CN113308447B - Application of arabidopsis UGT74F2 in catalyzing phenyllactic acid to synthesize phenyllactyl glucose - Google Patents
Application of arabidopsis UGT74F2 in catalyzing phenyllactic acid to synthesize phenyllactyl glucose Download PDFInfo
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Abstract
The invention discloses application of Arabidopsis UGT74F2 in catalyzing phenyllactic acid to generate glycosyl transfer reaction to synthesize phenyllactyl glucose, and researches show that UGT74F2 from Arabidopsis can catalyze phenyllactic acid to synthesize phenyllactyl glucose, has higher catalytic activity than that of AbUGT1 reported at present for tropane alkaloid synthesis, can be used for tropane alkaloid metabolic engineering to improve alkaloid content, widens the application value of UGT74F2, and has important significance for medicinal TA metabolic engineering and synthetic biology.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of arabidopsis UGT74F2 in catalyzing phenyllactic acid to generate glycosyl transfer reaction to synthesize phenyllactyl glucose.
Background
Tropane Alkaloids (TA) are a group of anticholinergic agents that act on the parasympathetic nerves. The representative drugs of hyoscyamine, scopolamine and their derivatives have been widely usedThe market demand is huge in the aspects of anesthetics, treating Parkinson, and various organ cramps and asthma caused by smooth muscle spasm. Tropane alkaloids are still completely dependent on extraction from a few solanaceae medicinal plants, including belladonna (Atropa belladonna), Datura stramonium (Datura stramnonium) and hyoscyami (Hyoscyamus niger). Belladonna is a commercial cultivated medicinal source plant for producing hyoscyamine and scopolamine recorded and specified in Chinese pharmacopoeia. The weight fraction of the hyoscyamine in the wild belladonna plant is 0.02-0.17% (dry weight), and the content of the scopolamine is only 0.01-0.08% of the dry weight. Therefore, the cultivation of medicinal plants with high tropane alkaloid yield is always a long-sought goal in the industry. With the establishment of a yeast engineering platform and the development of synthetic biology, a possible strategy is provided for solving the shortage of tropine alkaloid resources on the market. However, in the prior tropine alkaloid total synthesis yeast strains, the maximum yield of the hyoscyamine is 80 mu g -1 The highest yield of scopolamine is 30 mu g.L -1 Far below the commercial production demand (Prashath Srinivasan 1)&Christina D.Smolke.Biosynthesis of medicinal tropane alkaloids in yeast.Nature.2020)。
Tropane alkaloid biosynthesis starts with ornithine metabolism and phenylalanine metabolism. Ornithine is synthesized into Tropine (Tropine) through multi-step enzymatic reaction. Phenylalanine is used for synthesizing phenyllactic acid (Phenyllactate) through multi-step enzymatic reaction, and phenyllactic acid is catalyzed by Phenyllactate glucosyltransferase (UGT 1) to generate glycosyl transfer reaction to synthesize Phenyllactylglucose (Phenyllactylglucose). The tropine and the phenyllactyl glucose are subjected to esterification reaction under the action of Littorine Synthase (LS) to synthesize the Littorine. The conchioline is subjected to multi-step enzymatic reaction to synthesize Hyoscyamine (Hyoscyamine), Anisodamine (Anisodamine) and Scopolamine (Scopolamine).
Uridine diphosphate glucosyltransferase (UGT) catalyzes glucosyl transfer reactions, transferring sugar groups from an activated donor molecule, Uridine diphosphate glucose (UDPG), to an acceptor molecule. UGT-mediated glycosylation of natural products is a widely-existing modification in biosynthesis of secondary metabolites and is one of the most important biochemical reactions. The UGT gene family has high polymorphism, the number of family members is large, and the diversity of the substrate pockets enables the UGT family members to form different functional differentiation. Previous studies found that the reported belladonna AbUGT1 has very weak catalytic activity, so that the synthesis of phenyllactoyl glucose is a very critical rate-limiting step both in TA-derived plants and in TA de novo yeast, and the accumulation of downstream hyoscyamine and scopolamine is severely limited by insufficient supply of phenyllactoyl glucose (Prashanth Srinivasan1& Christina d. smear. biosynthessis of medicinal tropane alkaloids in yeast. nature. 2020). Therefore, the screening of the high-activity phenyllactic acid glucosyltransferase has very important significance for TA metabolic engineering and synthetic biology of medicine.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an application of arabidopsis UGT74F2 in catalyzing phenyllactic acid to perform glycosyl transfer reaction to synthesize phenyllactyl glucose; the invention also aims to provide the application of Arabidopsis UGT74F2 in improving the content of tropane alkaloid in Solanaceae TAs resource plants; the invention also aims to provide a method for improving the content of tropane alkaloid in a solanaceae TAs resource plant.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the application of Arabidopsis UGT74F2 in catalyzing phenyllactic acid to generate glycosyl transfer reaction to synthesize phenyllactyl glucose is disclosed, wherein the amino acid sequence of the Arabidopsis UGT74F2 is shown as SEQ ID No. 3.
