A utp-glucose-1-phosphate uridylyltransferase mutant and use thereof

Abstract

This invention discloses a UTP-glucose-1-phosphouridine transferase mutant and its application, belonging to the field of bioengineering technology. It solves the problem of low rhodioloside yield in existing technologies. This invention provides a UTP-glucose-1-phosphouridine transferase mutant, which is obtained by mutating any one of the following positions: 6, 126, 134, 140, 177, 180, 214, 249, 284, 289, 296, 298, and 300, using the amino acid sequence shown in SEQ ID NO.2 as the starting sequence. It is mainly used for high-efficiency UTP-glucose-1-phosphouridine transferase and its application in the de novo synthesis of rhodioloside.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a UTP-glucose-1-phosphate uridine transferase mutant and its application. Background Technology

[0002] Salidroside is the core active ingredient of Rhodiola rosea plants, belonging to the natural phenylethanoid glycoside class of compounds. It uses tyrosol as its core, linked to glucose groups via β-glycosidic bonds to form a water-soluble small molecule. Its most significant characteristic lies in its multi-target physiological activity: it can enhance cellular energy metabolism (anti-fatigue), scavenge free radicals (antioxidant), protect nerve cells, regulate mood (anti-depression), and also possess cardiovascular protective and immune-enhancing effects. Due to its low toxicity and well-defined mechanism of action, it is widely used in anti-fatigue drugs, antidepressants, and functional foods, and is hailed as the "golden molecule of altitude adaptation."

[0003] Rhodiolosides are derived from Rhodiola rosea. The core issue in the supply of rhodiolosides lies in the structural imbalance between resource scarcity, technological maturity, and the rapid growth of market demand. The annual harvest of wild Rhodiola rosea resources is limited, and while artificial cultivation bases are expanding, they still cannot meet demand. Chemical synthesis can reduce the consumption of natural resources but poses serious environmental pollution problems. Bio-fermentation offers advantages such as high yield, ease of scaling up, environmental friendliness, and low cost, making it a relatively economical, environmentally friendly, and sustainable production method. Summary of the Invention

[0004] The purpose of this invention is to solve the problem of low yield of rhodioloside in the prior art.

[0005] This invention provides a UTP-glucose-1-phosphouridine transferase mutant, which is obtained by mutating any one of the following positions: 6, 126, 134, 140, 177, 180, 214, 249, 284, 289, 296, 298, and 300, starting from the amino acid sequence shown in SEQ ID NO.2.

[0006] This invention provides a UTP-glucose-1-phosphate uridine transferase mutant, wherein the UTP-glucose-1-phosphate uridine transferase mutant is any one of the following: (1) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the threonine at position 6 with arginine; (2) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace valine at position 126 with isoleucine; (3) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace isoleucine at position 134 with valine; (4) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the leucine at position 140 with isoleucine; (5) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the alanine at position 177 with serine; (6) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace valine at position 180 with isoleucine; (7) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace isoleucine at position 214 with valine; (8) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace isoleucine at position 249 with methionine; (9) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the aspartic acid at position 284 with proline; (10) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the glutamic acid at position 289 with aspartic acid; (11) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the glutamic acid at position 296 with glutamine; (12) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the methionine at position 298 with isoleucine; (13) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the isoleucine at position 300 with alanine.

[0007] The present invention provides a gene encoding the above-mentioned UTP-glucose-1-phosphate uridine transferase mutant.

[0008] This invention provides a recombinant vector containing the above-mentioned genes.

[0009] Further specifying, the launch carriers are the pRSFDuet series and the pETDuet series.

[0010] This invention provides a recombinant microbial cell containing the above-mentioned genes.

[0011] Further specifying, the overexpression in *E. coli* originates from *E. coli* ( Escherichia coli ) phosphoglucose mutase gene pgm and UTP-glucose-1-phosphate uridine transferase mutant gene galU Mut 、 Derived from brewer's yeast ( Saccharomycescerevisiae) phenylpyruvate decarboxylase gene ARO10 and alcohol dehydrogenase gene ADH6 The recombinant plasmid pRSFDuet- pgm-galU-ARO10-ADH6 and overexpression of a mutant 3-deoxy-D-arabinohepulose-7-phosphate (DAHP) synthase gene derived from Escherichia coli. aroG fbr Derived from motile fermentation monosporus ( Zymomonas mobile ) cyclohexadiene dehydrogenase gene tyrC and derived from Arabidopsis thaliana ( Arabidopsis thaliana ) glycosyltransferase gene AtUGT85A1 Recombinant plasmid pETDuet- aroG fbr -tyrC-AtUGT85A1 .

