A recombinant Pantoea algina and its application in fermentation production of exopolysaccharides

Abstract

The present invention discloses a recombinant Pantoea alpina strain and its use in the fermentation production of exopolysaccharides. The recombinant Pantoea alpina strain has the gene mdoG encoding a transcription factor knocked out; wherein the amino acid sequence of the transcription factor is shown in SEQ ID NO.2. The present invention constructs a recombinant strain with high exopolysaccharide production using CRISPR technology. The gene encoding the transcription factor can be used to direct the improvement of the exopolysaccharide-producing strain, making its exopolysaccharide production significantly higher than that of the wild-type strain, laying the foundation for further metabolic engineering of Pantoea alpina strain to produce polysaccharides. The recombinant strain construction method provided by the present invention is simple and easy to use. The exopolysaccharide produced by the recombinant Pantoea alpina strain constructed by the present invention has a static viscosity of 10.32 to 24.11 Pa·s at room temperature for a 10 g / L exopolysaccharide aqueous solution prepared using the exopolysaccharide, showing good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to a recombinant Pantoea alginate and an application thereof in fermentation production of exopolysaccharides. Background Art

[0002] Exopolysaccharides (EPS) are a class of carbohydrate polymers produced by microbial fermentation. Due to their diversity in monosaccharide composition and glycosidic bonds, they have rich physical, chemical and biological activities and are widely used in food, medicine, polymer materials, cosmetics and agriculture. High molecular weight exopolysaccharides tend to have better thickening and stability. Specifically, in the food industry, exopolysaccharides can be used as excellent thickeners and stabilizers to improve the texture and taste of food and extend the shelf life; in the pharmaceutical field, they help to slowly release drugs, improve the efficacy and bioavailability of drugs; in polymer materials, they can enhance the mechanical properties and durability of materials. High-viscosity exopolysaccharides can increase the viscosity of products in food and give them a unique taste; in cosmetics, they can form a protective film to moisturize and protect the skin; in agriculture, they can be used to prepare water-retaining agents to improve the water and fertilizer retention capacity of the soil. Pantoea thornii ( Pantoea alhagi ) has been used for the industrial production of EPS. Since the initial publication of its genome sequence, its versatile metabolic pathways, along with their genetic components and regulatory mechanisms, have been extensively studied. To improve the efficiency of biotechnological production, genetic tools and omics-based analytical methods, including transcriptomics, proteomics, metabolomics, and rheology, have been developed based on genomic sequence information and are widely used to understand metabolic pathways and their post-transcriptional regulation.

[0003] Transcription factors (TFs) are important players in the regulatory gene expression network. Related studies have shown that transcription factors directly or indirectly affect the synthesis of polysaccharides by regulating the transcription levels of related genes in polysaccharide synthesis. They are an important component of polysaccharide synthesis regulation and are crucial for maintaining the balance of polysaccharide synthesis in cells and adapting to environmental changes. Current studies have shown that transcription factors can affect the synthesis of extracellular polysaccharides by regulating extracellular polysaccharide synthesis gene clusters; regulating extracellular polysaccharide synthesis gene clusters; affecting the two-component regulatory system, etc. The mdoG transcription factor involved in this invention has never been directly reported in any article on its role in extracellular polysaccharide synthesis, and is of great research value. Summary of the Invention

[0004] Purpose of the Invention: This invention addresses the technical problems and deficiencies of existing technologies by providing a recombinant Pantoea alkekengi and its use in the fermentation production of exopolysaccharides. The recombinant Pantoea alkekengi is a deletion mutant of a transcription factor-encoding gene. The transcription factor-encoding gene, mdoG, is knocked out from the genome of Pantoea alkekengi NX-11, redirecting carbon metabolism toward exopolysaccharide (EPS) production. This improves the EPS production capacity of Pantoea alkekengi NX-11, ultimately increasing EPS yield by 50%. This has significant economic and social significance.

[0005] To solve the above technical problems, the present invention discloses a recombinant Pantoea acuminata and its application in the fermentation production of exopolysaccharides. The specific technical solution is as follows:

[0006] A recombinant Pantoea alginate, wherein the gene mdoG encoding a transcription factor is knocked out of the recombinant Pantoea alginate; wherein the amino acid sequence of the transcription factor is shown in SEQ ID NO.2.