Preferably, the nucleotide sequence of the Arabidopsis UGT74F2 is shown in SEQ ID NO. 4.
2. The application of Arabidopsis UGT74F2 in improving the tropine alkaloid content in Solanaceae TAs resource plants is disclosed, wherein the amino acid sequence of the Arabidopsis UGT74F2 is shown as SEQ ID NO. 3.
Preferably, the Solanaceae TAs resource plant is belladonna, stramonium or scopolia; the tropane alkaloid is at least one of hypusine, hyoscyamine, anisodamine and scopolamine.
3. A method for improving the tropine alkaloid content of a solanaceae TAs resource plant is characterized in that an Arabidopsis thaliana UGT74F2 gene is overexpressed in the solanaceae TAs resource plant, and an amino acid sequence coded by the Arabidopsis thaliana UGT74F2 gene is shown as SEQ ID NO. 3.
Preferably, the method for over-expressing the Arabidopsis thaliana UGT74F2 gene is as follows: cloning an arabidopsis thaliana UGT74F2 gene, then constructing a plant expression vector, obtaining a recombinant plant expression vector containing the arabidopsis thaliana UGT74F2 gene, transforming agrobacterium tumefaciens by using the obtained recombinant plant expression vector to obtain engineering bacteria, finally transforming solanaceae TAs resource plants by using the engineering bacteria, and screening a transgenic regenerant to obtain the solanaceae TAs resource plants with improved tropane alkaloid content.
Preferably, the plant expression vector is formed by connecting the nucleotide shown in SEQ ID NO.4 between a CaMV 35S promoter driver and a Nos terminator of a pBI121 vector.
The invention has the beneficial effects that: the invention discloses that UGT74F2 from Arabidopsis thaliana can catalyze phenyllactic acid to synthesize phenyllactyl glucose, and the catalytic activity is higher than that of AbUGT1 reported at present for synthesizing tropane alkaloid. The enzyme expands the function of synthesizing the Salicylic acid glucose ester by taking UDPG as a glycosyl donor and Salicylic acid (Salicylic acid) as an acceptor, which is discovered at present, expands the application value of UGT74F2, can be used for tropane alkaloid metabolic engineering to improve the alkaloid content, and has important significance for medicinal TA metabolic engineering and synthetic biology.
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In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the UGT74F2/AbUGT1 model with phenyllactic acid and uridine diphosphate glucose complexes.
FIG. 2 shows the purification of UGT74F2 recombinant protein (Marker: protein molecular weight standard).
FIG. 3 shows the activity analysis of phenyllactyl transferase (A: phenyllactyl glucose ion flow diagram B: UGT74F2 catalysis sample phenyllactyl glucose mass spectrum).
FIG. 4 shows the detection of the expression level of the target gene of belladonna hairy roots.
FIG. 5 is a belladonna hairy root tropane alkaloid content analysis (A: conchioline content; B: hyoscyamine content; C: anisodamine content; D: scopolamine content, each transgenic event containing 15 independent transformed hairy roots, all statistical analyses tested using independent samples T, representing P value <0.01, representing P value < 0.001).
Figure 6 is the relative abundance of phenyllactoyl glucose in yeast fermentation media (3 biological replicates per yeast strain, all statistical analyses were performed using independent sample T-tests, representing P value <0.01, and P value < 0.001).
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 molecular docking screening of potential phenyllactic acid glucosyltransferases
909 glucosyltransferase protein crystal structures were downloaded from the protein database PDB. The protein structure model was obtained by homologous modeling belladonna AbUGT1 using Swiss model. Three-dimensional structures of D-Phenyllactic acid (PLA) and Uridine Diphosphate Glucose (UDPG) were downloaded from PubChem database. PLA and UDPG were docked separately by Autodock Vina software using PDB database-derived glycosyltransferase and the abutt 1 protein model, with 10 replicates per docking calculation and their binding free energies calculated. Finally, a complex model of glycosyltransferase, PLA and UDPG was generated using pymol and analyzed for substrate binding pockets.
