A phenyllactic acid udp-glycosyltransferase mutant and application thereof

Abstract

The application discloses a phenyllactic acid UDP-glycosyltransferase mutant and application thereof, and belongs to the technical field of biological medicine. A phenyllactic acid UDP-glycosyltransferase 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. Metabolic products are detected in yeast cells, and the mutant shows a catalytic efficiency significantly higher than that of a wild type. Further functional verification shows that the PLA-UGT-M5 can significantly promote the biosynthesis of scopolamine and hyoscyamine in a yeast cell factory and a belladonna hairy root system, has a good metabolic engineering application prospect, provides a key tool for the efficient biosynthesis of scopolamine and hyoscyamine, and has important industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a phenyllactic acid UDP-glycosyltransferase mutant, and its application in promoting the biosynthesis of hyoscyamine, scopolamine, and spiroquialine. Background Technology

[0002] Tropine alkaloids (TAs) are a class of natural alkaloids containing a tropane ring skeleton in their structure. The most representative examples include hyoscyamine and scopolamine, primarily synthesized from plants in the Solanaceae family. These compounds belong to the muscarinic acetylcholine receptor antagonist class and are widely used in clinical anesthesia, analgesia, cough suppression, asthma relief, motion sickness treatment, and organophosphate poisoning, possessing extremely high medicinal value. Although hyoscyamine and scopolamine can now be obtained through chemical synthesis, industrial production still relies mainly on extraction from natural plants due to the complex synthetic routes, numerous reaction steps, and low yields. Their main sources include a few Solanaceae plants such as Atropa belladonna, Datura stramonium, Hyoscyamus niger, and Anisodus acutangulus. However, these plants contain low levels of hyoscyamine and scopolamine. For example, belladonna, widely cultivated in my country, contains approximately 0.25% hyoscyamine in its leaf dry weight, and even less, only 0.03%. Because hyoscyamine and scopolamine are listed as core medicines in the World Health Organization's (WHO) Essential Medicines List, their market demand continues to grow. Therefore, the limited natural yield is currently a key bottleneck restricting their widespread application and commercial production.

[0003] To address the challenges of resource scarcity and high costs, researchers both domestically and internationally have explored strategies such as plant metabolic engineering and synthetic biology to enhance the synthetic efficiency of hyoscyamine and scopolamine in biological systems. The key to this process lies in elucidating their biosynthetic pathways and identifying and optimizing their rate-limiting enzymes. Currently, the complete biosynthetic pathways of hyoscyamine and scopolamine have been elucidated, with phenyllactate UDP-glycosyltransferase (PLA-UGT), the enzyme that catalyzes the combination of phenyllactic acid and UDP-glucose (UDPG) to produce phenyllactic acid glycosyl ester, identified as one of the rate-limiting enzymes in this pathway. The low natural catalytic efficiency of PLA-UGT is considered a significant factor limiting the yield of hyoscyamine and scopolamine. Therefore, obtaining PLA-UGT variants with higher catalytic efficiency is crucial for improving the synthetic levels of hyoscyamine and scopolamine in plants and microbial cell factories.

[0004] Directed evolution of enzymes is a strategy that simulates natural evolution in the laboratory. Combining random mutation with high-throughput screening, it can optimize enzyme properties such as activity, substrate selectivity, and stability, and has been widely applied to improve the performance of industrial and pharmaceutical enzymes. For example, Yu et al. significantly improved the enantioselectivity of P450-BM3 for styrene epoxidation through iterative saturation mutation; Ouyang et al. modified Cota laccase using DNA shuffling technology to improve its catalytic efficiency and thermal stability; TmTrpB (the β subunit of tryptophan synthase from Thermophyton floccosum) showed significantly enhanced activity after more than 100 rounds of evolution; and Keasling's team achieved a significant increase in enzyme activity by saturating mutations at 19 sites in the active site of γ-thiazolinone synthase, screening fewer than 2500 mutants. Currently, no research has been found on the enhancement of enzyme activity through PLA-UGT mutation. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide a phenyllactic acid UDP-glycosyltransferase mutant; a second objective of the present invention is to provide a nucleic acid encoding the phenyllactic acid UDP-glycosyltransferase mutant; a third objective of the present invention is to provide biological materials containing the nucleic acid; a fourth objective of the present invention is to provide the application of the nucleic acid or the biological material in promoting the biosynthesis of hyoscyamine, scopolamine, or spiroscopolamine; and a fifth objective of the present invention is to provide a method for preparing transgenic belladonna with high yields of hyoscyamine, scopolamine, or spiroscopolamine.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. A phenyllactic acid UDP-glycosyltransferase mutant, wherein the phenyllactic acid UDP-glycosyltransferase mutant is obtained by any one of the following mutations in 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.