[0007] Wherein, the nucleotide sequence of the gene mdoG encoding the transcription factor is shown as SEQ ID NO.1.

[0008] Wherein, the starting bacteria of the recombinant Pantoea algina is Pantoea algina Pantoea alhagi NX-11. Pantoea acuminata Pantoea alhagi NX-11, with the deposit number CGMCC NO.15525, was deposited in the General Microbiology Center of China Culture Collection Administration on March 29, 2018.

[0009] Wherein, the knockout is achieved using CRISPR-Cas9 technology.

[0010] In a second aspect, the present invention provides a method for constructing the recombinant Pantoea algina described in the first aspect, comprising the following steps:

[0011] (1) Construction of recombinant plasmid pTarget-mdoG: amplification of the sgRNA fragment targeting the mdoG, the upstream homology arm UP fragment, and the downstream homology arm DN fragment, and the gene fragment sgRNA-UP-DN was obtained by overlapping PCR, and cloned into the plasmid pTarget to obtain the recombinant plasmid pTarget-mdoG;

[0012] (2) Introducing the pCas9 plasmid into the starting bacteria to obtain a starting bacteria carrying the pCas9 plasmid, and then introducing the recombinant plasmid pTarget-mdoG prepared in step (1) to obtain the recombinant Pantoea acuminata. Preferably, the recombinant plasmid pTarget-mdoG is introduced into the starting bacteria carrying the pCas9 plasmid by electroporation.

[0013] Wherein, in step (1), the nucleotide sequence of the sgRNA fragment is shown as SEQ ID NO. 3. Further preferably, the sgRNA fragment, the upstream homology arm UP fragment and the downstream homology arm DN fragment are respectively amplified according to the following methods:

[0014] Using the starting bacteria as a template, the sgRNA fragment was amplified using the primer nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.7;

[0015] Using the starting bacteria as a template, the upstream homology arm UP fragment was amplified using the primer nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9;

[0016] Using the starting bacteria as a template, the downstream homology arm DN fragment was amplified using the primer nucleotide sequences shown in SEQ ID NO.10 and SEQ ID NO.11.

[0017] The starting bacteria is preferably Pantoea acuminata Pantoea alhagi NX-11.

[0018] Further preferably, in step (2), the recombinant plasmid pTarget-mdoG prepared in step (1) is introduced into the starting bacteria carrying the pCas9 plasmid, and the resulting recombinant bacteria are induced by high temperature to delete the pCas9 plasmid and the pTarget-mdoG recombinant plasmid, thereby obtaining an mdoG gene-directed deletion strain, namely the recombinant Pantoea truncatula.

[0019] In a third aspect, the present invention provides the use of the recombinant Pantoea algina described in the first aspect in the fermentation production of exopolysaccharides.

[0020] The recombinant Pantoea algina is inoculated into a fermentation medium for fermentation and culture.

[0021] Wherein, the fermentation medium comprises: a carbon source of 10-100 g / L, a nitrogen source of 1-30 g / L and an inorganic salt of 0.01-50 g / L; the carbon source is any one or a combination of several of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol, starch and molasses; the nitrogen source is any one or a combination of several of yeast powder, beef extract, peptone, yeast extract, corn steep liquor, soybean cake powder, cottonseed cake powder, urea, ammonium sulfate, ammonium chloride, diammonium hydrogen phosphate and ammonium nitrate; the inorganic salt is any one or a combination of several of sulfate, phosphate, dihydrogen phosphate, dihydrogen phosphate, sodium salt and hydrochloride; preferably, the fermentation medium comprises sucrose 10-100 g / L, sodium chloride 0.01-50 g / L, yeast powder 1-15 g / L, peptone 1-15 g / L; further preferably, the formula of the fermentation medium is sucrose 60 g / L, sodium chloride 10 g / L, yeast powder 5 g / L, peptone 10 g / L.

[0022] The fermentation culture has a culture temperature of 25-32°C and a culture time of 20-30 hours. Preferably, the fermentation is carried out at 30°C for 30 hours.

