Phenyllactic acid UDP-glycosyltransferase mutant and application thereof

Abstract

The invention discloses a phenyllactic acid UDP-glycosyl transferase mutant and application thereof, and belongs to the technical field of biological medicine, the phenyllactic acid UDP-glycosyl transferase mutant PLA-UGT-M5 with improved catalytic efficiency is obtained through a directed evolution method, the mutant simultaneously contains five amino acid substitutions F19S, G21E, N128S, I209L and I292V, metabolites are detected in yeast cells, and the activity of the metabolites is improved. The catalytic efficiency is obviously higher than that of a wild type. Further functional verification shows that the PLA-UGT-M5 can remarkably promote biosynthesis of the scopolamine and the scopolamine in a yeast cell factory and a belladonna hairy root system, has a good metabolic engineering application prospect, provides a key tool for efficient biosynthesis of the scopolamine and the scopolamine, and has important industrial application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a phenyllactic acid UDP-glycosyltransferase mutant, and application thereof in promoting the biosynthesis of hyoscyamine, scopolamine and hyoscine. BACKGROUND

[0002] Tropane alkaloids (TAs) are a class of natural alkaloids containing tropane ring skeleton in structure, among which the most representative include hyoscyamine and scopolamine, which are mainly synthesized by Solanaceae plants. This kind of compounds belongs to muscarine type acetylcholine receptor antagonists, and is widely used in clinical anesthesia analgesia, antitussive asthma, treatment of motion sickness and organophosphorus poisoning, etc., and has extremely high medicinal value. Although hyoscyamine and scopolamine can be obtained by chemical synthesis at present, due to the complex synthesis route, numerous reaction steps and low yield, the industrial production is still mainly based on natural plant extraction. The main sources include Atropa belladonna, Datura stramonium, Hyoscyamus niger and Anisodus acutangulus, etc. However, the content of hyoscyamine and scopolamine in these plants is low, for example, the content of hyoscyamine in the leaf dry weight of A. belladonna widely cultivated in China is about 0.25%, and the content of scopolamine is even lower, only 0.03%. Since hyoscyamine and scopolamine are listed as the core drugs in the WHO Essential Medicine List, the market demand continues to grow, and therefore the limited natural production has become a key bottleneck restricting its wide application and commercial production.

[0003] In order to solve the problems of resource shortage and high cost, domestic and foreign researchers have explored strategies such as plant metabolic engineering and synthetic biology to improve the synthesis efficiency of hyoscyamine and scopolamine in biological systems. The key to this process lies in the analysis of the biosynthetic pathway and the mining and optimization of the rate-limiting enzyme. At present, the complete biosynthetic pathway of hyoscyamine and scopolamine has been elucidated, and the enzyme phenyllactate UDP-glycosyltransferase (PLA-UGT) catalyzing the combination of phenyllactic acid and UDP-glucose (UDPG) to generate phenyllactyl glucose ester is confirmed as one of the rate-limiting enzymes in the synthesis pathway. Since PLA-UGT has low natural catalytic efficiency, it is considered to be an important factor limiting the production of hyoscyamine and scopolamine. Therefore, obtaining PLA-UGT variants with higher catalytic efficiency is of great significance to improve the synthesis level of hyoscyamine and scopolamine in plant and microbial cell factories.

[0004] Directed evolution of enzymes is a strategy to mimic natural evolution in the laboratory, combining random mutation with high-throughput screening, which can achieve optimization of enzyme activity, substrate selectivity, stability and other properties, and has been widely used in performance improvement of industrial and medical enzymes. For example, Yu et al. significantly improved the enantiomeric selectivity of P450-BM3 for styrene epoxidation through iterative saturation mutagenesis; Ouyang et al. used DNA shuffling technology to modify Cota laccase, improving its catalytic efficiency and thermal stability; TmTrpB (marine thermophiles tryptophan synthase beta subunit) was significantly enhanced in activity after more than 100 rounds of evolution; Keasling team achieved significant enzyme activity improvement by performing saturation mutation on 19 sites in the active center of gamma-rulene synthase, and only screened less than 2500 mutants. Currently, there is no related research on the mutation of PLA-UGT to improve enzyme activity. SUMMARY

[0005] Therefore, one of the purposes of the present application is to provide a phenyllactic acid UDP-glycosyltransferase mutant; the second purpose of the present application is to provide a nucleic acid encoding the phenyllactic acid UDP-glycosyltransferase mutant; the third purpose of the present application is to provide a biological material containing the nucleic acid; the fourth purpose of the present application is to provide an application of the nucleic acid or the biological material in promoting the biosynthesis of petrinine, scopolamine or hyoscyamine; and the fifth purpose of the present application is to provide a method for preparing a transgenic belladonna with high yield of petrinine, scopolamine or hyoscyamine.

