Mfs-1 transporter protein gene and method for increasing pullulan production

Abstract

The application discloses an MFS-1 transport protein gene and a method for improving the yield of pullulan, and belongs to the technical field of biology. The method comprises the following steps: obtaining the MFS-1 transport protein gene, wherein the nucleic acid sequence of the MFS-1 transport protein gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; transferring the MFS-1 transport protein gene into Aureobasidium pullulans to obtain an Aureobasidium pullulans pLH454-MFS-1 recombinant strain; and finally, fermenting the Aureobasidium pullulans pLH454-MFS-1 recombinant strain to obtain a fermentation liquor containing pullulan. The MFS-1 transport protein gene is obtained, the recombinant strain is constructed and then fermented, the fermentation time is shortened, the ability of the Aureobasidium pullulans to synthesize pullulan is improved, the pullulan is more efficiently, rapidly and intensively expressed and synthesized, the production cost of enterprises can be reduced, and therefore, the pullulan has a wider application space and prospect.

Description

Technical Field

[0001] This invention relates to an MFS-1 transporter protein gene and a method for increasing pullulan production, belonging to the field of biotechnology. Background Technology

[0002] MFS transporters function primarily by switching between two conformations during substance transport: one open to the outside and the other open to the inside. When in the open-outside conformation, they bind extracellular substrate molecules. Subsequently, they undergo a conformational change to open to the inside, releasing the substrate into the cell. Conversely, they can also transport substances from inside to outside the cell. MFS transporters participate in the transmembrane transport of signaling molecules. In the regulation of pullulan synthesis in *Brachystomata*, signaling pathways such as cAMP-PKA, TORC1, HOG1, and Snf1 play crucial roles. MFS transporters indirectly regulate the activity of these signaling pathways by transporting signaling molecules associated with them, ultimately influencing the synthesis and secretion of pullulan.

[0003] Pullulan is an extracellular polysaccharide produced by the fermentation of *Brachystomata*. It is non-toxic, edible, and biodegradable, and has wide applications in the food, pharmaceutical, and chemical industries. However, its current fermentation yield and production efficiency have become bottlenecks for large-scale industrial application. Traditional fermentation processes result in low pullulan yields, leading to high production costs. This puts cost pressure on many companies during large-scale production, limiting the widespread promotion and application of pullulan in the market. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an MFS-1 transporter protein gene and a method for increasing pullulan polysaccharide production. By obtaining the MFS-1 transporter protein gene, constructing a recombinant strain and fermenting it, the ability of *Brachystomata buddingis* to synthesize pullulan polysaccharide is improved, resulting in more efficient, rapid, and concentrated expression and synthesis of pullulan polysaccharide.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] An MFS-1 transporter gene, the nucleic acid sequence of which is shown in SEQ ID NO.1 and the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] Furthermore, the amino acid sequence shown in SEQ ID NO.2 is based on the original sequence of the MFS transporter gene with the following mutations: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is modified by replacing Y with S at position 28, E with A at position 102, S with I at position 119, R with Q at position 145, D with V at position 201, F with C at position 227, N with D at position 286, V with E at position 408, and E with A at position 427.

[0008] Furthermore, the MFS-1 transporter gene is obtained by site-directed mutation of the MFS transporter gene.

[0009] Furthermore, the MFS transporter gene has an accession number of 40751144 in the NCBI database.

[0010] A method for increasing pullulan yield using the MFS-1 transporter gene includes the following steps:

[0011] (1) Construction of recombinant plasmid pLH454-MFS-1: The MFS-1 gene was inserted into plasmid pLH454 to obtain recombinant plasmid pLH454-MFS-1;

[0012] (2) Preparation of recombinant Escherichia coli E. coli BL21 / pLH454-MFS-1: The recombinant plasmid was introduced into competent E. coli BL21 cells by heat shock method and sequenced to obtain recombinant E. coli E. coli BL21 / pLH454-MFS-1 containing the correctly sequenced recombinant plasmid pLH454-MFS-1;

[0013] (3) Preparation of recombinant strain pLH454-MFS-1 of budding short-term causal agent: The plasmid pLH454-MFS-1 with correct sequencing was extracted from recombinant Escherichia coli. The plasmid was transformed into competent cells of budding short-term causal agent S1 by electroporation to obtain recombinant strain pLH454-MFS-1 of budding short-term causal agent.