[0023] Wherein, at room temperature, the static viscosity of a 10 g / L exopolysaccharide aqueous solution prepared using the exopolysaccharide is 10.32-24.11 Pa·s.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides an mdoG transcription factor and a method for knocking out the mdoG transcription factor. P. alhagi NX-11 strain, which is a recombinant strain capable of increasing the production of extracellular polysaccharide EPS. The present invention increases the extracellular accumulation of EPS by knocking out the transcription factor mdoG involved in carbon metabolism regulation, with the highest concentration reaching 25.9 g / L, which is higher than that of the original strain. P. alhagi NX-11 increased by 50%, paving the way for further metabolic engineering P alhagi The method for constructing the recombinant strain provided by the present invention is simple, easy to use, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0027] Figure 1 This is the plasmid map of the recombinant plasmid pTarget-mdoG.

[0028] Figure 2 This is a graph showing the production of exopolysaccharides (EPS) obtained by fermentation of different strains.

[0029] Figure 3 The molecular weight of exopolysaccharide of NX-11 strain and mdoG knockout strain NX-11 (ΔmdoG).

[0030] Figure 4 The rheological properties of extracellular polysaccharides of NX-11 strain and mdoG knockout strain NX-11 (ΔmdoG). DETAILED DESCRIPTION

[0031] The present invention is further illustrated below by describing specific implementation methods, but this is not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the basic ideas of the present invention, but as long as they do not deviate from the basic ideas of the present invention, they are all within the scope of the present invention.

[0032] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0033] In the following examples, the Pantoea algina NX-11 is classified as Pantoea alhagi The deposit number is CGMCC NO.15525, and it was deposited in the General Microbiology Center of China Culture Collection Administration on March 29, 2018.

[0034] In the following examples, the mdoG is a transcription factor derived from Pantoea acuminata, the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the transcription factor mdoG is shown in SEQ ID NO.1.

[0035] Example 1 Construction of ΔmdoG engineered strain based on CRISPR / Cas9

[0036] Using the pTarget plasmid as a template, linearized primers (F-pTarget framework-CAT / M13, R-pTarget framework-CAT / M13) were used to amplify the pTarget plasmid to obtain a linearized vector.

[0037] The genome of Pantoea acuminata NX-11 was used as a template and PCR amplification was performed with the corresponding primer pairs (F-mdoG-sgRNA-CAT / M13 and R-mdoG-sgRNA-CAT / M13, F-mdoG-UP-CAT / M13 and R-mdoG-UP-CAT / M13, F-mdoG-DN-CAT / M13 and R-mdoG-DN-CAT / M13) to obtain the mdoG-sgRNA, upstream homology arm mdoG-UP and downstream homology arm mdoG-DN fragments, respectively, which were used to ligate the linearized vector pTarget to obtain the recombinant plasmid pTarget-mdoG.

[0038] The specific construction method is as follows:

[0039] The primer pairs F-pTarget framework-CAT / M13 and R-pTarget framework-CAT / M13 were used, and the pTarget plasmid was used as a template. The PCR conditions were as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 1 min; annealing at 55°C for 30 s; extension at 72°C for 1 min, for 30 cycles; and a final extension at 72°C for 10 min to obtain the linearized vector pTarget.

[0040] Using primer pairs F-mdoG-sgRNA-CAT / M13 and R-mdoG-sgRNA-CAT / M13, the genome of Pantoea acuminata NX-11 was used as a template. The PCR conditions were as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 1 min; annealing at 55°C for 30 s; extension at 72°C for 10 s, for 30 cycles; and finally extension at 72°C for 10 min to obtain the mdoG-sgRNA fragment. The nucleotide sequence of the mdoG-sgRNA fragment designed in this example is shown in SEQ ID NO. 3.

[0041] Primer pairs F-mdoG-UP-CAT / M13 and R-mdoG-UP-CAT / M13, and primer pairs F-mdoG-DN-CAT / M13 and R-mdoG-DN-CAT / M13 were used, respectively, with the genome of Pantoea acuminata NX-11 as the template. The PCR conditions were as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 1 min; annealing at 55°C for 30 s; extension at 72°C for 15 s, for 30 cycles; and finally extension at 72°C for 10 min, to obtain the mdoG-UP fragment and mdoG-DN fragment, respectively.

[0042] The PCR product fragments obtained above were recovered using a DNA purification kit (TianGen) to obtain the linearized pTarget plasmid vector, mdoG-sgRNA fragment, mdoG-UP fragment, and mdoG-DN fragment.

[0043] Overlap PCR was performed using primer pairs F-mdoG-sgRNA-CAT / M13 and R-mdoG-DN-CAT / M13, and the mdoG-UP fragment, mdoG-DN fragment, and mdoG-sgRNA fragment purified and recovered as described above. The PCR conditions were as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 1 min; annealing at 55°C for 30 s; and extension at 72°C for 15 s for 30 cycles. The PCR product was recovered using a DNA purification kit (TianGen) to obtain the fusion fragment mdoG-sgRNA-UP-DN with vector homology ends.

[0044] The ClonExpress II One Step Cloning Kit from Novozymes Biotech was used for ligation. The linearized vector pTarget obtained by PCR and the fusion fragment mdoG-sgRNA-UP-DN with homology ends of the vector were mixed at a molar ratio of 2:1. 5×CE II Buffer 4 μL and Exnase II 2 μL were added at the same time. Then ddH2O was added to make the total volume of the ligation system reach 20 μL. The reaction was incubated at 37°C for 30 min and kept at 4°C. Then 10 μL of the ligation system was transformed into E. coli BL21 (DE3) competent cells (for details on competent preparation, please refer to the instructions of the Takara E. coli competent kit). Select the correct transformants by colony PCR and send them to Anhui General Biotechnology Co., Ltd. for sequencing verification to obtain the recombinant plasmid pTarget-mdoG (such as Figure 1 The plasmid was extracted using the FastPure Plasmid Mini Kit from Novagen Biotech Co., Ltd., and the extracted pTarget-mdoG plasmid was transformed into P alhagi The NX-11(pCas) strain was subsequently induced with 0.1 mM IPTG for 4 hours and then incubated at 40°C to remove the pCas plasmid and pTarget-mdoG plasmid. Positive clones were selected using primers F-mdoG-OUT-CAT / M13 and R-mdoG-OUT-CAT / M13. PCR conditions were as follows: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 1 minute; annealing at 55°C for 30 seconds; extension at 72°C for 1 minute 20 seconds for 30 cycles; and a final extension at 72°C for 10 minutes. The strain was then sent to Anhui General Biotechnology Co., Ltd. for sequencing verification, resulting in the mdoG gene-deleted strain NX-11 (ΔmdoG).

[0045] Detailed information on the NX-11 (pCas) strain and the gene knockout method described in this example can be found in reference [Sun L, Cheng L, Ma Y, et al. Exopolysaccharides from Pantoea alhagi NX-11 specifically improve its root colonization and rice salt resistance. International Journal of Biological Macromolecules, 209: 396-404 (2022).].

[0046] The primer sequences involved in this example are shown in Table 1.

[0047] Table 1 Primer sequence list

[0048]

[0049] Example 2 Fermentation culture of recombinant bacteria

[0050] Using wild-type NX-11 without a plasmid as a control, the recombinant strain NX-11 (ΔmdoG) constructed in Example 1 and the starting strain NX-11 were inoculated into liquid LB medium, cultured overnight at 37°C and 200 rpm, and then transferred to a fermentation medium (60 g / L sucrose, 10 g / L sodium chloride, 5 g / L yeast powder, and 10 g / L peptone) at a 4% v / v inoculum. Fermentation was carried out at 30°C and 200 rpm for 30 h to obtain a fermentation broth. Three volumes of 95% v / v ethanol were added to the fermentation broth, and the precipitate was collected after centrifugation and freeze-dried. The extracellular polysaccharide (EPS) yield obtained by fermentation of the starting strain was 17.6 g / L, while the EPS yield obtained by fermentation of the mdoG gene-deficient strain NX-11 (ΔmdoG) was 25.9 g / L (as shown in Table 2). Figure 2 The EPS yield was quantified using the phenol-sulfuric acid method.

[0051] Example 3 Analysis of the properties of recombinant bacterial exopolysaccharides

[0052] The EPS prepared in Example 2 was added with ultrapure water to prepare a diluent, and the excess cells were removed by centrifugation. The solution was filtered through a 0.22 μm PES filter and freeze-dried. The sample was reconstituted to 10 g / L to obtain the test sample. The mobile phase (2 mM CH3COONH4, pH = 4.5) was then prepared and the sample was analyzed using Ultrahydrogel. TM 500 column and Ultrahydrogel TMThe molecular weight of the sample was detected by Agilent 1260 HPLC system. The results were as follows: Figure 3 As shown in Figure 2, the exopolysaccharide obtained by fermentation of NX-11 (ΔmdoG) showed a peak at 4 min, with a large molecular weight. The above sample was then analyzed for flow properties by a Discovery Hybrid Rheometer HR30 (condition: 25°C). The results are shown in Figure 2. Figure 4 As shown in the figure, at the same shear rate, the viscosity of the exopolysaccharide fermented with NX-11 (ΔmdoG) was consistently higher than that of the exopolysaccharide fermented with the starting strain NX-11. Furthermore, the static viscosity of the exopolysaccharide fermented with NX-11 (ΔmdoG) ranged from 10.32 to 24.11 Pa·s.

[0053] The present invention provides a method and concept for recombinant Pantoea camelids and its application in the fermentation production of exopolysaccharides. Numerous methods and approaches exist for implementing this technical solution. The foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A recombinant Pantoea algina, characterized in that: The recombinant Pantoea acuminata has the gene mdoG knocked out; wherein, the amino acid sequence encoded by the gene mdoG is shown in SEQ ID NO.

2.

2. The recombinant Pantoea algina according to claim 1, characterized in that The nucleotide sequence of the gene mdoG is shown in SEQ ID NO.

1.

3. The recombinant Pantoea algina according to claim 1, characterized in that The starting bacteria of the recombinant Pantoea algina is Pantoea algina Pantoea alhagi NX-11.

4. The recombinant Pantoea algina according to claim 1, characterized in that The knockout is achieved using CRISPR-Cas9 technology.

5. The method for constructing the recombinant Pantoea algina according to any one of claims 1 to 4, characterized in that: The steps include: (1) Construction of recombinant plasmid pTarget-mdoG: amplifying the sgRNA fragment targeting mdoG, the upstream homology arm UP fragment, and the downstream homology arm DN fragment, and obtaining the gene fragment sgRNA-UP-DN by overlapping PCR, and cloning it into the plasmid pTarget to obtain the recombinant plasmid pTarget-mdoG; the nucleotide sequence of the sgRNA fragment is shown in SEQ ID NO.3; (2) The pCas9 plasmid is introduced into the starting bacteria to obtain the starting bacteria carrying the pCas9 plasmid, and then the recombinant plasmid pTarget-mdoG prepared in step (1) is introduced to obtain the recombinant Pantoea truncatula.

6. Use of the recombinant Pantoea algina according to any one of claims 1 to 4 in the fermentation production of exopolysaccharides.

7. The use according to claim 6, characterized in that The recombinant Pantoea algina is inoculated into a fermentation medium for fermentation and culture.

8. The use according to claim 7, characterized in that The fermentation medium comprises: a carbon source of 10-100 g / L, a nitrogen source of 1-30 g / L, and an inorganic salt of 0.01-50 g / L; the carbon source is any one or a combination of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol, starch, and molasses; the nitrogen source is any one or a combination of yeast powder, beef extract, peptone, yeast extract, corn steep liquor, soybean meal, cottonseed meal, urea, ammonium sulfate, ammonium chloride, diammonium hydrogen phosphate, and ammonium nitrate; and the inorganic salt is any one or a combination of sulfate, phosphate, dihydrogen phosphate, dihydrogen phosphate, and hydrochloride. The fermentation culture has a culture temperature of 25-32°C and a culture time of 20-30 hours.

9. The use according to claim 6, characterized in that At room temperature, the viscosity of a 10 g / L exopolysaccharide aqueous solution prepared using the exopolysaccharide is 10.32-24.11 Pa·s in a static state.

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