[0006] To achieve the above-mentioned purposes, the present application provides the following technical solutions: 1. A phenyllactic acid UDP-glycosyltransferase mutant, wherein the phenyllactic acid UDP-glycosyltransferase mutant is obtained by any one of the following mutations of wild-type phenyllactic acid UDP-glycosyltransferase: F19S, F19S / N128S, F19S / G21E / N128S, F19S / G21E / N128S / I209L, F19S / G21E / N128S / I209L / I292V, and the amino acid sequence of the wild-type phenyllactic acid UDP-glycosyltransferase is shown in SEQ ID NO. 1.

[0007] In some embodiments of the present application, the nucleotide sequence encoding the wild-type phenyllactic acid UDP-glycosyltransferase is shown in SEQ ID NO. 2.

[0008] 2. A nucleic acid encoding the phenyllactic acid UDP-glycosyltransferase mutant.

[0009] 3. A biological material containing the nucleic acid, wherein the biological material is a vector or a host cell.

[0010] 4. Use of the nucleic acid or the biological material in promoting biosynthesis of hyoscyamine, scopolamine or hyoscine.

[0011] 5. A method for preparing a transgenic belladonna plant with high yield of hyoscyamine, scopolamine or hyoscine, comprising the following steps: constructing a nucleic acid sequence encoding a phenyllactic acid-UDP-glucose glycosyltransferase mutant of a mutation type of F19S / G21E / N128S / I209L / I292V on a plant expression vector, transforming a belladonna plant under mediation of Agrobacterium, and screening to obtain a belladonna plant or a hairy root with high yield of hyoscyamine, scopolamine or hyoscine.

[0012] In some embodiments of the present application, the nucleic acid sequence encoding the phenyllactic acid-UDP-glucose glycosyltransferase mutant of the mutation type of F19S / G21E / N128S / I209L / I292V is shown in SEQ ID NO. 25.

[0013] In some embodiments of the present application, the plant expression vector is pBI121.

[0014] The present application has the beneficial effect that, in the present application, in view of the bottleneck problem of low catalytic efficiency of PLA-UGT, the inventors take the substrate binding pocket of the enzyme as the core, transform the key amino acid residues through single-point saturation mutation, and screen by using a yeast eukaryotic expression system, and finally obtain a mutant PLA-UGT-M5 with enhanced activity, which contains five amino acid substitutions F19S, G21E, N128S, I209L, I292V. After transforming the yeast cells, the fermentation product is detected, which shows a significantly higher catalytic efficiency than the wild type. Further functional verification shows that PLA-UGT-M5 can significantly promote the biosynthesis of scopolamine and hyoscine in yeast cell and belladonna hairy root systems, and has good metabolic engineering application prospect.

[0015] In summary, the present application constructs and screens a PLA-UGT mutant with improved catalytic efficiency by a directed evolution method, which provides a key tool for efficient biosynthesis of scopolamine and hyoscine, and has important industrial application potential. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application provides the following drawings for illustration: Figure 1 The contents of hyoscyamine (A) and scopolamine (B) in the yeast transformed with wild-type PLA-UGT and mutant PLA-UGT_M5; Figure 2 The expression level of PLA-UGT gene in the transgenic hairy root; Figure 3To determine the contents of anisodine (A), hyoscyamine (B) and scopolamine (C) in transgenic hairy roots. DETAILED DESCRIPTION

[0017] The present application is further described in the following drawings and examples, which provide those skilled in the art with better understanding of the present application and enable them to carry out the present application. The examples are not intended to limit the present application. Unless otherwise specified, the experimental procedures in the following examples were carried out according to the conventional conditions, for example, the conditions described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturers.

[0018] In the present application, the amino acid and nucleic acid sequences are defined as follows: 1. Nomenclature of amino acid and DNA nucleic acid sequences The amino acid residues are represented using the IUPAC-recognized three-letter or one-letter code; the nucleic acid base sequences are labeled according to the IUPAC standard symbol system.

