MFS-1 transporter gene and method for increasing yield of pullulan

Abstract

The invention discloses an MFS-1 transporter gene and a method for increasing the yield of pullulan, and belongs to the technical field of biology. The method comprises the following steps: obtaining an MFS-1 transporter gene of which the nucleotide sequence is shown as SEQ ID NO.1 and the amino acid sequence is shown as SEQ ID NO.2, then transferring the MFS-1 transporter gene into aureobasidium pullulans to obtain an aureobasidium pullulans pLH4454-MFS-1 recombinant strain, and finally fermenting the aureobasidium pullulans pLH4454-MFS-1 recombinant strain to obtain fermentation liquor containing pullulan. The MFS-1 transporter gene is obtained, the MFS-1 transporter gene is used for constructing a recombinant strain and fermentation is performed, so that the fermentation time is shortened, the pullulan synthesis capability of aureobasidium pullulans is improved, the pullulan is expressed and synthesized more efficiently, quickly and intensively, the production cost of an enterprise can be reduced, and the production efficiency is improved. Therefore, the pullulan polysaccharide has wider application space and prospect.

Description

Technical Field

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

[0002] During the process of material transport, MFS transporters mainly achieve their functions by switching between two conformations: open to the extracellular side and open to the intracellular side. When in the conformation open to the extracellular side, it can bind to extracellular substrate molecules, and then through conformational changes, it turns into the conformation open to the intracellular side, thereby releasing the substrate into the cell. Conversely, it can also complete the material transport from inside the cell to the outside. MFS transporters are involved in the transmembrane transport of signaling molecules. In the regulation of pullulan polysaccharide synthesis in Aureobasidium pullulans, signaling pathways such as cAMP-PKA, TORC1, HOG1, and Snf1 play important roles. MFS transporters can indirectly regulate the activity of signaling pathways by transporting signaling molecules related to these pathways, and ultimately affect the synthesis and secretion of pullulan polysaccharide.

[0003] Pullulan polysaccharide is an extracellular polysaccharide produced by the fermentation of Aureobasidium pullulans, which has the characteristics of non-toxicity, edibility, biodegradability, etc., and has a wide range of applications in the fields of food, medicine, chemical industry, etc. However, its current fermentation yield and production efficiency have become bottlenecks for large-scale industrial applications. Under traditional fermentation processes, the yield of pullulan polysaccharide is low, resulting in high production costs. This causes many enterprises to face cost pressures during large-scale production, restricting the wide promotion and application of pullulan polysaccharide in the market. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an MFS-1 transporter protein gene and a method for increasing the production of pullulan polysaccharide. By obtaining the MFS-1 transporter protein gene, constructing a recombinant strain and fermenting it, the ability of Aureobasidium pullulans to synthesize pullulan polysaccharide is improved, and the synthesis of pullulan polysaccharide is expressed more efficiently, rapidly, and intensively.

[0005] To solve the above technical problems, the technical solutions provided by the present invention are as follows: An MFS-1 transporter protein gene, whose nucleic acid sequence is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.2.

[0006] Further, the amino acid sequence shown in SEQ ID NO.2 has the following mutations based on the original sequence of the nucleic acid sequence of the MFS transporter gene: the 28th amino acid in the polypeptide amino acid sequence corresponding to the nucleic acid sequence is replaced by S from Y, the 102nd amino acid is replaced by A from E, the 119th amino acid is replaced by I from S, the 145th amino acid is replaced by Q from R, the 201st amino acid is replaced by V from D, the 227th amino acid is replaced by C from F, the 286th amino acid is replaced by D from N, the 408th amino acid is replaced by E from V, and the 427th amino acid is replaced by A from E.

[0007] Further, the MFS-1 transporter gene is obtained by site-directed mutagenesis of the MFS transporter gene.

[0008] Further, the accession number of the MFS transporter gene in the NCBI database is: 40751144.

