Recombinant agrobacterium, preparation method thereof and microbial polysaccharide

Abstract

The invention belongs to the technical field of microbial oil displacement, and discloses a salt-resistant / temperature-resistant xanthan gum-producing recombinant agrobacterium and a preparation method thereof. The invention relates to a salt-resistant / temperature-resistant xanthan gum-producing recombinant agrobacterium, which is selected from any one of the following components: a) the recombinant agrobacterium comprises a xanthan gum-producing DNA fragment with a nucleotide sequence as shown in SEQ ID No.1, a salt-resistant DNA fragment with a nucleotide sequence as shown in SEQ ID No.3 and / or a temperature-resistant DNA fragment with a nucleotide sequence as shown in SEQ ID No.5; and b) the recombinant agrobacterium expresses a xanthan gum-producing peptide fragment with an amino acid sequence shown as SEQ ID No.2, a salt-resistant peptide fragment with an amino acid sequence shown as SEQ ID No.4 and / or a temperature-resistant peptide fragment with an amino acid sequence shown as SEQ ID No.6. The recombinant agrobacterium provided by the invention is high in xanthan gum yield, and the fermentation liquor containing the product xanthan gum has good zero shear viscosity.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial enhanced oil recovery. More specifically, the present invention relates to a salt / temperature-tolerant recombinant Agrobacterium producing xanthan gum, a preparation method thereof, and a microbial polysaccharide. Background Art

[0002] Xanthan gum (XG), also known as gum hansen and xanthomonas campestris gum, is an acidic polysaccharide. At present, it is mainly obtained by aerobic deep fermentation of Xanthomonas campestris using carbohydrates such as corn starch and sucrose as the main raw materials. The basic structure of xanthan gum is a repeating unit composed of three monosaccharides, namely glucose, mannose, and glucuronic acid, with a ratio of 2:2:1. Due to its unique structure, it has good thickening, suspending, and emulsifying stability, and has become a multifunctional microbial polysaccharide with great commercial benefits and potential value, playing an important role in the preparation of oil displacement agents in the microbial enhanced oil recovery technology in oil exploitation.

[0003] However, due to the limiting factors of the microbial enhanced oil recovery technology itself, and most oil fields having poor geological conditions and great development difficulties, there are still some problems to be solved in the application of the microbial enhanced oil recovery technology in oil fields. For example, the production process of xanthan gum mainly uses microbial fermentation, including a series of processes such as seed culture, strain fermentation, and post-treatment of the fermentation broth. However, the yield of existing strains is limited, affecting its application and popularization. Moreover, even if microorganisms producing xanthan gum with increased yield are developed, due to environmental limitations, the strains are difficult to survive in some extreme environments and cannot adapt to the oil field environment.

[0004] In view of this, there is an urgent need to provide a solution to improve the yield of xanthan gum and be applicable in extreme environments such as high salt / high temperature. Summary of the Invention

[0005] In order to solve at least one or more of the above-mentioned technical problems, embodiments of the present invention provide a salt-tolerant recombinant Agrobacterium producing xanthan gum, selected from any one of the following: a) the recombinant Agrobacterium contains a DNA fragment producing xanthan gum with the nucleotide sequence shown in SEQ ID No.1 and a salt-tolerant DNA fragment with the nucleotide sequence shown in SEQ ID No.3; b) the recombinant Agrobacterium expresses a peptide segment producing xanthan gum with the amino acid sequence shown in SEQ ID No.2 and a salt-tolerant peptide segment with the amino acid sequence shown in SEQ ID No.4.

[0006] According to an embodiment of the present application, the recombinant Agrobacterium producing xanthan gum with salt tolerance is constructed by using a recombinant Agrobacterium with a DNA fragment for producing xanthan gum from Xanthomonas campestris as a starting strain and implanting a salt-tolerant DNA fragment from Bacillus halodurans.

[0007] The embodiments of the present invention also provide a recombinant Agrobacterium producing xanthan gum with temperature tolerance, selected from any one of the following: a) the recombinant Agrobacterium contains a DNA fragment for producing xanthan gum with the nucleotide sequence shown in SEQ ID No.1 and a temperature-tolerant DNA fragment with the nucleotide sequence shown in SEQ ID No.5; b) the recombinant Agrobacterium expresses a peptide segment for producing xanthan gum with the amino acid sequence shown in SEQ ID No.2 and a temperature-tolerant peptide segment with the amino acid sequence shown in SEQ ID No.6.

[0008] According to an embodiment of the present application, the recombinant Agrobacterium producing xanthan gum with temperature tolerance is constructed by using a recombinant Agrobacterium with a DNA fragment for producing xanthan gum from Xanthomonas campestris as a starting strain and implanting a temperature-tolerant DNA fragment from Bacillus thermophilus.

