Recombinant pantoea alhagi XK 11 having filamented cell morphology, and construction method therefor and use thereof

Abstract

Disclosed in the present invention is recombinant Pantoea alhagi XK 11 having filamented cell morphology, achieved by constructing an expression vector to simultaneously overexpress genes slmA and sulA in starting strain Pantoea alhagi XK 11. In the present invention, cell morphology regulating genes slmA and sulA in Pantoea alhagi XK 11 are induced and expressed to construct recombinant strain, so that the obtained recombinant strain has significantly increased cell length which is 15-20 times greater than that of the starting strain Pantoea alhagi XK 11. The recombinant Pantoea alhagi XK 11 can achieve more effective strain collection, thereby simplifying a downstream processing flow, and reducing the cost of downstream processing.

Description

Recombinant pantoea alhagi with cell morphology of filamentation and construction method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of microbial engineering, in particular to a recombinant pantoea alhagi with cell morphology of filamentation and construction method and application thereof. BACKGROUND

[0002] Pantoea alhagi (XK-11) is a gram-negative bacterium widely present in natural environment, which has attracted widespread attention due to its ability to produce abundant extracellular polysaccharide (EPS). Extracellular polysaccharide has various industrial application values, including as thickening agent, stabilizer and carrier material in biological medicine, etc. However, the extracellular polysaccharide produced by pantoea alhagi in the fermentation process faces many challenges in the downstream separation and purification process, mainly because the bacterial cells are mixed with the extracellular polysaccharide, which is difficult to separate effectively. The existing technology usually relies on traditional centrifugation and filtration technology, which is difficult to solve the problem of mixing of extracellular polysaccharide and bacterial cells, thereby increasing the complexity and cost of the separation process. Therefore, filamentation of cell morphology of pantoea alhagi is one of the measures to reduce the complexity and cost of the separation process.

[0003] CN117965413A discloses a recombinant pantoea alhagi with cell morphology of filamentation and construction method and application thereof, which is achieved by exchanging minc gene, and the recombinant pantoea alhagi has a cell morphology more filamentous than the starting strain XK-11, with a length of two to several times of the starting strain, which can be better used for preparation of pantoea alhagi polysaccharide, facilitating separation and improving the efficiency of downstream processing.

[0004] Therefore, under the premise of not affecting the growth condition of bacterial cells and polysaccharide yield, the bacterial cells can be better applied to downstream processing by regulating the morphology of bacterial cells. SUMMARY

[0005] The present application aims to solve the technical problems of the prior art, and provides a recombinant pantoea alhagi with cell morphology of filamentation and construction method and application thereof, so as to facilitate the separation of extracellular polysaccharide, significantly improve the downstream separation efficiency of pantoea alhagi extracellular polysaccharide, reduce the separation cost, and improve the production efficiency.

[0006] In order to solve the above technical problems, the application discloses a recombinant Pantoea alhagi with cell morphology filamentation, which is obtained by simultaneously overexpressing slmA gene and sulA gene in a starting strain Pantoea alhagi XK 11 with a preservation number of CGMCC NO.15526, wherein the nucleotide sequence of the slmA gene is shown as SEQ ID NO:3, and the nucleotide sequence of the sulA gene is shown as SEQ ID NO:4.

[0007] The recombinant Pantoea alhagi contains an expression vector, wherein the expression vector contains a resistance gene promoter-KanR resistance gene, an araBAD promoter, an slmA gene and a sulA gene; the nucleotide sequence of the resistance gene promoter-KanR resistance gene is shown as SEQ ID NO:1; the nucleotide sequence of the araBAD promoter is shown as SEQ ID NO:2; and the araBAD promoter exists twice on the expression vector, and is located at the 5' end of the slmA gene and the sulA gene, respectively.

[0008] The application further provides a construction method of the recombinant Pantoea alhagi, which comprises the following steps:

[0009] (1) preparing competent cells of the starting strain Pantoea alhagi XK 11;

[0010] (2) constructing an expression vector, wherein the plasmid of the expression vector is inserted with the slmA gene and the sulA gene, and the expression vector is electrically transformed into the prepared competent cells of the starting strain XK-11 to obtain a recombinant strain containing the expression vector; preferably, the plasmid of the expression vector is a pBAD plasmid;

[0011] (3) culturing the recombinant strain by L-arabinose induction to obtain the recombinant Pantoea alhagi with simultaneous overexpression of the slmA gene and the sulA gene.