[0009] A method for increasing the production of pullulan by the MFS-1 transporter gene, comprising the following steps: (1) Construction of recombinant plasmid pLH454-MFS-1: Insert the MFS-1 gene into plasmid pLH454 to obtain recombinant plasmid pLH454-MFS-1; (2) Preparation of recombinant Escherichia coli E.coli BL21 / pLH454-MFS-1: Introduce the recombinant plasmid into competent cells E.coli BL21 by heat shock method, and perform sequencing verification to obtain recombinant Escherichia coli E.coli BL21 / pLH454-MFS-1 containing the correctly sequenced recombinant plasmid pLH454-MFS-1; (3) Preparation of Aureobasidium pullulans pLH454-MFS-1 recombinant strain: Extract the correctly sequenced plasmid pLH454-MFS-1 from the recombinant Escherichia coli, and transfer the plasmid into the competent cells of Aureobasidium pullulans S1 by electroporation transformation method to obtain the Aureobasidium pullulans pLH454-MFS-1 recombinant strain; (4) Preparation of seed liquid: Inoculate the Aureobasidium pullulans recombinant strain on a YPD solid medium plate, culture until the bacteria cover the plate, collect the mycelia and inoculate them into the seed medium, and culture to obtain the seed liquid; (5) Preparation of fermentation broth containing pullulan: Inoculate the seed liquid into the fermentation medium, ferment and culture for 42 h to obtain the fermentation broth containing pullulan, and the fermentation yield > 90 g / L.

[0010] Further, in step (4), the composition of the seed culture medium is as follows: sucrose 50 g / L, sodium chloride 1 g / L, magnesium sulfate heptahydrate 0.2 g / L, dipotassium hydrogen phosphate anhydrous 6.3 g / L, ammonium sulfate 0.6 g / L, yeast extract 2 g / L, and the pH is adjusted to 6.5.

[0011] Further, in step (4), the culture temperature is 28 °C, the culture rotation speed is 250 rpm, and the culture time is 16 h.

[0012] Further, in step (5), the composition of the fermentation medium is as follows: glucose 100 g / L, sodium chloride 1 g / L, magnesium sulfate heptahydrate 0.2 g / L, dipotassium hydrogen phosphate anhydrous 6.3 g / L, ammonium sulfate 0.6 g / L, yeast extract 2 g / L, Tween-80 0.05%, antifoaming agent added 3%, and the pH is adjusted to 5.5.

[0013] Further, in step (5), the inoculum amount of the seed liquid is 10 - 20% (V / V) of the fermentation medium.

[0014] Further, in step (5), the fermentation temperature is 28 °C, the fermentation rotation speed is 400 rpm, and the aeration rate of the fermentation is 1 vvm.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention performs site-directed mutagenesis on the MFS transporter protein gene to obtain the MFS-1 transporter protein gene, and constructs a recombinant strain, which can improve the ability of the strain to produce pullulan polysaccharide, reduce the production cost of pullulan polysaccharide, and increase the market share of pullulan polysaccharide.

[0016] 2. The present invention modifies Aureobasidium pullulans through genetic engineering, improves the production capacity of the strain, shortens the fermentation time to 42 h. Compared with the original strain, under the same fermentation time, the fermentation yield of the recombinant strain is increased by more than 55%, and there is no need to change the original fermentation production conditions and processes, and the operation is simple and convenient. Specific Embodiments

[0017] Example 1 Preparation of MFS-1 Transporter Protein Gene: The MFS-1 transporter protein gene is obtained by performing site-directed mutagenesis on the MFS transporter protein gene. Its nucleic acid sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

[0018] Example 2 Preparation of Aureobasidium pullulans pLH454-MFS-1 recombinant strain, including the following steps: (1)Construction of recombinant plasmid pLH454-MFS-1: Insert the MFS-1 gene into plasmid pLH454 to obtain the recombinant plasmid pLH454-MFS-1; (2)Preparation of recombinant Escherichia coli E. coli BL21 / pLH454-MFS-1: Introduce the recombinant plasmid pLH454-MFS-1 into competent cells of E. coli BL21 by heat shock method, and perform sequencing verification to obtain recombinant Escherichia coli E. coli BL21 / pLH454-MFS-1 containing the correctly sequenced recombinant plasmid pLH454-MFS-1; (3)Preparation of Aureobasidium pullulans pLH454-MFS-1 recombinant strain: Extract the correctly sequenced plasmid pLH454-MFS-1 from recombinant Escherichia coli E. coli BL21 / pLH454-MFS-1, and transfer the plasmid pLH454-MFS-1 into the competent cells of Aureobasidium pullulans S1 by electroporation transformation to obtain the Aureobasidium pullulans pLH454-MFS-1 recombinant strain.

