Sugar transporter gene RkMAL1 and application thereof

Abstract

This invention discloses a sugar transporter gene. RkMAL1 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 invention utilizes *Rhodotorula rubrum* (…). Rhodosporidium kratochvilovae Glycotransporter gene isolated from ) RkMAL1 uridine diphosphate glucose pyrophosphorylase RkUGPP After ligating the two components to a vector, they were transferred into *Rhodotorula rubrum* to achieve gene transfer. RkMAL1、 Gene RkMAL1+RkUGPP Overexpression of the gene significantly improves the synthesis efficiency of extracellular polysaccharides in recombinant strains. This invention provides a new method for producing extracellular polysaccharides, which is simple, easy to operate, and offers good application prospects for the large-scale production of extracellular polysaccharides.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a sugar transporter gene. RkMAL1 and its effect on improving Rhodotorula rubrum ( Rhodosporidium kratochvilovae Applications in the production of extracellular polysaccharides. Background Technology

[0002] Sugar transporter MAL1, a member of the maltose permease family, plays a crucial role in the transport of carbohydrates in yeast cells. Its main function is to actively transport exogenous sugars from the extracellular space into the cell, where they are further converted into glucose-1-phosphate (Glc-1-P) to enter the glycogenosynthesis pathway, providing precursors for polysaccharide synthesis. Urate-1-phosphate glucose pyrophosphorylase (UGPP) is a vital enzyme widely found in yeast and other organisms. Its core function is to catalyze the reaction of uridine triphosphate (UTP) and Glc-1-P to generate a key glycosyl donor, uridine-1-phosphate glucose (UDP-Glc).

[0003] Yeast extracellular polysaccharides are polysaccharide substances secreted by yeast during its growth and metabolism. These natural biological macromolecules have a variety of physiological activities, such as anti-oxidation, anti-tumor, antiviral, immunomodulatory, and cholesterol-lowering effects. As an important component of microbial polysaccharides, they have advantages such as convenient extraction, low cost, non-toxicity, and environmental friendliness. They can be used for the development of immune stimulation and anti-tumor drugs, and have a very broad market prospect.

[0004] Microbial extracellular polysaccharides are natural macromolecules with a variety of biological activities. Through gene editing, metabolic pathway reconstruction and other means, the performance of strains can be efficiently modified to achieve a certain degree of yield increase. However, the synthesis pathway and key enzyme characteristics of fungal polysaccharides have not been fully elucidated, which greatly limits the improvement of their yield.

[0005] Currently, regarding sugar transporter genes RkMAL1 There are few reports on promoting the production of extracellular polysaccharides by *Rhodotorula rubrum*. Summary of the Invention

[0006] This invention provides a sugar transporter gene. RkMAL1 It comes from Rhodotorula rubrum ( Rhodosporidium kratochvilovae It was obtained from YM25235. RkMAL1The nucleotide sequence and the encoding amino acid sequence are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The gene sequence is 1686 bp long and the encoding amino acid sequence is 561 bp long. When the gene is ligated to a vector and transformed into Rhodotorula rubrum cells, the increased expression level of the gene promotes the synthesis of extracellular polysaccharides in Rhodotorula rubrum.

[0007] In addition, this invention also utilizes *Rhodotorula rubrum* (… Rhodosporidium kratochvilovae The uridine diphosphate glucose pyrophosphorylase gene was isolated from YM25235. RkUGPP Its nucleotide sequence and encoding amino acid sequence are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. The gene sequence is 1410 bp long, and the encoding amino acid sequence is 469 bp long. This gene was ligated to a vector and transformed into an expression gene. RkMAL1 In Rhodotorula rubrum cells, it can enhance the synthesis of extracellular polysaccharides in Rhodotorula rubrum.

