Recombinant halomonas for producing hydroxyectoine as well as preparation method and application of recombinant halomonas

Abstract

The invention discloses a recombinant halomonas for producing hydroxyectoine as well as a preparation method and application of the recombinant halomonas. The recombinant halomonas disclosed by the invention does not contain an ectoine hydrolase (DoeA) gene, and can be used for expressing ectoine hydroxylase and glutamate dehydrogenase. Experiments prove that when the salt concentration of the recombinant halomonas is reduced to 1.4 M, fermentation can produce 1.02 g / L of hydroxyectoine, and compared with wild halomonas, the yield of hydroxyectoine under the salt concentration of 1.4 M is increased by 10 times. According to the recombinant halomonas disclosed by the invention, the hydroxyectoine is successfully produced under the condition that the salt concentration is reduced, chassis bacteria for efficiently producing the hydroxyectoine under the medium-salt condition are successfully constructed, and theoretical guidance is provided for further reducing the salt concentration for producing the hydroxyectoine; meanwhile, an experimental basis is provided for improving the biosynthesis of a high-added-value chemical hydroxyl ectoine.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and relates to a recombinant halomonas used for producing hydroxyectoine, a preparation method and an application thereof. Background Art

[0002] Hydroxyectoine (1,4,5,6-tetrahydro-2-methyl-5-hydroxy-4-pyrimidinecarboxylic acid, Hydroxyectoine) is a heterocyclic amino acid with a molecular formula of C 6 H 10 N 2 O 3 , the structural formula is as Formula I, the relative molecular mass is 158.16, CAS: 165542-15-4, it has almost the same chemical properties as tetrahydropyrimidine, but the hydroxyl group (-OH) gives it additional protective properties. It has important functions such as stabilizing proteins and DNA, protecting cell and tissue activity, and promoting protein folding in the body, so that cells can resist extreme environmental stresses such as hypertonicity, high temperature or dryness. Therefore, it is widely used in biomedicine, biotechnology, cosmetics and other fields.

[0003]

[0004] Halomonas salifedinae is one of the important representative bacterial groups of halophilic microorganisms. It has a scalable high cell density and carries the ectABC / D cluster in its genome. It is a host for the efficient production of hydroxyectoine. In the traditional fermentation process of Halomonas, high salt conditions can significantly improve the biosynthesis efficiency of hydroxyectoine, but there are problems such as fermentation tank corrosion and high-salt wastewater treatment. Therefore, reducing the salt concentration has a positive effect on large-scale fermentation. However, under low-salt conditions, the yield of hydroxyectoine is low, and its biosynthesis is affected by limiting factors such as ectD expression and α-ketoglutaric acid supply. In addition, Halomonas lacks a complete gene editing platform, strain transformation is limited, and the number of available genetic elements is limited. It is necessary to test and screen promoter elements of different strengths to ensure gene synergistic expression. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to prepare hydroxyectoine under the condition of reducing the salt concentration.

[0006] In order to solve the above technical problems, the present invention first provides a recombinant Halomonas, wherein the recombinant Halomonas comprises the following characteristics:

[0007] A1) does not contain ectoine hydrolase gene or contains substances that inhibit the expression of ectoine hydrolase gene or contains substances that reduce the content or activity of ectoine hydrolase;

[0008] A2) A substance containing a gene capable of expressing ectoinehydroxyectoine or a substance promoting the expression of ectoinehydroxyectoine or a substance increasing the activity or content of ectoinehydroxyectoine.

[0009] In the above-mentioned recombinant Halomonas, the substance promoting the expression of the tetrahydropyrimidine hydroxylase gene in A2) may be the pUP119 promoter, and the pUP119 promoter is b1) or b2) or b3):

[0010] b1) a DNA molecule shown in SEQ ID No. 4 in the sequence list;

[0011] b2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) and having the function of the pUP119 promoter;

[0012] b3) A DNA molecule which hybridizes with the nucleotide sequence defined in b1) or b2) under stringent conditions and has the function of the pUP119 promoter.

[0013] Furthermore, the tetrahydropyrimidine hydroxylase may be EctD.

[0014] In one embodiment of the present invention, EctD is shown as SEQ ID No. 3 in the sequence list. The EctD gene is shown as SEQ ID No. 2 in the sequence list.

[0015] The above-mentioned recombinant salt mononas may also include the following characteristics:

[0016] A3) A substance containing a substance capable of expressing a glutamate dehydrogenase (GDH) gene, a substance promoting the expression of a glutamate dehydrogenase gene, or a substance increasing the activity or content of glutamate dehydrogenase.

