situation Figure 8 The results showed that DT01 IM3 could grow well in the medium with an isobutanol concentration of 8 g / L compared with DT01.

[0108] (2) 8 g / L n-butanol and 4 g / L isoamyl alcohol were added to the culture medium to perform growth tests with other higher alcohols. After culturing at 37°C for 4 h, 8 h, 12 h, 24 h, and 48 h, the OD 600 Situation Fig. 9 and Fig.10 shown.

[0109] The results show that DT01 IM3 can tolerate not only isobutanol, butanol up to 8 g / L, and isoamyl alcohol up to 4 g / L, and can tolerate higher alcohols including but not limited to isobutanol.

[0110] Example 4: Comparison of isobutanol production by Jejubacter sp. DT01 and DT01 IM3 in high-salt non-sterile fermentation medium and high-salt sterile fermentation medium

[0111] Construction of isobutanol production plasmid:

[0112] (1) Acetolactate synthase (AlsS) encoding gene (Gene ID: 936852) was obtained from Bacillus subtilis by PCR. pUC19 was selected as the vector plasmid, on which there was ColE1 ori replicon and ampicillin resistance gene. 20bp overlap was introduced as a linker at both ends of the AlsS gene by primers, and the AlsS gene was connected to the pUC19 vector using Gibson seamless cloning enzyme. 10μL of the ligation product was transferred into 100μL JM109 competent medium, ice bathed for 30min, heat shocked at 42℃ for 90s, placed on ice for 2min, and added with 1ml LB to recover at 37℃ for 45min. Centrifuged at 5000rpm for 2min, all coated on Amp resistance plates, and placed in a 37℃ incubator for 12h. Select colonies on the plate for colony PCR verification, use specific primers to verify whether the joint is successfully connected, select PCR products with correct band size for sequencing, and inoculate single colonies with correct sequencing results into 5ml LB containing 5μL Amp for overnight culture. After extracting the plasmid, it is named pUC19-AlsS.

[0113] (2) The acetolactate isomerase (IlvC) encoding gene (Gene ID: 948286) and acetohydroxyacid dehydratase (IlvD) encoding gene (Gene ID: 948277) were obtained from Escherichia coli MG1655 by PCR. Using pUC19-AlsS as a template plasmid, a 20 bp overlap was introduced as a linker by primers, and the IlvC gene and IlvD gene were connected to the pUC19-AlsS vector using Gibson seamless cloning enzyme. 10 μL of the ligation product was transferred into 100 μL JM109 competent medium, ice bathed for 30 minutes, heat-shocked at 42°C for 90 seconds, placed on ice for 2 minutes, and 1 ml of LB was added to recover at 37°C for 45 minutes. Centrifuged at 5000 rpm for 2 minutes, all coated on Amp-resistant plates, and placed in a 37°C incubator for 12 hours. Select colonies on the plate for colony PCR verification, use specific primers to verify whether the joint is successfully connected, select PCR products with correct band size for sequencing, and inoculate single colonies with correct sequencing results into 5 ml LB containing 5 μL Amp for overnight culture. After extracting the plasmid, it is named pUC19-AlsS-IlvC-IlvD.

[0114] (3) Exogenous synthesis of ketoisovalerate decarboxylase (Kivd) encoding gene (Gene ID: 61109570) and alcohol dehydrogenase (AdhA) encoding gene (Gene ID: 4989476). Using pUC19-AlsS-IlvC-IlvD as the template plasmid, the primers introduced a 20bp overlap as a linker, and the Kivd gene and AdhA gene were connected to the pUC19-AlsS-IlvC-IlvD vector using Gibson seamless cloning enzyme. 10μL of the ligation product was transferred into 100μL JM109 competent medium, ice bathed for 30min, heat shocked at 42℃ for 90s, placed on ice for 2min, and added with 1ml LB to recover at 37℃ for 45min. Centrifuged at 5000rpm for 2min, all coated on Amp resistant plates, and placed in a 37℃ incubator for 12h. Select colonies on the plate for colony PCR verification, use specific primers to verify whether the connector is successfully connected, select PCR products with correct band size for sequencing, and inoculate single colonies with correct sequencing results into 5 ml LB containing 5 μL Amp for overnight culture. After extracting the plasmid, it is named pUC19-AlsS-IlvC-IlvD-Kivd-AdhA.

[0115] (4) Using the pUC19-AlsS-IlvC-IlvD-Kivd-AdhA plasmid as a template, PCR was performed with primers to replace the promoter, and the promoter on the original plasmid was replaced with the gadA60 promoter (gene sequence:

[0116] The plasmid with the replaced promoter was named pDT192. This expression vector is the isobutanol production plasmid. Fig.11 shown.

[0117] The pDT192 plasmid was transferred into DT01 and DT01 IM3, respectively. The transformation was performed by electroporation at a voltage of 2600V, and the culture was revived at 37°C for 1 hour. All the clones were spread on Amp plates to construct strains DT01 / pDT192 and DT01 IM3 / pDT192, respectively. Three single clones were picked from each Amp plate and inoculated into LB medium containing Amp. The culture was incubated at 37°C and 220rpm in a shaker for 12 hours to obtain 6 tubes of seed solution as a replicate group. The seed solution was transferred at a ratio of 1% to high-salt non-sterile M9 fermentation medium and high-salt sterile M9 fermentation medium for fermentation and culture to produce isobutanol.