[0009] The embodiments of the present invention also provide a recombinant Agrobacterium producing xanthan gum with salt and temperature tolerance, selected from any one of the following: a) the recombinant Agrobacterium contains a DNA fragment for producing xanthan gum with the nucleotide sequence shown in SEQ ID No.1, a salt-tolerant DNA fragment with the nucleotide sequence shown in SEQ ID No.3, and a temperature-tolerant DNA fragment with the nucleotide sequence shown in SEQ ID No.5; b) the recombinant Agrobacterium expresses a peptide segment for producing xanthan gum with the amino acid sequence shown in SEQ ID No.2, a salt-tolerant peptide segment with the amino acid sequence shown in SEQ ID No.4, and a temperature-tolerant peptide segment with the amino acid sequence shown in SEQ ID No.6.

[0010] According to an embodiment of the present application, the recombinant Agrobacterium producing xanthan gum with salt and temperature tolerance is constructed by using a recombinant Agrobacterium with a DNA fragment for producing xanthan gum from Xanthomonas campestris as a starting strain and implanting a salt-tolerant DNA fragment from Bacillus halodurans and a temperature-tolerant DNA fragment from Bacillus thermophilus.

[0011] The embodiments of the present invention also provide a microbial polysaccharide, which is prepared by fermentation of the aforementioned recombinant Agrobacterium.

[0012] An embodiment of the present invention also provides a method for preparing salt-tolerant recombinant Agrobacterium, which includes: first implanting a DNA fragment producing xanthan gum into Agrobacterium through a recombinant plasmid to obtain recombinant Agrobacterium Agrobacterium-XG; obtaining a salt-tolerant DNA fragment of Bacillus halodurans; implanting the salt-tolerant DNA fragment into recombinant Agrobacterium Agrobacterium-XG through a recombinant plasmid to obtain salt-tolerant recombinant Agrobacterium Salt-AXG.

[0013] An embodiment of the present invention also provides a method for preparing temperature-tolerant recombinant Agrobacterium, which includes: first implanting a DNA fragment producing xanthan gum into Agrobacterium through a recombinant plasmid to obtain recombinant Agrobacterium Agrobacterium-XG; obtaining a temperature-tolerant DNA fragment of Bacillus thermophilus; implanting the temperature-tolerant DNA fragment into recombinant Agrobacterium Agrobacterium-XG through a recombinant plasmid to obtain temperature-tolerant recombinant Agrobacterium Heat-AXG.

[0014] An embodiment of the present invention also provides a method for preparing salt-tolerant and temperature-tolerant recombinant Agrobacterium, which includes: first implanting a DNA fragment producing xanthan gum into Agrobacterium through a recombinant plasmid to obtain recombinant Agrobacterium Agrobacterium-XG; obtaining a salt-tolerant DNA fragment of Bacillus halodurans; obtaining a temperature-tolerant DNA fragment of Bacillus thermophilus; implanting the salt-tolerant DNA fragment and the temperature-tolerant DNA fragment into recombinant Agrobacterium Agrobacterium-XG through a recombinant plasmid to obtain salt-tolerant and temperature-tolerant recombinant Agrobacterium SH-AXG.

[0015] The recombinant Agrobacterium provided above has stable genetic traits, high xanthan gum yield, and can be applied in extreme environments such as high salt / high temperature. Its fermentation broth containing xanthan gum has good zero-shear viscosity. Detailed implementation manners

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

[0017] It should be understood that the terms "include" and "comprise" used in the description and claims of the present invention indicate the presence of the described features, wholes, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

[0018] It should also be understood that the terms used in the specification of the present invention are merely for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the specification and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include the plural forms. It should be further understood that the term "and / or" used in the specification and claims of the present invention refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0019] As used in this specification and the claims, the term "if" may be construed, depending on the context, as "when", "once", "in response to determining", or "in response to detecting". Similarly, the phrase "if determined" or "if [the described condition or event] is detected" may be construed, depending on the context, as meaning "once determined", "in response to determining", "once [the described condition or event] is detected", or "in response to detecting [the described condition or event]".

[0020] In the present invention, the materials or reagents used, unless otherwise specified, can be obtained from conventional commercial channels.

[0021] In the present invention, a xanthan gum-producing DNA fragment from a Xanthomonas campestris strain was obtained by means of CRISPR / Cas technology, implanted into Agrobacterium through a recombinant plasmid to obtain Agrobacterium with high yield, and this recombinant Agrobacterium strain was named Agrobacterium-XG. Source of Xanthomonas campestris: Wuhan Huizao Biotechnology Co., Ltd., product number: HZB140304.

[0022] In the present invention, salt-tolerant DNA was obtained from Bacillus halodurans, and its gene fragment was transferred to Agrobacterium-XG to obtain salt-tolerant Agrobacterium, and this salt-tolerant recombinant Agrobacterium strain was named Salt-AXG.