[0019] Example 3 Verification of the production of pullulan polysaccharide by the recombinant strain: (1)Inoculate the Aureobasidium pullulans pLH454-MFS-1 recombinant strain on a YPD solid medium plate and incubate it in an inverted position in an incubator at 28 °C until the medium plate is covered with the mycelium; (2)Collect the mycelium of the Aureobasidium pullulans pLH454-MFS-1 recombinant strain and inoculate it into the seed medium, and culture it at 28 °C and 250 rpm for 16 h to obtain the seed liquid; (3)Transfer 300 mL of the seed liquid to a 5 L fermenter containing 3 L of the fermentation medium, and culture it at 28 °C, 400 rpm, and an aeration rate of 1 vvm for 42 h to obtain a fermentation broth containing pullulan polysaccharide.

[0020] (4)Fermentation yield determination: Take 50 mL of the fermentation broth, dilute it with 1 volume of water, centrifuge it at 10000 r / min for 20 min, collect the supernatant, add 3 volumes of 95% ethanol to the supernatant to wash and precipitate the polysaccharide. After the washing is completed, filter to remove the supernatant, dry the precipitate at 60 °C and weigh it to obtain the content of crude polysaccharide, and the fermentation yield is calculated to be 93.9 g / L after conversion.

[0021] Example 4 Verification of the production of pullulan polysaccharide by the recombinant strain: (1)Inoculate the Aureobasidium pullulans pLH454-MFS-1 recombinant strain on a YPD solid medium plate and incubate it in an inverted position in an incubator at 28 °C until the medium plate is covered with the mycelium; (2) The mycelia of the Aureobasidium pullulans pLH454-MFS-1 recombinant strain were collected and inoculated into the seed medium, and cultured at 28 °C and 250 rpm for 16 h to obtain a seed solution. (3) 2 L of the seed solution was transferred to a 20 L fermenter containing 12 L of the fermentation medium, and cultured at 28 °C and 400 rpm with an aeration rate of 1 vvm for 42 h to obtain a fermentation broth containing pullulan polysaccharide.

[0022] (4) Determination of fermentation yield: 50 mL of the fermentation broth was diluted with 1-fold volume of water and centrifuged at 10000 r / min for 20 min. The supernatant was collected, and 3-fold volume of 95% ethanol was added to the supernatant to wash and precipitate the polysaccharide. After washing, the supernatant was filtered off, and the precipitate was dried at 60 °C and weighed to obtain the content of crude polysaccharide. After conversion, the fermentation yield was 92.9 g / L.

[0023] Example 5 Verification of the yield of pullulan polysaccharide produced by the recombinant strain: (1) The Aureobasidium pullulans pLH454-MFS-1 recombinant strain was inoculated on a YPD solid medium plate and incubated in an inverted position in an incubator at 28 °C until the medium plate was covered with the cells. (2) The mycelia of the Aureobasidium pullulans pLH454-MFS-1 recombinant strain were collected and inoculated into the seed medium, and cultured at 28 °C and 250 rpm for 16 h to obtain a seed solution, which was then transferred to a seed tank containing 6 L of the seed medium at an inoculation amount of 5% and cultured at 28 °C for 12 h to obtain a seed solution. (3) 6 L of the seed solution was transferred to a 100 L fermenter containing 60 L of the fermentation medium, and cultured at 28 °C, 300 rpm, and an aeration rate of 1 vvm for 42 h to obtain a fermentation broth containing pullulan polysaccharide.

[0024] (4) Determination of fermentation yield: 50 mL of the fermentation broth was diluted with 1-fold volume of water and centrifuged at 10000 r / min for 20 min. The supernatant was collected, and 3-fold volume of 95% ethanol was added to the supernatant to wash and precipitate the polysaccharide. After washing, the supernatant was filtered off, and the precipitate was dried at 60 °C and weighed to obtain the content of crude polysaccharide. After conversion, the fermentation yield was 94.6 g / L.

[0025] Comparative Example 1 The difference from Example 3 was only that: the strain was not modified, and the original strain was directly used for subsequent treatment, and fermentation culture was carried out for 42 h.

[0026] Comparative Example 2 The difference from Example 4 was only that: the strain was not modified, and the original strain was directly used for subsequent treatment, and fermentation culture was carried out for 42 h.

[0027] Comparative Example 3 The difference from Example 5 is only that: the strain is not modified, and the original strain is directly used for subsequent treatment, and fermentation culture is carried out for 42 h.

[0028] Experimental Example 1 The yields of pullulan polysaccharide in Examples 3 - 5 and Comparative Examples 1 - 3 were measured, and the measurement results are listed in Table 1.