The molecular docking calculation result shows that the binding free energy of AbUGT1 of belladonna to PLA is-5.5 kcal -1 The binding free energy to UDPG was-10.8 kcal -1 (ii) a The binding free energy to PLA of Arabidopsis derived UGT74F2 was-5.6 kcal -1 The binding free energy to UDPG was-10.4 kcal -1 And the two combined free energy analysis have no significant difference. UGT74F2 is from the modulusThe plant Arabidopsis thaliana has been studied, and only the enzyme has found that UDPG is taken as glycosyl donor, Salicylic acid (Salicylic acid) is taken as acceptor to synthesize glucose salicylate, and the enzyme participates in the biological process of Salicylic acid signal path, and has no ability of catalyzing other secondary metabolites (Alayna M. etc. Differencens in Salicylic acid glycosides by UGT74F1 and UGT74F2 from Arabidopsis thaliana. scientific reports.2017.). Complex models of AbUGT1 or UGT74F2 with two substrates were obtained by pymol software, respectively, and the results are shown in FIG. 1. The substrate binding pockets were found to be substantially identical, and it was predicted that a similar catalytic mechanism might exist, with the carboxy terminus of the PLA molecule adjacent to the glucosyl group of UDPG. The analysis provides theoretical support for the potential phenyllactic acid glucosyltransferase activity of UGT74F 2.
Example 2 enzyme Activity assay
(1) Total RNA extraction from Arabidopsis thaliana
Taking a proper amount of arabidopsis plant tissues, placing the arabidopsis plant tissues in liquid nitrogen for quick freezing, grinding the arabidopsis plant tissues into fine powder at low temperature, adding the arabidopsis plant tissues into a 1.5mL Eppendorf (EP) centrifuge tube containing 1mL of lysate, fully oscillating the arabidopsis plant tissues and uniformly mixing the arabidopsis plant tissues and the Eppendorf (EP) centrifuge tube, and extracting total RNA according to the instruction of a TIANGEN kit. The integrity of total RNA was checked by agarose gel electrophoresis and the concentration of extracted RNA was analyzed on a ultramicro UV spectrophotometer.
(2) Gene cloning and prokaryotic expression vector construction
Synthesizing cDNA by using the extracted total RNA as a template according to the specification of a first strand synthesis kit of the Tiangen FastKing cDNA; the UGT74F2 sequence is derived from NCBI public database, and recombinant primers are designed by combining an expression vector pET-28a according to the electronic sequence of UGT74F2, wherein the specific primers are as follows:
pET28a-UGT74F2-F：5’-aatgggtcgcggatccatggagcataagagaggaca-3’(SEQ ID NO.1)；
pET28a-UGT74F2-R：5’-gtggtggtggtggtgctcgagctatttgctctgaacccttgat-3’(SEQ ID NO.2)；
firstly, taking Arabidopsis thaliana cDNA as a template and pET28a-UGT74F2-F and pET28a-UGT74F2-R as primers, and carrying out PCR amplification to obtain a UGT74F2 gene fragment with a pET28a homologous sequenceAnd cutting a PCR product, recovering and sequencing to obtain a UGT74F2 gene sequence shown as SEQ ID NO.3, and a coded amino acid sequence shown as SEQ ID NO. 4. The pET28a (+) was linearized with EcoRI and XhoI endonucleases and recovered after digestion
And carrying out recombination reaction by using the PCR one-step directional cloning kit. The product is transformed into colon bacillus, and after the plasmid is extracted and sequenced, the prokaryotic protein expression vector pET28a-UGT74F2 is obtained.
The AbUGT1 prokaryotic expression plasmid pET28a-AbUGT1 was cloned and constructed from belladonna by the same method, and the primers were designed as follows:
pET28a-AbUGT1-F：5’-aatgggtcgcggatccatgggatctcaaggtaccaa-3’(SEQ ID NO.5)；
pET28a-AbUGT1-R：5’-gtggtggtgctcgagctaattggatagaggtgcta-3’(SEQ ID NO.6)。
(3) recombinant protein induced expression and purification
The plasmid pET28a-UGT74F2 is transferred into a Rosseta competent cell, and a prokaryotic protein expression strain Rosetta-pET28a-UGT74F2 is successfully obtained. Inoculating Rosetta-pET28a-UGT74F2 bacterial solution into 400ml LB medium of Carna resistance in a 1L triangular flask, culturing to OD at 37 ℃ 600 The value is 0.6, IPTG is added to a final concentration of 0.5 mol. L -1 After the induction at 18 ℃ for 17 hours, the cells were collected by centrifugation at 8000rpm at room temperature. The collected thallus is resuspended by using 40mL of Phosphate Buffer Saline (PBS), and then the lysozyme is added into the thallus mixed liquor until the final concentration of the lysozyme is 0.3 mg/mL -1 Gently stirring with a glass rod, standing on ice for more than 30min, crushing in an ultrasonicator for 30min (power 60%, ultrasonication for 3 s, and stopping for 7 s), centrifuging the ultrasonicated product at 11000rpm at 4 ℃ for 40min, filtering the obtained supernatant with a 0.45-micrometer filter, and purifying the filtered protein supernatant.