[0023] Bacillus halodurans is a microorganism found in high-salt oil reservoirs. After external cultivation, it was proved to be Bacillus halodurans; in high-salt oil reservoirs, the salt tolerance salinity is as high as 200,000 - 230,000 ppm, the brine is mainly NaCl, followed by CaCl2 and MgCl2, and a small amount of other salts. Bacillus halodurans is a facultative anaerobic microorganism, which is relatively easy to cultivate externally. The relatively strong strains cultivated after several rounds of subculture were first used for salt tolerance tests, and the strains with a survival rate exceeding 90% at a salinity of 200,000 ppm were reserved. A total of 8 strains were screened out after 6 rounds of subculture.

[0024] The present invention obtains temperature-resistant DNA from Bacillus thermophilus, transfers its gene fragment to Agrobacterium-XG to obtain temperature-resistant Agrobacterium, and names the temperature-resistant recombinant Agrobacterium strain Heat-AXG.

[0025] Bacillus thermophilus is a commercially available bacterial strain. Mainly targeting those with a temperature resistance of up to 75°C, after culturing, strains with a survival rate exceeding 80% at 75°C are screened and reserved. A total of 12 strains are screened out after 3 rounds of subculture.

[0026] The present invention obtains salt-resistant DNA from Bacillus halodurans and heat-resistant DNA from Bacillus thermophilus, and simultaneously transfers their gene fragments to Agrobacterium-XG to obtain salt-resistant and temperature-resistant Agrobacterium, and names the salt-resistant and temperature-resistant recombinant Agrobacterium strain SH-AXG.

[0027] The method for constructing a recombinant Agrobacterium strain includes:

[0028] The first step S1, culturing the Xanthomonas campestris strain:

[0029] Inoculate a loop of Xanthomonas campestris strain into 5 mL of YT medium (Order NO.A507016, Sangon Biotech (Shanghai) Co., Ltd.), culture overnight at 30°C until OD 600 = 0.6, take a loop of colonies into YT liquid culture medium to obtain a bacterial suspension.

[0030] Take 2.0 mL of the bacterial suspension into a clean and sterile polypropylene tube, centrifuge at 10000 r / min for 5 min, discard the supernatant, and store the spare bacteria at -20°C.

[0031] The second step S2, extracting the DNA of the Xanthomonas campestris strain by the SDS / CTBA method:

[0032] Take the spare bacteria obtained in the first step, add 567 μL of TE buffer, resuspend it by repeatedly pipetting with a pipette, add 30 μL of 10% SDS, mix well, add 3 μL of 20 mg / mL proteinase K, mix well, incubate at 37°C for 1 h to digest the histone bound to DNA.

[0033] Add 100 μL of 5 mol / L NaCl, mix well, then add 80 μL of CTAB / NaCl, mix well, incubate at 65°C for 10 min. CTAB / NaCl can form complexes with proteins and polysaccharides but will not precipitate nucleic acids, thereby purifying the DNA.

[0034] Add an equal volume of chloroform / isoamyl alcohol, mix well, extract DNA, then centrifuge at 15,000 r / min for 10 min. Transfer the supernatant to a new centrifuge tube. The DNA is in the supernatant.

[0035] Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) to the supernatant, mix well, and extract DNA for the second time. Then centrifuge at 15,000 r / min for 10 min. Keep the supernatant. The DNA after the second extraction is still in the supernatant.

[0036] Add 0.6 times the volume of isopropanol to the supernatant, mix well to precipitate DNA, centrifuge at 15,000 r / min for 10 min, collect the DNA precipitate, and discard the supernatant. Wash the DNA precipitate by centrifugation with 70% ethanol. Ethanol will remove the water molecules around the DNA, causing the DNA to lose water and polymerize more easily, resulting in the secondary precipitation of DNA. Discard the supernatant and keep the DNA precipitate. Dry it at room temperature, dissolve the DNA with 100 μL of TE, add RNase A with a final concentration of 20 μg / mL for digestion, and store it at -20°C. TE helps dissolve and stabilize the DNA, and RNase A can digest RNA molecules, making the finally extracted DNA completely free of any RNA.

[0037] The third step S3, restriction enzyme digestion of the DNA of Xanthomonas campestris pv. campestris strains:

[0038] The extracted DNA is digested with EcoRI. The EcoRI restriction enzyme can specifically recognize the GAATTC sequence and cut this sequence between G and A. The ends of the cut small fragments are sticky ends with a 5'-end overhang.

[0039] Specifically, in a 20 μL reaction volume, add in sequence:

[0040] 8.5 μL of ddH2O,

[0041] 2 μL of 10×Buffer restriction enzyme buffer, 8 μL of DNA, 1.5 μL of EcoRI, mix well. After digestion at 37°C for 5 h, treat at 65°C for 10 min to terminate the restriction enzyme reaction and obtain crude DNA.

[0042] The components of the 10×Buffer restriction enzyme buffer include:

[0043] 50 mmol / L Tris / HCl (pH 7.5), 10 mmol / L MgCl2, 100 mmol / L NaCl, 0.02% Triton X-100, 0.1 mg / mL BSA.