[0008] The objective of this invention is achieved through the following technical solution: 1. Total RNA was extracted from *Rhodotorula rubrum* YM25235 and then reverse transcribed into cDNA. Using the synthesized cDNA as a template, amplification was performed... RkMAL1 and RkUGPP Specific primers were used to amplify the target sequence via polymerase chain reaction. Vectors pRH2034 and pRZ2034 were double-digested and recovered. A one-step cloning method was then used to clone the target sequence. RkMAL1 The target fragment was ligated to the pRH2034 vector; RkUGPP The target fragment was ligated with the pRZ2034 vector to obtain the ligation product, recombinant plasmid pRH- RkMAL1 and pRZ- RkUGPP ; recombinant plasmid pRH- RkMAL1 and pRZ- RkUGPP The plasmid was transferred into E. coli, and positive single clones were selected by PCR. RkMAL1 and pRZ- RkUGPP use Bam HⅠ、 Eco Enzyme digestion with two restriction endonucleases (R and V) was performed for verification. After culturing positive clones, plasmids were extracted and sequenced, yielding a 1686 bp fragment of a sugar transporter gene. RkMAL1 And the uridine diphosphate glucose pyrophosphorylase gene with a fragment size of 1410 bp RkUGPP ; 2. Using the PEG-mediated protoplast method to reconstitute the pRH- RkMAL1 Transformed into *Rhodotorula rubrum* YM25235, transformants were screened to obtain those containing pRH- RkMAL1 Overexpression strain M (YM25235 / pRH- RkMAL1 ); and again, the recombinant vector pRZ- was used via the PEG-mediated protoplast method. RkUGPP Transformed into strain M, transformants were screened to obtain strains containing pRH- RkMAL1 and pRZ- RkUGPP Co-overexpression strain M1 (YM25235 / pRH- RkMAL1 +pRZ- RkUGPP The M strain and M1 strain were cultured in YPD medium (1% yeast extract, 2% peptone, 2% glucose), and extracellular polysaccharides were extracted. The yield of extracellular polysaccharides was determined by the phenol-sulfuric acid method.

[0009] The *Rhodotorula rubrum* strain YM25235 used in this invention has advantages such as short production cycle, genetic stability, and production safety. A sugar transporter gene was isolated from the cDNA of this strain. RkMAL1 uridine diphosphate glucose pyrophosphorylase RkUGPP After ligating the two components to a vector, they were transferred into *Rhodotorula rubrum* to achieve gene transfer. RkMAL1、 Gene RkMAL1+RkUGPP Overexpression of the gene significantly improves the synthesis efficiency of extracellular polysaccharides in recombinant strains. This invention provides a novel method for producing extracellular polysaccharides, which is simple, easy to operate, and offers promising applications for the large-scale production of extracellular polysaccharides. Attached Figure Description

[0010] Figure 1 The red syringomyelia YM25235 of the present invention RkMAL1 PCR amplification diagram of the gene; 1. DNA molecular weight marker DL2000; 2. Gene RkMAL1 3. cDNA fragment; negative control; Figure 2 The red syringomyelia YM25235 of the present invention RkUGPP PCR amplification diagram of the gene; 1. DNA molecular weight marker DL2000; 2. Gene RkUGPP 3. cDNA fragment; negative control; Figure 3 Electrophoresis image for colony PCR verification; 1. DNA molecular weight marker DL2000; 2. Gene RkMAL1 cDNA fragments; 3-4 are transformants; Figure 4 Electrophoresis image for colony PCR verification; 1. DNA molecular weight marker DL2000; 2. Gene RkUGPP cDNA fragments; 3-4 are transformants; Figure 5 For recombinant plasmid pRH- RkMAL1Restriction enzyme digestion analysis; including: 1. DNA molecular weight marker DL10000; 2. plasmid pRH2034. Bam HⅠ and Eco RV double digestion; 3. Recombinant plasmid pRH- RkMAL1 of Bam HⅠ、 Eco RV double digestion; 4. Gene RkMAL1 5. cDNA fragment; 6. DNA molecular weight marker DL2000; 7. Negative control; Figure 6 For recombinant plasmid pRZ- RkUGPP Restriction enzyme digestion analysis; including: 1. DNA molecular weight marker DL10000; 2. Negative control; 3. Plasmid pRZ2034. BamH I and EcoR V double enzyme digestion; 4. Recombinant plasmid pRZ- RkUGPP of Bam HⅠ、 Eco RV double enzyme digestion; 5. Gene RkUGPP 6. cDNA fragment; DNA molecular weight marker DL2000; Figure 7 For recombinant plasmid pRH- RkMAL1 Verification of positive clones transformed with *Rhodotorula rubrum* YM25235; 1. DNA molecular marker concentration 5000; 2. Negative control; 3. Using recombinant plasmid pRH- RkMAL1 4. PCR product amplified from the genome of strain M; 5. PCR product amplified from the genome of YM25235; Figure 8 For recombinant plasmid pRH- RkMAL1 and pRZ- RkUGPP Verification of positive clones transformed with *Rhodotorula rubrum* YM25235; 1. DNA molecular marker level DL5000; 2. Using recombinant plasmid pRZ- RkUGPP 1. Template PCR product; 2. PCR product amplified from the M1 strain genome; 3. PCR product amplified from the YM25235 genome; 4. DNA molecular marker level DL5000; 5. PCR product amplified from the recombinant plasmid pRH- RkMAL1 7. PCR product of template; 8. PCR product of M1 strain genome amplification; 9. PCR product of YM25235 genome amplification; Figure 9 The results show the comparison of extracellular polysaccharide production among strain M, strain M1, and control strain YM25235. Detailed Implementation

[0011] 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.