[0012] This invention provides the application of the above-mentioned UTP-glucose-1-phosphate uridine transferase mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cells in the production of rhodioloside.

[0013] The present invention provides a method for producing rhodioloside, the steps of which are as follows: fermenting the above-mentioned recombinant microbial cells at 30°C for 72 hours.

[0014] Further specified, the fermentation medium consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract, with 10 g / L CaCO3 used to maintain the pH of the fermentation medium. The culture medium was supplemented with 0.1% by volume of trace elements, the composition of which was: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O and 15 g / L ZnSO4·7H2O. The pH was adjusted to 7.0 using NaOH.

[0015] Beneficial Effects: UTP-glucose-1-phosphate uridine transferase plays a crucial role in the biosynthesis of rhodiolosides, acting as a sugar donor activator. Its function is to combine glucose-1-phosphate with UTP to generate UDP-glucose (UDPG), which serves as a glycosyl donor. The final step in rhodioloside biosynthesis is catalyzed by glycosyltransferase, transferring the glucose group from UDPG to the C8 hydroxyl group of tyrosol, thus forming rhodioloside. Insufficient UDPG supply directly limits the efficiency of rhodioloside synthesis. Therefore, modifying UTP-glucose-1-phosphate uridine transferase to obtain highly efficient UTP-glucose-1-phosphate uridine transferase mutants is another effective approach for the efficient biosynthesis of rhodiolosides.

[0016] The genetically engineered bacteria of this invention contain UTP-glucose-1-phosphate uridine transferase genes with different site-directed mutations. galU phenylpyruvate decarboxylase gene ARO10 Ethanol dehydrogenase gene ADH6 and phosphoglucose mutase gene pgm Overexpression plasmids and genes containing mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase. aroG fbr Cyclohexadiene dehydrogenase gene tyrC and glycosyltransferase gene AtUGT85A1 The overexpression plasmids of these genetically engineered bacteria can use glucose as a carbon source to efficiently catalyze the synthesis of rhodioloside, showing promising application prospects and providing valuable resources for constructing efficient rhodioloside-producing strains. Detailed Implementation

[0017] In the examples, the fermentation medium for shake-flask fermentation consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, 7 g / L yeast extract, 10 g / L CaCO3, 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O, and 15 g / L ZnSO4·7H2O, with the pH adjusted to 7.0.

[0018] Example 1: Construction of genetically engineered bacteria containing different UTP-glucose-1-phosphate uridine transferase mutants Overexpression originates from Escherichia coli (E. coli) Escherichia coli ) phosphoglucose mutase gene pgm and UDP-glucose pyrophosphorylase gene galU, Derived from brewer's yeast ( Saccharomycescerevisiae ) phenylpyruvate decarboxylase gene ARO10 and alcohol dehydrogenase gene ADH6 The recombinant plasmid pRSFDuet- pgm-galU-ARO10-ADH6 and overexpression of a mutant 3-deoxy-D-arabinohepulose-7-phosphate (DAHP) synthase gene derived from Escherichia coli. aroG fbr Derived from motile fermentation monosporus ( Zymomonas mobilis ) cyclohexadiene dehydrogenase gene tyrC and derived from Arabidopsis thaliana ( Arabidopsis thaliana ) glycosyltransferase gene AtUGT85A1 Recombinant plasmid pETDuet- aroG fbr -tyrC-AtUGT85A1 From Zhou Jingwen's team at Jiangnan University (Engineering) Escherichia coli forEfficient De Novo Synthesis of Salidroside ） .