[0044] The fourth step S4, TA cloning to construct a gene library:

[0045] The above-extracted crude DNA was uniformly fragmented using the Klenow fragmentase (Cat.No.RK20525) provided by ABclonal, and then the ends of the above DNA fragments were repaired using Taq DNA polymerase (Cat.No.RK20600). After the fragmentation, end repair, and A-tailing steps, the structure of the DNA fragments became a structure with an A-tail at the 3' end.

[0046] The end-repaired DNA fragments and the adapters were ligated by TA cloning under the action of T4 DNA ligase (Cat.No.RK21500).

[0047] After the adapter ligation was completed, the Y-shaped adapter was a complete long adapter with an Index, and the library yield met the requirements for on-machine sequencing, so library amplification could be omitted; if a truncated adapter without an Index was used, the library still needed to be enriched using a Primer with an Index sequence before on-machine sequencing. When library amplification was required, a Jieli Mei QX96M real-time fluorescence quantitative PCR instrument was used for amplification.

[0048] Step S5, transforming Agrobacterium:

[0049] Plasmid minipreparation was carried out according to the instructions of the endotoxin-free plasmid miniprep midiprep kit (Tiangen, DP118). The plasmid pRK293 was selected, and a 10-20 kb exogenous DNA fragment was inserted at the single SalI cleavage site of pRK293, and the recombinant plasmid was used to transform Agrobacterium.

[0050] Agrobacterium was cultured until OD 600 = 0.6, and the transformation solution was prepared: The Agrobacterium strain stored in the ultra-low temperature freezer was streaked on a solid LB culture dish containing 50 mg / L and cultured in a 28°C constant temperature incubator for 48 h for resuscitation. A single colony was picked and inoculated into a liquid LB medium containing 50 mg / L and cultured on a shaker at 28°C and 200 rpm for 24 h (temporarily stored at 4°C after taking out). 2 ml of the bacterial solution was transferred into 300 ml of liquid LB medium and cultured until OD 600 = 0.6.

[0051] Transfer all the bacterial solution into a 50 ml centrifuge tube, place it in an ice bath for 30 min, and at the same time, place 0.02 mol / L CaCl2 in an ice bath. Put the centrifuge tube into a 4 °C centrifuge, centrifuge at 5000 x g for 5 min, pour off the supernatant, and dry the residual liquid with sterilized absorbent paper. Add 2 ml of pre-cooled 0.02 mol / L CaCl2 to each centrifuge tube to resuspend the Agrobacterium, and place it on ice for 20 min. Centrifuge at 4 °C and 5000 x g for 5 min, remove the supernatant, add 2 ml of CaCl2 + 15% glycerol mixture, mix well with a 1 ml pipette, collect the bacterial solutions in multiple centrifuge tubes, freeze them on ice, then place them at 4 °C for 24 h, and aliquot and store them in a -80 °C ultra-low temperature refrigerator.

[0052] Mix the vector carrying the DNA fragment with Agrobacterium to obtain a transformation solution. First, refrigerate the transformation solution, then perform a heat shock at 42 °C, and then refrigerate it again. Add the transformation solution to the medium for recovery culture.

[0053] The sixth step S6, screening the strong colonies producing xanthan gum:

[0054] Cultivate the transformed Agrobacterium-XG until OD 600 = 0.6, inoculate it on a YT solid plate and culture it at 27 °C for 24 h to screen for strong colonies. Denote the Agrobacterium-XG with the highest yield among the strong colonies in each batch as Agrobacterium-XG-1, Agrobacterium-XG-2, and Agrobacterium-XG-3 of the colonies in that batch. Cultivate them separately in YT liquid medium until OD 600 = 0.6, measure the xanthan gum concentration in each bacterial solution, and select the group with the best concentration for continuous isolation and culture until a stable high-yield strain is obtained.

[0055] After obtaining the recombinant Agrobacterium producing xanthan gum, sequence the part of the sequence involved in xanthan gum production to obtain a DNA fragment producing xanthan gum with the nucleotide sequence shown in SEQ ID No.1, and sequence the part of the protein involved in xanthan gum production to obtain a peptide segment producing xanthan gum with the amino acid sequence shown in SEQ ID No.2.

[0056] The recombinant Agrobacterium-XG is constructed by using Agrobacterium as the starting strain and implanting a DNA fragment producing xanthan gum from Xanthomonas campestris pv. campestris.

[0057] DNA fragment producing xanthan gum:

[0058]

[0059] Peptides producing xanthan gum:

[0060] MNNEQAAEILQDIFQHAVNSARAGPVTLANLPEKPRGRCVVIGAGKASAAMAAAVDAAWPDVAVSGVVVTRYGYAVPAGRIRIIEAAHPVSDAMSEVAAMLIVETLRGLTADDLVLALISGGGSALMALPAPGLTLADKQTITRALLHSGASIKEMNLVRRHLSAVKGGKLATMAQPARIVSLIISDVPGDNPTDVASGPTVADNSAPRDALRVLQRYGIAIPKPVSERLNQPAGPVENAATGEVRLIATPAMALAAAALAARQHGFTPLILGDAIEGESREVAVVMAGMAKSAKQYGHPISGPAVLLSGGETTVTVNNTQPGKGGRNTEFLLSLACALQGEHGIWAMAGDSDGIDGTEDAAGAIVFPDTLARGKLSGLNAVQYLDGHDSYCYFHALNDLLITGPTLTNVNDIRAILIA(SEQ ID No.2).