[0017] Specifically, the substance capable of expressing the glutamate dehydrogenase gene in A3) may be a glutamate dehydrogenase gene expression cassette.

[0018] The promoter in the expression cassette may be a pJ23119 promoter, and the pJ23119 promoter is c1) or c2) or c3):

[0019] c1) the DNA molecule shown in positions 1 to 64 of SEQ ID No. 5 in the sequence listing;

[0020] c2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in c1) and having the promoter function of pJ23119;

[0021] c3) A DNA molecule which hybridizes with the nucleotide sequence defined in c1) or c2) under stringent conditions and has the function of the pJ23119 promoter.

[0022] The glutamate dehydrogenase may be derived from Halomonas longata;

[0023] The glutamate dehydrogenase may be GDH.

[0024] In one embodiment of the present invention, GDH is shown as SEQ ID No. 6 in the sequence listing. The GDH gene is shown as positions 65-4906 of SEQ ID No. 5. The glutamate dehydrogenase gene expression cassette is shown as SEQ ID No. 5.

[0025] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0026] Specifically, more than 80% identity may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or more identity.

[0027] The stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, rinse at 50°C, 2×SSC, 0.1% SDS; may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5MNaPO4 and 1mM EDTA, rinse at 50°C, 1×SSC, 0.1% SDS; may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, rinse at 50°C, 0.5×SSC, 0.1% SDS; may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, rinse at 50°C, 0.1×SSC, 0.1% SDS; may also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, rinse at 50°C, 0.1×SSC, 0.1% SDS; The membrane can be hybridized in a mixed solution of NaPO4 and 1mM EDTA, and rinsed in 0.1×SSC, 0.1% SDS at 65°C. Alternatively, the membrane can be hybridized in a solution of 6×SSC, 0.5% SDS at 65oC, and then washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS. Alternatively, the membrane can be hybridized in a solution of 2×SSC, 0.1% SDS at 68°C and washed twice for 5 minutes each time, and then hybridized in a solution of 0.5×SSC, 0.1% SDS at 68°C and washed twice for 15 minutes each time. Alternatively, the membrane can be hybridized and washed in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS at 65°C.

[0028] The present invention also provides a method for preparing recombinant Halomonas, the method comprising:

[0029] M1) knocking out the tetrahydropyrimidine hydrolase gene in the recipient Halomonas or reducing the content or activity of the tetrahydropyrimidine hydrolase in the recipient Halomonas;

[0030] M2) expressing the tetrahydropyrimidine hydroxylase gene in the recipient Halomonas or promoting the expression of the tetrahydropyrimidine hydroxylase gene or increasing the activity or content of the tetrahydropyrimidine hydroxylase.

[0031] In the above method, M2) can be achieved by utilizing the pUP119 promoter to drive the expression of the ectoine hydroxylase gene.

[0032] The above method may further include: M3) expressing the glutamate dehydrogenase gene in the recipient salt mononas or promoting the expression of the glutamate dehydrogenase gene or increasing the activity or content of the glutamate dehydrogenase.

[0033] The recombinant Halomonas obtained by the method for preparing recombinant Halomonas also falls within the protection scope of the present invention.

[0034] The present invention also provides a method for preparing hydroxyectoine, which comprises: culturing the recombinant Halomonas to prepare hydroxyectoine.

[0035] The present invention also provides a product, which contains (or its component is) the recombinant Halomonas.

[0036] The product may be a bacterial agent.

[0037] The present invention also provides any of the following applications of the recombinant Halomonas or the product:

[0038] P1) production of hydroxyectoine;

[0039] P2) Preparation of products for producing hydroxytetrahydropyrimidine.

[0040] Experiments have shown that the recombinant Halomonas of the present invention can produce 1.02 g / L hydroxyectoine by fermentation when the salt concentration is reduced to 1.4 M, which is 10 times higher than the yield of hydroxyectoine of the wild-type Halomonas at a salt concentration of 1.4 M. The recombinant Halomonas of the present invention successfully achieves the production of hydroxyectoine at reduced salt concentrations, and successfully constructs a chassis bacteria for efficiently producing hydroxyectoine under medium salt conditions, providing theoretical guidance for further reducing the salt concentration of hydroxyectoine production, and also providing an experimental basis for improving the biosynthesis of high value-added chemicals hydroxyectoine.

[0041] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The production of ectoine and hydroxyectoine by Halomonas at different salt concentrations. ns indicates no significant difference, *(p<0.05), ****(p<0.0001) indicates significant difference.