[0019] 2. Identification of PLA-UGT high-activity mutants In the present application, the mutation sites of PLA-UGT mutants are represented in the form of “original amino acid position replaced by mutant amino acid”. The positions of the mutation sites are numbered according to the amino acid sequence of the wild-type PLA-UGT mature peptide (SEQ ID NO. 1). For example, Phe19 indicates that the 19th amino acid of the PLA-UGT mature peptide is phenylalanine (Phe), and Phe19Ser indicates that the position is mutated from Phe to Ser. The single-letter abbreviation of the amino acid can also be used for representation, such as F19S. If there are multiple mutation sites, the mutations are connected using “ / ”, and the amino acids are listed in the order of their positions in the sequence, for example, F19S / G21E / N128S / I209L / I292V indicates that the 19th, 21st, 128th, 209th and 292nd amino acids of PLA-UGT are replaced by S, E, S, L and V, respectively, from the wild-type F, G, N, I and I.

[0020] Example 1. Obtaining key amino acid residues based on PLA-UGT protein modeling According to the PLA-UGT protein sequence (SEQ ID NO. 1), a protein model with structural similarity or high sequence homology was obtained as a template in the PDB library (https: / / www.rcsb.org / ), and a three-dimensional model of the PLA-UGT protein was constructed by homology modeling through AlphaFold3 tool; using the AutoDock program, the three-dimensional model of the PLA-UGT protein was subjected to molecular docking with the substrates phenyllactic acid and UDP-glucose, and the docking results were analyzed by visual analysis using the PyMOL program, and the amino acid residues within 5 Å of the substrate phenyllactic acid molecule were selected as the target for directed evolution, a total of ten amino acid sites were selected, including F19, G21, N128, F130, Q151, F195, L205, I209, I292, and W389.

[0021] Wild-type PLA-UGT protein sequence MGSQGTNIDSIIHVFLISFPGQGHVNPLLRLGKRLASKGVLVSFCAPECVGKDMRAANNNIISDEPTPYGDGFIRFEFFDGWEYTQPKENRQLEIELANLEVVGRAVLPAMLKENEAKGRPVSCLINNPFIPWVCDVADSLGIPCAVLWVQSCASFSAYYHYHFNLAPFPNESNPNIDVHLPNMPILKWDELPSFLLPSNPYPALANAILRQFNYLSKPIRIFIESFDELEKDIVDYMSDFLPIKTVGPLLVEDPKIEQVVRADLVKADSSITQWLNSKPPSSVVYISFGSIVVPSQEQVDEIAYGILNSGLNFLWIMKPPRKNSSFPTVVLPQGYLDKIGDKGKVVEWCLQEQVLAHPSLACFVTHCGWNSSMEVIANGVPIVAFPQWGDQVTDAKYLVDEFKIGVRLSRGVTENRVIPRDEVERSLHDVTSGPKVAEMKENALKWKMKATEAVAEGGSSDLNLKSFVDELRTLQNSNKNLAKLAPLSN (SEQ ID NO. 1) Wild-type PLA-UGT DNA sequence (after nucleic acid codon optimization) Example 2. Construction of PLA-UGT key catalytic residue single point saturation mutant library The codon-optimized wild-type PLA-UGT gene sequence (SEQ ID NO. 2) was used as a template, and primers p416-GPD-PLA-UGT-F and p416-GPD-PLA-UGT-R were used, as well as NNK single point saturation mutation primers designed for mutation sites F19, G21, N128, F130, Q151, F195, L205, I209, I292, and W389 (Table 1). The PLA-UGT gene was amplified by fusion PCR using a high-fidelity DNA polymerase and introducing the corresponding mutations.

[0022] The PCR product and the yeast expression vector p416-GPD were digested with BamHI and EcoRI restriction enzymes, respectively, and then purified using a DNA clean-up recovery kit. Subsequently, the mutated PLA-UGT gene was ligated to the p416-GPD vector using T4 DNA ligase, and the recombinant plasmid was transformed into competent E. coli DH5a.

[0023] The transformed bacterial solution of 1 mL was not plated but directly inoculated into a 10 mL EP tube containing 7 mL of LB medium for liquid culture. After activation culture, the plasmid was extracted, and a single point saturation mutant library containing the above-mentioned 10 key amino acid residue sites was constructed.