[0061] Using a similar method described above, the recombinant Agrobacterium Salt-AXG was constructed by taking the recombinant Agrobacterium Agrobacterium-XG as the starting strain and implanting a salt-tolerant DNA fragment from Bacillus halodurans.

[0062] Salt-tolerant DNA fragment:

[0063]

[0064] Salt-tolerant peptide segment:

[0065] MMSSPTPRLRWGILGAAKINQRLVPAFQKSATADLRAIASRSDDKAKQAAAEAGIARGVGSYEALLEDPDIDAIYIPLPNHLHAEWTRKAADAGKHILCEKPLCPDAADAAALIAYCRAKNVRLMDGFMWPHHPRTAKIRQMLDAGAIGKVQRVNTAFTFNLNPLNDSNIRMHRNMGGGALLDVGCYCVYGIRWAFQAEPVKVYAEAKLLNDVDVSLSAMLWFADGRTAFLDTGFVSPLRGWLEIVGESGTIHIPDLWLPSADAAFTLTSDEQPAQTITVPGHDQIVCMLDDFAAAVHEQREAWPNPDEAVKSLKVLNAIDRSARSGQIEFVN (SEQ ID No.4).

[0066] Using a similar method described above, the recombinant Agrobacterium Heat-AXG was constructed by taking the recombinant Agrobacterium Agrobacterium-XG as the starting strain and implanting a DNA fragment with high temperature tolerance from Bacillus thermophilus.

[0067] DNA fragment with high temperature tolerance:

[0068] gtgaatatgttagtgtcgatgaaggacatgcttcagcatgccctgcgggacggctacgccgtcggtcagttcaacattaataatctggaatgggtcggcgccgtattaagcaccgcccagcagtgccgctcaccggttattttgggggtgtccggcggcacggttaagcatatgctcgggttaaaatgtattcatgacattgtggttaacgccatggagtatttgcatattgatgttccggtggcgctgcatctggatcatggcacctcccgggaggcctgcgaagcggcgatcgccgccggcttcagttccattatgtttgatggctcgcatctgccgttcagggaaaacctggccattactcgccacctggtgacgctggcccacagcaaaggtatctctgtggaggccgaactggggaccatcgccggcagtgaagacggcattgtcaattccgaagtcatctacgccgatccgcaggagtgctacaccctggtgacagaaacccaggtggattgcctcgccgccgcgctgggctccacccatggtctgtataaaggtaaagccaggctgggatttaccgagatgaaagccattgccgagcaggtgaaggttccgctggtgctgcatggcggcaccggtattgccgatgaggatatgcgccgggcgattgcctgcggcaccgccaagattaacgttaataccgaaaatatgtacgcctggtgccaacaggtgaaagcaattttcgccgccgacaccggacacgatgtgaacgatccgcggaaagtcatcgcccagggactgcagccggtacgcgagatgattgcccgccgcatggcgctctttggctcagagcagcgctattga(SEQ ID No.5).

[0069] Peptide segments with high temperature resistance:

[0070] MNMLVSMKDMLQHALRDGYAVGQFNINNLEWVGAVLSTAQQCRSPVILGVSGGTVKHMLGLKCIHDIVVNAMEYLHIDVPVALHLDHGTSREACEAAIAAGFSSIMFDGSHLPFRENLAITRHLVTLAHSKGISVEAELGTIAGSEDGIVNSEVIYADPQECYTLVTETQVDCLAAALGSTHGLYKGKARLGFTEMKAIAEQVKVPLVLHGGTGIADEDMRRAIACGTAKINVNTENMYAWCQQVKAIFAADTGHDVNDPRKVIAQGLQPVREMIARRMALFGSEQRY(SEQIDNo.6).

[0071] Using a similar method described above, the recombinant Agrobacterium SH-AXG was constructed by using the recombinant Agrobacterium Agrobacterium-XG as the starting strain and implanting the salt-tolerant DNA fragment from Bacillus halodurans and the heat-tolerant DNA fragment from Bacillus thermophilus.

[0072] According to another aspect of the present invention, there is also provided a microbial polysaccharide prepared by fermentation of the aforementioned recombinant Agrobacterium.