[0019] Overexpression plasmid pRSFDuet- pgm-galU-ARO10-ADH6 Wild-type UTP-glucose-1-phosphate uridine transferase gene galU Replace with UTP-glucose-1-phosphate uridine transferase mutant genes with different site-directed mutations galU Mut Fragment (mutant UTP-glucose-1-phosphate uridine transferase gene) galU A2S Mutant UTP-glucose-1-phosphate uridine transferase gene galU I4Y Mutant UTP-glucose-1-phosphate uridine transferase gene galU T6R Mutant UTP-glucose-1-phosphate uridine transferase gene galU V8I Mutant UTP-glucose-1-phosphate uridine transferase gene galU K9R Mutant UTP-glucose-1-phosphate uridine transferase gene galU T55N Mutant UTP-glucose-1-phosphate uridine transferase gene galU V58I Mutant UTP-glucose-1-phosphate uridine transferase gene galU D92E Mutant UTP-glucose-1-phosphate uridine transferase gene galU V94I Mutant UTP-glucose-1-phosphate uridine transferase gene galU L111I Mutant UTP-glucose-1-phosphate uridine transferase gene galU V119I Mutant UTP-glucose-1-phosphate uridine transferase gene galU L120M Mutant UTP-glucose-1-phosphate uridine transferase gene galU V125L Mutant UTP-glucose-1-phosphate uridine transferase gene galU V126I Mutant UTP-glucose-1-phosphate uridine transferase gene galU V131F Mutant UTP-glucose-1-phosphate uridine transferase gene galU I134V Mutant UTP-glucose-1-phosphate uridine transferase gene galU L140I Mutant UTP-glucose-1-phosphate uridine transferase gene galU S148K Mutant UTP-glucose-1-phosphate uridine transferase gene galU Q149K Mutant UTP-glucose-1-phosphate uridine transferase gene galU A153S Mutant UTP-glucose-1-phosphate uridine transferase gene galU I156L Mutant UTP-glucose-1-phosphate uridine transferase gene galU R157Q Mutant UTP-glucose-1-phosphate uridine transferase gene galU F159Y Mutant UTP-glucose-1-phosphate uridine transferase gene galU D160E Mutant UTP-glucose-1-phosphate uridine transferase gene galU E161Q Mutant UTP-glucose-1-phosphate uridine transferase gene galU A173P Mutant UTP-glucose-1-phosphate uridine transferase gene galU T176S Mutant UTP-glucose-1-phosphate uridine transferase gene galU A177S Mutant UTP-glucose-1-phosphate uridine transferase gene galU V180I Mutant UTP-glucose-1-phosphate uridine transferase gene galU K184Q Mutant UTP-glucose-1-phosphate uridine transferase gene galU V186Y Mutant UTP-glucose-1-phosphate uridine transferase gene galU E187D Mutant UTP-glucose-1-phosphate uridine transferase gene galU A189Q Mutant UTP-glucose-1-phosphate uridine transferase gene galU E192D Mutant UTP-glucose-1-phosphate uridine transferase gene galU V194A Mutant UTP-glucose-1-phosphate uridine transferase gene galU V199I Mutant UTP-glucose-1-phosphate uridine transferase gene galU V207E Mutant UTP-glucose-1-phosphate uridine transferase gene galU I214V Mutant UTP-glucose-1-phosphate uridine transferase gene galU I249M Mutant UTP-glucose-1-phosphate uridine transferase gene galU M259L Mutant