[0024] Table 1. NNK single point saturation mutation primers designed for mutation sites Primer name Sequence 5'→3' p416-GPD-PLA-UGT-F cgcGGATCCATGGGTAGCCAGGGTACGAA (SEQ ID NO. 3) p416-GPD-PLA-UGT-R cgcGAATTCCTAATTGCTCAGCGGTGCCA (SEQ ID NO. 4) PLA-UGT_F19-F TGATCAGCNNKCCGGGTCA (SEQ ID NO. 5) PLA-UGT_F19-R ACCCGGMNNGCTGATCAGAA (SEQ ID NO. 6) PLA-UGT_G21-F TTTCCGNNKCAGGGCCATGTT (SEQ ID NO. 7) PLA-UGT_G21-R ATGGCCCTGMNNCGGAAAGCTGA (SEQ ID NO. 8) PLA-UGT_N128-F GCCTGATTAATNNKCCGTTTATCCC (SEQ ID NO. 9) PLA-UGT_N128-R AACGGMNNATTAATCAGGCAGC (SEQ ID NO. 10) PLA-UGT_F130-F ATCCGNNKATCCCTTGGGTGT (SEQ ID NO. 11) PLA-UGT_F130-R CCAAGGGATMNNCGGATTATTAATCA (SEQ ID NO. 12) PLA-UGT_Q151-F GTGGGTGNNKTCATGTGCAAG (SEQ ID NO. 13) PLA-UGT_Q151-R CACATGAMNNCACCCACAGAAC (SEQ ID NO. 14) PLA-UGT_F195-F GAGCNNKCTGCTGCCGTCTAAT (SEQ ID NO. 15) PLA-UGT-F195-R ACGGCAGCAGMNNGCTCGGCAGTTCAT (SEQ ID NO. 16) PLA-UGT_L205-F CTGCCNNKGCCAATGCGATT (SEQ ID NO. 17) PLA-UGT_L205-R CGCATTGGCMNNGGCAGGATA (SEQ ID NO. 18) PLA-UGT_I209-F CAATGCGNNKCTGCGTCAGT (SEQ ID NO. 19) PLA-UGT_I209-R TGACGCAGMNNCGCATTGGCCA (SEQ ID NO. 20) PLA-UGT_I292-F TGGTAGCNNKGTTGTTCCGAGC (SEQ ID NO. 21) PLA-UGT_I292-R CGGAACAACMNNGCTACCAAAGGA (SEQ ID NO. 22) PLA-UGT_W389-F TTTCCGCAGNNKGGTGATCAG (SEQ ID NO. 23) PLA-UGT_W389-R ATCACCMNNCTGCGGAAATG (SEQ ID NO. 24) Example 3. Screening for high-activity PLA-UGT mutants The single point mutant plasmid library prepared in Example 2 was transformed into the Saccharomyces cerevisiae BY4742 strain, and the transformants were screened on a solid plate of uracil-deficient medium (SD-U) to obtain the transformants. The well-grown yeast single colonies were picked from the petri dish for expansion culture and enzyme activity comparison. The details are as follows: 1. A well-grown single colony was picked into a 1.5 mL EP tube containing 200 μL of SD-U liquid medium and placed in a 200 rpm, 30°C constant temperature incubator for 36-48 h of shaking culture. 2. The cultured bacterial solution was inoculated into a 10 mL EP tube containing 2 mL of SD-U liquid culture at a ratio of 1:50, and placed in a 200 rpm, 30°C constant temperature incubator for 12 h of shaking culture. 3. Add phenyllactic acid substrate to the cultured bacterial solution to a final concentration of 1 mM, and incubate at 200 rpm and 30°C in a shaker for 72 h. Then, centrifuge 1 mL of the bacterial solution at 12000 rpm for 2 min at room temperature, filter through a syringe filter, and transfer to a sample vial. Detect the content of the product phenyllactic acid glucoside using UPLC-MS. The UPLC-MS was performed using a Thermo ultra-high performance liquid chromatography-tandem electrostatic field track trap high-resolution mass spectrometer. Compound separation was performed using the Thermo Scientific™ HypersilGOLD. ™ A C18 column (2.1 mm × 50 mm, 1.9 μm) was used. The mobile phase consisted of 0.1% (w / v) formic acid water (mobile phase A) and methanol (mobile phase B). The flow rate was set to 0.3 mL / min, and the column oven temperature was set to 35 °C. Gradient elution was used: 0–1 min, 10% (volume fraction) (mobile phase B); 1–3.5 min, 10%–95% (volume fraction) (mobile phase B); 4–4.2 min, 95%–10% (volume fraction) (mobile phase B); 4.2–6 min, 10% (volume fraction) (mobile phase B). Mass spectrometry detection was performed using electrospray ionization (ESI-) mode, with a spray voltage of 2500 V, a sheath gas flow rate of 40 Arb, an auxiliary gas flow rate of 10 Arb, a backflush gas flow rate of 2 Arb, an ion transfer tube temperature of 325 °C, and a nebulizer temperature of 350 °C.