[0043] Figure 2 The promoter strength of different promoters in Halomonas salinarum IM328. In the horizontal axis, pBAD represents pBAD (araC), the concentration represents the added concentration of inducer arabinose, which is 0 mg / mL, 0.02 mg / mL, 0.2 mg / mL, 2 mg / mL, and 20 mg / mL, and the vertical axis represents the relative fluorescence intensity (relative fluorescence unit).

[0044] Figure 3The doeA knockout strain ΔdoeA and the pUP119 promoter-inserted strain ΔdoeA::P UP119 -ectD, recombinant Halomonas strain ΔdoeA p-gdh, recombinant Halomonas strain ΔdoeA::P UP119 -ectD p-gdh PCR identification map. ΔdoeA::gdh represents ΔdoeAp-gdh, ΔdoeA::ectD represents ΔdoeA::P UP119 -ectD, ΔdoeA::gdh-ectD represents ΔdoeA::P UP119 -ectDp-gdh.

[0045] Figure 4 The synthesis of ectoine and hydroxyectoine by different recombinant Halomonas strains. ΔdoeA::gdh represents ΔdoeAp-gdh, ΔdoeA::ectD represents ΔdoeA::P UP119 -ectD, ΔdoeA::gdh-ectD represents ΔdoeA::P UP119 -ectDp-gdh. ns indicates no significant difference, *(p<0.05), ****(p<0.0001) indicates significant difference. DETAILED DESCRIPTION

[0046] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0047] Halomonas alifodinae IM328 is recorded in the article "Zhang M., Qiong X., et al. Development of whole-cell catalyst system for sulfide biotreatment based on the engineered haloalkaliphilic bacterium. AMB Express, 2021, 11: 142." The public can obtain the biomaterial from the Institute of Microbiology, Chinese Academy of Sciences. The biomaterial is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0048] The vectors pBBR1-pTac-RFP-TrrnB, pBBR1-pJ23100-RFP-TrrnB, pBBR1-pJ23110-RFP-TrrnB, pBBR1-pJ23111-RFP-TrrnB, pBBR1-pJ23119-RFP-TrrnB, pBBR1-pUP119-RFP-TrrnB, and pBBR1-pBAD(araC)-RFP-TrrnB are all recorded in the article "Jiang M, Zeng Y., et al. A red fluorescent protein reporter system developed for measuring gene expression inphotosynthetic bacteria under anaerobic conditions. Microorganisms, 2022, 10(2): 201." The public can obtain the biological material from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0049] The vector pJQ-200KS is described in the article “Quandt J., Hynes MF Versatile suicide vectors which allow direct selection for gene replacement in Gram-Negative bacteria. Gene. 1993; 127: 15–21.” The public can obtain the biological material from the Institute of Microbiology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0050] Halomonas elongata DSM 2581: China General Microbiological Culture Collection Center, CGMCC1.6329.

[0051] Example 1. Construction of a recombinant Halomonas capable of biosynthesizing hydroxyectoine at reduced production salt concentrations. 1. Preparation of hydroxyectoine in Halomonas and the effect of salt concentration on hydroxyectoine production

[0052] 1. Fermentation conditions and methods

[0053] The triangular baffle shake flask fermentation was used. First, the Halomonas IM328 was pre-cultured in 80LB medium, and the OD 600When the concentration of NaCl reaches 2.5±0.2, the seed solution is obtained and inoculated into MMD fermentation medium or MME fermentation medium with different salt (NaCl) concentrations at a 5% inoculum, and cultured at 37°C and 200 rpm in a shaking incubator for 48 hours to determine the yield of hydroxyectoine. The NaCl concentration is set to 0.2M, 0.8M, 1.4M, 2.0M, and 3.0M.

[0054] Preparation of 80LB medium: 10.0 g tryptone, 5.0 g yeast extract, 80.0 g NaCl, add distilled water to 1 L, and sterilize by autoclave at 121°C for 20 min;

[0055] Preparation of MMD fermentation medium: glucose 30g / L, NaCl (different salt concentrations), yeast extract 1g / L, (NH 4 ) 2 SO 4 0.25g / L, MgSO 4 0.2g / L, Na 2 HPO 4 12H 2 O 9.65g / L, KH 2 PO 4 1.5 g / L, trace element solution I 10 mL / L and trace element solution II 1 mL / L, 100 mM sodium aspartate, 100 mM KCl, 1.7 g / L citric acid and 0.9% (mass percentage) beef extract, pH 8.0 (adjust pH with 2 M NaOH), the balance is water. Trace element solution I: Fe(III)-NH 4 -citrate 5g / L, CaCl 2 2g / L, HCl 1M, the balance is water. Trace element solution II: ZnSO 4 7H 2 O 100mg / L, MnCl 2 ·4H 2 O 30mg / L, H 3 BO 3 300mg / L, CoCl 2 6H 2 O 200mg / L, CuSO 4 ·5H 2 O 10mg / L, NiCl 2 6H 2 O 20mg / L and NaMoO 4 ·2H 2 O 30mg / L, the balance is water. Salt concentration is added according to experimental needs.