[0012] Example 1: Isolation of a sugar transporter gene from Rhodotorula rubrum YM25235 RkMAL1、 uridine diphosphate glucose pyrophosphorylase gene RkUGPP and the overexpression vector pRH- RkMAL1、 pRZ- RkUGPP Construction Total RNA was extracted from *Rhodotorula rubrum* YM25235 using the UNlQ-10 Trizol column-based 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), and the RNA was extracted based on findings from transcriptome sequencing. RkMAL1 and RkUGPP Sequence, design specific primers RkMAL1 -F、 RkMAL1 -R and RkUGPP -F、 RkUGPP -R, using the cDNA template obtained above, respectively, primers... RkMAL1 -F and RkMAL1 -R 、RkUGPP -F and RkUGPP -R, PCR amplification was performed on a PCR instrument. The primers, amplification system, and amplification conditions used for the reaction are as follows: RkMAL1 -F: 5'-CACTCACCATGGC GGATCC GATGACTGCTCCTGCTGGC-3' (single underscore) BamH I. Enzyme cleavage site); RkMAL1 -R:5'-CGGTCGGCATCTAC GATATC CTAAACGTGCTCCTTCGAGATC-3' (single underscore) EcoR V restriction site); The PCR amplification system is as follows (50 μL): Template cDNA 2.5 μL, Forward Primer ( RkMAL1 -F) 2μL, Reverse Primer ( RkMAL1-R) 2μL, dNTP Mix 1μL, Vazyme2×phanta Max Buffer 25μL, VazymePhanta Max Super-Fidelity Polymerase 1μL, ddH2O added to 50μL; Amplification conditions: pre-denaturation at 95℃ for 3 min, followed by denaturation at 95℃ for 15 s, annealing at 70℃ for 15 s, and extension at 72℃ for 1 min 50 s, with the extension and annealing temperatures decreasing by 1℃ for 10 cycles. Then, denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, and extension at 72℃ for 1 min 50 s for 20 cycles, for a total of 30 cycles. Finally, complete extension at 72℃ for 5 min.

[0013] RkUGPP -F:5'- CACTCACCATGGC GGATCC GATGCGCAACGAGCTCAACGC-3' (single underscore) BamH I. Enzyme cleavage site); RkUGPP -R:5'- CGGTCGGCATCTAC GATATC TTAGTGGTCGAGGAGGGAGAGC-3' (Single underscore is) EcoR V restriction site); The PCR amplification system is as follows (50 μL): Template cDNA 2.5 μL, Forward Primer ( RkUGPP -F) 2μL, Reverse Primer ( RkUGPP -R) 2μL, dNTP Mix 1μL, Vazyme2×phanta Max Buffer 25μL, VazymePhanta Max Super-Fidelity Polymerase 1μL, ddH2O added to 50μL; Amplification conditions: pre-denaturation at 95℃ for 3 min, followed by denaturation at 95℃ for 15 s, annealing at 70℃ for 15 s, and extension at 72℃ for 1 min 30 s, with the extension and annealing temperatures decreasing by 1℃ for 10 cycles. Then, denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, and extension at 72℃ for 1 min 30 s for 20 cycles, for a total of 30 cycles. Finally, complete extension at 72℃ for 5 min.

[0014] 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 , Figure 2 As shown; fragments of approximately 1686 bp and 1423 bp in size were amplified and named respectively. RkMAL1and RkUGPP pRH2034 and pRZ2034 were processed BamH I. EcoR V. Double digestion with two restriction endonucleases; recover the above four fragments using a multifunctional DNA recovery kit (Beijing Biotech Biotechnology Co., Ltd., product number: DP1502), and then collect the two sets of recovered fragments ( RkMAL1 and pRH2034, RkUGPP pRZ2034 and pRZ2034 were cloned using the Seamless Cloning Kit (LightNing). TM DNAAssembly Mix Plus (Jiangsu Baishimei Biotechnology Co., Ltd.) was used for ligation to obtain recombinant plasmid pRH- RkMAL1 and pRZ- RkUGPP The connection system is as follows (10µL): LightNing TM DNA Assembly Mix Plus 5μL, linearized pRH2034 or linearized pRZ2034 3μL, RkMAL1 fragments or RkUGPP Fragment 2μL; 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℃ for 30 min; cool to 4℃ or immediately place on ice to cool.