[0025] By comparing the relative content of phenyllactic acid glucoside in the fermentation broth, mutant strains with significantly enhanced PLA-UGT activity were screened. Plasmid DNA was extracted from these mutant strains and sequenced to obtain information on the PLA-UGT mutants with enhanced activity. Further iterative mutations were performed on the sites of enhanced activity to obtain the mutant with the highest activity in this invention, namely PLA-UGT-M5. Yeast fermentation results showed that its phenyllactic acid glucoside yield was 26.26 times higher than that of the wild-type protein. Specific mutation information and specific activity fold changes are shown in Table 2 below.

[0026] Table 2. Specific activity folds of different mutation types Gene name Specific activity fold Wild-type PLA-UGT 1 PLA-UGT-M1 (F19S) 1.89 PLA-UGT-M2 (F19S / N128S) 3.83 PLA-UGT-M3 (F19S / G21E / N128S) 8.11 PLA-UGT-M4 (F19S / G21E / N128S / I209L) 15.59 PLA-UGT-M5 (F19S / G21E / N128S / I209L / I292V) 26.26 DNA sequence of mutant PLA-UGT-M5 (after codon optimization) Mutant PLA-UGT-M5 protein sequence MGSQGTNIDSIIHVFLISSPEQGHVNPLLRLGKRLASKGVLVSFCAPECVGKDMRAANNNIISDEPTPYGDGFIRFEFFDGWEYTQPKENRQLEIELANLEVVGRAVLPAMLKENEAKGRPVSCLINSPFIPWVCDVADSLGIPCAVLWVQSCASFSAYYHYHFNLAPFPNESNPNIDVHLPNMPILKWDELPSFLLPSNPYPALANALLRQFNYLSKPIRIFIESFDELEKDIVDYMSDFLPIKTVGPLLVEDPKIEQVVRADLVKADSSITQWLNSKPPSSVVYISFGSVVVPSQEQVDEIAYGILNSGLNFLWIMKPPRKNSSFPTVVLPQGYLDKIGDKGKVVEWCLQEQVLAHPSLACFVTHCGWNSSMEVIANGVPIVAFPQWGDQVTDAKYLVDEFKIGVRLSRGVTENRVIPRDEVERSLHDVTSGPKVAEMKENALKWKMKATEAVAEGGSSDLNLKSFVDELRTLQNSNKNLAKLAPLSN (SEQ ID NO. 26) Example 4. PLA-UGT-M5 mutants significantly promote the accumulation of escobiline and hyoscyamine in yeast The ability of PLA-UGT and PLA-UGT-M5 to synthesize hyoscyamine and scopolamine was compared in the yeast strain CEN.PK2-1C by feeding substrate. The primers pESC-PLA-UGT-F (primer sequence: 5'-cgcGCGGCCGCATGGGTAGCCAGGGTACGAAC-3', SEQ ID NO. 27), pESC-PLA-UGT-R (primer sequence: 5'-cgcGAGCTCCTAATTGCTCAGCGGTGCCAG-3', SEQ ID NO. 28) were used to amplify the genes PLA-UGT and PLA-UGT-M5, and the restriction endonucleases NotI and SacI were used to cut the genes and the vector pESC-LEU, respectively. After agarose gel electrophoresis detection and recovery, ligation, transformation of E. coli DH5α, and activation and expansion culture of positive single colony with correct sequencing, the pESC-PLA-UGT and pESC-PLA-UGT-M5 plasmids were obtained. The Ls gene (Genbank: MN256146.1) of the Solanum dulcamara hyoscyamine synthase and the DsRED2 red fluorescent protein (Genbank: AJ851284.1) in corals were assembled into a DNA fragment using fusion PCR technology. The primers pESC-DsRed-LS-EcoRI-F (primer sequence: 5'-ttcGAATTCATGGCCTCCTCCGAGAACGT-3', SEQ ID NO. 29), DsRED2-LS-Overlap-R (primer sequence: 5'-ACAATTGTTTTCTTCATGGACCCACCTCCGCCCAGGAACAGGTGGTGGCGGCC-3', SEQ ID NO. 30), DsRED2-LS-Overlap-F (primer sequence: 5'-CCACCACCTGTTCCTGGGCGGAGGTGGGTCCATGAAGAAAACAATTGTGGTTC-3', SEQ ID NO. 31), and pESC-DsRed-LS-EcoRI-R (primer sequence: 5'-agtGCGGCCGCTTATAGAGGTTGATAATATATCCAT-3', SEQ ID NO. 32) were used to amplify the DNA fragment of the DsRED2 and LS gene fusion, which was constructed into the EcoRI and NotI enzyme cutting sites of the yeast expression vector pESC-URA to obtain the pESC-URA-DsRED2-AbLS plasmid.The constructed pESC-PLA-UGT and pESC-PLA-UGT_M5 plasmids were co-transformed with pESC-URA-DsRED2-AbLS into yeast strain CEN.PK2-1C, respectively, and screened on uracil and leucine-deficient medium (SD-U-L) solid plates to obtain transformants labeled as CEN.PK PLA-UGT and CEN.PK PLA-UGT-M5, respectively. To test the effect of PLA-UGT mutants on the synthesis of scopine in yeast, the CYP80F1 (Genbank: KF267456.1), HDH (Genbank: MT981110.1) and CPR genes (Genbank: NM_118585.4) in the scopine biosynthesis pathway were integrated into the CEN.PK2-1C strain to obtain the yeast strain CEN.PK-004, and then the pESC-PLA-UGT and pESC-PLA-UGT_M5 plasmids were co-transformed with the pESC-URA-DsRED2-AbLS plasmid into the yeast strain CEN.PK-004 to obtain transformants labeled as CEN.PK-004 PLA-UGT and CEN.PK-004 PLA-UGT-M5, respectively.