[0056] The MME fermentation medium is a medium obtained by replacing 100 mM sodium aspartate in the MMD fermentation medium with 100 mM sodium glutamate, and the remaining components and concentrations are consistent with those of the MMD fermentation medium.

[0057] 2. Hydroxytetrahydropyrimidine sample preparation and detection method

[0058] After the fermentation, 1 mL of the fermentation broth was centrifuged for 2 min, and the supernatant was transferred to a new EP tube (sample A1). The bacteria were washed with 1 mL of ddH 2 O and stored at -20°C (sample A2).

[0059] Preparation of extracellular samples: Dilute sample A1 20 times with water, then mix the diluted sample with acetonitrile in a volume ratio of 3:7, filter it through a 0.22 μm microporous filter membrane into a brown liquid phase bottle to obtain the sample B1 to be tested, and perform high performance liquid chromatography detection.

[0060] Preparation of intracellular samples: Place sample A2 in a -80℃ freezer for 15 minutes, then heat and melt in a 65℃ water bath. Repeat this step three times to rupture the cells, and centrifuge at 12000rpm for 10 minutes. Take the supernatant in a clean EP tube, mix the supernatant with acetonitrile in a volume ratio of 3:7, and filter it through a 0.22μm microporous filter membrane into a brown liquid phase bottle to obtain the sample B2 to be tested, and perform high performance liquid chromatography detection.

[0061] High performance liquid chromatography detection: A 25 cm amino column from GL Sciences was used, the mobile phase was an acetonitrile / water mixture (70:30, v / v), the mobile phase was a flow rate of 1 ml per minute, the running time for each sample was 20 min, and the ultraviolet detector (210 nm) was used for the detection of ectoine and hydroxyectoine. The results are expressed as the mean ± standard deviation (SD) of 3 replicates. The standards used were ectoine (Beijing Puyihua Technology Co., Ltd., 81619-1G-F) and hydroxyectoine (Beijing Leco Biotech Co., Ltd., H923952-1g).

[0062] 3. Effect of salt concentration on hydroxytetrahydropyrimidine production

[0063] Halomonas IM328 was fermented in MME fermentation medium with salt concentrations of 0.2M, 0.8M, 1.4M, 2.0M, and 3.0M. Ectoine and hydroxyectoine were not detected in the extracellular samples, and the detection results of the intracellular samples were as follows Figure 1As shown, the synthesis of hydroxyectoine could not be detected at a salt concentration of 0.2 M, and only 0.11 g / L of hydroxyectoine was produced at a salt concentration of 1.4 M. At a salt concentration of 3 M, the production of hydroxyectoine was as high as 0.96 g / L, which has the potential for high production of hydroxyectoine. It also shows that high salt is an important condition for significantly improving the biosynthesis efficiency of hydroxyectoine. In order to enable Halomonas IM328 to synthesize hydroxyectoine under conditions of reduced salt concentration, it is necessary to perform metabolic engineering on it.

[0064] 2. Characterization of the expression strength of different constitutive and inducible promoters in Halomonas

[0065] The recombinant vectors pBBR1-pTac-RFP-TrrnB, pBBR1-pJ23100-RFP-TrrnB, pBBR1-pJ23110-RFP-TrrnB, pBBR1-pJ23111-RFP-TrrnB, pBBR1-pJ23119-RFP-TrrnB, pBBR1-pUP119-RFP-TrrnB, and pBBR1-pBAD (araC) -RFP-TrrnB expressing different promoters of red fluorescent protein RFP (specifically mCherry in RFP in this embodiment) were introduced into Escherichia coli by chemical transformation. In S17-1 competent cells, the obtained Escherichia coli recombinant strain was used as the conjugation donor strain, and Halomonas IM328 was used as the recipient strain. Recombinant Halomonas strains containing the above different promoters (pTac, pJ23100, pJ23110, pJ23111, pJ23119, pUP119, pBAD (araC)) driving the expression of the RFP gene were constructed by conjugation. Each group of Halomonas strains was cultured in 80LB medium to the late exponential phase. The strain containing pBAD (araC) was induced to express the protein using different concentrations of inducer arabinose, and the promoter strength of different promoters in Halomonas was compared. The results are shown in Figure 2 As shown, the pUP119 promoter has the highest promoter strength in Halomonas IM328.