[0012] In step (2), the expression vector has kanamycin resistance, and the expression vector is electrically transformed into the starting strain under the condition of a voltage of 12-14 kv and a time of 3-5 ms, and then the recombinant strain containing the expression vector is finally obtained through kanamycin resistance screening.

[0013] In an embodiment, the expression vector is pBADF1 slmA-sulA, which is constructed by the following method: 1) PCR amplification of slmA gene fragment and sulA gene fragment by using the genome of the starting strain as a template through primers (pBAD-slmA-F / R and pBAD-sulA-F / R), and transforming the resistance gene AmpR of the original pBAD / His plasmid into KanR, so that the plasmid has kanamycin resistance, and the reformed empty plasmid is named as pBADH;

[0014] 2) Double enzyme digestion of the empty plasmid pBADH at the enzyme cutting sites by using SacI and HindIII endonucleases to obtain vector framework 1;

[0015] 3) Linking the slmA gene fragment and the sulA gene fragment obtained in step 1) to the above-mentioned vector framework 1 through ligase Exnase II to obtain two expression vectors pBADF slmA and pBADF sulA , i.e., the expression vector pBADF is a plasmid for expressing one piece of foreign DNA inserted into the empty plasmid pBADH.

[0016] 4) Using the plasmid pBADF slmA containing the slmA gene (and the plasmid pBADF slmA containing the sulA gene) as a template, cutting the enzyme cutting sites by using XmnI endonuclease to obtain vector framework 2 (and vector framework 3), and then using the plasmid pBADF sulA containing the sulA gene as a template, PCR amplification and purification of the PCR product through primers (KAN-sulA-F / R) to obtain an araBAD-sulA nucleotide fragment, which is a nucleotide fragment containing the araBAD promoter and the sulA gene; the nucleotide sequence of the araBAD-sulA fragment is SEQ ID NO: 7;

[0017] 5) Linking the vector framework 2 and the araBAD-sulA nucleotide fragment through ligase Exnase II to obtain the expression vector pBADF1 slmA-sulA .

[0018] In step (3), before adding L-arabinose, the recombinant strain carrying the expression vector is inoculated into a kanamycin-resistant culture medium for culture, and then L-arabinose is added for induction after the OD600 of the bacterial liquid is cultured to 0.4-0.6.

[0019] Preferably, the working concentration of the L-arabinose is 10 mM-20 mM.

[0020] Meanwhile, according to the construction method, the mreB gene fragment is obtained by PCR amplification with primers (pBAD-mreB-F / R), wherein the nucleotide sequence of the mreB gene is shown as SEQ ID NO: 5. mreB The expression vector pBADF1 is obtained by the method of step 4) with the plasmid pBADF mreB as a template, and a araBAD-mreB nucleotide fragment containing an araBAD promoter and an mreB gene is obtained by PCR amplification with primers (KAN-mreB-F / R) and purification of the PCR product, wherein the nucleotide sequence of the araBAD-mreB fragment is SEQ ID NO: 9; and the expression vector pBADF1 slmA-mreB and the expression vector pBADF1 sulA-mreB .

[0021] The application further provides application of the recombinant S. camels in fermentation preparation of S. camels extracellular polysaccharide.

[0022] Specifically, in application, after fermentation, the bacteria are separated by centrifugation, and the centrifugal force is greater than or equal to 5000g.

[0023] Beneficial effects: the production strain of the extracellular polysaccharide constructed in the application significantly reduces the mixing degree of the bacteria and the extracellular polysaccharide in the fermentation process through the filamentation of the cell morphology, so that the separation process of the extracellular polysaccharide is more simple and efficient. In the prior art, the complex centrifugation and filtration process leads to high separation cost. The application simplifies the separation steps by changing the physical morphology, thereby greatly reducing the production cost. Due to the simplification and efficiency improvement of the separation process, the downstream processing time is significantly shortened, and the overall production efficiency is improved. The application is not only suitable for S. camels, but also can be popularized and applied to other microorganisms that need to improve the separation efficiency of extracellular polysaccharide, and has wide industrial application prospect. It has great application potential in food, medicine, cosmetics and other industries.

[0024] Table of drawings

[0025] The above and / or other aspects of the application will become more apparent by describing in detail the preferred embodiments thereof with reference to the attached drawings table and specific embodiments.