UTP-glucose-1-phosphate uridine transferase gene galU K262R Mutant UTP-glucose-1-phosphate uridine transferase gene galU I281M Mutant UTP-glucose-1-phosphate uridine transferase gene galU N284P Mutant UTP-glucose-1-phosphate uridine transferase gene galU T288K Mutant UTP-glucose-1-phosphate uridine transferase gene galU E289D Mutant UTP-glucose-1-phosphate uridine transferase gene galU E295Q Mutant UTP-glucose-1-phosphate uridine transferase gene galU E296Q Mutant UTP-glucose-1-phosphate uridine transferase gene galU E297T Mutant UTP-glucose-1-phosphate uridine transferase gene galU M298I Mutant UTP-glucose-1-phosphate uridine transferase gene galU I300A ), obtained 50 kinds containing galU The mutant plasmid pRSFDuet- pgm-galU A2S -ARO10-ADH6 pRSFDuet- pgm-galU I4Y -ARO10-ADH6 pRSFDuet- pgm-galU T6R -ARO10-ADH6 pRSFDuet- pgm- galU V8I -ARO10-ADH6 pRSFDuet- pgm-galU K9R -ARO10-ADH6 pRSFDuet- pgm-galU T55N -ARO10- ADH6 pRSFDuet- pgm-galU V58I -ARO10-ADH6 pRSFDuet- pgm-galU D92E -ARO10-ADH6 pRSFDuet- pgm-galU V94I -ARO10-ADH6 pRSFDuet- pgm-galU L111I -ARO10-ADH6 pRSFDuet- pgm-galU V119I - ARO10-ADH6 pRSFDuet- pgm-galU L120M -ARO10-ADH6 pRSFDuet- pgm-galU V125L -ARO10-ADH6 pRSFDuet- pgm-galU V126I -ARO10-ADH6 pRSFDuet- pgm-galU V131F -ARO10-ADH6 pRSFDuet- pgm-galU I134V -ARO10-ADH6 pRSFDuet- pgm-galUL140I -ARO10-ADH6 、pRSFDuet- pgm-galU S148K - ARO10-ADH6 、pRSFDuet- pgm-galU Q149K -ARO10-ADH6 、pRSFDuet- pgm-galU A153S -ARO10-ADH6 、pRSFDuet- pgm-galU I156L -ARO10-ADH6 、pRSFDuet- pgm-galU R157Q -ARO10-ADH6 、pRSFDuet- pgm-galU F159Y -ARO10-ADH6 、pRSFDuet- pgm-galU D160E -ARO10-ADH6 、pRSFDuet- pgm-galU E161Q - ARO10-ADH6 、pRSFDuet- pgm-galU A173P -ARO10-ADH6 、pRSFDuet- pgm-galU T176S -ARO10-ADH6 、pRSFDuet- pgm-galU A177S -ARO10-ADH6 、pRSFDuet- pgm-galU V180I -ARO10-ADH6 、pRSFDuet- pgm-galU K184Q -ARO10-ADH6 、pRSFDuet- pgm-galU V186Y -ARO10-ADH6 、pRSFDuet- pgm-galU E187D - ARO10-ADH6 、pRSFDuet- pgm-galU A189Q - ARO10-ADH6 、pRSFDuet- pgm-galU E192D -ARO10-ADH6 、pRSFDuet- pgm-galU V194A -ARO10-ADH6 、pRSFDuet- pgm-galU V199I -ARO10-ADH6pRSFDuet- pgm-galU V207E -ARO10-ADH6 pRSFDuet- pgm-galU I214V -ARO10-ADH6 pRSFDuet- pgm-galU I249M - ARO10-ADH6 pRSFDuet- pgm-galU M259L -ARO10-ADH6 pRSFDuet- pgm-galU K262R -ARO10-ADH6 pRSFDuet- pgm-galU I281M -ARO10-ADH6 pRSFDuet- pgm-galU N284P -ARO10-ADH6 pRSFDuet- pgm-galU T288K -ARO10-ADH6 pRSFDuet- pgm-galU E289D -ARO10-ADH6 pRSFDuet- pgm-galU E295Q - ARO10-ADH6 pRSFDuet- pgm-galU E296Q -ARO10-ADH6 pRSFDuet- pgm-galU E297T -ARO10-ADH6 pRSFDuet- pgm-galU M298 -ARO10-ADH6 pRSFDuet- pgm-galU I300A -ARO10-ADH6 The above 50 plasmids were respectively combined with pETDuet- aroG fbr -tyrC-AtUGT85A1 Plasmid combinations were introduced into the chassis strain *Escherichia coli* BL21(DE3) to obtain 50 genetically engineered bacteria containing different UTP-glucose-1-phosphate uridine transferase mutants. The bacteria were named according to the mutated amino acid sites.