The recombinant protein was purified with reference to Proteinlso TM Ni-NTA Resin application manual, as follows: eluting with different concentrations of imidazole in equilibration buffer solution, and collecting 10 mmol.L -1 、20mmol·L -1 、40mmol·L -1 、60mmol·L -1 The effluent of imidazole equilibration buffer eluting the target protein. The results of detection on a 12% (w/v) polyacrylamide gel by SDS-PAGE analysis are shown in FIG. 2. The results showed that the concentration of L was 20mmol -1 The recombinant UGT74F2-6xHis protein size is consistent with prediction, overnight dialysis is carried out to remove salt, the BCA method is used for determining the protein concentration, and a fresh protein sample is immediately used for an in vitro enzyme activity detection experiment.
AbUGT1 was expressed and purified according to the same method.
(4) Enzyme Activity assay
The purified UGT74F2 recombinant protein was added to a 200. mu.l reaction mix (10. mu.g UGT74F2 recombinant protein, 1mM phenyllactic acid, 5mM UDP-glucose, 5mM MnCl) 2 Tris-HCl pH 7.2). After two hours at 30 ℃ the reaction was stopped by adding an equal amount of pure methanol, and the boiled UGT74F2 recombinant protein was used as a negative control, and AbUGT1 obtained in the same manner as above was used as a positive control. The reaction products were identified in a full-scan negative ion mode using Thermo Fisher Scientific UPLC-Q active ultra-high pressure liquid-mass spectrometer with electrospray Ionization (ESI).
The instrument parameters were set as follows: the flow rate of the sheath gas is 35, the flow rate of the auxiliary gas is 10, the spray voltage is 3.00kV, the temperature of the capillary tube is 350 ℃, the radio frequency level of the S lens is 50, and the temperature of the auxiliary gas heater is 350 ℃. The flow rate of the system is 0.3mL/min, and the temperature of the oven is 35 ℃; all standards were purchased from Sigma Aldrich. The content of the target compound was identified and analyzed using an ACQUITY UPLC BEN HILIC chromatography column (2.1 mm. times.100 mm, 1.7 μm). Sample analysis was performed using gradient elution, mobile phase a: 100mM ammonium formate and 1% aqueous formic acid, mobile phase B is acetonitrile; the volume of each injection was 5. mu.L.
The elution procedure is as follows:
the results of the enzyme activity measurement are shown in FIG. 3. The results show that phenyllactyl glucose can be detected in the UGT74F2 catalytic reaction sample, and the peak time is completely consistent with that of AbUGT1, the peak time is 7.03min, and the nucleus ratio m/z 327.1088 is the main charged form under negative ions, namely, the reaction sample is charged with a negative charge after one H is lost. While the compound was not detected in the negative control sample, it was demonstrated that UGT74F2 catalyzes the conversion of phenyllactic acid to phenyllactyl glucose ester. In addition, the abundance of the phenyllactyl glucose in the UGT74F2 catalytic sample is 72% higher than that in the AbUGT1 catalytic sample, which shows that the catalytic efficiency of UGT74F2 is higher than that of AbUGT 1.
Example 3 role of UGT74F2 in plant Metabolic engineering of tropane alkaloids
(1) Construction of plant overexpression vectors
In vitro enzyme activity experiments have shown that UGT74F2 has higher catalytic efficiency than AbUGT1, suggesting that UGT74F2 may have better promotion effect on the biosynthesis of downstream conchine. Therefore, in order to understand the promotion effect of UGT74F2 and AbUGT1 on the downstream alkaloid synthesis in vivo, the research evaluates the significance of the two genes in tropane alkaloid biosynthesis by constructing an overexpression vector and performing plant metabolic engineering comparison by using belladonna hairy roots.
Recombinant primers were designed in conjunction with the overexpression vector pBI121 based on the UGT74F2 coding region sequence obtained above. The primers were designed as follows:
121-UGT74F2-F：5’-cgggggactctagaggatccatggagcataagagaggaca-3’(SEQ ID NO.7)；
121-UGT74F2-R：5’-gatcggggaaattcgagctcctatttgctctgaacccttg-3’(SEQ ID NO.8)。
the gene was amplified using KOD-Plus using UGT74F2 gene fragment as a template. The pBI121 is linearized by utilizing BamHI and SacI double enzyme digestion, UGT74F2 is recombined between a CaMV 35S promoter driver and an Nos terminator of the over-expression vector pBI121 through a recombinase, and the plant over-expression vector pBI121-UGT74F2 is successfully constructed after sequencing is correct.
The same procedure was used to construct pBI121-AbUGT1 with the following primers:
121-AbUGT1-F：5’-cgggggactctagaggatccatgggatctcaaggtaccaa-3’(SEQ ID NO.9)；
121-AbUGT1-R：5’-gatcggggaaattcgagctcctaattggatagaggtgcta-3’(SEQ ID NO.10)。
(2) agrobacterium rhizogenes mediated belladonna hairy root transformation
The constructed plant overexpression vectors pBI121-UGT74F2 and pBI121-AbUGT1 are respectively transformed into agrobacterium rhizogenes C58C1 by a freeze-thaw method, and PCR verification is carried out to successfully obtain an engineering strain for subsequent genetic transformation.