[0026] Fig. 1 is a structure diagram of the expression plasmid pBADF;

[0027] Fig. 2 is a structure diagram of the expression plasmid pBADF1;

[0028] Figure 3 is the cell morphology of the recombinant strain P. alhagi XK 11-pBADF SulA (WT / sulA), P. alhagi XK 11-pBADF SlmA (WT / slmA), P. alhagi XK 11-pBADF slmA-sulA (WT / slmA-sulA) and the cell morphology of the empty starting strain P. alhagi XK 11-pBADH (WT / pBADF);

[0029] Figure 4 is the cell morphology of the recombinant strain P. alhagi XK 11-pBADF mreB (WT / mreB), P. alhagi XK 11-pBADF sulA-mreB (WT / sulA-mreB), P. alhagi XK 11-pBADF slmA-mreB (WT / slmA-sulA) and the cell morphology of the empty starting strain P. alhagi XK 11-pBADH (WT / pBADF);

[0030] Figure 5 is the growth curve of the recombinant strain P. alhagi XK 11-pBADF / pBADF1 overexpressing the morphological regulatory genes and the empty starting strain P. alhagi XK 11-pBADH;

[0031] Figure 6 is the state of the shake flask fermentation broth of the recombinant strain P. alhagi XK 11-pBADF1 slmA-sulA overexpressing slmA and sulA and the empty starting strain P. alhagi XK 11-pBADH under the condition of ≥5000g centrifugation for 20min; wherein the control group is P. alhagi XK 11-pBADH and the experimental group is P. alhagi XK 11-pBADF1 slmA-sulA . DETAILED DESCRIPTION

[0032] The primer list used in the following examples is shown in Table 1.

[0033] Table 1 Primers used in the study

[0034] Example 1 Construction of expression vector pBADF.

[0035] The expression vector contains conventional expression elements, resistance gene promoter- kanamycin resistance gene (KanR), araBAD promoter (araBAD promoter), and slmA or (sulA and mreB) nucleotide fragments.

[0036] The nucleotide fragment of the resistance gene promoter - kanamycin resistance gene (KanR) is shown as SEQ ID NO: 1:

[0037] The nucleotide fragment of the araBAD promoter is shown as SEQ ID NO: 2:

[0038] The nucleotide fragment of the slmA gene is shown as SEQ ID NO: 3:

[0039] The nucleotide fragment of the sulA gene is shown as SEQ ID NO: 4:

[0040] The mreB nucleotide fragment is shown as SEQ ID NO: 5:

[0041] First, the slmA, sulA and mreB gene fragments were obtained by PCR using the genome of the starting strain as a template and designing primers (shown in Table 1); meanwhile, the resistance gene and promoter (AmpR and AmpR promoter) of the original pBAD / His plasmid were transformed into KanR and KanR promoter, and the transformed empty plasmid was named pBADH. The primers are shown in Table 1.

[0042] On this basis, the empty plasmid pBADH was used as a template, and double digestion was performed at the enzyme cutting site by SacI and HindIII endonuclease to obtain vector framework 1.

[0043] Then, the slmA (or sulA, mreB) gene fragments obtained by purifying the PCR products were respectively connected with the above-mentioned vector framework 1 by ligase Exnase II to obtain the expression vector pBADF (pBADF slmA , pBADF sulA and pBADF mreB ) carrying the above-mentioned target genes. The expression vector pBADF is a plasmid in which an exogenous DNA is inserted into the empty plasmid pBADH for expression. The expression vector pBADF is shown in Figure 1.

[0044] Example 2 Construction of expression vector pBADF1.

[0045] The routine expression elements contained in the expression vector pBADF1, the kanamycin resistance gene (KanR), the promoter of the kanamycin resistance gene (KanR promoter), and the araBAD promoter (araBAD promoter) are the same as those in the above-mentioned expression vector pBADF, except that the target gene contained in the cloning site is a double gene fragment, such as a slmA-sulA nucleotide fragment, a slmA-mreB nucleotide fragment, and a sulA-mreB nucleotide fragment.

[0046] The slmA-sulA nucleotide fragment contains a slmA gene fragment, an araBAD-sulA nucleotide fragment, and a sulA gene fragment; the slmA-sulA nucleotide fragment is shown in SEQ ID NO: 6; and the araBAD-sulA nucleotide fragment is shown in SEQ ID NO: 7.

[0047] Specifically, the slmA-sulA fragment nucleotide sequence SEQ ID NO: 6 is as follows:

[0048] The araBAD-sulA fragment nucleotide sequence SEQ ID NO: 7 is as follows:

[0049] The slmA-mreB nucleotide fragment contains a slmA gene fragment, an araBAD-mreB nucleotide fragment, and a mreB gene fragment; the slmA-mreB nucleotide fragment is shown in SEQ ID NO: 8; and the araBAD-mreB nucleotide fragment is shown in SEQ ID NO: 9.