[0066] The above experiment was performed to determine the strength of the constitutive promoter as follows: The fluorescent protein mCherry was used to characterize the strength of the promoter. An appropriate amount of cells were collected by centrifugation at 12000 rpm, resuspended in 10 mM PBS buffer and centrifuged twice. The cells were resuspended in 1 mL 10 mM PBS buffer and 200 μL of the sample was taken to a transparent 96-well plate to measure the OD 600nm At the same time, take 200 μL of sample into a black 96-well plate to measure the fluorescence intensity RFU (excitation wavelength 587 nm, emission wavelength 610 nm), and use the fluorescence intensity of the unit cell RFU / OD600nm Indicates the promoter strength.

[0067] 3. Construction of recombinant Halomonas for biosynthesis of hydroxytetrahydropyrimidine

[0068] 1. Construction of doeA gene knockout vector

[0069] First, the gentamicin resistance gene of pJQ-200KS was replaced with chloramphenicol resistance: pJQ-F and pJQ-R were used as primers to PCR amplify the vector pJQ-200KS, and then the plasmid template was digested with DpnI to obtain the vector backbone; using the plasmid pBBR1-pTac-RFP-TrrnB as a template, pJQ-cat-F and pJQ-cat-R were used as primers to PCR amplify the chloramphenicol cat resistance gene fragment, and the product was cleaned and recovered to obtain the cat resistance gene. The above two fragments (vector backbone and cat resistance gene) were assembled by T5 nuclease according to the method of the following document "Xia Y, Li K, Li J, et al. T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis [J]. Nucleic Acids Research, 2019, 47." to obtain a vector in which the pJQ-200KS gentamicin resistance gene was replaced with a chloramphenicol resistance gene, recorded as pJQ.

[0070] The cat resistance gene sequence is as follows:

[0071] .

[0072] The upstream and downstream homologous arm fragments of the doeA gene, each about 1000 bp, were amplified from Halomonas IM328: Using Halomonas IM328 genomic DNA as a template, pJQ-doeA-up-F and doeA-up-R were used as primers to PCR amplify the upstream homologous arm gene, and doeA-up-F and pJQ-doeA-do-R were used as primers to PCR amplify the downstream homologous arm gene, and the products were cleaned and recovered to obtain two PCR products of the upstream homologous arm and downstream homologous arm of the doeA gene.

[0073] The pJQ vector was double-digested with restriction endonucleases XhoI and BamHI, and then assembled with the above two fragments by T5 exonuclease according to the method of the following document "Xia Y, Li K, Li J, et al. T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis [J]. Nucleic Acids Research, 2019, 47." The PCR products of the upstream homology arm and the downstream homology arm of the doeA gene were connected to the pJQ vector and the obtained recombinant vector with the correct sequence was recorded as pJQ-ΔdoeA. pJQ-ΔdoeA is a recombinant vector obtained by replacing the DNA sequence (5′-GTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGG-3′) between the two restriction sites of XhoI and BamHI in the pJQ plasmid with the PCR products of the upstream and downstream homology arm fragments of the doeA gene.

[0074] The primer sequences used are as follows:

[0075] pJQ-F: The bold part is the homologous sequence complementary to the cat gene;

[0076] pJQ-R: The bold part is the homologous sequence complementary to the cat gene.

[0077] pJQ-cat-F: The bold part is the homologous sequence complementary to the vector;

[0078] pJQ-cat-R: The bold part is the homologous sequence complementary to the vector.

[0079] pJQ-doeA-up-F: The bold part is the homologous sequence complementary to the vector, and the underlined part is the added XhoI restriction site;

[0080] doeA-up-R: The bold part is the homologous sequence complementary to the downstream homology arm.

[0081] doeA-up-F: The bold part is the homologous sequence complementary to the upstream homology arm.

[0082] pJQ-doeA-do-R: The bold part is the homologous sequence complementary to the vector, and the underlined part is the added BamHI restriction site.

[0083] The sequences of the upstream homology arm and downstream homology arm of the doeA gene are as follows:

[0084]

[0085] 2. Construction of recombinant Halomonas with knockout of doeA gene

[0086] The knockout doeA vector (pJQ-ΔdoeA) was transferred into Escherichia coli S17-1 competent cells by chemical transformation to obtain a recombinant Escherichia coli strain. Then it was transferred into Halomonas IM328 by conjugation transfer to achieve single exchange. The obtained single exchange bacterial solution was streaked onto a medium containing 100 g / L sucrose to force the occurrence of double exchange. Then a single clone was picked on this sucrose plate, and streaked and enriched on a medium containing 100 g / L sucrose and a medium containing 100 g / L sucrose plus Cm resistance (control). The colonies that only grew in a medium containing 100 g / L sucrose were picked, and finally the strain with the correct knockout of the doeA gene was obtained by PCR identification, hereinafter referred to as recombinant Halomonas ΔdoeA. Among them, the culture medium is MMD fermentation medium.