[0008] In some embodiments of the present invention, the nucleotide sequence encoding wild-type phenyllactic acid UDP-glycosyltransferase is shown in SEQ ID NO.2.

[0009] 2. The nucleic acid encoding the phenyllactic acid UDP-glycosyltransferase mutant.

[0010] 3. A biological material containing the nucleic acid, wherein the biological material is a carrier or a host cell.

[0011] 4. The application of the nucleic acid or the biological material in promoting the biosynthesis of scopolamine, hyoscyamine, or spiroquialine.

[0012] 5. A method for preparing transgenic belladonna with high yields of hyoscyamine, scopolamine, or spiroscopolamine, the method comprising the following steps: constructing the nucleic acid sequence encoding a phenyllactic acid-UDP glucosyltransferase mutant with mutation type F19S / G21E / N128S / I209L / I292V into a plant expression vector, transforming belladonna plants under Agrobacterium-mediated transformation, and screening to obtain belladonna plants or hairy roots with high yields of hyoscyamine, scopolamine, or spiroscopolamine.

[0013] In some embodiments of the present invention, the nucleic acid sequence of the phenyllactic acid UDP-glycosyltransferase mutant encoding mutation type F19S / G21E / N128S / I209L / I292V is shown in SEQ ID NO.25.

[0014] In some embodiments of the present invention, the plant expression vector is pBI121.

[0015] The beneficial effects of this invention are as follows: Addressing the bottleneck problem of low catalytic efficiency in PLA-UGT, the inventors focused on the enzyme's substrate-binding pocket, modifying key amino acid residues through single-point saturation mutagenesis, and screening using a yeast eukaryotic expression system. This resulted in the acquisition of a mutant with enhanced activity, PLA-UGT-M5. This mutant contains five amino acid substitutions: F19S, G21E, N128S, I209L, and I292V. After transformation into yeast cells, the fermentation products showed significantly higher catalytic efficiency than the wild type. Further functional verification demonstrated that PLA-UGT-M5 significantly promoted the biosynthesis of hyoscyamine and scopolamine in both yeast cells and belladonna hairy root systems, indicating promising applications in metabolic engineering.

[0016] In summary, this invention has constructed and screened PLA-UGT mutants with improved catalytic efficiency through directed evolution, providing a key tool for the efficient biosynthesis of hyoscyamine and scopolamine, and has significant potential for industrial application. Attached Figure Description

[0017] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0018] Figure 1 The content of spirochetine (A) and hyoscyamine (B) in yeast transformed from wild-type PLA-UGT and mutant PLA-UGT_M5;

[0019] Figure 2 The expression level of the PLA-UGT gene in transgenic hairy roots;

[0020] Figure 3 The content of hyoscyamine (A), scopolamine (B), and scopolamine (C) in transgenic hairy roots. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments are not intended to limit the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

[0022] In this invention, amino acid and nucleic acid sequences are defined as follows:

[0023] 1. Nomenclature of amino acids and DNA nucleic acid sequences

[0024] Amino acid residues are represented using IUPAC-recognized three-letter or single-letter codes; nucleic acid base sequences are labeled according to the IUPAC standard symbol system.

[0025] 2. Identification of PLA-UGT high-activity mutants

[0026] In this invention, the mutation sites of the PLA-UGT mutant are represented in the form of "original amino acid positions replaced by mutant amino acids". 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. Single-letter abbreviations of amino acids can also be used, such as F19S. If multiple mutation sites exist, each mutation is connected by " / " and listed in order of amino acid position 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, representing the replacement of F, G, N, I, and I of the wild-type.

[0027] Example 1. Obtaining key amino acid residues based on PLA-UGT protein modeling

[0028] Based on the PLA-UGT protein sequence (SEQ ID NO.1), structurally similar or highly homologous protein models were obtained from the PDB library (https: / / www.rcsb.org / ) as templates. A three-dimensional model of the PLA-UGT protein was constructed using the AlphaFold3 tool for homology modeling. The PLA-UGT protein three-dimensional model was molecularly docked with the substrates phenyllactic acid and UDP-glucose using the AutoDock program. The docking results were visualized and analyzed using the PyMOL program. Amino acid residues within 5 Å of the substrate phenyllactic acid molecule were selected as targets for directed evolution. A total of ten amino acid sites were selected, including F19, G21, N128, F130, Q151, F195, L205, I209, I292, and W389.