[0118] Preparation of high salt non-sterile M9 fermentation medium (130ml):

[0119] Weigh 1.456g 5×M9 Mix, 0.65g Yeast Extract, 7.8g NaCl, dissolve in distilled water, and dilute to 117mL. Take 13mL of 40% glucose solution sterilized at 115℃ for 15min, 130μL 1M MgSO 4 Solution, 130 μL 0.1M CaCl 2 Solution: add 130μL 10mg / mL vitamin B1 solution and 130μL Amp to 117mL M9 saline solution and mix well.

[0120] Preparation of high salt sterilized M9 fermentation medium (130ml):

[0121] Weigh 1.456g 5×M9 Mix, 0.65g Yeast Extract, 7.8g NaCl and dissolve in distilled water, and dilute to 117mL. Sterilize at 121℃ for 20min. Take 13mL of 40% glucose solution sterilized at 115℃ for 15min, 130μL of 1MM MgSO 4 Solution, 130 μL 0.1 M CaCl 2 Solution: add 130 μL of 10 mg / mL vitamin B1 solution and 130 μL of Amp to 117 mL of sterilized M9 saline solution and mix well.

[0122] Each experiment was repeated three times, and each fermentation bottle was filled with 20 mL of fermentation medium. No sugar source and nitrogen source needed to be supplemented during the fermentation process.

[0123] Fermentation conditions: 30°C, 220 rpm; 1 mL sample was taken at 12 h, 24 h, 36 h and 48 h.

[0124] Sample processing: First, 200 μL of bacterial solution was taken from each sample and OD was measured using a microplate reader (BioTek Cytation 3). 600 The remaining bacterial solution was centrifuged at 12000rpm for 10 minutes. 100μL of supernatant was added to the sleeve of the gas phase vial, and then 100μL of 1g / L n-pentanol (final concentration of 0.5g / L) was added as the internal standard for gas chromatography detection. The cap of the gas phase vial was screwed on and fully shaken.

[0125] Preparation of standard: Add 160 μL of water to the cannula of the gas phase vial, and add 20 μL of 10 g / L n-pentanol (final concentration is 1 g / L) and 20 μL of 10 g / L isobutyl alcohol (final concentration is 1 g / L) respectively. Screw on the cap of the gas phase vial and shake it thoroughly.

[0126] Gas chromatography detection procedure: An A91 gas chromatograph (GC) and a DB-FFAP capillary column (30 m × 0.32 mm × 0.2 μm; Agilent Technologies) from Changzhou Pannuo Instrument Co., Ltd. were used for quantitative determination of isobutanol. The GC column temperature was initially maintained at 78 °C for 1.5 min; then, it was increased to 94 °C at a gradient of 40 °C per minute and maintained for 0.2 min; it was increased to 100 °C at a gradient of 10 °C per minute and maintained for 0.2 min; it was increased to 235 °C at a gradient of 115 °C per minute and maintained for 0.5 min. The carrier gas was nitrogen with a split ratio of 50:1, and the injector and detection temperatures were maintained at 250 °C and 280 °C, respectively. The injector volume was 0.2 μL, and the injection was performed using a microinjector produced by Agilent Technologies.

[0127] The results are as follows Fig.12 As shown in the figure, after 48 hours of fermentation, the yields of isobutanol produced by DT01 / pDT192 and DT01 IM3 / pDT192 using glucose in high-salt non-sterile M9 fermentation medium were 4.53 g / L and 8.28 g / L, respectively. The results showed that the isobutanol yield obtained by DT01IM3 / pDT192 under high-salt non-sterile conditions was higher than that of DT01 / pDT192. On the one hand, it shows that DT01 IM3 can increase the yield of higher alcohols as a base strain compared with DT01, and it also proves the feasibility of non-sterile fermentation production of host DT01 IM3 in a high-salt environment.

[0128] The results of high salt sterilization M9 fermentation to produce isobutanol are as follows Fig.13 As shown, after 48 hours of fermentation, the yields of isobutanol produced by DT01 / pDT192 and DT01 IM3 / pDT192 using glucose in high-salt sterilized M9 fermentation medium were 5.45 g / L and 9.28 g / L, respectively.

[0129] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A Jeju bacillus, It is characterized in that The Jejubacter sp. DT01 IM3 is specifically described, and its deposit number is CGMCC No. 28463.

2. Use of Jejubacter sp. DT01 IM3 according to claim 1.

3. The use according to claim 2, It is characterized in that It is used in the production of higher alcohols.

4. The use according to claim 3, It is characterized in that The higher alcohols include, but are not limited to, n-propanol, isopropanol, n-butanol, isobutanol, n-pentanol, and isopentanol.

5. The use according to claim 2, It is characterized in that It is used in non-sterile fermentation in high-salt medium.

6. The use according to claim 5, It is characterized in that The salt concentration in the high-salt culture medium is no higher than 120 g / L.

7. The use according to claim 2, It is characterized in that The invention is used as a base bacteria for constructing a strain for producing higher alcohols.

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