[0073] According to another aspect of the present invention, there is also provided a method for preparing a salt-tolerant recombinant Agrobacterium, comprising: first implanting the DNA fragment for producing xanthan gum into Agrobacterium through a recombinant plasmid to obtain the recombinant Agrobacterium Agrobacterium-XG; obtaining the salt-tolerant DNA fragment from Bacillus halodurans; implanting the salt-tolerant DNA fragment into the recombinant Agrobacterium Agrobacterium-XG through a recombinant plasmid to obtain the salt-tolerant recombinant Agrobacterium Salt-AXG.

[0074] According to another aspect of the present invention, there is also provided a method for preparing a heat-tolerant recombinant Agrobacterium, comprising: first implanting the DNA fragment for producing xanthan gum into Agrobacterium through a recombinant plasmid to obtain the recombinant Agrobacterium Agrobacterium-XG; obtaining the heat-tolerant DNA fragment from Bacillus thermophilus; implanting the heat-tolerant DNA fragment into the recombinant Agrobacterium Agrobacterium-XG through a recombinant plasmid to obtain the heat-tolerant recombinant Agrobacterium Heat-AXG.

[0075] According to another aspect of the present invention, there is also provided a method for preparing salt- and temperature-tolerant recombinant Agrobacterium, comprising: first implanting a DNA fragment producing xanthan gum into Agrobacterium through a recombinant plasmid to obtain recombinant Agrobacterium Agrobacterium-XG; obtaining a salt-tolerant DNA fragment of Bacillus halodurans; obtaining a temperature-tolerant DNA fragment of Bacillus thermophilus; and implanting the salt-tolerant DNA fragment and the temperature-tolerant DNA fragment into recombinant Agrobacterium Agrobacterium-XG through a recombinant plasmid to obtain salt- and temperature-tolerant recombinant Agrobacterium SH-AXG.

[0076] Example 1

[0077] Cultivate the strain Salt-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 = 0.6, perform centrifugation, discard the liquid part, add the precipitate part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 25 °C, pH at 6, culture for 3 days, and collect the fermentation broth.

[0078] Example 2

[0079] Cultivate the strain Salt-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 = 0.6, perform centrifugation, discard the liquid part, add the precipitate part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 25 °C, pH at 10, culture for 3 days, and collect the fermentation broth.

[0080] Example 3

[0081] Cultivate the strain Salt-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 = 0.6, perform centrifugation, discard the liquid part, add the precipitate part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 25 °C, pH at 4, culture for 3 days, and collect the fermentation broth.

[0082] Example 4

[0083] Cultivate the strain Salt-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 = 0.6, perform centrifugation, discard the liquid part, add the precipitate part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 40 °C, pH at 6, culture for 3 days, and collect the fermentation broth.

[0084] Example 5

[0085] Cultivate the strain Salt-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to fresh TY liquid medium for fermentation culture. Set the fermentation temperature at 40 °C, pH at 10, culture for 3 days, and collect the fermentation broth.

[0086] Example 6

[0087] Cultivate the strain Salt-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to fresh TY liquid medium for fermentation culture. Set the fermentation temperature at 35 °C, pH at 6, culture for 3 days, and collect the fermentation broth.

[0088] TY liquid medium (COOLABER SCIENCE&TECHNOLOGY Co.,LTD, product number PM0762).

[0089] Example 7

[0090] Cultivate the strain Salt-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to fresh TY liquid medium for fermentation culture. Set the fermentation temperature at 35 °C, pH at 10, culture for 3 days, and collect the fermentation broth.

[0091] Example 8

[0092] Cultivate the strain Salt-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to fresh TY liquid medium for fermentation culture. Set the fermentation temperature at 35 °C, pH at 4, culture for 3 days, and collect the fermentation broth.

[0093] Example 9

[0094] Cultivate the strain Heat-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to fresh TY liquid medium for fermentation culture. Set the fermentation temperature at 25 °C, pH at 6, culture for 3 days, and collect the fermentation broth.

[0095] Example 10

[0096] Cultivate the strain Heat-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 25°C, pH at 10, culture for 3 days, and collect the fermentation broth.

[0097] Example 11

[0098] Culture the strain Heat-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 25°C, pH at 4, culture for 3 days, and collect the fermentation broth.

[0099] Example 12

[0100] Culture the strain Heat-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 40°C, pH at 6, culture for 3 days, and collect the fermentation broth.

[0101] Example 13

[0102] Culture the strain Heat-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 40°C, pH at 10, culture for 3 days, and collect the fermentation broth.

[0103] Example 14

[0104] Culture the strain Heat-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 35°C, pH at 6, culture for 3 days, and collect the fermentation broth.

[0105] TY liquid medium (COOLABER SCIENCE&TECHNOLOGY Co.,LTD, product number PM0762).

[0106] Example 15

[0107] Culture the strain Heat-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to a new TY liquid culture medium for fermentation culture, set the fermentation temperature at 35°C, pH at 10, culture for 3 days, and collect the fermentation broth.

[0108] Example 16

[0109] Cultivate the strain Heat-AXG in a TY liquid culture medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to a new TY liquid culture medium for fermentation culture, set the fermentation temperature at 35°C, pH at 4, culture for 3 days, and collect the fermentation broth.