[0029] Wild-type PLA-UGT protein sequence

[0030] MGSQGTNIDSIIHVFLISFPGQGHVNPLLRLGKRLASKGVLVSFCAPECVGKDMRAANNNIISDEPTPYGDGFIRFEFFDGWEYTQPKENRQLEIELANLEVVGRAVLPAMLKENEAKGRPVS CLINNPFIPWVCDVADSSLGIPCAVLWVQSCASFSAYYHYHFNLAPFPNESNPNIDVHLPNMPILKWDELPSFLLPSNPYPALANAILRQFNYLSKPIRIFIESFDELEKDIVDYMSDFLPIKTV GPLLVEDPKIEQVVRADLVKADSSITQWLNSKPPSSVVYISFGSIVVPSQEQVDEIAYGILNSGLNFLWIMKPPRKNSSFPTVVLPQGYLDKIGDKGKVVEWCLQEQVLAHPSLACFVTHCGW NSSMEVIANGVPIVAFPQWGDQVTDAKYLVDEFKIGVRLSRGVTENRVIPRDEVERSLHDVTSGPKVAEMKENALKWKMKATEAVAEGGSSDLNLKSFVDELRTLQNSNKNLAKLAPLSN (SEQ ID NO.1)

[0031] Wild-type PLA-UGT DNA sequence (after codon optimization)

[0032]

[0033] Example 2. Construction of a PLA-UGT key catalytic residue single-point saturation mutant library

[0034] Using the codon-optimized wild-type PLA-UGT gene sequence (SEQ ID NO.2) as a template, primers p416-GPD-PLA-UGT-F and p416-GPD-PLA-UGT-R, 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 high-fidelity DNA polymerase and the corresponding mutations were introduced.

[0035] The PCR product and the yeast expression vector p416-GPD were digested with BamHI and EcoRI restriction endonucleases, respectively, and then purified using a DNA cleaning and recovery kit. Subsequently, the mutant PLA-UGT gene was ligated into the p416-GPD vector using T4 DNA ligase, and the recombinant plasmid was transformed into competent Escherichia coli DH5α.

[0036] 1 mL of the transformed bacterial culture was not plated, but directly inoculated into a 10 mL EP tube containing 7 mL of LB medium for liquid culture. After activation culture, plasmids were extracted to construct a single-point saturated mutant library containing the aforementioned 10 key amino acid residue sites.

[0037] Table 1. NNK single-site saturation mutagenesis primers designed for mutation sites

[0038] 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)

[0039] Example 3. Screening to obtain PLA-UGT high-activity mutants

[0040] The single-point mutant plasmid library obtained in Example 2 was transformed into *Saccharomyces cerevisiae* strain BY4742, and transformants were obtained by screening on uracil-deficient medium (SD-U) solid plates. Healthy yeast single-clone colonies were picked from the culture dishes for scale-up culture and enzyme activity comparison. Details are as follows:

[0041] 1. Select a single colony with good growth into a 1.5 mL EP tube containing 200 μL of SD-U liquid medium and place it in a constant temperature shaker at 200 rpm and 30℃ for 36-48 h;

[0042] 2. Inoculate the cultured bacterial solution into a 10mL EP tube containing 2mL SD-U liquid culture at a ratio of 1:50, and place it in a constant temperature shaker at 200rpm and 30℃ for 12h.

[0043] 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.

[0044] 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.

[0045] Table 2. Specific activity folds of different mutation types

[0046] Gene name Vitality ratio 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

[0047] DNA sequence of mutant PLA-UGT-M5 (after codon optimization)

[0048]