[0015] 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℃ 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℃ and 90 rpm for 1 h with shaking, 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 it on LB agar plates (containing 100 µg / mL spectinomycin). Incubate at 37℃ upside down 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 two white colonies growing on the plates and number them 1-2. Verify positive clones by colony PCR. The results are shown in the figure. Figure 3 , Figure 4 As can be seen from the figure, the selected monoclonal strains all amplified specific bands of the same size as the target fragment by colony PCR, indicating that the recombinant plasmid was successfully transformed into all four selected DH5α strains. Plasmid extraction (Star Prep Rapid Plasmid Mini-Prep Kit, Beijing Kangrun Chengye Biotechnology Co., Ltd.), using BamH I. EcoR V for pRH- RkMAL1 and pRZ- RkUGPP Double enzyme digestion was performed for verification; results are shown below. Figure 5 , Figure 6 The results showed that the recombinant plasmid pRH- RkMAL1 and pRZ- RkUGPP After double enzyme digestion, four bands of approximately 1686bp and 10kb, and 1423bp and 10kb were produced. Figure 5 Lane 3; Figure 6 (lane 4), these four bands are respectively with RkMAL1 Fragments RkUGPP The fragments from the pRH2034 and pRZ2034 vectors were of the same size after double enzyme digestion, which preliminarily indicates that the recombinant plasmid pRH- RkMAL1 and pRZ- RkUGPP Construction successful; the plasmid, verified by enzyme digestion, was sent for further sequencing for validation. Sequencing (Shanghai Sangon Biotech Co., Ltd.) showed that the amplified fragment was identical to the target sequence, indicating successful construction of the expression vector pRH- RkMAL1 and pRZ- RkUGPP .

[0016] Example 2: Genes RkMAL1、 Gene RkMAL1+ Gene RkUGPP Analysis of extracellular polysaccharide production in overexpressed Rhodotorula rubrum yeast YM25235 1. Overexpressed genes RkMAL1 Construction of strain M Successfully selected and transferred into the correct recombinant vector pRH- RkMAL1 Single clones of the DH5α strain 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 the concentration was measured. The plasmids were stored at -20℃ for later use. A single colony of *Rhodotorula rubrum* YM25235 was picked and inoculated into 5 mL of YPD liquid medium and cultured overnight at 28℃ with shaking at 160 rpm. An appropriate amount of the overnight culture was transferred to 50 mL of YPD liquid medium and cultured at 28℃ with shaking at 160 rpm until the bacterial growth rate reached OD. 600The pH was set to 0.58. The culture was centrifuged at 4℃ and 4500 rpm for 5 min to collect the bacterial cells. The cells were washed with 10 mL of a pre-prepared citrate buffer (200 mL citrate buffer containing 1.155 g citric acid, 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, place on ice for later use; prepare lysin solution (0.075 g snailase, 0.03 g lysozyme, bring to 5 mL with citrate buffer), 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, 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; resuspend the bacterial cells in 1 mL of STC buffer, aliquot YM25235 competent yeast cells into 5 mL sterile centrifuge tubes at 100 μL each for later use; add 100 μL of STC buffer to the centrifuge tube. Add 10 μL of pRH to YM25235 competent cells. RkMAL1 Add the recombinant plasmid and mix gently (the volume of the added fragment should generally not exceed 10 μL). Incubate on ice for 10 min. Add 200 μL of pre-chilled PTC (50% PEG, 10 mM Tris-HCl, 100 mM CaCl2), incubate on ice for 10 min, add another 200 μL of pre-chilled PTC, incubate on ice for 10 min, and finally add 800 μL of pre-chilled PTC and mix gently. Heat shock at 42 °C for 30 min. After the water bath, centrifuge at 4 °C and 1500 rpm for 11 min to collect the bacterial cells. Add 1 mL of... 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 collected by centrifugation at 4500 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in the remaining 100 μL of medium. Finally, strain M was spread on YPD solid medium (containing 40 μg / mL hygromycin B). 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.

[0017] 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 7 As shown in the figure, using the genome of a yeast transformant as a template, PCR can amplify a variant similar to pRH- RkMAL1The fact that the fragments amplified from the template are the same size indicates that the gene verification of the recombinant transformants is correct, suggesting that... RkMAL1 It has been successfully incorporated into the yeast genome.