Belladonna seed is soaked in gibberellin solution overnight, washed with tap water overnight for 1 day, and then sterilized. Respectively sterilizing with 75% ethanol for 1min and 50% NaClO solution for 20min, fully shaking during the period to ensure full sterilization, and finally cleaning with sterile water for 5-6 times. Placing the sterilized belladonna seeds on sterile absorbent paper by using tweezers to suck excessive water, inoculating the belladonna seeds on a solid culture medium of MS +200mg/L Cef at 25 ℃, culturing for about 15 days under 16h/8h (light/dark) illumination conditions, and shearing sterile seedling leaves and hypocotyl explants for genetic transformation.
Adding the explant into a resuspension (MS + AS100 mu mol/L) of the agrobacterium rhizogenes engineering bacteria of the activated overexpression vector, fully contacting the bacteria liquid with the explant for 5 minutes, transferring to a co-culture solid culture medium (MS + AS100 mu mol/L), and performing dark culture at 28 ℃ for 2 days.
Transferring the belladonna explants cultured for 2d in the co-culture to a screening culture medium (MS + Kan 100mg/L + Cef 400mg/L) for dark culture at 25 ℃, subculturing once a week, and obtaining Kan resistant hairy roots after 1-2 subcultures. The well-grown hairy roots are cut off and transferred to a culture medium (MS + Cef 200mg/L) to be cultured to be completely sterile, thereby obtaining Kan resistant belladonna hairy roots.
(3) Positive identification of belladonna hairy root and detection of target gene expression quantity
In order to identify the positive clone of transgenic belladonna hairy root, the genomic DNA of the belladonna hairy root is extracted by using a conventional CTAB method, and the positive transgenic hairy root is screened by using PCR. And taking over-expression material of positive belladonna hairy roots and control group material, extracting RNA and carrying out reverse transcription to obtain cDNA, carrying out fluorescent quantitative PCR, and measuring gene expression quantity. The qPCR primer sequences were as follows:
q-AbUGT1-F:5‘-cttcgatggttgggaatac-3’(SEQ ID NO.11)；
q-AbUGT1-R:5‘-ggacccataggacagcaca-3’(SEQ ID NO.12)；
q-UGT74F2-F:5‘-ctgtcaaacaaagccatcg-3’SEQ ID NO.13)；
q-UGT74F2-R:5‘-ccttccacacatcttgtat-3’(SEQ ID NO.14)。
the results of expression level measurement are shown in FIG. 4. The results showed that the UGT74F2 gene was detected only in UGT74F2 overexpressing hairy roots, not in control hairy roots and abutt 1 overexpressing hairy roots; whereas the AbUGT1 gene was significantly elevated in AbUGT1 overexpressing hairy roots, which was not detected in the control group and UGT74F2 overexpressing hairy roots. The above results indicate that we have obtained transgenic hairy roots with successfully over-expressed target genes.
(4) LC-MS (liquid chromatography-Mass Spectrometry) determination of content of tropane alkaloid in belladonna
For each independent positive hairy root line, about 100mg of fresh hairy roots are inoculated into 100mL of MS liquid culture medium. 15 independently transformed hairy root lines were cultured per group, and 15 hairy root lines independently transformed with pBI121 served as empty plasmid control groups. Harvesting the hairy roots after culturing for 30 days, washing culture solution on the surfaces of the hairy roots with running water, wrapping fresh materials in tinfoil, and performing low-temperature cold drying in a freeze dryer for next extraction.
The freeze-dried hairy roots were loaded into a 2mL EP tube previously loaded with 2-4 small 3mm steel balls and ground into fine powder in a fully automatic sample rapid-speed grinder set at 55Hz for 90 s. Weighing 25mg of dry powder, placing the dry powder in a 2mL EP tube, adding 1mL of alkaloid extract into the tube, placing the tube at 25 ℃, shaking the tube for 3h at 200rpm, centrifuging the tube for 5min at 11000rpm, and absorbing supernatant. Filtering the supernatant with a 0.22 μm filter head, diluting ten times, and performing on-machine analysis.
The alkaloid content was analyzed and determined in a full-scan positive ion mode using a Thermo Fisher Scientific UPLC-Q active ultra-high pressure liquid-mass spectrometer with electrospray Ionization (ESI) as the ion source, and the specific instrument parameters were the same as above.