[0029] Table 1. Yield of pullulan polysaccharide As can be seen from Table 1, the yield of pullulan polysaccharide in Comparative Example 1 was 58.6 g / L, and the yield of pullulan polysaccharide in Example 3 was 93.9 g / L, an increase of 60.2% compared with Comparative Example 1; the yield of pullulan polysaccharide in Comparative Example 2 was 59.0 g / L, and the yield of pullulan polysaccharide in Example 4 was 92.9 g / L, an increase of 57.4% compared with Comparative Example 1; the yield of pullulan polysaccharide in Comparative Example 3 was 59.3 g / L, and the yield of pullulan polysaccharide in Example 5 was 94.6 g / L, an increase of 59.5% compared with Comparative Example 1. It shows that the Aureobasidium pullulans recombinant strain constructed by mutating the MFS transporter gene in the present invention is excellent, can produce high - yield pullulan polysaccharide, and shorten the fermentation time. In fermentation systems of different scales, after fermentation culture for 42 h, the fermentation yield of the recombinant strain was increased by more than 55% compared with that of the original strain, greatly improving the yield of pullulan polysaccharide production and the fermentation efficiency, laying a foundation for realizing industrial fermentation production of pullulan polysaccharide.

Claims

1. A MFS-1 transporter gene, characterized in that: The nucleic acid sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The MFS-1 transporter gene according to claim 1, characterized in that: The amino acid sequence shown in SEQ ID NO.2 has the following mutations on the basis of the nucleic acid sequence being the original sequence of the MFS transport protein gene: the polypeptide amino acid sequence corresponding to the nucleic acid sequence has the 28th amino acid replaced by Y to S, the 102nd amino acid replaced by E to A, the 119th amino acid replaced by S to I, the 145th amino acid replaced by R to Q, the 201st amino acid replaced by D to V, the 227th amino acid replaced by F to C, the 286th amino acid replaced by N to D, the 408th amino acid replaced by V to E, and the 427th amino acid replaced by E to A.

3. The MFS-1 transporter gene according to claim 1, characterized in that: The MFS-1 transporter gene is obtained by site-directed mutation of the MFS transporter gene.

4. The MFS-1 transporter gene according to claim 3, characterized in that: The accession number of the MFS transporter gene in the NCBI database is: 40751144.

5. A method for increasing pullulan production by using the MFS-1 transporter gene according to claim 1, characterized in that: The following steps are involved: (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 E. coli BL21 / pLH454-MFS-1: The recombinant plasmid was introduced into competent cells E. coli BL21 by heat shock method, and sequencing was performed to obtain recombinant E. coli BL21 / pLH454-MFS-1 containing the recombinant plasmid pLH454-MFS-1 with correct sequencing; (3) Preparation of the recombinant strain of A. pullulans pLH454-MFS-1: The correctly sequenced plasmid pLH454-MFS-1 was extracted from the recombinant Escherichia coli, and the plasmid was transformed into the competent cells of A. pullulans S1 by electroporation to obtain the recombinant strain of A. pullulans pLH454-MFS-1; (4) Preparation of seed solution: The recombinant strain of Aureobasidium pullulans pLH454-MFS-1 was inoculated on a YPD solid culture medium plate, cultured until the mycelium covered the entire plate, and the mycelium was collected and inoculated into a seed culture medium to obtain a seed solution; (5) Preparation of fermentation broth containing pullulan: The seed liquid was inoculated into the fermentation medium and fermented for 42 h to obtain a fermentation broth containing pullulan with a fermentation yield of >90 g / L.

6. The method for increasing pullulan production by using the MFS-1 transporter gene according to claim 5, characterized in that: In step (4), the composition of the seed culture medium is: 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 the pH is adjusted to 6.

5.

7. The method for increasing pullulan production by using the MFS-1 transporter gene according to claim 5, characterized in that: In step (4), the culture temperature is 28°C, the culture speed is 250 rpm, and the culture time is 16 h.

8. The method for increasing pullulan production by using the MFS-1 transporter gene according to claim 5, characterized in that: In step (5), the fermentation medium comprises the following components: 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% defoaming agent, and the pH is adjusted to 5.

5.

9. The method for increasing pullulan production by using the MFS-1 transporter gene according to claim 5, characterized in that: In step (5), the fermentation temperature is 28° C., the fermentation speed is 400 rpm, and the fermentation ventilation volume is 1 vvm.

Images