[0087] Identification of doeA knockout strain ΔdoeA Figure 3 The recombinant Halomonas ΔdoeA lacks the doeA gene shown in SEQ ID No. 1 in the sequence table.

[0088] 3. Construction of vector for inserting pUP119 promoter

[0089] Amplify the upstream and downstream fragments of pUP119 promoter from Halomonas IM328 (about 1000 bp): Use the genomic DNA of Halomonas IM328 as template and pJQ-P UP119 -up-F and P UP119 -up-R was used as the primer to PCR amplify the upstream homology arm gene, and the ectD gene was used as the downstream homology arm, and the primer P UP119 -do-F and pJQ-P UP119 -do-R PCR was used to amplify the downstream homology arm gene using the genomic DNA of Halomonas IM328 as a template and primers P UP119 -F and P UP119 -R used the vector pBBR1-pUP119-RFP-TrrnB as a template to PCR amplify the pUP119 promoter sequence, and cleaned and recovered each PCR product to obtain the upstream homology arm and downstream homology arm inserted into the pUP119 promoter gene and three PCR products of the pUP119 promoter.

[0090] The vector pJQ was double-digested with restriction endonucleases XhoI and BamHI, and then assembled with the three fragments using T5 exonuclease according to the above method. The three PCR products were connected into the pJQ vector, and the resulting recombinant vector with the correct sequence was recorded as pJQ-P UP119-ectD.pJQ-P UP119 -ectD is a recombinant vector obtained by replacing the DNA sequence between the XhoI and BamHI restriction sites in the pJQ vector (5′-GTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGG-3′) with the PCR product of the upstream and downstream fragments of the pUP119 promoter and the pUP119 promoter fragment.

[0091] The primer sequences used are as follows:

[0092] QP UP119 -up-F: The bold part is the homologous sequence complementary to the vector, and the underlined part is the added XhoI restriction site;

[0093] P UP119 -up-R: The bold part is the homologous sequence complementary to the pUP119 promoter gene sequence.

[0094] P UP119 -do-F: The bold part is the homologous sequence complementary to the pUP119 promoter gene sequence.

[0095] QP UP119 -do-R: The bold part is the homologous sequence complementary to the vector, and the underlined part is the added BamHI restriction site.

[0096] P UP119 -F: The bold part is the homologous sequence complementary to the downstream homology arm.

[0097] P UP119 -R: The bold part is the homologous sequence complementary to the upstream homology arm.

[0098] The sequences of the upstream and downstream fragments of the pUP119 promoter and the pUP119 promoter fragment are as follows:

[0099]

[0100] 4. Construction of recombinant Halomonas with pUP119 promoter inserted

[0101] pJQ-P UP119 The -ectD vector was transferred into E. coli S17-1 competent cells by chemical transformation to obtain a recombinant E. coli strain. The recombinant Halomonas strain ΔdoeA was used as the recipient strain, and the obtained recombinant E. coli strain was conjugated with the recipient strain. The screening of the recombinant strain was consistent with the screening method of the recombinant strain with knockout doeA gene. Finally, the recombinant Halomonas strain ΔdoeA::P was obtained by PCR identification. UP119 -ectD, which is the introduction of pJQ-P into the recombinant Halomonas strain ΔdoeA UP119 -ectD vector obtained recombinant bacteria, the strain pUP119 promoter (P UP119 ) drives the expression of ectD gene, the sequence of ectD gene is SEQ ID No.2 in the sequence list, encoding ectD protein shown in SEQ ID No.3, pUP119 promoter (P UP119 ) is SEQ ID No.4 in the sequence listing.

[0102] ΔdoeA::P UP119 -ectD identification Figure 3 shown.