[0014] (4) Preparation of seed liquid: The recombinant strain of budding short-stalked fungus was inoculated on YPD solid medium plates and cultured until the fungal cells covered the plates. The mycelium was collected and inoculated into seed medium to obtain seed liquid.

[0015] (5) Preparation of fermentation broth containing pullulan: The seed liquid was inoculated into the fermentation medium and fermented for 42 hours to obtain fermentation broth containing pullulan, with a fermentation yield of >90g / L.

[0016] Furthermore, in step (4), the seed culture medium consists of: 50 g / L sucrose, 1 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 6.3 g / L anhydrous dipotassium hydrogen phosphate, 0.6 g / L ammonium sulfate, 2 g / L yeast extract, and pH adjusted to 6.5.

[0017] Furthermore, in step (4), the culture temperature is 28°C, the culture speed is 250 rpm, and the culture time is 16 h.

[0018] Furthermore, in step (5), the fermentation medium consists of: 100 g / L glucose, 1 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 6.3 g / L anhydrous dipotassium hydrogen phosphate, 0.6 g / L ammonium sulfate, 2 g / L yeast extract, 0.05% Tween-80, 3% antifoaming agent, and pH adjusted to 5.5.

[0019] Furthermore, in step (5), the inoculation amount of the seed liquid is 10-20% (V / V) of the fermentation medium.

[0020] Furthermore, in step (5), the fermentation temperature is 28°C, the fermentation speed is 400 rpm, and the aeration rate is 1 vvm.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. This invention involves site-directed mutagenesis of the MFS transporter gene to obtain the MFS-1 transporter gene, and constructing a recombinant strain that can improve the strain's ability to produce pullulan, reduce the production cost of pullulan, and increase the market share of pullulan.

[0023] 2. This invention modifies the budding short-stem mold through genetic engineering, thereby improving the production capacity of the strain and shortening the fermentation time to 42 hours. Compared with the original strain, the fermentation yield of the recombinant strain is increased by more than 55% under the same fermentation time, and there is no need to change the original fermentation production conditions and processes, making the operation simple and convenient. Detailed Implementation

[0024] Example 1

[0025] Preparation of the MFS-1 transporter gene:

[0026] The MFS-1 transporter gene was obtained by site-directed mutagenesis of the MFS transporter gene, and its nucleic acid sequence is shown in SEQ ID NO.1 and its amino acid sequence is shown in SEQ ID NO.2.

[0027] Example 2

[0028] The preparation of the recombinant strain of *Bacillus buddingus* pLH454-MFS-1 includes the following steps:

[0029] (1) Construction of recombinant plasmid pLH454-MFS-1: The MFS-1 gene was inserted into plasmid pLH454 to obtain recombinant plasmid pLH454-MFS-1;

[0030] (2) Preparation of recombinant Escherichia coli E. coli BL21 / pLH454-MFS-1: The recombinant plasmid pLH454-MFS-1 was introduced into competent E. coli BL21 cells by heat shock method, and sequenced to verify that the recombinant E. coli E. coli BL21 / pLH454-MFS-1 containing the correctly sequenced recombinant plasmid pLH454-MFS-1 was obtained;

[0031] (3) Preparation of recombinant strain pLH454-MFS-1 of budding short-termilk: The plasmid pLH454-MFS-1 with correct sequencing was extracted from recombinant E. coli BL21 / pLH454-MFS-1. The plasmid pLH454-MFS-1 was transformed into competent cells of budding short-termilk S1 by electroporation to obtain the recombinant strain pLH454-MFS-1 of budding short-termilk.

