A glucose phosphate mutase gene RkPGM and its application

Abstract

This invention discloses a glucose phosphate mutase gene Rk PGM Its nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence encoded by this gene is shown in SEQ ID NO:2; this gene was isolated from *Rhodotorula rubrum* (…). Rhodosporidium kratochvilovae YM25235 was used to ligate the gene to a vector and transfected into Rhodotorula rubrum cells. Experimental results showed that overexpression of Rk... PGM The gene will increase the level of extracellular polysaccharide synthesis in this strain; this invention modifies microorganisms through genetic engineering to improve their ability to secrete extracellular polysaccharides, providing a theoretical basis for increasing the yield of extracellular polysaccharides in Rhodotorula rubrum.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a glucose phosphate mutase gene Rk. PGM and its effect on improving Rhodotorula rubrum ( Rhodosporidium kratochvilovae Applications in the production of extracellular polysaccharides. Background Technology

[0002] Phosphoglucose mutases (PGMs) are ubiquitous in the biological world and belong to the phosphohexose mutase family. PGMs possess important biological activities. Studies have found that PGMs play a role in carbon metabolism, bacterial cell morphology, bacterial pathogenicity, efficient bacterial hydrogen production, bacterial lipopolysaccharide biosynthesis, and maintaining intracellular calcium in yeast cells. 2+ Balance is affected. In particular, PGM can regulate the interconversion between glucose-1-phosphate (G-1-P) and glucose-6-phosphate (G-6-P). On one hand, G-6-P can be generated to produce ATP and reducing power through different catabolism pathways; on the other hand, G-1-P can also be generated to enter the glycogenosynthesis pathway, synthesizing UDP-glucose, providing precursors for the synthesis of polysaccharides and ribonucleotides. Simultaneously, polysaccharides are essential for cell wall synthesis. PGM mutants lead to abnormal phosphogluconate polymer synthesis, thereby affecting the synthesis and storage of intracellular carbohydrates. To maintain osmotic homeostasis, cell structure will be altered.

[0003] Currently, research on microbial polysaccharides largely focuses on their extraction, purification, structure, and function, with limited research on enzymes and pathways related to their synthesis and metabolism. Among reported studies on microbial polysaccharide synthesis pathways, most focus on bacteria. Fungal polysaccharide synthesis is more complex due to the greater number of enzymes, factors, and precursors involved, resulting in less research. Some studies have found that altering the activity or expression levels of enzymes related to fungal polysaccharide synthesis can increase polysaccharide production. In a study of *G. lucidum*, silencing the PGM gene led to a decrease in hyphal biomass, a reduction in extracellular polysaccharide to 20-40% of the wild-type, and a 1.7-fold increase in intracellular polysaccharide production. This indicates that the reversible reactions catalyzed by PGM in intermediate metabolites are species-specific and could serve as an effective target for constructing high-yielding fungal polysaccharide strains.

[0004] Yeast extracellular polysaccharides are polysaccharide substances secreted by yeast during metabolism. As a major class of microbial polysaccharides, they are easy to extract from culture media, low in cost, non-toxic and environmentally friendly. They have a variety of physiological activities such as anti-oxidation, antiviral, anti-tumor, lowering serum cholesterol and immunomodulation. They can be developed as immune stimulants and anti-tumor drugs and have broad market prospects.

[0005] Microbial extracellular polysaccharides are macromolecules with various biological activities. Researchers have already improved their yield through fermentation regulation optimization or strain modification. However, the synthetic pathways of fungal polysaccharide donors are not fully understood, and the characteristics of key enzymes are not yet fully elucidated, hindering significant increases in microbial polysaccharide yield. Therefore, using genetic engineering techniques to modify fermentation strains to prepare microbial extracellular polysaccharides has become a novel method for fermenting extracellular polysaccharides.

[0006] Currently, regarding the glucose phosphate mutase gene PGM There are few reports on promoting the production of extracellular polysaccharides by *Rhodotorula rubrum*. Summary of the Invention

[0007] This invention provides a glucose phosphate mutase gene Rk PGM This gene was derived from *Rhodotorula rubrum* (…). Rhodosporidium kratochvilovae The gene was isolated from YM25235, and its nucleotide sequence is shown in SEQ ID NO:1. The gene sequence is 1656 bp long and the encoded amino acid sequence is shown in SEQ ID NO:2. The gene was ligated to a vector and transformed into Rhodotorula rubrum cells. The increase in the expression level of this gene promoted the synthesis of extracellular polysaccharides in Rhodotorula rubrum.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] 1. Total RNA was extracted from *Rhodotorula rubrum* YM25235 and then reverse transcribed into cDNA. Using the synthesized cDNA as a template, Rk was amplified. PGM Specific primers were used to amplify the target sequence via polymerase chain reaction. The vector pRH2034 was double-digested and recovered. The target fragment and the vector were ligated using a one-step cloning method to obtain the ligation product, recombinant plasmid pRHRkPGM. The recombinant plasmid pRHRkPGM was transformed into E. coli, and positive single clones were screened by PCR. The recombinant plasmid pRHRkPGM was used... BamH I. EcoR V. Enzyme digestion with two restriction endonucleases was performed for verification. After culturing positive clones, plasmids were extracted and sequenced to obtain the glucose-phosphate mutase gene Rk with a fragment size of 1656 bp. PGM ;