The results of the content assays showed that the content of aspirine, hyoscyamine, anisodamine and scopolamine in the abutt 1 overexpression strains and UGT74F2 overexpression strains were significantly increased compared to the control group strains (fig. 5). Wherein the contents of AbUGT1 overexpression strain conchiolin, hyoscyamine, anisodamine and scopolamine were 126%, 66%, 80% and 106% higher than the control group (fig. 5). The contents of the UGT74F2 over-expression strains of conchioline, hyoscyamine, anisodamine and scopolamine are 168%, 109%, 119% and 158% higher than those of the control group (FIG. 5). Comparing the AbUGT1 overexpression strain and the UGT74F2 overexpression strain, the content of the four alkaloids in the UGT74F2 overexpression strain is obviously higher than that in the AbUGT1 overexpression strain.
The results show that the UGT74F2 is over-expressed in belladonna plants by utilizing plant metabolic engineering to promote the synthesis of the downstream products of tropane alkaloid. Compared with AbUGT1, UGT74F2 overexpression enables downstream products to be accumulated more strongly, and metabolic engineering effects are more excellent. Both in vivo and in vitro experiments confirm that UGT74F2 is a more ideal candidate gene for the engineering of tropane alkaloid metabolism.
Example 4 Effect of UGT74F2 in Yeast fermentation production of lactoyl glucose
(1) Construction of yeast total synthetic phenyllactyl glucose strain
To compare the effects of UGT74F2 and AbUGT1 in yeast synthetic biology. Firstly, constructing a yeast expression UGT74F2 plasmid: the plasmid pESC-URA is cut by restriction enzymes BamHI and KpnI, and the gel is cut and recovered. UGT74F2 was amplified using primers containing a 20bp homologous sequence to the vector multiple cloning site region. By using
The PCR one-step directional cloning kit performs recombination reaction on the plasmid skeleton and the target gene amplification product, transforms Escherichia coli, identifies positive clones by PCR, and after sequencing is correct, the plasmid is named as pESC-URA-UGT74F 2. PCR primer designThe following were used:
ESC-UGT74F2-F:5’-ggagaaaaaaccccggatccatggagcataagagaggaca-3’(SEQ ID NO.15)；
ESC-UGT74F2-R:5’-tcttagctagccgcggtaccctatttgctctgaacccttg-3’(SEQ ID NO.16)；
the same procedure was used to construct the yeast expression plasmid pESC-URA-AbUGT 1. The PCR primers were designed as follows:
ESC-AbUGT1-F:5’-ggagaaaaaaccccggatccatgggatctcaaggtaccaa-3’(SEQ ID NO.17)；
ESC-AbUGT1-R:5’-tcttagctagccgcggtaccctaattggatagaggtgcta-3’(SEQ ID NO.18)；
and respectively transforming the two yeast expression plasmids into Saccharomyces cerevisiae BY4742 competent cells BY referring to a conventional lithium acetate transformation method to obtain yeast engineering strains BY4742-UGT74F2 and BY4742-AbUGT 1.
(2) LC-MS (liquid chromatography-mass spectrometry) for determining content of phenyllactyl glucose in culture medium
The BY4742 original yeast strain and the two engineered yeast strains are respectively inoculated into YPD liquid culture medium and cultured overnight with shaking at 30 ℃. Transferred to Erlenmeyer flasks containing 50ml of SC-U liquid medium (containing 1% raffinose and 2% lactose) and 3 biological replicates per group, and shake-cultured at 30 ℃ to OD 600 Adding phenyl lactic acid and UDP-glucose with the final concentration of 1mM to 0.6, continuously culturing for 72 hours, filtering 1ml of the culture medium after fermentation by a 0.22 mu m filter head, diluting ten times, and analyzing the content of the phenyllactyl glucose by using an ultrahigh pressure liquid-mass spectrometer. Relative content analysis of phenyllactyl glucose was performed in a full-scan negative ion mode using a Thermo Fisher Scientific UPLC-Q active ultra-high pressure liquid-mass spectrometer with an electrospray ion source (ESI).