[0018] 2. Overexpressed genes RkMAL1、 Gene RkUGPP Construction of strain M1 Successfully selected and transferred into the correct recombinant vector pRZ- RkUGPP Single clones of DH5α strain 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, and their concentrations were measured. The plasmids were stored at -20°C for later use. Single colonies of strain M were picked and inoculated into 5 mL of YPD liquid medium and cultured overnight at 28°C with shaking at 160 rpm. An appropriate amount of the overnight culture was transferred to 50 mL of YPD liquid medium and cultured at 28°C with shaking at 160 rpm until the bacterial growth rate reached OD. 600 The concentration was 0.58. The culture was centrifuged at 4°C and 4500 rpm for 5 min to collect the bacterial cells. The cells were washed with 10 mL of pre-prepared citrate buffer placed on ice, and then centrifuged again at 4°C and 4500 rpm for 5 min. 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 (0.075 g snailase, 0.03 g lysozyme, diluted to 5 mL with citrate buffer), filter the enzyme solution through a 0.22 μm sterile filter membrane, and place it 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 with STC on ice to prepare M competent cells; resuspend the bacterial cells in 1 mL of STC buffer, and aliquot the M competent cells into 100 μL 5 mL sterile centrifuge tubes for later use; add 10 μL of pRZ- to 100 μL of M competent cells. RkUGPPThe recombinant plasmid was gently mixed and incubated on ice for 10 min. 200 μL of pre-chilled PTC 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. After the water bath, the cells were collected by centrifugation at 4 °C and 1500 rpm for 11 min. 1 mL of 0.4 M sucrose YPD liquid medium was added to suspend the cells, and the cells were cultured at 28 °C and 90 rpm for 12 h to revive them. The revived cells were collected by centrifugation at 4500 rpm for 5 min. The supernatant was discarded, and the cells were suspended in 100 μL of medium. The M1 strain was spread on YPD solid medium (containing 12.5 μg / mL bleomycin) and cultured upside down at 28 °C for 3 days. The transformants obtained after spreading were transferred to YPD solid medium (containing 12.5 μg / mL bleomycin) and cultured upside down at 28 °C for 2 days.

[0019] 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 8 As shown in the figure, the PCR amplification using the genome of a yeast transformant as a template can be seen. RkUGPP or RkMAL1 Fragments and plasmid pRZ- RkUGPP or pRH- RkMAL1 The fact that the fragments amplified by template PCR are the same size indicates that the gene verification of the recombinant transformants is correct, which means... RkUGPP、RkMAL1 The gene has been successfully inserted into the yeast genome.

[0020] 3. Analysis of extracellular polysaccharide production in strains M and M1 Strains M and M1 were cultured separately in YPD liquid medium at 28°C for 168 h. The fermentation products were centrifuged at 5000 rpm for 5 min to separate the cells from the supernatant. Then, 2 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 polysaccharides, yielding crude polysaccharides. An appropriate amount of distilled water was added to dissolve the crude polysaccharides, and the insoluble matter was removed by centrifugation, retaining the supernatant to obtain an aqueous solution of crude polysaccharides. Simultaneously, using the original Rhodotorula rubrum strain YM25235 as a control, the yield of extracellular polysaccharides was determined using the phenol-sulfuric acid method. The results are as follows Figure 9 As shown in the figure, the extracellular polysaccharide synthesis of the overexpressing strain was significantly higher than that of the wild-type *Rhodotorula rubrum* strain YM25235. The extracellular polysaccharide production of the wild-type *Rhodotorula rubrum* strain YM25235 was 0.165 ± 0.01 g / L. The overexpressing gene... RkMAL1 The extracellular polysaccharide yield of strain M was 0.303 ± 0.02; overexpression RkMAL1+RkUGPPThe extracellular polysaccharide yield of strain M1 was 0.580 ± 0.02 g / L, and the overexpression strain YM25235 / pRH- RkMAL1 +pRZ- RkUGPP The extracellular polysaccharide production was 3.51 times that of the control strain; the results showed that the sugar transporter gene... RKMAL1 and uridine diphosphate glucose pyrophosphorylase gene RkUGPP It is related to the synthesis of extracellular polysaccharides in Rhodotorula rubrum.

Claims

1. A sugar transporter gene RkMAL1 Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The sugar transporter gene according to claim 1 RkMAL1 In promoting the growth of Rhodotorula rubrum ( Rhodosporidium short-lived Applications in the production of extracellular polysaccharides.

3. The sugar transporter gene according to claim 1 RkMAL1 and uridine diphosphate glucose pyrophosphorylase gene RkUGPP In promoting the growth of Rhodotorula rubrum ( Rhodosporidium kratochvilovae Its application in the production of extracellular polysaccharides is characterized by: uridine diphosphate glucose pyrophosphorylase gene RkUGPP The nucleotide sequence is shown in SEQ ID NO:3.

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