[0010] 2. The recombinant vector pRHRkPGM was transformed into Rhodotorula rubrum YM25235 using the PEG-mediated protoplast method. Transformants were screened to obtain overexpression strains containing pRHRkPGM. The overexpression strains containing pRHRkPGM were cultured in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) to extract extracellular polysaccharides. The yield of extracellular polysaccharides was determined using the phenol-sulfuric acid method.

[0011] This invention provides a novel method for producing extracellular polysaccharides. The method utilizes genetic engineering to modify microorganisms, thereby increasing the yield of extracellular polysaccharides. The glucose-phosphoryltransferase gene Rk is isolated from cDNA reverse-transcribed from total RNA extracted from *Rhodotorula rubrum* YM25235. PGM Rk in YM25235 of Rhodotorula rubrum PGM Overexpression of the gene leads to an increase in the transcription level of this gene within the cell. *Rhodotorula rubrum* YM25235 has advantages such as short production cycle, genetic stability, and safe production. The results of this study help to elucidate the mechanism of extracellular polysaccharide production in *Rhodotorula rubrum* YM25235, providing a reference for revealing the mechanism by which microorganisms increase extracellular polysaccharide production. This offers promising application prospects and economic benefits for extracellular polysaccharide production and provides a basis for the efficient formulation of fermentation strategies for extracellular polysaccharide synthesis. The method of this invention is simple and easy to operate. Attached Figure Description

[0012] Figure 1 Rk of the red syringomyelia YM25235 of the present invention PGM PCR amplification diagram of the gene; where 1. DNA molecular weight marker DL2000; 2. Negative control; 3. Gene Rk PGM cDNA fragments;

[0013] Figure 2 Electrophoresis image for colony PCR verification; 1. DNA molecular weight marker DL2000; 2. Gene Rk PGM cDNA fragments; 3-7 are transformants;

[0014] Figure 3 Restriction enzyme digestion analysis of recombinant plasmid pRHRkPGM; including 1. DNA molecular weight marker DL10000; 2. negative control; 3. plasmid pRH2034. BamH I and EcoR V double enzyme digestion; 4. Recombinant plasmid pRHRkPGM BamH I. EcoR V double enzyme digestion; 5. Gene Rk PGM 6. cDNA fragment; DNA molecular weight marker DL2000;

[0015] Figure 4 The plasmid map of the recombinant plasmid pRHRkPGM;

[0016] Figure 5To validate positive clones of *Rhodotorula rubrum* YM25235 transformed with recombinant plasmid pRHRkPGM; 1. DNA molecular scale DL2000; 2. Negative control; 3. PCR product amplified with the YM25235 genome; 4. PCR product amplified with plasmid pRHRkPGM; 5. PCR product amplified with the YM25235 / pRHRkPGM strain genome;

[0017] Figure 6 The results show the comparison of extracellular polysaccharide production between the overexpression strain YM25235 / pRHRkPGM and the control strain YM25235. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the reagents and methods used in the embodiments are all conventional reagents and conventional methods.

[0019] Example 1: Isolation of glucose-phosphoryltransferase gene Rk from Rhodotorula rubrum YM25235 PGM Construction of the overexpression vector pRHRkPGM

[0020] Total RNA was extracted from *Rhodotorula rubrum* YM25235 using the UNlQ-10 Trizol Total RNA Extraction Kit (product number: SK1321) from Sangon Biotech (Shanghai) Co., Ltd. Then, cDNA was synthesized via reverse transcription according to the instructions of the Vazyme HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper). 1 μL of cDNA was used as a template for polymerase chain reaction (PCR). Based on the Rk sequence found in the transcriptome sequencing... PGM Based on the obtained cDNA sequence, specific primers RkPGM-F and RkPGM-R were designed. Using the cDNA template obtained above, PCR amplification was performed on a PCR instrument using primers RkPGM-F and RkPGM-R. The primers, amplification system, and amplification conditions used are as follows:

[0021] RkPGM-F: 5'-CATGGC GGATC CGATGCAGGCCGGCACGTCGT-3' (single underscore) BamH I) Enzyme cleavage site

[0022] RkPGM-R: 5'-CGGTCGGCATCTACG ATATC CTACGCCTTCTCCAGCCCGTTC-3' (single underscore) EcoR V restriction site);