The content measurement result shows that no phenyllactyl glucose is detected in the culture medium after the fermentation of the BY4742 original yeast, and the relative abundance of phenyllactyl glucose in the culture medium after the fermentation of the BY4742-UGT74F2 is 78% higher than that of the BY4742-AbUGT1 (figure 6). The results show that UGT74F2 has higher catalytic efficiency and higher application value than AbUGT1 in yeast fermentation biosynthesis.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of southwest
Application of <120> Arabidopsis UGT74F2 in catalyzing phenyllactic acid to synthesize phenyllactyl glucose
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aatgggtcgc ggatccatgg agcataagag aggaca 36
<210> 2
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtggtggtgg tggtgctcga gctatttgct ctgaaccctt gat 43
<210> 3
<211> 1350
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 3
atggagcata agagaggaca tgtattagca gtgccgtacc caacgcaagg acacatcaca 60
ccattccgcc aattctgcaa acgacttcac ttcaaaggtc tcaaaaccac tctcgctctc 120
accactttcg tcttcaactc catcaatcct gacctatccg gtccaatctc catagccacc 180
atctccgatg gctatgacca tgggggtttc gagacagctg actccatcga cgactacctc 240
aaagacttta aaacttccgg ctcgaaaacc attgcagaca tcatccaaaa acaccagact 300
agtgataacc ccatcacttg tatcgtctat gatgctttcc tgccttgggc acttgacgtt 360
gctagagagt ttggtttagt tgcgactcct ttctttacgc agccttgtgc tgttaactat 420
gtttattatc tttcttacat aaacaatgga agcttgcaac ttcccattga ggaattgcct 480
tttcttgagc tccaagattt gccttctttc ttctctgttt ctggctctta tcctgcttac 540
tttgagatgg tgcttcaaca gttcataaat ttcgaaaaag ctgatttcgt tctcgttaat 600
agcttccaag agttggaact gcatgagaat gaattgtggt cgaaagcttg tcctgtgttg 660
acaattggtc caactattcc atcaatttac ttagaccaac gtatcaaatc agacaccggc 720
tatgatctta atctctttga atcgaaagat gattccttct gcattaactg gctcgacaca 780
aggccacaag ggtcggtggt gtacgtagca ttcggaagca tggctcagct gactaatgtg 840
cagatggagg agcttgcttc agcagtaagc aacttcagct tcctgtgggt ggtcagatct 900
tcagaggagg aaaaactccc atcagggttt cttgagacag tgaataaaga aaagagcttg 960
gtcttgaaat ggagtcctca gcttcaagtt ctgtcaaaca aagccatcgg ttgtttcttg 1020
actcactgtg gctggaactc aaccatggag gctttgacct tcggggttcc catggtggca 1080
atgccccaat ggactgatca accgatgaac gcaaagtaca tacaagatgt gtggaaggct 1140
ggagttcgtg tgaagacaga gaaggagagt gggattgcca agagagagga gattgagttt 1200
agcattaagg aagtgatgga aggagagagg agcaaagaga tgaagaagaa cgtgaagaaa 1260
tggagagact tggctgtcaa gtcactcaat gaaggaggtt ctacggatac taacattgat 1320
acatttgtat caagggttca gagcaaatag 1350
<210> 4
<211> 449
<212> PRT
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 4
Met Glu His Lys Arg Gly His Val Leu Ala Val Pro Tyr Pro Thr Gln
1 5 10 15
Gly His Ile Thr Pro Phe Arg Gln Phe Cys Lys Arg Leu His Phe Lys
20 25 30
Gly Leu Lys Thr Thr Leu Ala Leu Thr Thr Phe Val Phe Asn Ser Ile
35 40 45
Asn Pro Asp Leu Ser Gly Pro Ile Ser Ile Ala Thr Ile Ser Asp Gly
50 55 60
Tyr Asp His Gly Gly Phe Glu Thr Ala Asp Ser Ile Asp Asp Tyr Leu
65 70 75 80
Lys Asp Phe Lys Thr Ser Gly Ser Lys Thr Ile Ala Asp Ile Ile Gln
85 90 95
Lys His Gln Thr Ser Asp Asn Pro Ile Thr Cys Ile Val Tyr Asp Ala
100 105 110
Phe Leu Pro Trp Ala Leu Asp Val Ala Arg Glu Phe Gly Leu Val Ala
115 120 125
Thr Pro Phe Phe Thr Gln Pro Cys Ala Val Asn Tyr Val Tyr Tyr Leu
130 135 140
Ser Tyr Ile Asn Asn Gly Ser Leu Gln Leu Pro Ile Glu Glu Leu Pro
145 150 155 160
Phe Leu Glu Leu Gln Asp Leu Pro Ser Phe Phe Ser Val Ser Gly Ser
165 170 175
Tyr Pro Ala Tyr Phe Glu Met Val Leu Gln Gln Phe Ile Asn Phe Glu
180 185 190
Lys Ala Asp Phe Val Leu Val Asn Ser Phe Gln Glu Leu Glu Leu His
195 200 205
Glu Asn Glu Leu Trp Ser Lys Ala Cys Pro Val Leu Thr Ile Gly Pro
210 215 220
Thr Ile Pro Ser Ile Tyr Leu Asp Gln Arg Ile Lys Ser Asp Thr Gly
225 230 235 240
Tyr Asp Leu Asn Leu Phe Glu Ser Lys Asp Asp Ser Phe Cys Ile Asn
245 250 255
Trp Leu Asp Thr Arg Pro Gln Gly Ser Val Val Tyr Val Ala Phe Gly
260 265 270
Ser Met Ala Gln Leu Thr Asn Val Gln Met Glu Glu Leu Ala Ser Ala
275 280 285
Val Ser Asn Phe Ser Phe Leu Trp Val Val Arg Ser Ser Glu Glu Glu
290 295 300
Lys Leu Pro Ser Gly Phe Leu Glu Thr Val Asn Lys Glu Lys Ser Leu
305 310 315 320
Val Leu Lys Trp Ser Pro Gln Leu Gln Val Leu Ser Asn Lys Ala Ile
325 330 335
