Enzymatic preparation of scopoletin

Abstract

This invention relates to an enzymatic method for the preparation of scopolamine, specifically comprising: first, preparing a recombinant genetically engineered bacterium containing the F6'H1 gene and the feruloyl-CoA synthase gene; then, activating and fermenting the recombinant genetically engineered bacterium, followed by further fermentation and transformation using ferulic acid as a substrate to obtain scopolamine. After gene sequence optimization, this invention is the first to simultaneously construct feruloyl-CoA synthase and the F6'H1 enzyme in *Escherichia coli* and achieve simultaneous and efficient functional co-expression of both. A method for the rapid, efficient, and convenient synthesis of the high-value-added product scopolamine using inexpensive, safe, low-toxicity, and sustainable ferulic acid as a substrate through a "two-enzyme, two-step catalysis, one-pot synthesis" approach has been established. This invention achieves a molar yield of 86.5% after 12 hours of catalysis using 1 mM ferulic acid as a substrate, obtaining a maximum yield of 166.28 mg / L of scopolamine produced from ferulic acid as a substrate. The catalytic process is simple, environmentally friendly, and has broad prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an enzymatic method for the preparation of scopolamine lactone. Background Technology

[0002] Scopolamine, also known as scopolamine, is chemically named 7-hydroxy-6-methoxycoumarin. It belongs to the coumarin class of compounds and has the molecular formula C0. 10 H8O4, with a relative molecular mass of 192.17, is an important phenolic antitoxin found in plants. Scopolamine is widely distributed in various medicinal plants, weeds, and crops; currently, over 110,000 plant species have been identified as containing scopolamine. Scopolamine has been proven to possess a variety of biological activities, including clinical effects such as anticancer, anti-inflammatory, hepatoprotective, and antispasmodic properties. Studies have also shown that scopolamine can lower blood pressure, regulate blood lipids, and inhibit aortic contraction, thus combating cardiovascular diseases. It also has a good therapeutic effect on Alzheimer's disease and exhibits antiplatelet aggregation, anti-HIV, and antioxidant activities.

[0003] As the activity of scopolamine is continuously explored and market demand increases, the production of scopolamine has become a research hotspot. However, currently, scopolamine is mainly obtained through isolation and extraction from the tissues of the medicinal plant Artemisia annua. However, the content of scopolamine in plants is very low, and traditional extraction methods have many drawbacks in the current context of promoting an environmentally friendly society. Secondly, many scopolamine analogues in plants are difficult to remove in a low-cost and environmentally friendly manner. Chemical synthesis currently faces unresolved problems such as long reaction times, high energy consumption, toxic byproducts, and environmental pollution. The development of synthetic biology has provided possibilities for the synthesis of many plant secondary metabolites. It mainly utilizes the fermentation of recombinant microbial engineered strains. These microbial strains can easily be used for large-scale industrial production from the laboratory, and have advantages such as short production cycles, low cultivation costs, simple product purification, and less environmental pollution, thus possessing broader application prospects.

[0004] In recent years, there have been few reports on the use of genetic engineering techniques to modify Escherichia coli to biosynthesize scopolamine. Lin et al. [1]Tyrosine ammonia-lyase (TAL), two-component flavin-dependent monoxygenase (HpaBC), caffeoyl-CoA-3-O-methyltransferase (CcoOMT), 4-coumaroyl-CoA ligase (4CL), and feruloyl-CoA-6'-hydroxylase (F6'H) were co-expressed in *Escherichia coli*, enabling the production of scopolamine at a concentration of 31 mg / L using glucose and glycerol as carbon sources. Yang et al. [2] Fusing F6'H with the GST-tagged protein enhanced its stability and solubility, leading to the production of 80 mg / L scopolamine using ferulic acid as a substrate. This demonstrates that the microbial preparation of scopolamine is still in its early stages, with many problems remaining to be solved. Developing and optimizing a complete scopolamine synthesis system will undoubtedly have a significant positive impact on the scopolamine industry. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an enzymatic method for the synthesis of scopolamine with high efficiency. This method can produce high yields of scopolamine, and the catalytic process is simple, environmentally friendly, and has broad prospects for industrial application.

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

[0007] 1. An enzymatic method for preparing scopolamine lactone, comprising the following steps:

[0008] A. Preparation of recombinant genetically engineered bacteria containing the F6'H1 gene and the feruloyl-CoA synthase gene;

[0009] B. After activating and fermenting the recombinant genetically engineered bacteria, scopolamine was obtained by further fermentation and transformation using ferulic acid as a substrate.

[0010] Furthermore, in the enzymatic preparation method of scopolamine provided by the present invention, the sequence of the F6'H1 gene is shown in SEQ ID NO.1, and the sequence of the feruloyl-CoA synthase gene is shown in SEQ ID NO.3.

[0011] Furthermore, in the enzymatic preparation method of scopolamine lactone provided by the present invention, the expression strain of the recombinant genetically engineered bacteria prepared in step A is Escherichia coli strain BL21(DE3).

[0012] Furthermore, in the enzymatic preparation method of scopolamine lactone provided by this invention, the specific preparation method of the recombinant genetically engineered bacteria in step A is as follows:

[0013] (1) The artificially synthesized F6'H1 gene was constructed into plasmid pUC57 to obtain plasmid pUC57-F6'H1 containing the target gene; an upstream primer F6'H1-BamHI-F containing a Bam HI restriction site and a downstream primer F6'H1-EcoRI-R containing an EcoRI restriction site were designed. Using pUC57-F6'H1 as a template, the F6'H1 gene containing double restriction sites was amplified by PCR: ggatcc+F6'H1+gaattc; the vector pGEX-6p-1 was also digested with BamHI and EcoRI respectively; the above target fragment and vector were ligated with T4 ligase; after ligation, the fragments were transformed into competent host cells; positive clones containing the F6'H1 gene were selected.

[0014] (2) The artificially synthesized feruloyl-CoA synthase gene was constructed into plasmid pUC57 to obtain plasmid pUC57-FCS containing the target gene; upstream primer FCS-EcoRI-rbs-F containing EcoRI restriction site and ribosome binding site rbs and downstream primer FCS-XhoI-R containing XhoI restriction site were designed. Using pUC57-FCS as template, the FCS gene fragment containing double restriction site and ribosome binding site was amplified by PCR: ggatcc+aaggagatatacca+FCS+ctcgag; similarly, the vector pGEX-F6'H1 was digested with EcoRI and XhoI respectively; the above target fragment and vector were ligated with T4 ligase; after ligation, it was transformed into competent host cells; finally, recombinant genetically engineered bacteria containing F6'H1 gene and FCS gene were obtained.

[0015] