Recombinant Pantoea alhagi and application of recombinant Pantoea alhagi in fermentation production of exopolysaccharides

Abstract

The invention discloses recombinant pantoea alhagi and application of the recombinant pantoea alhagi in fermentation production of exopolysaccharides. A gene mdoG for coding a transcription factor is knocked out of the recombinant pantoea alhagi; wherein the amino acid sequence of the transcription factor is as shown in SEQ ID NO.2. A recombinant strain of high-yield exopolysaccharide is constructed through a CRISPR (clustered regularly interspaced short palindromic repeats) technology, and the gene for coding the transcription factor can be used for directionally improving an exopolysaccharide production strain, so that the yield of the exopolysaccharide is obviously higher than that of a wild strain; and a foundation is laid for further producing the polysaccharide by transforming the pantoea alhagi through metabolic engineering. The recombinant strain provided by the invention is simple in construction method and convenient to use, the viscosity of a 10 g / L extracellular polysaccharide aqueous solution prepared from the extracellular polysaccharide at normal temperature in a static state is 10.32-24.11 Pa.s, and the recombinant strain has a very good application prospect.

Description

Technical Field

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

[0002] Exopolysaccharide (EPS) is 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 often 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 medical field, they help with the slow release of drugs, improving the efficacy and bioavailability of drugs; in polymer materials, they can enhance the mechanical properties and durability of materials. And high-viscosity exopolysaccharides can increase the viscosity of products in food, giving a unique taste; in cosmetics, they can form a protective film to play a role in moisturizing and skin care; in agriculture, they can be used to prepare water retainers to improve the water and fertilizer retention capacity of the soil. Pantoea alhagi ( Pantoea alhagi ) has been used in the industrial production of EPS. Since the first publication of its genomic sequence, its multifunctional metabolic pathways, genetic components, and regulatory mechanisms have been widely studied. To improve the efficiency of biotechnological production, genetic tools and omics-based analysis 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 (TF) are important participants in the regulation of gene expression networks. Relevant studies have shown that transcription factors directly or indirectly affect polysaccharide synthesis by regulating the transcriptional levels of related genes in polysaccharide synthesis. They are an important part of the regulation of polysaccharide synthesis and are crucial for maintaining the balance of intracellular polysaccharide synthesis and adapting to environmental changes. Currently, there are studies showing that transcription factors can affect exopolysaccharide synthesis by regulating the exopolysaccharide synthesis gene cluster; regulating the exopolysaccharide synthesis gene cluster; affecting the two-component regulatory system, etc. The mdoG transcription factor involved in the present invention has never been directly reported in terms of its role in exopolysaccharide synthesis, and it has great research value. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide, in view of the deficiencies of the prior art, a recombinant Pantoea alhagi and its application in the fermentative production of exopolysaccharide. The recombinant Pantoea alhagi is a deletion mutant of the transcription factor-encoding gene. The transcription factor-encoding gene mdoG is knocked out from the genome of Pantoea alhagi NX-11, so that more carbon metabolic flux flows towards the production of exopolysaccharide (EPS), thereby improving the ability of Pantoea alhagi NX-11 to produce EPS. Finally, the EPS yield is increased by 50%, which has important economic value and social significance.

[0005] To solve the above technical problems, the present invention discloses a recombinant Pantoea alhagi and its application in the fermentative production of exopolysaccharide. The specific technical solutions are as follows: A recombinant Pantoea alhagi, wherein the recombinant Pantoea alhagi has knocked out the gene mdoG encoding a transcription factor; wherein, the amino acid sequence of the transcription factor is as shown in SEQ ID NO.2.

[0006] Wherein, the nucleotide sequence of the gene mdoG encoding the transcription factor is as shown in SEQ ID NO.1.

[0007] Wherein, the starting strain of the recombinant Pantoea alhagi is Pantoea alhagi Pantoea alhagi NX-11. The Pantoea alhagi Pantoea alhagi NX-11, with the preservation number of CGMCC NO.15525, was preserved in the China General Microbiological Culture Collection Center on March 29, 2018.

[0008] Wherein, the knockout is achieved by using the CRISPR-Cas9 technology.

[0009] In the second aspect, the present invention provides a method for constructing the recombinant Pantoea alhagi described in the first aspect, comprising the following steps: (1) Construct a recombinant plasmid pTarget-mdoG: Amplify the sgRNA fragment targeting mdoG, the upstream homologous arm UP fragment, and the downstream homologous arm DN fragment, and obtain the gene fragment sgRNA-UP-DN by overlapping PCR, and clone it into the plasmid pTarget to obtain the recombinant plasmid pTarget-mdoG; (2) Introduce the pCas9 plasmid into the starting strain to obtain the starting strain carrying the pCas9 plasmid, and then introduce the recombinant plasmid pTarget-mdoG prepared in step (1) to obtain the recombinant Pantoea alhagi. Preferably, the recombinant plasmid pTarget-mdoG is introduced into the starting strain carrying the pCas9 plasmid by electroporation.

[0010] Among them, in step (1), the nucleotide sequence of the sgRNA fragment is as shown in 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 by the following methods: Using the starting bacterium as a template, the sgRNA fragment is amplified by using the primer nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.7; Using the starting bacterium as a template, the upstream homology arm UP fragment is amplified by using the primer nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9; Using the starting bacterium as a template, the downstream homology arm DN fragment is amplified by using the primer nucleotide sequences shown in SEQ ID NO.10 and SEQ ID NO.11.

[0011] The starting bacterium is preferably Pantoea alhagi Pantoea alhagi NX-11.

[0012] Further preferably, in step (2), after the recombinant plasmid pTarget-mdoG prepared in step (1) is introduced into the starting bacterium carrying the pCas9 plasmid, the obtained recombinant bacterium is induced at high temperature to delete the pCas9 plasmid and the pTarget-mdoG recombinant plasmid, and a strain with targeted deletion of the mdoG gene, that is, the recombinant Pantoea alhagi, is obtained.

[0013] In a third aspect, the present invention provides the use of the recombinant Pantoea alhagi described in the first aspect in the fermentation production of exopolysaccharides.

[0014] Among them, the recombinant Pantoea alhagi is inoculated into a fermentation medium for fermentation culture.

[0015] Among them, the fermentation medium includes: carbon source 10-100 g / L, nitrogen source 1-30 g / L, and inorganic salts 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 salts are any one or a combination of several of sulfates, phosphates, dihydrogen phosphates, hydrogen phosphates, sodium salts, and hydrochlorides; preferably, the fermentation medium includes sucrose 10-100 g / L, sodium chloride 0.01-50 g / L, yeast powder 1-15 g / L, and 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, and peptone 10 g / L.

[0016] 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.

[0017] Wherein, at room temperature, the viscosity of a 10 g / L extracellular polysaccharide aqueous solution prepared by using the extracellular polysaccharide is 10.32-24.11 Pa·s in a static state.

[0018] Beneficial effects: Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides an mdoG transcription factor and provides 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, and its concentration can reach up to 25.9 g / L, which is higher than that of the original strain. P. alhagi NX-11 increased by 50%, providing opportunities for further metabolic engineering P alhagi NX-11 lays the foundation for producing EPS. The recombinant strain construction method provided by the invention is simple, easy to use, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0020] Figure 1 This is the plasmid map of the recombinant plasmid pTarget-mdoG.

[0021] Figure 2 This is a graph showing the production of extracellular polysaccharides (EPS) obtained by fermentation of different strains.

[0022] Figure 3 The molecular weight of extracellular polysaccharide of NX-11 strain and mdoG knockout strain NX-11 (ΔmdoG).

[0023] Figure 4 The rheological properties of extracellular polysaccharides of NX-11 strain and mdoG knockout strain NX-11 (ΔmdoG). DETAILED DESCRIPTION

[0024] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention, but as long as they do not depart from the basic idea of the present invention, they are all within the scope of the present invention.

[0025] In the following examples, the experimental methods are all conventional methods unless otherwise specified; the reagents and materials can all be obtained from commercial sources unless otherwise specified.

[0026] In the following examples, the Pantoea alhagi NX-11 has the taxonomic name of Pantoea alhagi , and its deposit number is CGMCC NO.15525, and it was deposited in the China General Microbiological Culture Collection Center on March 29, 2018.

[0027] In the following examples, the mdoG is a transcription factor derived from Pantoea alhagi, and its amino acid sequence 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.

[0028] Example 1 Construction of ΔmdoG engineering strain based on CRISPR / Cas9 Using the pTarget plasmid as a template, the linearized vector was obtained by amplifying the pTarget plasmid with linearization primers (F-pTarget framework-CAT / M13, R-pTarget framework-CAT / M13).

[0029] Using the genome of Pantoea alhagi NX-11 as a template, PCR amplification was carried out 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 homologous arm mdoG-UP and downstream homologous arm mdoG-DN fragments respectively for ligating with the linearized vector pTarget to obtain the recombinant plasmid pTarget-mdoG.

[0030] The specific construction method is as follows: Using primer pairs F-pTarget framework-CAT / M13 and R-pTarget framework-CAT / M13, with the pTarget plasmid 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 1 min, with 30 cycles of reaction; finally, extension at 72 °C for 10 min to obtain the linearized vector pTarget; Using primer pairs F-mdoG-sgRNA-CAT / M13 and R-mdoG-sgRNA-CAT / M13, with the genome of Pantoea alhagi 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 10 s, with 30 cycles of reaction; 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.

[0031] Using primer pairs F-mdoG-UP-CAT / M13 and R-mdoG-UP-CAT / M13, primer pairs F-mdoG-DN-CAT / M13 and R-mdoG-DN-CAT / M13 respectively, with the genome of Pantoea alhagi 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, with 30 cycles of reaction; finally, extension at 72 °C for 10 min to obtain the mdoG-UP fragment and the mdoG-DN fragment respectively.

[0032] The above-obtained PCR product fragments 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.

[0033] Using primer pairs F-mdoG-sgRNA-CAT / M13 and R-mdoG-DN-CAT / M13, and adding the above-purified and recovered mdoG-UP fragment, mdoG-DN fragment, and mdoG-sgRNA fragment for overlap PCR. 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, with 30 cycles of reaction; the PCR product was recovered using a DNA purification kit (TianGen) to obtain the fusion fragment mdoG-sgRNA-UP-DN with vector homologous ends.

[0034] Ligation was performed using the ClonExpress II One Step Cloning Kit rapid cloning kit from Novoprotein Scientific Inc. The linearized vector pTarget obtained by PCR and the fusion fragment mdoG-sgRNA-UP-DN with vector homologous ends were mixed at a molar ratio of 2:1. At the same time, 4 μL of 5×CE II Buffer and 2 μL of Exnase II were added, and then ddH2O was added to make the total volume of the ligation system reach 20 μL. The reaction was carried out at 37 °C for 30 min and incubated at 4 °C. Then, 10 μL of the ligation system was taken for transformation E.coli into BL21(DE3) competent cells (for the method of preparing competent cells, please refer to the instruction manual of Takara Escherichia coli competent cell kit). Transformants with correct colony PCR were selected and then sent to Anhui General Biosciences Co., Ltd. for sequencing verification to obtain the recombinant plasmid pTarget-mdoG (as Figure 1 shown). The plasmid extraction was performed using the FastPure Plasmid Mini Kit plasmid extraction kit from Novoprotein Scientific Inc. The extracted pTarget-mdoG plasmid was transformed into P alhagi NX-11(pCas) strain by electroporation. Subsequently, it was induced with 0.1 mM IPTG for 4 h and cultured at 40 °C at high temperature to lose the pCas plasmid and pTarget-mdoG plasmid. Using the F-mdoG-OUT-CAT / M13 and R-mdoG-OUT-CAT / M13 primers, positive clones were selected. The PCR conditions were: 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 20 s, for 30 cycles; and finally extension at 72 °C for 10 min. Then it was sent to Anhui General Biosciences Co., Ltd. for sequencing verification, and finally the strain NX-11 (ΔmdoG) with mdoG gene deletion was obtained.

[0035] Among them, the detailed information of the NX-11(pCas) strain and the gene knockout method described in this example can be found in the 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). ].

[0036] The primer sequences involved in this example are shown in Table 1.

[0037] Table 1 Primer Sequence Table

[0038]

[0039] Example 2 Fermentation Culture of Recombinant Bacteria Taking the wild-type NX-11 without plasmid as a control, the recombinant bacterium NX-11 (ΔmdoG) constructed in Example 1 and the starting bacterium NX-11 were respectively 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, 10 g / L peptone) at an inoculation amount of 4% v / v, and fermented 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 alcohol precipitation was carried out overnight at 4 °C. After centrifugation, the precipitate was collected, and after freeze-drying, the extracellular polysaccharide (EPS) yield obtained by fermenting the starting bacterium was measured to be 17.6 g / L, while the EPS yield obtained by fermenting the mdoG gene-deleted strain NX-11 (ΔmdoG) was 25.9 g / L (as Figure 2 shown), which was nearly 50% higher than that of the starting bacterium. The EPS yield was quantitatively detected by the phenol-sulfuric acid method.

[0040] Example 3 Characterization Analysis of Extracellular Polysaccharide of Recombinant Bacteria The EPS prepared in Example 2 was added with ultrapure water to make a dilution, centrifuged to remove excess bacteria, filtered through a 0.22 μm PES filter membrane, freeze-dried, and redissolved to 10 g / L to obtain a sample to be tested. Then, a mobile phase (2 mM CH3COONH4, pH = 4.5) was prepared, and an Ultrahydrogel TM 500 column and an Ultrahydrogel TM column water-soluble gel column series chromatographic column were used, and the molecular weight of the sample was detected by an Agilent 1260 HPLC system. The results were as Figure 3 shown. The extracellular polysaccharide obtained by fermenting NX-11 (ΔmdoG) showed a peak at 4 min, indicating a large molecular weight. Then, the flow characteristics of the above sample were analyzed by a Discovery hybrid rheometer HR30 (condition: 25 °C). The results were as Figure 4 shown. At the same shear rate, the viscosity of the extracellular polysaccharide obtained by fermenting NX-11 (ΔmdoG) was always higher than that of the extracellular polysaccharide obtained by fermenting the starting bacterium NX-11. And the viscosity of the extracellular polysaccharide obtained by fermenting NX-11 (ΔmdoG) under static conditions was 10.32 - 24.11 Pa·s.

[0041] The present invention provides an idea and method for a recombinant Pantoea alhagi and its application in the fermentation production of exopolysaccharides. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A recombinant Pantoea alhagi, characterized in that, The described Pantoea alhagi has knocked out the gene mdoG encoding a transcription factor; wherein, the amino acid sequence of the transcription factor is as shown in SEQ ID NO.

2.

2. The recombinant Pantoea alhagi according to claim 1, wherein The nucleotide sequence of the gene mdoG encoding the transcription factor is as shown in SEQ ID NO.

1.

3. The recombinant Pantoea alhagi according to claim 1, wherein The starting strain of the recombinant Pantoea alhagi is Pantoea alhagi Pantoea alhagi NX-11.

4. The recombinant Pantoea alhagi according to claim 1, wherein The knockout is achieved by using the CRISPR-Cas9 technology.

5. The construction method of the recombinant Pantoea alhagi according to any one of claims 1 to 4, characterized in that It includes the following steps: (1) Construct the recombinant plasmid pTarget-mdoG: Amplify the sgRNA fragment targeting the mdoG, the upstream homologous arm UP fragment, and the downstream homologous arm DN fragment, and obtain the gene fragment sgRNA-UP-DN by overlapping PCR, and clone it into the plasmid pTarget to obtain the recombinant plasmid pTarget-mdoG; (2) Introduce the pCas9 plasmid into the starting bacterium to obtain the starting bacterium carrying the pCas9 plasmid, and then introduce the recombinant plasmid pTarget-mdoG prepared in step (1) to obtain the described Pantoea alhagi.

6. The construction method according to claim 5, characterized in that In step (1), the nucleotide sequence of the sgRNA fragment is as shown in SEQ ID NO.

3.

7. Use of the Pantoea alhagi according to any one of claims 1 to 4 in the fermentation production of exopolysaccharides.

8. The application according to claim 7, wherein Inoculate the described Pantoea alhagi into a fermentation medium for fermentation culture.

9. The application according to claim 8, wherein The fermentation medium includes: carbon source 10 - 100 g / L, nitrogen source 1 - 30 g / L, and inorganic salts 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 salts are any one or a combination of several of sulfates, phosphates, dihydrogen phosphates, hydrogen phosphates, sodium salts, and hydrochlorides; For the fermentation culture, the culture temperature is 25 - 32 °C, and the culture time is 20 - 30 h.

10. The application according to claim 7, wherein At room temperature, the viscosity of a 10 g / L aqueous solution of the exopolysaccharide prepared using the exopolysaccharide is 10.32 - 24.11 Pa·s under static conditions.

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