[0027] The transformed single colonies were picked into 2 mL of liquid SD-U-L medium and cultured in a shaker at 200 rpm and 30°C for 24-48 h. The above bacterial liquid was inoculated into 20 mL of liquid medium (without glucose, containing 2% galactose) in a 250 mL conical flask, and the bacterial liquid concentration was adjusted to OD600 of 0.2. After being cultured in a constant temperature shaker at 200 rpm and 30°C for 24 h, 1 mM of substrate phenyllactic acid and tropine was added, and the culture was continued in a constant temperature shaker at 200 rpm and 30°C for about 76 h. 1 mL of bacterial liquid was centrifuged at 12000 rpm and room temperature for 2 min, filtered, diluted with anhydrous methanol to 1-fold volume, and then the contents of escopine and scopine were detected using UPLC-MS mass spectrometer. The UPLC-MS was Orbitrap Exploris 120 mass spectrometer (Thermo), the chromatographic column was Hypersil GOLD™ C18 reversed-phase silica gel column (2.1 mm x 100, 1.9 μm) of Thermo company, the mobile phase A was 0.1% (W / V) formic acid aqueous solution, the mobile phase B was acetonitrile, and gradient elution was adopted, and the UPLC elution program was shown in Table 3 below.

[0028] Table 3. UPLC elution program Elution time (min) Mobile phase A (%) Mobile phase B (%) 0 99 1 2 99 1 4.5 85 15 7 75 25 8.5 50 50 9 5 95 10 2 98 11 99 1 12 99 1 The column temperature was set at 35 °C, the flow rate was 0.3 mL / min, the injection volume was 3 μL, and the mass spectrometer detector used an electrospray ion source (ESI) in positive ion mode. The spray voltage was 3000 V, the sheath gas was 40 Arb, the auxiliary gas was 10 Arb, the backflush gas was 2 Arb, the ion transfer tube temperature was 325 °C, and the nebulizer temperature was 350 °C. The contents of scopine and hyoscyamine in the yeast fermentation samples were calculated by establishing an external standard curve with the standard substances of hyoscyamine and scopine. The results were the average of three repetitions, and the error bars represented the standard deviation. Statistical analysis was performed using t-test.

[0029] The results are shown in Table 1. Figure 1 As shown in Table 1, in the CEN.PK2-1C yeast transformed with PLA-UGT or PLA- UGT-M5 and DsRED2-LS, the hyoscyamine titer in the transformed PLA-UGT was 1.29 μg / L, while the hyoscyamine titer in the transformed PLA-UGT-M5 was 8.84 μg / L. Compared with the transformed wild-type PLA-UGT, the transformed PLA-UGT-M5 significantly improved the ability to synthesize hyoscyamine, with a 5.83-fold increase in titer. In the modified CEN.PK-004 yeast strain, the hyoscyamine titer synthesized by the transformed PLA-UGT was 1.02 μg / L, and the hyoscyamine titer synthesized by the transformed PLA-UGT-M5 was 10.44 μg / L. Compared with the transformed wild-type PLA-UGT, the ability of PLA-UGT-M5 to synthesize hyoscyamine was improved by 9.19-fold. In addition, the scopine titer in the transformed PLA-UGT was 0.49 μg / L, and the scopine titer in the transformed PLA-UGT-M5 was 5.96 μg / L. Compared with the transformed wild-type PLA-UGT, the ability of the transformed PLA-UGT-M5 to synthesize scopine was improved by 11.12-fold.