[0050] Specifically, the slmA-mreB fragment nucleotide sequence SEQ ID NO: 8 is as follows:

[0051] The araBAD-mreB fragment nucleotide sequence SEQ ID NO: 9 is as follows:

[0052] The sulA-mreB nucleotide fragment contains a sulA gene fragment, an araBAD-mreB nucleotide fragment, and a mreB gene fragment; the sulA-mreB nucleotide fragment is shown in SEQ ID NO: 10.

[0053] Specifically, the sulA-mreB fragment nucleotide sequence SEQ ID NO: 10 is as follows:

[0054] First, the vector framework 2 and the vector framework 3 were obtained by cutting the XmnI endonuclease cutting site as templates of the plasmid pBADF slmA ( and pBADF sulA ) containing the slmA (and sulA) gene, and then purified by PCR to obtain an araBAD-sulA (and araBAD-mreB) nucleotide fragment as templates of the plasmid pBADF sulA ( and pBADF mreB ) containing the sulA (and mreB) gene.

[0055] The araBAD-sulA nucleotide fragment is a nucleotide fragment containing the araBAD promoter and the sulA gene.

[0056] The araBAD-mreB nucleotide fragment is a nucleotide fragment containing the araBAD promoter and the mreB gene.

[0057] Finally, the above vector framework 2 and the araBAD-sulA (or araBAD-mreB) nucleotide fragment were connected by the ligase Exnase II to obtain the expression vector pBADF1 (pBADF1 slmA-sulA , pBADF1 slmA-mreB , pBADF1 sulA-mreB ), which is a plasmid for expressing a foreign DNA inserted on the basis of the expression vector pBADF. The structure of the expression vector pBADF1 is shown in FIG. 2, thereby obtaining three different expression vectors.

[0058] Example 3 Construction of Recombinant Leptospirillum.

[0059] First, the competent cells of the starting strain XK-11 were prepared by inoculating the starting strain XK-11 into 5 mL of LB culture solution for overnight culture to obtain a primary seed solution, and then inoculating the primary seed solution into 100 mL of LB culture solution at an inoculation amount of 1% for 2-3 h (OD 600(Approximately 0.4-0.6), then incubate the bacterial culture on ice for 10 minutes, aliquot into pre-chilled 50mL centrifuge tubes, centrifuge at 4℃, 4000rpm for 5 minutes, discard the supernatant, resuspend in pre-chilled ddH2O and add to a final volume of 30mL, mix by inverting, centrifuge at 4℃, 4000rpm for 5 minutes, discard the supernatant, resuspend in ddH2O containing 10% glycerol and add to a final volume of 30mL, mix by inverting, centrifuge at 4℃, 4000rpm for 5 minutes, and finally use... XK-11 competent cells were obtained by resuspending bacterial sludge in 1-2 mL of ddH2O containing 10% glycerol. The constructed expression vector was then introduced into the starting strain XK-11 via electroporation, with a volume ratio of expression vector to competent cells of 0.5 / 10 to 1 / 10. The mixture was introduced into the starting strain XK-11 under conditions of 12-14 kV and 3-5 ms. Recombinant strains were obtained after kanamycin resistance screening. These recombinant strains carried pBADF... slmA pBADF sulA pBADF mreB pBADF1 slmA-sulA pBADF1 slmA-mreB pBADF1 sulA-mreB The XK-11 strain expressing the vector was used. The recombinant strain was inoculated into kanamycin-resistant medium and cultured. The OD of the bacterial culture was... 600 The culture was induced by adding L-arabinose at a working concentration of 10mM to 20mM when the culture temperature reached 0.4-0.6; finally, recombinant pantotheca camelidii with filamentous cell morphology was obtained.

[0060] Example 4: Morphological observation of recombinant Pantotheca acuminata.