[0020] Based on the amino acid sequence shown in SEQ ID NO: 2, the corresponding base substitutions are A2S, I4Y, T6R, V8I, K9R, T55N, V58I, D92E, V94I, L111I, V119I, L120M, V125L, V126I, V131F, I134V, L140I, S148K, Q149K, A153S, I156L, R157Q, F159Y, D1 60E, E161Q, A173P, T176S, A177S, V180I, K184Q, V186Y, E187D, A189Q, E192D, V194A, V199I, V207E, I214V, I249M, M259L, K262R, I281M, N284P, T288K, E289D, E295Q, E296Q, E297T, M298I, or I300A. The letters preceding the numbers represent the original amino acids, and the letters following the numbers represent the mutated amino acids.

[0021] SEQ ID NO: 1 (nucleotide sequence of wild-type UTP-glucose-1-phosphate uridine transferase GalU): ATGGCTGCCATTAATACGAAAGTCAAAAAAGCCGTTATCCCCGTTGCGGGATTAGGAACCAGGATGTTGCCGGCGACGAAAGCCATCCCGAAAGAGATGCTGCCACTTGTCGATAAGCCATTAATTCAATACGTCGTGAATGAATGTATTGCGGCTGGCATTACTGAAATTGTGCTGGTTACACACTCATCTAAAAACTCTATTGAAAACCACTTTGATACCAGTTTTGAACTGGAAGCAATGCTGGAAAAACGTGTAAAACGTCAACTGCTTGATGAAGTGCAGTCTATTTGTCCGCCGCACGTGACTATTATGCAAGTTCGTCAGGGGCTGGCGAAAGGTCTGGGACACGCGGTATTGTGTGCTCACCCGGTAGTGGGTGATGAACCTGTGGCTGTTATTTTGCCTGACGTTATTCTGGATGAATATGAATCCGATTTGTCACAGGATAACCTGGCAGAGATGATCCGCCGCTTTGATGAAACGGGTCATAGCCAGATCATGGTTGAACCGGTTGCTGATGTGACCGCATATGGCGTTGTGGATTGCAAAGGCGTTGAATTAGCGCCGGGTGAAAGCGTACCGATGGTTGGCGTGGTTGAAAAACCAAAAGCGGATGTTGCGCCGTCTAATCTCGCTATTGTGGGTCGTTACGTACTTAGTGCGGATATTTGGCCGTTGCTGGCAAAAACCCCTCCGGGAGCTGGTGATGAAATTCAGCTCACCGACGCAATTGATATGCTAATCGAAAAAGAAACGGTTGAAGCCTATCATATGAAAGGGAAGAGCCATGACTGCGGTAATAAATTAGGTTACATGCAGGCCTTCGTTGAATACGGTATTCGTCATAACACCCTTGGCACGGAATTTAAAGCCTGGCTTGAAGAAGAGATGGGCATTAAGAAGTAA; SEQ ID NO: 2 (Amino acid sequence of wild-type UTP-glucose-1-phosphate uridylyltransferase GalU): MAAINTKVKKAVIPVAGLGTRMLPATKAIPKEMLPLVDKPLIQYVVNECIAAGIETIVLVTHSSKNSIENHFDTSFELEAMLEKRVKRQLLDEVQSICPPHVTIMQVRQGLAKGLGHAVLCAHPVVGDEPVAVILPDVILDEYESDLSQDNL AEMIRRFDETGHSQIMVEPVADVTAYGVVDCKGVELAPGESVPMVGVVEKPKADVAPSNLAIVGRYVLSADIWPLLAKTPPGAGDEIQLTDAIDMLIEKETVEAYHMKGKSHDCGNKLGYMQAFVEYGIRHNTLGETFKAWLEEEMGIKK*.

[0022] Table 1 Genetically engineered bacteria containing different UTP-glucose-1-phosphate uridine transferase mutants

[0023] Example 2. Fermentation experiment of rhodioloside from genetically engineered bacteria containing different UTP-glucose-1-phosphate uridine transferase mutants. These 50 genetically engineered bacteria and the control strain (strain genotype: E.coli BL21 (pRSFDuet- pgm- galU-ARO10-ADH6 + pETDuet- aroG fbr -tyrC-AtUGT85A1 The culture medium was inoculated into LB liquid medium containing antibiotic resistance. The culture system consisted of 10 mL of medium dispensed into a 100 mL Erlenmeyer flask, supplemented with 50 μg / mL kanamycin and 100 μg / mL ampicillin. The culture conditions were constant temperature shaking incubator at 37°C and 220 rpm overnight to obtain the seed culture. This seed culture was then inoculated into fresh fermentation medium at a volume of 2%. The culture system consisted of 50 mL of medium dispensed into a 250 mL Erlenmeyer flask, supplemented with 50 μg / mL kanamycin and 100 μg / mL ampicillin. The culture conditions were constant temperature shaking incubator at 37°C and 220 rpm. 600 When the concentration reaches 0.6-0.8, add 0.1 mM IPTG to induce fermentation. Fermentate at 30℃ and 220 rpm for 72 h to prepare samples.