[0049] mutant PLA-UGT-M5 protein sequence

[0050] MGSQGTNIDSIIHVFLISSPEQGHVNPLLRLGKRLASKGVLVSFCAPECVGKDMRAANNNIISDEPTPYGDGFIRFEFFDGWEYTQPKENRQLEIELANLEVVGRAVLPAMLKENEAKGRPVS CLINSPFIPWVCDVADSLGIPCAVLWVQSCASFSAYYHYHFNLAPFPNESNPNIDVHLPNMPILKWDELPSFLLPSNPYPALANALLRQFNYLSKPIRIFIESFDELEKDIVDYMSDFLPIKTV GPLLVEDPKIEQVVRADLVKADSSITQWLNSKPPSSVVYISFGSVVVPSQEQVDEIAYGILNSGLNFLWIMKPPRKNSSFPTVVLPQGYLDKIGDKGKVVEWCLQEQVLAHPSLACFVTHCGW NSSMEVIANGVPIVAFPQWGDQVTDAKYLVDEFKIGVRLSRGVTENRVIPRDEVERSLHDVTSGPKVAEMKENALKWKMKATEAVAEGGSSDLNLKSFVDELRTLQNSNKNLAKLAPLSN (SEQ ID NO.26)

[0051] Example 4. The PLA-UGT-M5 mutant in yeast significantly promoted the accumulation of spirochetine and hyoscyamine.

[0052] The ability of PLA-UGT and PLA-UGT-M5 to synthesize spirochetine and hyoscyamine was compared in the Saccharomyces cerevisiae strain CEN.PK2-1C by feeding substrate. The genes PLA-UGT and PLA-UGT-M5 were amplified using primers pESC-PLA-UGT-F (primer sequence: 5′-cgcGCGGCCGCATGGGTAGCCAGGGTACGAAC-3′, SEQ ID NO.27) and pESC-PLA-UGT-R (primer sequence: 5′-cgcGAGCTCCTAATTGCTCAGCGGTGCCAG-3′, SEQ ID NO.28). The genes and vector pESC-LEU were digested with restriction endonucleases NotI and SacI, respectively. After detection and recovery by agarose gel electrophoresis, the gene was ligated and transformed into Escherichia coli DH5α. The positive single colonies with correct sequencing were activated and expanded to obtain plasmids pESC-PLA-UGT and pESC-PLA-UGT-M5. The belladonna serotonin synthase LS gene (Genbank: MN256146.1) and the DsRED2 red fluorescent protein from corals (Genbank: AJ851284.1) were assembled into a single DNA fragment using fusion PCR. 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), and DsRED2-LS-Overlap-F (primer sequence: 5′-CCACCACCTGTTCCTGGGCGGAGGTGGGTCCATGAAGAAAACAATTGTGGTTC-3′, SEQ ID NO. 30 ... The DNA fragment fused with the DsRED2 and LS genes was amplified using pESC-DsRed-LS-EcoRI-R (primer sequence: 5′-agtGCGGCCGCTTATAGAGGTTGATAATATATCCAT-3′, SEQ ID NO.32), and then constructed between the EcoRI and NotI restriction sites of the yeast expression vector pESC-URA to obtain the pESC-URA-DsRED2-AbLS plasmid.The constructed plasmids pESC-PLA-UGT and pESC-PLA-UGT_M5 were co-transformed with pESC-URA-DsRED2-AbLS into yeast strain CEN.PK2-1C. The transformants were screened on uracil- and leucine-deficient medium (SD-UL) solid plates to obtain transformants labeled CEN.PKPLA-UGT and CEN.PKPLA-UGT-M5, respectively. To test the effect of the PLA-UGT mutant on hyoscyamine synthesis in yeast, yeast strain CEN.PK-004 was obtained by integrating the CYP80F1 (Genbank: KF267456.1), HDH (Genbank: MT981110.1), and CPR genes (Genbank: NM_118585.4) from the hyoscyamine biosynthesis pathway into strain CEN.PK2-1C. Then, the pESC-PLA-UGT and pESC-PLA-UGT-M5 plasmids were co-transformed into yeast strain CEN.PK-004 with the pESC-URA-DsRED2-AbLS plasmid, respectively. The resulting transformants were labeled CEN.PK-004 PLA-UGT and CEN.PK-004 PLA-UGT-M5, respectively.

[0053] Transformed single colonies were picked and cultured in 2 mL of SD-UL liquid medium at 200 rpm and 30 °C for 24–48 h. The bacterial culture was then inoculated into 250 mL Erlenmeyer flasks containing 20 mL of SD-UL liquid medium (glucose-free, 2% galactose), and the bacterial concentration was adjusted to an OD600 of 0.2. After culturing in a shaker at 200 rpm and 30 °C for 24 h, phenyllactic acid and tropine were added to a final concentration of 1 mM, and the culture was continued in a shaker at 200 rpm and 30 °C for approximately 76 h. 1 mL of the bacterial culture was centrifuged at 12000 rpm at room temperature for 2 min, filtered, and diluted 1 / 2 volume with anhydrous methanol. The contents of spirochetine and hyoscyamine were then detected using UPLC-MS mass spectrometry. The UPLC-MS was performed using an Orbitrap Exploris 120 mass spectrometer (Thermo). The chromatographic column was a Hypersil GOLD™ C18 reversed-phase silica column (2.1 mm × 100, 1.9 μm) from Thermo. Mobile phase A was 0.1% (W / V) formic acid aqueous solution, and mobile phase B was acetonitrile. Gradient elution was used, and the UPLC elution program is shown in Table 3 below.