[0023] The PCR amplification system is as follows (50 μL):

[0024]

[0025] Amplification conditions: 95℃ pre-denaturation for 3 min, followed by 95℃ denaturation for 15 s, 62℃ annealing for 15 s, and 72℃ extension for 1 min 45 s, for a total of 30 cycles, with a final extension at 72℃ for 5 min; after the reaction, 2 μL of the product was taken and analyzed by electrophoresis on a 1.5% agarose gel. The results are as follows. Figure 1 As shown; the amplified fragment was approximately 1700 bp in size, named RkPGM pRH2034 was processed BamH I. EcoR V. Double digestion with two restriction endonucleases; the two fragments were then recovered using a multifunctional DNA recovery kit (Beijing Biotech Biotechnology Co., Ltd., product number: DP1502), and the recovered fragments were then processed using a seamless cloning kit (LightNing). TM The DNA Assembly Mix Plus (Jiangsu Bestmate Biotechnology Co., Ltd.) was used to ligate the recombinant plasmid pRHRkPGM. The ligation system was as follows (10µL):

[0026]

[0027] Gently mix the mixture using a pipette, briefly centrifuge to collect the reaction solution to the bottom of the tube, and then react it in a PCR instrument (Beijing Liuyi Biotechnology Co., Ltd.) at 50°C for 30 min; cool to 4°C or immediately place on ice to cool.

[0028] Add 10 µL of the obtained ligation product to 50 µL of DH5α competent cells, gently tap the tube wall to mix, incubate on ice for 30 min, heat shock at 42°C for 90 s, and immediately place on ice to cool for 2-3 min. Add 900 µL of LB liquid medium to the ligation system, incubate at 37°C with shaking at 90 rpm for 1 h, centrifuge at 5000 rpm for 10 min, discard 900 µL of supernatant, and gently pipette the remaining approximately 100 µL of LB medium to suspend the cells and spread on LB agar plates (containing 100 µg / mL spectinomycin). Incubate upside down at 37°C for 12-16 h. Pick white colonies growing on the plates and verify positive clones by colony PCR. Inoculate the verified positive clones into LB liquid medium (containing 100 µg / mL spectinomycin) and incubate overnight. Randomly pick 5 white colonies growing on the plates and number them 1-5. Verify positive clones by colony PCR. The results are shown in the figure. Figure 2As shown in the figure, all five selected monoclonal strains amplified specific bands of the same size as the target fragment by colony PCR, indicating that the recombinant plasmid was successfully transformed into all five selected DH5α strains; plasmid was extracted (Star Prep Rapid Plasmid Mini-Prep Kit, Beijing Kangrun Chengye Biotechnology Co., Ltd.) using... BamH I. EcoR V. Double digestion of pRHRkPGM was performed for verification; results are shown below. Figure 3 The results showed that the recombinant plasmid pRHRkPGM produced two bands of approximately 1700 bp and 10 kb after double enzyme digestion. Figure 3 Lane 4), these two bands are respectively with RkPGM The fragments were the same size as those from the pRH2034 vector after double enzyme digestion, initially indicating successful construction of the recombinant plasmid pRHRkPGM. The plasmid, verified by enzyme digestion, was further sequenced for validation. Sequencing (Shanghai Sangon Biotech Co., Ltd.) showed that the amplified fragment matched the target sequence, confirming successful construction of the expression vector pRHRkPGM. The plasmid map of the recombinant vector pRHRkPGM is shown below. Figure 4 .

[0029] Example 2: RkPGM Analysis of extracellular polysaccharide production in *Rhodotorula rubrum* YM25235 with gene overexpression