[0030] Example 5. Overexpression of PLA-UGT-M5 promotes the accumulation of tropane alkaloids in Atropa belladonna (1) Construction of PLA-UGT and PLA-UGT-M5 plant overexpression vectors To evaluate whether the PLA-UGT-M5 gene can increase the content of tropane alkaloids in Atropa belladonna, the plant overexpression vectors pBI121-PLA-UGT / pBI121-PLA-UGT-M5 of PLA-UGT and PLA-UGT-M5 were constructed, and the plant basic expression vector was pBI121. The primers used for gene amplification were PLA-UGT-BamHI-F (primer sequence: 5'-cgcGGATCCATGGGTAGCCAGGGTACGAAC-3', SEQ ID NO. 33) and PLA-UGT-SacI-R (primer sequence: 5'-cgcGAGCTCCTAATTGCTCAGCGGTGCCAGT-3', SEQ ID NO. 34), and the high-fidelity enzyme was used to amplify the PLA-UGT wild-type gene and the PLA-UGT-M5 mutant gene obtained by the application. After the amplification product and the pBI121 vector were digested and connected using BamHI and SacI, the target gene replaced the GUS gene on the pBI121 vector, and after sequencing verification, the plant overexpression vectors pBI121-PLA-UGT and pBI121-PLA-UGT-M5 were obtained.

[0031] (2) Obtain Agrobacterium rhizogenes engineering bacteria for genetic transformation of Atropa belladonna The recombinant plasmids pBI121-PLA-UGT and pBI121-PLA-UGT-M5 were transformed into Agrobacterium rhizogenes C58C1 competent cells, respectively, and after single clone colonies were obtained by screening at 28°C, positive single clone colonies were screened by PCR technology, and engineering bacteria for genetic transformation of Atropa belladonna hairy roots were obtained.

[0032] (3) Obtain transgenic Atropa belladonna hairy roots A. Co-culture of Agrobacterium and explants The single clone engineering bacteria were inoculated into 15 mL YEP liquid medium containing rifampicin (Rif) and kanamycin (Kan) resistance for expansion culture until OD 600 was 0.5-0.6, the bacterial cells were collected by centrifugation and resuspended in the transformation resuspension liquid (MS liquid medium + 100 μM acetosyringone) to OD 600 was 0.5; Atropa belladonna sterile seedling leaves were cut into 1 cm 2 in size, and appropriate wounds were made on the leaves with scissors, the sterile explants were soaked in the aforementioned bacterial solution for 8 minutes, and then the explants were transferred to the co-culture solid medium (MS + 100 μM acetosyringone), and cultured at 25°C in the dark for 2 days.

[0033] (4) Screening and identification of resistant hairy roots The Solanum capsicoides explants co-cultured for 2 days were transferred to the screening medium (MS+Kan 100 mg / L+Cef 400 mg / L) and cultured at 25°C in the dark, and subcultured once a week. After 2-3 subcultures, Kan-resistant hairy roots were obtained. The well-grown hairy roots were cut and transferred to the medium (MS+Cef 200 mg / L) for culture until completely sterile, thereby obtaining Kan-resistant Solanum capsicoides hairy roots. The 35S promoter region upstream of the expression cassette of the target gene and PLA-UGT were used to design forward primers and reverse primers, respectively, for genomic PCR detection of the target gene. The hairy roots positive for genomic identification were further analyzed for the expression level of the target gene by fluorescent quantitative PCR. The wild-type Solanum capsicoides hairy roots were used as the control for the gene expression level, and the results are shown in Table 2. Figure 2 As shown in Table 2, the expression levels of the target gene in the two transgenic hairy roots were significantly improved. In the material overexpressing PLA-UGT, the expression level of the target gene was 5.56-11.68 times that of the control. In the hairy roots overexpressing PLA-UGT-M5, the expression level of the UGT1 gene was 6.91-11.68 times that of the control.