[0054] Table 3. UPLC elution program

[0055] Washing 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

[0056] The column temperature was set at 35℃, the flow rate at 0.3 mL / min, and the injection volume at 3 μL. Electrospray ionization (ESI) was used as the mass spectrometer detector 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℃, and the nebulizer temperature was 350℃. External standard curves were established using spirochetine and hyoscyamine standards to calculate the contents of spirochetine and hyoscyamine in the yeast fermentation samples. The results are the average of three replicates, and the error bars represent the standard deviation. Statistical analysis was performed using the t-test.

[0057] The results are as follows Figure 1 As shown, in CEN.PK2-1C yeast transformed with PLA-UGT or PLA-UGT-M5 and DsRED2-LS, the titer of spirochetine in PLA-UGT was 1.29 μg / L, while the titer in PLA-UGT-M5 was 8.84 μg / L. Compared with wild-type PLA-UGT, PLA-UGT-M5 significantly promoted the ability of spirochetine synthesis, with a 5.83-fold increase in titer. In the modified CEN.PK-004 yeast strain, the titer of spirochetine synthesized by PLA-UGT was significantly higher. The concentration of hyoscyamine was 1.02 μg / L, and the titer of hyoscyamine synthesized by PLA-UGT-M5 was 10.44 μg / L, which is 9.19 times higher than that of wild-type PLA-UGT. In addition, the titer of hyoscyamine in yeast transformed with PLA-UGT was 0.49 μg / L, and the titer of hyoscyamine in yeast transformed with PLA-UGT-M5 was 5.96 μg / L, which is 11.12 times higher than that of wild-type PLA-UGT.

[0058] Example 5. Overexpression of PLA-UGT-M5 promotes the accumulation of tropane alkaloids in belladonna.

[0059] (1) Construction of PLA-UGT and PLA-UGT-M5 plant overexpression vectors

[0060] To assess whether the PLA-UGT-M5 gene can increase the content of tropane alkaloids in belladonna, plant overexpression vectors pBI121-PLA-UGT / pBI121-PLA-UGT-M5 were constructed, using the plant-based expression vector pBI121. 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). High-fidelity enzymes were used to amplify the wild-type PLA-UGT gene and the PLA-UGT-M5 mutant gene obtained in this invention. After the amplified products and pBI121 vector were ligated by digestion with BamHI and SacI, the GUS gene on the pBI121 vector was replaced with the target gene. After sequencing verification, the plant overexpression vectors pBI121-PLA-UGT and pBI121-PLA-UGT-M5 were obtained.

[0061] (2) Obtaining Agrobacterium rhizogenes engineered strains for the genetic transformation of belladonna.

[0062] Recombinant plasmids pBI121-PLA-UGT and pBI121-PLA-UGT-M5 were transformed into Agrobacterium rhizogenes C58C1 competent cells, respectively. After obtaining single-clone colonies by screening at 28℃, positive single-clone colonies were screened by PCR technology to obtain engineered bacteria for the genetic transformation of belladonna hairy roots.

[0063] (3) Obtaining transgenic belladonna hairy roots

[0064] A. Co-culture of Agrobacterium and explants

[0065] Single-clone engineered bacteria were inoculated into 15 mL of YEP liquid medium containing rifampicin (Rif) and kanamycin (Kan) resistance and cultured to OD. 600 The concentration was 0.5–0.6. The cells were collected by centrifugation and resuspended in transformation resuspension (MS liquid medium + 100 μM acetylsuccine) to OD0.5. 600 The value is 0.5; cut the leaves of the aseptic belladonna seedlings into 1cm pieces. 2 Size the explants and make appropriate wounds on the leaves with scissors. Soak the sterile explants in the aforementioned bacterial solution for 8 minutes, and then transfer the explants to co-culture solid medium (MS + 100 μM acetylsuccinone) and incubate in the dark at 25°C for 2 days.