[0110] Example 17

[0111] Cultivate the strain SH-AXG in a TY liquid culture medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to a new TY liquid culture medium for fermentation culture, set the fermentation temperature at 25°C, pH at 6, culture for 3 days, and collect the fermentation broth.

[0112] Example 18

[0113] Cultivate the strain SH-AXG in a TY liquid culture medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to a new TY liquid culture medium for fermentation culture, set the fermentation temperature at 25°C, pH at 10, culture for 3 days, and collect the fermentation broth.

[0114] Example 19

[0115] Cultivate the strain SH-AXG in a TY liquid culture medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to a new TY liquid culture medium for fermentation culture, set the fermentation temperature at 25°C, pH at 4, culture for 3 days, and collect the fermentation broth.

[0116] Example 20

[0117] Cultivate the strain SH-AXG in a TY liquid culture medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 When OD = 0.6, perform centrifugation, discard the liquid part, add the precipitated part to a new TY liquid culture medium for fermentation culture, set the fermentation temperature at 40°C, pH at 6, culture for 3 days, and collect the fermentation broth.

[0118] Example 21

[0119] Cultivate the strain SH-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 = 0.6, perform centrifugation, discard the liquid part, add the precipitate part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 40 °C, pH at 10, culture for 3 days, and collect the fermentation broth.

[0120] Example 22

[0121] Cultivate the strain SH-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 = 0.6, perform centrifugation, discard the liquid part, add the precipitate part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 35 °C, pH at 6, culture for 3 days, and collect the fermentation broth.

[0122] TY liquid medium (COOLABER SCIENCE&TECHNOLOGY Co.,LTD, product number PM0762).

[0123] Example 23

[0124] Cultivate the strain SH-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 = 0.6, perform centrifugation, discard the liquid part, add the precipitate part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 35 °C, pH at 10, culture for 3 days, and collect the fermentation broth.

[0125] Example 24

[0126] Cultivate the strain SH-AXG in TY liquid medium under the conditions described in the instruction manual. When the number of cultured bacteria reaches OD 600 = 0.6, perform centrifugation, discard the liquid part, add the precipitate part to fresh TY liquid medium for fermentation culture, set the fermentation temperature at 35 °C, pH at 4, culture for 3 days, and collect the fermentation broth.

[0127] Take the fermentation broths of Examples 1 to 8, sample and measure the concentration of xanthan gum and the shear viscosity of the produced xanthan gum.

[0128] Table 1 shows the survival rate %, the concentration of xanthan gum, and the measurement results of the shear viscosity of the produced xanthan gum in Examples 1 - 8 at a salinity of 200,000 ppm.

[0129] Table 1

[0130]

[0131] As can be seen from Table 1, during the fermentation process of strain Salt-AXG: the fermentation temperature range is 25 - 40 °C, and the best effect is achieved at 35 °C. In particular, when the fermentation temperature is 35 °C, the survival rate % of strain Salt-AXG at a salinity of 200,000 ppm can reach over 94%.

[0132] Take the fermentation broths of Examples 9 to 16, and sample and measure the concentration of xanthan gum and the shear viscosity of the produced xanthan gum.

[0133] Table 2 shows the measurement results of the survival rate %, the concentration of xanthan gum, and the shear viscosity of the produced xanthan gum of Heat-AXG in Examples 9 - 16 at 75 °C.

[0134] Table 2

[0135]

[0136]

[0137] As can be seen from Table 2, during the fermentation process of strain Heat-AXG: the fermentation temperature range is 25 - 40 °C, and the best effect is achieved at 35 °C. In particular, when the fermentation temperature is 35 °C, the survival rate % of strain Heat-AXG at 75 °C can reach over 90%.

[0138] Take the fermentation broths of Examples 17 to 24, and sample and measure the concentration of xanthan gum and the shear viscosity of the produced xanthan gum.

[0139] Table 3 shows the measurement results of the survival rate % of SH-AXG in Examples 17 - 24 at a salinity of 200,000 ppm, the survival rate % at 75 °C, the concentration of xanthan gum, and the shear viscosity of the produced xanthan gum.

[0140] Table 3

[0141]

[0142] As can be seen from Table 3, during the fermentation process of strain SH-AXG: the fermentation temperature range is 25 - 40 °C, and the best effect is achieved at 35 °C. In particular, when the fermentation temperature is 35 °C, the survival rate % of strain SH-AXG at a salinity of 200,000 ppm can reach over 91%, and at the same time, the survival rate % at 75 °C can reach over 78%.

[0143] The recombinant Agrobacterium provided above has stable genetic traits and high xanthan gum production. The fermentation broth containing the product xanthan gum has good zero-shear viscosity.

[0144] Although several embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes and alternative forms may be contemplated by those skilled in the art without departing from the spirit and scope of the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The appended claims are intended to define the scope of the present invention and thus cover equivalents or alternatives within the scope of these claims.