[0061] The recombinant P.alhagi was cultured in 0.2% L-arabinose for 24 hours, 2 mL of bacterial solution was collected by centrifugation at 5000 g for 2 min, and the bacterial cells were washed with ddH2O three times, centrifuged to discard the supernatant, 2.5% glutaraldehyde was added to the 2 mL centrifuge tube, and the sample was fixed at 4°C overnight. Then, the phosphate buffer was washed for 3 times, 1% osmium acid was added for 4-6 hours, and then the phosphate buffer was washed, ethanol gradient dehydration was used, and then the cell sample was freeze-dried as described above. The cell morphology of the strain was observed by scanning electron microscopy, and the cell morphology of the strain is shown in Figure 3. As can be seen from Figure 3, overexpression of sulA and slmA genes in P.alhagi can make P.alhagi filamentous, and the cell length is increased by 10-15 times compared with the starting strain. And overexpression of sulA and slmA genes in P.alhagi makes the filamentous degree more obvious, and the cell length is increased by 15-20 times compared with the starting strain. As shown in Figure 4, overexpression of mreB gene makes the cell morphology tend to be round, and overexpression of sulA and mreB genes and overexpression of slmA and mreB genes make the cell morphology increase by 1-1.5 times or tend to be round. In summary, the best combination of filamentous cell morphology is overexpression of sulA and slmA genes.

[0062] Example 5 Analysis of the morphology and growth of recombinant P.alhagi

[0063] The activated recombinant P.alhagi cultured on solid plates was inoculated into 10 mL of fresh LB liquid medium, and was cultured at 37°C, 200 rpm overnight to obtain a primary seed liquid; P.alhagi XK 11-pBADF, P.alhagi XK 11-pBADF1 and P.alhagi XK 11 primary seed liquids were each inoculated into three 50 mL LB liquid medium 250 mL shake flasks at a 1% inoculation amount, and were cultured at 37°C, 200 rpm for 24 hours. When the OD600 value of the bacterial solution was 0.4-0.6, L-arabinose with a working concentration of 10 mM-20 mM was added for induction, and the OD600 value was measured every 2 hours from the start of inoculation to compare and analyze the growth as shown in Figure 5. As can be seen from Figure 5, the recombinant P.alhagi P.alhagi XK 11 pBADF1 slmA-sulA , P.alhagi XK 11 pBADF sulA , P.alhagi XK 11 pBADF1 slma , P.alhagi XK 11 pBADF mreB , P.alhagi XK 11 pBADF1 slmA-mreB , P.alhagi XK 11 pBADF1sulA-mreB The growth of the bacteria was affected.

[0064] Example 6 Fermentation preparation of recombinant P.alhagi XK 11-pBADF polysaccharide

[0065] P.alhagi XK 11-pBADF1 polysaccharide was obtained by the above method. slmA-sulA The primary seed liquid was inoculated into 200 mL of LB medium at an inoculation amount of 1% to 4%, and cultured at 37°C and 200 rpm for 12 h to obtain a secondary seed liquid.

[0066] The secondary seed liquid was inoculated into a fermenter containing 2 L of fermentation medium (sucrose 60 g / L, peptone 10 g / L, sodium chloride 5 g / L, potassium phosphate dibasic 1 g / L, pH = 7) at an inoculation amount of 4% to 10%, and fermented for 24 h under the conditions of aeration amount of 2 to 2.5 vvm and stirring speed of 200 rpm, to obtain a fermentation liquid of recombinant P.alhagi XK 11-pBADF slmA-sulA ) polysaccharide.

[0067] The alcohol precipitation method is the mainstream process for extracting various polysaccharides. The fermentation liquid of 5000 g was centrifuged for 20 min, the supernatant was removed and 2 times the volume of 95% ethanol was added for alcohol precipitation, and then 5000 g centrifugation was performed for 5 min, and the supernatant was discarded and dried to obtain P.alhagi XK 11-pBADF1 polysaccharide. In addition, the fermentation preparation of P.alhagi XK 11 and other recombinant P.alhagi XK 11 polysaccharides was the same as the above steps. Finally, the polysaccharide yield of P.alhagi XK 11-pBADF slmA-sulA was 12.5 ± 1 g / L, and the polysaccharide yield of the original strain XK 11 was 11 ± 1 g / L, and there was no significant difference in polysaccharide yield.

[0068] Example 7 Analysis of the separation efficiency of P.alhagi filamentous strains

[0069] For the analysis of the separation efficiency of P.alhagi filamentous strains, first, 20 mL of P.alhagi XK 11-pBADF slmA-sulA and P.alhagi XK 11 fermentation liquid was taken into a 50 mL EP centrifuge tube, and centrifuged at 5000 g for 20 min. After centrifugation, the sedimentation degree of the two liquid bacteria was observed, as shown in Figure 6, and the control group was the strain P.alhagi XK 11, and the experimental group was the recombinant strain P.alhagi XK 11-pBADF slmA-sulAIt can be seen from the figure that under the centrifugal force of 5000g for 20min, the supernatant of the control strain P.alhagi XK 11 is relatively clear compared with the recombinant strain P.alhagi XK 11-pBADF slmA-sulA The supernatant is relatively turbid. That is, the recombinant strain can achieve better cell collection, which helps to improve the efficiency of downstream separation processing.