[0032] Example 3

[0033] Validation of pullulan production yield by recombinant strain:

[0034] (1) Inoculate the recombinant strain of budding short-stalked fungus pLH454-MFS-1 onto YPD solid medium plates and incubate them upside down in a 28℃ incubator until the bacteria cover the medium plates.

[0035] (2) Collect the mycelium of the recombinant strain of budding short-stalked dermal ...

[0036] (3) Transfer 300 mL of seed culture to a 5 L fermenter containing 3 L of fermentation medium and culture at 28 °C, 400 rpm and 1 vvm for 42 h to obtain a fermentation broth containing pullulan polysaccharide.

[0037] (4) Fermentation yield determination: Take 50 mL of fermentation broth, dilute it with 1 volume of water, centrifuge at 10000 r / min for 20 min, collect the supernatant, add 3 volumes of 95% ethanol to the supernatant, wash and precipitate the polysaccharide, filter to remove the supernatant after washing, dry the precipitate at 60℃ and weigh it to obtain the crude polysaccharide content, and the fermentation yield is 93.9 g / L after conversion.

[0038] Example 4

[0039] Validation of pullulan production by recombinant strain:

[0040] (1) Inoculate the recombinant strain of budding short-stalked fungus pLH454-MFS-1 onto YPD solid medium plates and incubate them upside down in a 28℃ incubator until the bacteria cover the medium plates.

[0041] (2) Collect the mycelium of the recombinant strain of budding short-stalked dermal ...

[0042] (3) Transfer 2L of seed culture to a 20L fermenter containing 12L of fermentation medium and culture at 28℃, 400rpm and 1vvm for 42h to obtain fermentation broth containing pullulan polysaccharide.

[0043] (4) Fermentation yield determination: Take 50 mL of fermentation broth, dilute it with 1 volume of water, centrifuge at 10000 r / min for 20 min, collect the supernatant, add 3 volumes of 95% ethanol to the supernatant, wash and precipitate the polysaccharide, filter to remove the supernatant after washing, dry the precipitate at 60℃ and weigh it to obtain the crude polysaccharide content, and the fermentation yield is 92.9 g / L after conversion.

[0044] Example 5

[0045] Validation of pullulan production by recombinant strain:

[0046] (1) Inoculate the recombinant strain of budding short-stalked fungus pLH454-MFS-1 onto YPD solid medium plates and incubate them upside down in a 28℃ incubator until the bacteria cover the medium plates.

[0047] (2) Collect the mycelium of the recombinant strain of budding short-stalked dermal fungus pLH454-MFS-1 and inoculate it into seed culture medium. Culture it at 28℃ and 250rpm for 16h to obtain seed liquid. Then, transfer it to a seed tank with a seed culture medium volume of 6L at a 5% inoculation rate and culture it at 28℃ for 12h to obtain seed liquid.

[0048] (3) Transfer 6L of seed culture to a 100L fermenter containing 60L of fermentation medium and culture it at 28℃, 300rpm and 1vvm for 42h to obtain fermentation broth containing pullulan polysaccharide.

[0049] (4) Fermentation yield determination: 50 mL of fermentation broth was diluted with 1 volume of water and centrifuged at 10000 r / min for 20 min. The supernatant was collected, and 3 volumes of 95% ethanol were added to the supernatant to wash and precipitate polysaccharides. After washing, the supernatant was removed by filtration. The precipitate was dried at 60℃ and weighed to obtain the crude polysaccharide content. The fermentation yield was calculated to be 94.6 g / L.

[0050] Comparative Example 1

[0051] The only difference from Example 3 is that the strain is not modified, and the original strain is used directly for subsequent treatment and fermentation culture for 42 hours.

[0052] Comparative Example 2

[0053] The only difference from Example 4 is that the strain is not modified, and the original strain is used directly for subsequent treatment and fermentation culture for 42 hours.

[0054] Comparative Example 3

[0055] The only difference from Example 5 is that the strain is not modified, and the original strain is used directly for subsequent treatment and fermentation culture for 42 hours.

[0056] Experimental Example 1

[0057] The yields of pullulan in Examples 3-5 and Comparative Examples 1-3 were determined, and the results are listed in Table 1.