Claims

1. A recombinant Agrobacterium that produces xanthan gum and is salt-tolerant, characterized in that, Selected from any one of the following: a) The recombinant Agrobacterium contains a xanthan gum-producing DNA fragment with the nucleotide sequence shown in SEQ ID No.1 and a salt-tolerant DNA fragment with the nucleotide sequence shown in SEQ ID No.3; b) The recombinant Agrobacterium expresses a xanthan gum-producing peptide segment with the amino acid sequence shown in SEQ ID No.2 and a salt-tolerant peptide segment with the amino acid sequence shown in SEQ ID No.

4.

2. The recombinant Agrobacterium according to claim 1, characterized in that, The recombinant Agrobacterium is constructed by using a recombinant Agrobacterium with a xanthan gum-producing DNA fragment from Xanthomonas campestris as the starting strain and implanting a salt-tolerant DNA fragment from Bacillus halodurans.

3. A recombinant Agrobacterium that can tolerate high temperature and produce xanthan gum, characterized in that, Selected from any one of the following: a) The recombinant Agrobacterium contains a xanthan gum-producing DNA fragment with the nucleotide sequence shown in SEQ ID No.1 and a temperature-tolerant DNA fragment with the nucleotide sequence shown in SEQ ID No.5; b) The recombinant Agrobacterium expresses a xanthan gum-producing peptide segment with the amino acid sequence shown in SEQ ID No.2 and a temperature-tolerant peptide segment with the amino acid sequence shown in SEQ ID No.

6.

4. The recombinant Agrobacterium according to claim 3, characterized in that, The recombinant Agrobacterium is constructed by using a recombinant Agrobacterium with a xanthan gum-producing DNA fragment from Xanthomonas campestris as the starting strain and implanting a temperature-tolerant DNA fragment from Bacillus thermophilus.

5. A recombinant Agrobacterium that is salt-tolerant and temperature-tolerant and produces xanthan gum, characterized in that, Selected from any one of the following: a) The recombinant Agrobacterium contains a xanthan gum-producing DNA fragment with the nucleotide sequence shown in SEQ ID No.1, a salt-tolerant DNA fragment with the nucleotide sequence shown in SEQ ID No.3, and a temperature-tolerant DNA fragment with the nucleotide sequence shown in SEQ ID No.5; b) The recombinant Agrobacterium expresses a xanthan gum-producing peptide segment with the amino acid sequence shown in SEQ ID No.2, a salt-tolerant peptide segment with the amino acid sequence shown in SEQ ID No.4, and a temperature-tolerant peptide segment with the amino acid sequence shown in SEQ ID No.

6.

6. The recombinant Agrobacterium tumefaciens according to claim 5, characterized in that, The recombinant Agrobacterium is constructed by using a recombinant Agrobacterium with a xanthan gum-producing DNA fragment from Xanthomonas campestris as the starting strain and implanting a salt-tolerant DNA fragment from Bacillus halodurans and a temperature-tolerant DNA fragment from Bacillus thermophilus.

7. A microbial polysaccharide, characterized in that the microbial polysaccharide is prepared by fermentation of the recombinant Agrobacterium according to any one of claims 1-6.

8. A method for preparing salt-tolerant recombinant Agrobacterium, characterized in that, Including: First, implant the xanthan gum-producing DNA fragment into Agrobacterium through a recombinant plasmid to obtain recombinant Agrobacterium Agrobacterium-XG; Obtain the salt-tolerant DNA fragment of Bacillus halodurans; Implant the salt-tolerant DNA fragment into recombinant Agrobacterium Agrobacterium-XG through a recombinant plasmid to obtain salt-tolerant recombinant Agrobacterium Salt-AXG.

9. A method for preparing temperature-resistant recombinant Agrobacterium, characterized in that, Including: First, implant the xanthan gum-producing DNA fragment into Agrobacterium through a recombinant plasmid to obtain recombinant Agrobacterium Agrobacterium-XG; Obtain the temperature-tolerant DNA fragment of Bacillus thermophilus; Implant the temperature-tolerant DNA fragment into recombinant Agrobacterium Agrobacterium-XG through a recombinant plasmid to obtain temperature-tolerant recombinant Agrobacterium Heat-AXG.

10. A method for preparing salt-tolerant and temperature-tolerant recombinant Agrobacterium, characterized in that, Including: First, implant the DNA fragment that produces xanthan gum into Agrobacterium through a recombinant plasmid to obtain recombinant Agrobacterium Agrobacterium-XG; Obtain the salt-tolerant DNA fragment of Bacillus halodurans; Obtain the temperature-tolerant DNA fragment of Bacillus thermophilus; Implant the salt-tolerant DNA fragment and the temperature-tolerant DNA fragment into recombinant Agrobacterium Agrobacterium-XG through a recombinant plasmid to obtain salt-tolerant and temperature-tolerant recombinant Agrobacterium SH-AXG.

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