Gly Cys Phe Leu Thr His Cys Gly Trp Asn Ser Thr Met Glu Ala Leu
340 345 350
Thr Phe Gly Val Pro Met Val Ala Met Pro Gln Trp Thr Asp Gln Pro
355 360 365
Met Asn Ala Lys Tyr Ile Gln Asp Val Trp Lys Ala Gly Val Arg Val
370 375 380
Lys Thr Glu Lys Glu Ser Gly Ile Ala Lys Arg Glu Glu Ile Glu Phe
385 390 395 400
Ser Ile Lys Glu Val Met Glu Gly Glu Arg Ser Lys Glu Met Lys Lys
405 410 415
Asn Val Lys Lys Trp Arg Asp Leu Ala Val Lys Ser Leu Asn Glu Gly
420 425 430
Gly Ser Thr Asp Thr Asn Ile Asp Thr Phe Val Ser Arg Val Gln Ser
435 440 445
Lys
<210> 5
<211> 36
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 5
aatgggtcgc ggatccatgg gatctcaagg taccaa 36
<210> 6
<211> 35
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 6
gtggtggtgc tcgagctaat tggatagagg tgcta 35
<210> 7
<211> 40
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 7
cgggggactc tagaggatcc atggagcata agagaggaca 40
<210> 8
<211> 40
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 8
gatcggggaa attcgagctc ctatttgctc tgaacccttg 40
<210> 9
<211> 40
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 9
cgggggactc tagaggatcc atgggatctc aaggtaccaa 40
<210> 10
<211> 40
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 10
gatcggggaa attcgagctc ctaattggat agaggtgcta 40
<210> 11
<211> 19
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 11
cttcgatggt tgggaatac 19
<210> 12
<211> 19
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 12
ggacccatag gacagcaca 19
<210> 13
<211> 19
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 13
ctgtcaaaca aagccatcg 19
<210> 14
<211> 19
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 14
ccttccacac atcttgtat 19
<210> 15
<211> 40
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 15
ggagaaaaaa ccccggatcc atggagcata agagaggaca 40
<210> 16
<211> 40
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 16
tcttagctag ccgcggtacc ctatttgctc tgaacccttg 40
<210> 17
<211> 40
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 17
ggagaaaaaa ccccggatcc atgggatctc aaggtaccaa 40
<210> 18
<211> 40
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 18
tcttagctag ccgcggtacc ctaattggat agaggtgcta 40
Claims (7)
1. The application of Arabidopsis UGT74F2 in catalyzing phenyllactic acid to generate glycosyl transfer reaction to synthesize phenyllactyl glucose is characterized in that: the amino acid sequence of the Arabidopsis UGT74F2 is shown in SEQ ID NO. 3.
2. Use according to claim 1, characterized in that: the nucleotide sequence of the Arabidopsis UGT74F2 is shown in SEQ ID NO. 4.
3. The application of Arabidopsis UGT74F2 in improving the content of tropane alkaloid in Solanaceae TAs resource plants is characterized in that: the amino acid sequence of the Arabidopsis UGT74F2 is shown in SEQ ID NO. 3.
4. Use according to claim 3, characterized in that: the Solanaceae TAs resource plant is belladonna, Datura stramonium or scopoletin; the tropane alkaloid is at least one of hypusine, hyoscyamine, anisodamine and scopolamine.
5. The method for improving the content of tropine alkaloid in a solanaceae TAs resource plant is characterized by comprising the following steps of: the arabidopsis thaliana UGT74F2 gene is overexpressed in a Solanaceae TAs resource plant, and an amino acid sequence coded by the arabidopsis thaliana UGT74F2 gene is shown as SEQ ID NO. 3.
6. The method of claim 5, wherein: the method for overexpression of the Arabidopsis thaliana UGT74F2 gene is as follows: cloning an arabidopsis thaliana UGT74F2 gene, then constructing a plant expression vector, obtaining a recombinant plant expression vector containing the arabidopsis thaliana UGT74F2 gene, transforming agrobacterium tumefaciens by using the obtained recombinant plant expression vector to obtain engineering bacteria, finally transforming solanaceae TAs resource plants by using the engineering bacteria, and screening a transgenic regenerant to obtain the solanaceae TAs resource plants with improved tropane alkaloid content.
7. The method of claim 6, wherein: the plant expression vector is formed by connecting nucleotides shown in SEQ ID NO.4 between a CaMV 35S promoter driver and an Nos terminator of a pBI121 vector.
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