[0066] (4) Screening and identification of resistant hairy roots

[0067] Belladonna explants co-cultured for 2 days were transferred to selection medium (MS + Kan 100 mg / L + Cef 400 mg / L) and cultured in the dark at 25°C. Subculture was performed weekly for 2-3 subcultures to obtain Kan-resistant hairy roots. Well-grown hairy roots were cut and transferred to medium (MS + Cef 200 mg / L) and cultured until completely sterile to obtain Kan-resistant belladonna hairy roots. For genomic PCR was performed using forward and reverse primers designed based on the 35S promoter region upstream of the target gene expression cassette and PLA-UGT, respectively. Roots with positive genomic identification were further analyzed for target gene expression levels using quantitative real-time PCR. Wild-type belladonna hairy roots were used as a control for gene expression levels. Results are as follows: Figure 2 As shown, the expression levels of the target gene were significantly increased in both types of transgenic hairy roots. In the material overexpressing PLA-UGT, the expression level of the target gene was 5.56 to 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 to 11.68 times that of the control.

[0068] (5) Extraction and detection of tropane alkaloids from hairy roots

[0069] Hairy roots with excellent growth and similar development were selected from hairy root lines with significantly increased gene expression levels and cultured in MS liquid medium with shaking for 30 days. After harvesting, the hairy roots were freeze-dried to constant weight. After grinding into a fine powder using a tissue homogenizer, 25 mg of the hairy root powder was weighed and 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 thoroughly mixed and placed in a shaker for continuous extraction for 3–4 h. The tube was then centrifuged at 12000 rpm for 10 min, and the supernatant was filtered into a new EP tube. The filtrate was diluted 20-fold with the alkaloid extraction solution and transferred to a sample vial. The content of tropane alkaloids in the hairy roots, including spirochetine, hyoscyamine, and scopolamine, was detected using UPLC-MS, following the same detection method as in Example 4. The alkaloid content was calculated using the external standard method, with each sample containing three biological replicates. The error bar represents the standard deviation. Statistical analysis was performed using the t-test.

[0070] alkaloid content test results as follows Figure 3As shown, overexpression of the PLA-UGT-M5 mutant obtained in this invention in hairy roots significantly promotes the accumulation of hyoscyamine, scopolamine, and spirochetine in belladonna hairy roots. The content of hyoscyamine in hairy roots overexpressing PLA-UGT-M5 is 1.74 to 4.38 times that of wild-type PLA-UGT; the content of scopolamine in hairy roots overexpressing PLA-UGT-M5 is 1.50 to 1.75 times that of wild-type PLA-UGT; and the content of scopolamine in hairy roots overexpressing PLA-UGT-M5 is 1.25 to 1.88 times that of wild-type PLA-UGT.

[0071] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention 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 by any of the following mutations: F19S, F19S / N128S, F19S / G21E / N128S, F19S / G21E / N128S / I209L, F19S / G21E / N128S / I209L / I292V. The amino acid sequence of the wild-type phenyllactic acid UDP-glycosyltransferase is shown in SEQ ID NO.

1.

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

3. A biomaterial containing the nucleic acid of claim 2, characterized in that, The biomaterial is a carrier or a host cell.

4. The application of the nucleic acid of claim 2 or the biomaterial of claim 3 in promoting the biosynthesis of hyoscyamine and spirochetine in yeast cells, wherein the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

25.

5. The application of the nucleic acid of claim 2 or the biomaterial of claim 3 in promoting the biosynthesis of hyoscyamine and scopolamine in belladonna, wherein the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

25.

6. A method for preparing transgenic belladonna with increased content of scopolamine, hyoscyamine, or hyoscyamine, characterized in that, The method includes the following steps: constructing the nucleic acid sequence encoding the phenyllactic acid UDP-glycosyltransferase mutant with mutation type F19S / G21E / N128S / I209L / I292V into a plant expression vector, transforming belladonna plants under Agrobacterium-mediated transformation, and screening for belladonna plants or hairy roots with increased content of hyoscyamine, scopolamine, or hyoscyamine.

7. The method according to claim 6, characterized in that, The nucleic acid sequence of the phenyllactic acid UDP-glycosyltransferase mutant encoding the mutation type F19S / G21E / N128S / I209L / I292V is shown in SEQ ID NO.

25.

8. The method according to claim 6, characterized in that, The plant expression vector is pBI121.

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