[0103] 5. Construction of gdh gene expression vector

[0104] The vector was constructed in E. coli S17-1, gdh from Halomonas elongata DSM 2581 was selected for overexpression, and the expression of gdh was initiated by pJ23119 promoter. Firstly, genomic DNA of Halomonas elongata DSM 2581 was used as a template, and p1-gdh-F and p1-gdh-R were used as primers to PCR amplify the gdh gene fragment, and the product was cleaned and recovered to obtain the PCR product of the gdh gene. Based on the pBBR1-pJ23119-RFP-TrrnB vector, the RFP gene was cut out with restriction endonucleases XhoI and NdeI, and then assembled with the above fragments using T5 exonuclease according to the method of the following literature: "Xia Y, Li K, Li J, et al. T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis[J]. Nucleic Acids Research, 2019, 47." The PCR product of the gdh gene was transferred into the pBBR1 vector. After obtaining the recombinant vector with the correct sequence, the p1-P J23119 -gdh-F and p1-P J23119 -gdh-R was used as primer to amplify the vector obtained above, thereby transferring the promoter sequence into the recombinant vector, and finally the plasmid template was digested with DpnI. The recombinant vector with the correct sequence was obtained by chemical transformation and was recorded as pBBR1-pJ23119-gdh.

[0105] pBBR1-pJ23119-gdh is a recombinant vector obtained by replacing the RFP gene sequence between the two restriction sites of XhoI and NdeI in the pBBR1-pJ23119-RFP-TrrnB vector with a gene fragment of gdh. pBBR1-pJ23119-gdh can express gdh protein, and the expression of gdh gene is driven by the pJ23119 promoter. The sequence of gdh gene is the 65th to 4906th position of SEQ ID No.5 in the sequence list, encoding the gdh protein shown in SEQ ID No.6, and the sequence of pJ23119 promoter is the 1st to 64th position of SEQID No.5 in the sequence list.

[0106] The primer sequences used are as follows:

[0107] p1-gdh-F: 5′-GATGTATGCTCTCTCGAGATGCTACACGTCGCACAGGAG-3′, the bold part is the homologous sequence complementary to the vector, and the underline is the added XhoI restriction site;

[0108] p1-gdh-R: 5′-CCCGGGGTCGACCATATGTCAGGCCTCGCTGTTGCTCTC-3′, the bold part is the homologous sequence complementary to the vector, and the underline is the added NdeI restriction site.

[0109] p1-P J23119 -gdh-F: 5′-TAGGTATAATGCTAGCTACTAGAGAAAGAGGAGAAATACATGCTACACGTCGCACAGG-3′, the bold part is the homologous sequence complementary to the pJ23119 promoter gene;

[0110] p1-P J23119 -gdh-R: 5′-AGTAGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAACTCGAGAGAGCATACATCTG-3′, the bold part is the homologous sequence complementary to the pJ23119 promoter gene.

[0111] 6. Construction of recombinant Halomonas expressing gdh

[0112] pBBR1-pJ23119-gdh was transformed into E. coli S17-1 competent cells by chemical transformation to obtain the recombinant E. coli strain. UP119 -ectD was used as the recipient bacteria, and the obtained recombinant Escherichia coli strain was conjugated with the two recipient strains respectively. The screening method of the recombinant strain was consistent with the screening method of the recombinant strain with knocked out doeA gene. Finally, the recombinant Halomonas strain ΔdoeAp-gdh and the recombinant Halomonas strain ΔdoeA::P were obtained by PCR identification. UP119 -ectD p-gdh. The recombinant Halomonas strain ΔdoeAp-gdh is a recombinant strain obtained by introducing pBBR1-pJ23119-gdh into the recombinant Halomonas strain ΔdoeA. The recombinant Halomonas strain ΔdoeA::P UP119 -ectD p-gdh is the recombinant Halomonas strain ΔdoeA::P UP119 In these two recombinant bacteria, the pJ23119 promoter drives the expression of the gdh gene.

[0113] Recombinant Halomonas strains ΔdoeA p-gdh, ΔdoeA::P UP119 Identification of -ectD p-gdh Figure 3 shown.

[0114] 7. Cultivation of recombinant Halomonas strains under conditions of reduced production salt concentration

[0115] Strains to be tested: Halomonas IM328 (wild type, WT), recombinant Halomonas ΔdoeA, ΔdoeAp-gdh, ΔdoeA::P UP119 -ectD、ΔdoeA::P UP119 -ectDp-gdh.

[0116] The synthesis of hydroxyectoine by each recombinant halomonas strain was evaluated in MME fermentation medium at 1.4 M salt concentration. The activated strains to be tested were inoculated into 10 ml of 80LB medium and cultured in a shaking incubator at 37°C and 200 rpm. The OD 600 When the concentration reaches 2.5±0.2, the seed solution is obtained. The obtained seed solution is inoculated into the MME fermentation medium with a concentration of 1.4M NaCl at a 5% inoculation amount, and cultured in a shaking incubator at 37°C and 200rpm for 48h. The obtained fermentation liquid is tested for hydroxyectoine and ectoine according to step 2 of step 1 above. The tested samples are intracellular samples. The experiment is repeated 3 times, and 3 flasks are used for each treatment each time.