[0058] Table 1. Yield of pullulan

[0059]

[0060] Table 1 shows that the pullulan yield in Comparative Example 1 was 58.6 g / L, and the pullulan yield in Example 3 was 93.9 g / L, representing a 60.2% increase compared to Comparative Example 1; the pullulan yield in Comparative Example 2 was 59.0 g / L, and the pullulan yield in Example 4 was 92.9 g / L, representing a 57.4% increase compared to Comparative Example 1; the pullulan yield in Comparative Example 3 was 59.3 g / L, and the pullulan yield in Example 5 was 94.6 g / L, representing a 59.5% increase compared to Comparative Example 1. This indicates that the recombinant strain of *Breezing Short-Stemmed Pulmonaria* constructed using the mutant MFS transporter gene is excellent, capable of high pullulan yield and shortened fermentation time. In fermentation systems of different scales, after 42 hours of fermentation, the fermentation yield of the recombinant strain was more than 55% higher than that of the original strain, which greatly increased the production of pullulan and improved fermentation efficiency, laying the foundation for the industrial-scale fermentation production of pullulan.

Claims

1. An MFS-1 transporter protein gene, characterized in that: Its nucleic acid sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.

2.

2. A method for increasing pullulan yield using the MFS-1 transporter gene as described in claim 1, characterized in that: Includes the following steps: (1) Construction of recombinant plasmid pLH454-MFS-1: The MFS-1 gene was inserted into plasmid pLH454 to obtain recombinant plasmid pLH454-MFS-1; (2) Preparation of recombinant Escherichia coli E. coli BL21 / pLH454-MFS-1: The recombinant plasmid was introduced into competent E. coli BL21 cells by heat shock method and sequenced to obtain recombinant E. coli E. coli BL21 / pLH454-MFS-1 containing the correctly sequenced recombinant plasmid pLH454-MFS-1; (3) Preparation of recombinant strain pLH454-MFS-1 of budding short-term causal agent: The plasmid pLH454-MFS-1 with correct sequencing was extracted from recombinant Escherichia coli. The plasmid was transformed into competent cells of budding short-term causal agent S1 by electroporation to obtain recombinant strain pLH454-MFS-1 of budding short-term causal agent. (4) Preparation of seed culture: The recombinant strain of budding short-stalked dermal fungus pLH454-MFS-1 was inoculated on YPD solid medium plates and cultured until the bacteria covered the plates. The mycelium was collected and inoculated into seed culture medium to obtain seed culture. (5) Preparation of fermentation broth containing pullulan: The seed liquid was inoculated into the fermentation medium and fermented for 42 hours to obtain fermentation broth containing pullulan, with a fermentation yield of >90g / L.

3. The method for increasing pullulan yield using the MFS-1 transporter gene according to claim 2, characterized in that: In step (4), the seed culture medium consists of: 50 g / L sucrose, 1 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 6.3 g / L anhydrous dipotassium hydrogen phosphate, 0.6 g / L ammonium sulfate, 2 g / L yeast extract, and pH adjusted to 6.

5.

4. The method for increasing pullulan yield using the MFS-1 transporter gene according to claim 2, characterized in that: In step (4), the culture temperature is 28℃, the culture speed is 250rpm, and the culture time is 16h.

5. The method for increasing pullulan yield using the MFS-1 transporter gene according to claim 2, characterized in that: In step (5), the fermentation medium consists of: 100 g / L glucose, 1 g / L sodium chloride, 0.2 g / L magnesium sulfate heptahydrate, 6.3 g / L anhydrous dipotassium hydrogen phosphate, 0.6 g / L ammonium sulfate, 2 g / L yeast extract, 0.05% Tween-80, 3% antifoaming agent, and pH adjusted to 5.

5.

6. The method for increasing pullulan yield using the MFS-1 transporter gene according to claim 2, characterized in that: In step (5), the fermentation temperature is 28°C, the fermentation speed is 400 rpm, and the aeration rate is 1 vvm.

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