[0034] (5) Extraction and detection of tropane alkaloids in hairy roots Single hairy roots with good growth and similar development were selected from the hairy root lines with significantly improved gene expression levels, and were cultured in a MS liquid medium for 30 days. After harvesting, the hairy roots were freeze-dried to constant weight in a freeze dryer. After being ground into fine powder with a tissue grinder, 25 mg of the hairy root powder was transferred to a 1.5 mL EP tube, 1 mL of an alkaloid extraction solution composed of 20% methanol and 0.01% formic acid was added, and the mixture was continuously shaken for 3-4 h in a shaking bed. After centrifugation at 12,000 rpm for 10 min, the supernatant was filtered into a new EP tube. The filtrate was diluted 20 times with the alkaloid extraction solution and transferred to a sample vial. The content of tropane alkaloids, including hyoscyamine, scopolamine and hyoscine, in the hairy roots was detected by UPLC-MS, and the detection method was the same as that in Example 4. The alkaloid content was calculated using the external standard method, and each sample included 3 biological replicates. The error bar represents the standard deviation. Statistical analysis was performed by t-test.

[0035] The results of the alkaloid content detection are shown in Table 3. Figure 3 CGGAACAACMNNGCTACCAAAGGA (SEQ ID NO. 22) PLA-UGT_W389-F TTTCCGCAGNNKGGTGATCAG (SEQ ID NO. 23) PLA-UGT_W389-R ATCACCMNNCTGCGGAAATG (SEQ ID NO. 24) Gene name Specific activity fold Wild-type PLA-UGT PLA-UGT-M1 (F19S) PLA-UGT-M2 (F19S / N128S) PLA-UGT-M3 (F19S / G21E / N128S) PLA-UGT-M4 (F19S / G21E / N128S / I209L) PLA-UGT-M5 (F19S / G21E / N128S / I209L / I292V) Elution time (min) Mobile phase A (%) Mobile phase B (%) Figure 1 Figure 2 Figure 3 CGGAACAACAs shown, overexpression of the PLA-UGT-M5 mutant obtained in the application in hairy roots can significantly promote the accumulation of scoline, hyoscyamine and scopine in Atropa belladonna hairy roots. The content of scoline in the hairy roots overexpressing PLA-UGT-M5 is 1.74-4.38 times that of the content of scoline in the hairy roots overexpressing wild-type PLA-UGT; the content of hyoscyamine in the hairy roots overexpressing PLA-UGT-M5 is 1.50-1.75 times that of the content of hyoscyamine in the hairy roots overexpressing wild-type PLA-UGT; and the content of scopine in the hairy roots overexpressing PLA-UGT-M5 is 1.25-1.88 times that of the content of scopine in the hairy roots overexpressing wild-type PLA-UGT.

[0036] The above-described embodiments are merely preferred embodiments of the present application and are not intended to limit the protection scope of the present application. Equivalent substitutions or changes that can be made by those skilled in the art on the basis of the present application are intended to fall within the protection scope of the present application. The protection scope of the present application is defined by the claims.

Claims

1. A phenyllactic acid UDP-glycosyltransferase mutant, characterized in that, The phenyllactic acid UDP-glycosyltransferase mutant is obtained by mutating the wild-type phenyllactic acid UDP-glycosyltransferase in any one of the following ways: F19S, F19S / N128S, F19S / G21E / N128S, F19S / G21E / N128S / I209L, F19S / G21E / N128S / I209L / I292V, and the amino acid sequence of the wild-type phenyllactic acid UDP-glycosyltransferase is shown as SEQ ID NO.

1.

2. A nucleic acid encoding the phenyllactic acid UDP-glycosyltransferase mutant of claim 1.

3. Biomaterials containing the nucleic acid according to claim 2, characterized in that, The biological material is a carrier or a host cell.

4. Use of the nucleic acid of claim 2 or the biological material of claim 3 in promoting the biosynthesis of hyoscyamine, scopolamine and scopine.

5. A method of making a transgenic Duboisia plant that produces high yields of hyoscyamine, scopolamine or hoscine, the method comprising, The method comprises the following steps: constructing a nucleic acid sequence encoding a phenyllactic acid UDP-glycosyltransferase mutant of mutation type F19S / G21E / N128S / I209L / I292V onto a plant expression vector, transforming a plant of Atropa belladonna under the mediation of Agrobacterium, and screening to obtain a plant of Atropa belladonna or a hairy root with high yield of hyoscyamine, scopolamine or scopine.

6. The method of claim 5, wherein, The nucleic acid sequence encoding the phenyllactic acid UDP-glycosyltransferase mutant of mutation type F19S / G21E / N128S / I209L / I292V is shown as SEQ ID NO.

25.

7. The method of claim 5, wherein, The plant expression vector is pBI121.

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