[0030] 1. Transformation of Rhodotorula rubrum YM25235

[0031] Single colonies of DH5α strain successfully transformed into the correct recombinant vector pRHRkPGM were inoculated into LB liquid medium (containing 100 µg / mL spectinomycin) and cultured overnight. Plasmids were extracted using the StarPrep rapid plasmid mini-prep kit (Beijing Kangrun Chengye Biotechnology Co., Ltd.), and their concentration was measured. The plasmids were stored at -20℃ for later use. A single colony of *Rhodotorula rubrum* YM25235 was inoculated into 5 mL of YPD liquid medium and cultured overnight at 28℃ with shaking at 160 rpm. The overnight culture was then transferred at a 1% inoculum to 50 mL of YPD liquid medium and cultured at 28℃ with shaking at 160 rpm until the bacterial growth rate reached OD. 600The concentration was set at 0.45. The culture was centrifuged at 4500 rpm for 5 min at 4℃ to collect the bacterial cells. The cells were washed with 10 mL of pre-prepared citrate buffer (200 mL citrate buffer containing 1.155 g citrate, 4.263 g sodium citrate, 21.909 g mannitol, and pH adjusted to 5.4 with NaOH) placed on ice. Centrifuge at rpm for 5 min, discard the supernatant, repeat this step once, collect the bacterial cells and resuspend them in 2 mL of citrate buffer, then place on ice for later use; prepare the lysin solution (0.1 g snailase, 0.4 g lysozyme, bring to a final volume of 10 mL with ddH2O), filter the enzyme solution through a 0.22 μm sterile filter membrane, and place in a 50 mL sterile centrifuge tube for later use; mix 4 mL of enzyme solution with 1 mL of bacterial solution and incubate at 28 °C and 90 rpm for 2.5 h with shaking, then centrifuge the culture at 4 °C and 1300 rpm for 11 min to collect the bacterial cells; wash the collected bacterial cells twice on ice with STC (1.2 M sorbitol, 10 mM Tris-HCl, 100 mM CaCl2) to prepare competent yeast cells; aliquot the competent yeast cells into 100 μL tubes in 5 mL sterile centrifuge tubes for later use; add 2.8 μg of lysin to each 100 μL of competent cells. The pRHRPGM recombinant plasmid was gently mixed (the volume of the added fragment should generally not exceed 10 μL), and incubated on ice for 10 min. Then, 200 μL of pre-chilled PTC (50% PEG, 10 mM Tris-HCl, 100 mM CaCl2) was added, and the mixture was incubated on ice for 10 min. Another 200 μL of pre-chilled PTC was added, and the mixture was incubated on ice for 10 min. Finally, 800 μL of pre-chilled PTC was added and gently mixed. The mixture was then heat-shocked at 42 °C for 30 min, and centrifuged at 4 °C and 1500 rpm for 11 min to collect the bacterial cells. 1 mL of the mixture was added... The cells were suspended in 0.4M sucrose YPD liquid medium and cultured at 28℃ with shaking at 90 rpm for 12 h to revive them. The revived cells were centrifuged at 4500 rpm for 5 min to collect the cells. The supernatant was discarded, and the cells were resuspended in the remaining 100 μL of medium. Finally, the cells were spread onto YPD solid medium (containing 40 μg / mL hygromycin B) and cultured upside down at 28℃ for 3 days. The transformants obtained after spreading were numbered and transferred to YPD solid medium (containing 150 μg / mL hygromycin B) and cultured upside down at 28℃ for 2 days.

[0032] The selected transformants were picked, and genomic DNA was extracted from the yeast transformants according to the instructions of the DNA extraction kit from Shanghai Sangon Biotech Co., Ltd. PCR verification was then performed, and the results are as follows: Figure 5 As shown in the figure, PCR can amplify genes that are similar to those of yeast transformants, using the yeast transformant genome as a template. RkPGM The presence of bands of the same cDNA fragment size confirms the correct gene verification in the recombinant transformant, indicating... RkPGM The fragment has been successfully ligated into the yeast genome.

[0033] 2. RkPGM Analysis of extracellular polysaccharide production in *Rhodotorula rubrum* YM25235 with gene overexpression

[0034] The overexpression strain containing pRHRkPGM was cultured in YPD liquid medium at 28°C for 120 h. The fermentation product was centrifuged at 5000 rpm for 5 min to separate the cells from the supernatant. Then, two volumes of 95% icy ethanol were added to the fermentation broth, mixed thoroughly, and incubated at 4°C for 12 h to precipitate the extracellular polysaccharide, yielding crude polysaccharide. An appropriate amount of distilled water was added to dissolve the polysaccharide, and the insoluble matter was removed by centrifugation, retaining the supernatant to obtain an aqueous solution of crude polysaccharide. Using the original Rhodotorula rubrum strain YM25235 as a control, the yield of extracellular polysaccharide was determined using the phenol-sulfuric acid method. The results are as follows: Figure 6 As shown in the figure, the extracellular polysaccharide synthesis of the overexpressing strain YM25235 / pRHRkPGM was significantly higher than that of the wild-type Rhodotorula rubrum strain YM25235. The extracellular polysaccharide yield of the wild-type Rhodotorula rubrum strain YM25235 was 0.09±0.00 g / L, while that of the overexpressing strain YM25235 / pRHRkPGM was 0.12±0.01 g / L, meaning that the extracellular polysaccharide yield of the overexpressing strain YM25235 / pRHRkPGM was 1.33 times that of the control strain. The results indicate that the glucose phosphate mutase gene... RkPGM Overexpression of [a substance] can promote the synthesis of extracellular polysaccharides in *Rhodotorula rubrum*.

Claims

1. A glucose phosphomutase gene Rk PGM having a nucleotide sequence as shown in SEQ ID NO:

1.

2. The glucose phosphomutase gene Rk of claim 1 PGM In the production of exocellular polysaccharide by Rhodotorula glutinis Rhodosporidium kratochvilovae ).

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