[0117] The fermentation results are as follows Figure 4 As shown in Figure 2, the production of hydroxyectoine increased from 0.1 g / L in the wild-type strain to 1.02 g / L (ΔdoeA::P UP119 -ectDp-gdh), which increased by 10 times and was equal to the yield of the wild-type strain at 3M salt concentration (0.96g / L), showing good production potential. The present invention successfully enables Halomonas IM328 to obtain the ability to produce hydroxyectoine at a salt concentration of 1.4M, and the yield increase is obvious at a production salt concentration reduced by half. The present invention constructs a chassis cell suitable for producing hydroxyectoine at medium salt concentration, further alleviating the problem of high-salt wastewater generated during the fermentation process and corrosion of the fermentation tank.

[0118] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A recombinant Halomonas, comprising the following characteristics: A1) does not contain the ectoine hydrolase gene or contains a substance that inhibits the expression of the ectoine hydrolase gene or contains a substance that reduces the content or activity of ectoine hydrolase; A2) Contains a substance capable of expressing the ectoine pyrimidine hydroxylase gene, a substance promoting the expression of the ectoine pyrimidine hydroxylase gene, or a substance increasing the activity or content of ectoine pyrimidine hydroxylase.

2. The recombinant Halomonas according to claim 1, Features: The substance promoting the expression of the ectoine hydroxylase gene in A2) is the pUP119 promoter, and the pUP119 promoter is b1) or b2) or b3): b1) a DNA molecule shown in SEQ ID No. 4 in the sequence list; b2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) and having the function of the pUP119 promoter; b3) a DNA molecule that hybridizes with the nucleotide sequence defined in b1) or b2) under stringent conditions and has the function of the pUP119 promoter; Furthermore, the tetrahydropyrimidine hydroxylase is EctD.

3. The recombinant Halomonas according to claim 1 or 2, Features: The recombinant salt mononas also includes the following features: A3) Contains a substance capable of expressing glutamate dehydrogenase gene, a substance promoting the expression of glutamate dehydrogenase gene, or a substance increasing the activity or content of glutamate dehydrogenase.

4. The recombinant Halomonas according to claim 3, Features: The substance capable of expressing the glutamate dehydrogenase gene in A3) is a glutamate dehydrogenase gene expression cassette.

5. The recombinant Halomonas according to claim 4, Features: The promoter in the expression cassette is the pJ23119 promoter, and the pJ23119 promoter is c1) or c2) or c3): c1) the DNA molecule shown in positions 1 to 64 of SEQ ID No. 5 in the sequence listing; c2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in c1) and having the promoter function of pJ23119; c3) a DNA molecule that hybridizes with the nucleotide sequence defined in c1) or c2) under stringent conditions and has the function of the pJ23119 promoter; And / or, the glutamate dehydrogenase is derived from Halomonas longata; Furthermore, the glutamate dehydrogenase is GDH.

6. A method for preparing recombinant Halomonas, include: M1) knocking out the tetrahydropyrimidine hydrolase gene in the recipient Halomonas or reducing the content or activity of the tetrahydropyrimidine hydrolase in the recipient Halomonas; M2) expressing the tetrahydropyrimidine hydroxylase gene in the recipient Halomonas or promoting the expression of the tetrahydropyrimidine hydroxylase gene or increasing the activity or content of the tetrahydropyrimidine hydroxylase.

7. The method according to claim 6, Features: M2) is achieved by utilizing the pUP119 promoter described in claim 2 to drive the expression of the ectoine hydroxylase gene.

8. The method according to claim 6 or 7, Features: The method further comprises: M3) expressing a glutamate dehydrogenase gene in the recipient Halomonas or promoting the expression of the glutamate dehydrogenase gene or increasing the activity or content of glutamate dehydrogenase.

9. The recombinant Halomonas obtained by the method described in any one of claims 6 to 8.

10. A method for preparing hydroxytetrahydropyrimidine, include: Cultivate the recombinant Halomonas described in any one of claims 1-5 and 9 to prepare hydroxytetrahydropyrimidine.

11. A product comprising the recombinant Halomonas according to any one of claims 1 to 5 and 9.

12. Any of the following uses of the recombinant Halomonas according to any one of claims 1 to 5 and 9 or the product according to claim 11: P1) production of hydroxyectoine; P2) Preparation of products for producing hydroxytetrahydropyrimidine.

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