[0070] The present application provides a camel thorn bacteria cell morphology filamentous construction ideas and methods, the method and the way of realizing the technical scheme are many, the above-mentioned is only the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the principle of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application. The components not explicitly described in the present embodiment can be realized by existing technology

Claims

1. A recombinant species of Camelot spicatus with cell morphology filamentation, characterized by, The recombinant Pantoea alhagi is obtained by simultaneously overexpressing slmA gene and sulA gene in the starting strain Pantoea alhagi, wherein the starting strain is Pantoea alhagi XK 11 with the preservation number of CGMCC NO.15526, the nucleotide sequence of the slmA gene is shown in SEQ ID NO: 6 from the 1st to the 597th position, and the nucleotide sequence of the sulA gene is shown in SEQ ID NO: 7 from the 314th to the 820th position.

2. The recombinant H. marinum of claim 1, wherein, The recombinant Pantoea alhagi comprises an expression vector, wherein the expression vector comprises a resistance gene promoter-KanR resistance gene, an araBAD promoter, a slmA gene and a sulA gene, the nucleotide sequence of the resistance gene promoter-KanR resistance gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the araBAD promoter is shown in SEQ ID NO: 2; the araBAD promoter exists twice on the expression vector, and the positions are respectively at the 5' ends of the slmA gene and the sulA gene.

3. The method of constructing a recombinant of the genus Camelovalgatus according to claim 1 or 2, characterized in that, The method comprises the following steps: (1) preparing competent cells of the starting strain Pantoea alhagi XK 11; (2) constructing an expression vector, inserting the slmA gene and the sulA gene into the plasmid of the expression vector, and electrically transforming the expression vector into the competent cells of the starting strain Pantoea alhagi XK 11 prepared in step (1) to obtain a recombinant strain containing the expression vector; (3) culturing the recombinant strain by induction of L-arabinose to obtain a recombinant Pantoea alhagi overexpressing the slmA gene and the sulA gene.

4. The construction method according to claim 3, characterized in that, In step (2), the expression vector has kanamycin resistance, the expression vector is electrically transformed into the starting strain under the condition of a voltage of 12-14 kv and a time of 3-5 ms, and finally the recombinant strain containing the expression vector is obtained through screening of kanamycin resistance.

5. The construction method of claim 3, wherein, The expression vector is pBADF1 slmA-sulA which is constructed by the following method: 1) obtaining the slmA gene and the sulA gene by PCR with the genome of the starting strain as a template, and transforming the resistance gene AmpR of the original pBAD / His plasmid into KanR so that the plasmid has kanamycin resistance, and naming the transformed empty plasmid as pBADH; 2) obtaining the vector framework 1 by double enzyme digestion at the enzyme cutting site of SacI and HindIII with the empty plasmid pBADH as a template; 3) The slmA gene and sulA gene obtained from step 1) are respectively connected with the above-mentioned vector framework 1 by ligase to obtain two expression vectors pBADF slmA , pBADF sulA ; 4) Using the expression vector pBADF slmA As a template, the vector framework 2 was obtained by cutting the enzyme cutting site, and then using the expression vector pBADF sulA As a template, an araBAD-sulA nucleotide fragment containing an araBAD promoter and a sulA gene was obtained by PCR and product purification; the nucleotide sequence of the araBAD-sulA fragment is SEQ ID NO: 7; 5) The nucleotide fragment of araBAD-sulA is ligated to the above-mentioned vector frame 2 by ligase to obtain an expression vector pBADF1 slmA-sulA .

6. The method of construction of claim 3, wherein, In step (3), before adding L-arabinose, the recombinant strain carrying the expression vector is inoculated into a kanamycin-resistant culture medium for culture, and then L-arabinose is added for induction after the OD600 of the bacterial liquid is cultured to 0.4-0.

6.

7. The construction method according to claim 3 or 5, characterized in that, The working concentration of the L-arabinose is 10-20 mM.

8. Application of the recombinant Pantoea alhagi of claim 1 or 2 in fermentation for preparing Pantoea alhagi exopolysaccharide.

9. Use according to claim 8, characterized in that, After fermentation, the bacterial bodies are separated by centrifugation with a centrifugal force of ≥5000 g.

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