[0024] Example 3. Extraction and liquid chromatography detection of rhodioloside Transfer 1 mL of fermentation broth to a 1.5 mL centrifuge tube and centrifuge at 12000 rpm for 5 min. Mix 200 μL of the supernatant with 800 μL of mobile phase A. After diluting the sample 5-fold, determine the rhodioloside yield using a Shimadzu high-performance liquid chromatograph. Quantification was performed using a reversed-phase C18 column. Mobile phase A was water containing 1% trifluoroacetic acid (TFA); mobile phase B was acetonitrile containing 1% TFA. The flow rate was 1 mL / min. -1 The absorbance of the sample was detected at 274 nm. The HPLC program was as follows: 10% B: 0-5 min, 60% B: 5-10 min, 90% B: 10-12 min, 2% B: 12-16 min, 0% B: 16-20 min. Using strains containing unmutated UTP-glucose-1-phosphate uridine transferase as a control (with a shake-flask fermentation yield as high as 4.0 g / L), the statistical results of the increase in rhodioloside yield during shake-flask fermentation of 50 different site-directed mutations of UTP-glucose-1-phosphate uridine transferase GalU mutants are shown in Table 2.

[0025]

[0026] Note: +% indicates the increase in rhodioloside production; - indicates no increase.

[0027] Increase (%) = (Rhodioloside yield of mutant strain (g / L) - Rhodioloside yield of control strain (g / L)) / Rhodioloside yield of control strain (g / L) × 100%.

[0028] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present invention provides protection against bacteria derived from Escherichia coli (E. coli). Escherichia coli The UTP-glucose-1-phosphate uridine transferase GalU was site-directedly modified to create the UTP-glucose-1-phosphate uridine transferase mutant GalU. V180I Overexpression in genetically engineered bacteria significantly increased the yield of rhodioloside, reaching 5 g / L in shake-flask fermentation, a 25% increase compared to the control strain, providing valuable resources for constructing efficient rhodioloside-producing strains.

[0029] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A UTP-glucose-1-phosphate uridine transferase mutant, characterized in that, The UTP-glucose-1-phosphate uridine transferase mutant is based on the amino acid sequence shown in SEQ ID NO.2, with valine at position 180 replaced by isoleucine.

2. The gene encoding the UTP-glucose-1-phosphate uridine transferase mutant of claim 1.

3. A recombinant vector containing the gene of claim 2.

4. The recombinant vector according to claim 3, characterized in that, The launch carriers are the pRSFDuet series and the pETDuet series.

5. A recombinant microbial cell containing the gene of claim 2.

6. The recombinant microbial cell according to claim 5, characterized in that, Overexpression in Escherichia coli originates from Escherichia coli (E. coli) Escherichia coli ) phosphoglucose mutase gene pgm and UTP-glucose-1-phosphate uridine transferase mutant gene galU Mut 、 Derived from brewer's yeast ( Saccharomyces cerevisiae phenylpyruvate decarboxylase gene ARO10 and alcohol dehydrogenase gene ADH6 The recombinant plasmid pRSFDuet- pgm-galU-ARO10-ADH6 and overexpression of a mutant 3-deoxy-D-arabinohepulose-7-phosphate (DAHP) synthase gene derived from Escherichia coli. aroG fbr Derived from motile fermentation monosporus ( Zymomonas mobilis ) cyclohexadiene dehydrogenase gene tyrC and derived from Arabidopsis thaliana ( Arabidopsis thaliana ) glycosyltransferase gene AtUGT85A1 Recombinant plasmid pETDuet- aroG fbr -tyrC- AtUGT85A1 .

7. The use of the UTP-glucose-1-phosphate uridine transferase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, or the recombinant microbial cell of claim 5 or 6 in the production of rhodioloside.

8. A method for producing rhodioloside, characterized in that, The method involves the following steps: fermenting the recombinant microbial cells described in claim 6 at 30°C for 72 hours.

9. The method according to claim 8, characterized in that, The fermentation medium consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract. The pH of the fermentation medium was maintained using 10 g / L CaCO3. 0.1% by volume of trace elements were added to the medium, consisting of: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O, and 15 g / L... ZnSO4·7H2O, pH adjusted to 7.0 using NaOH.

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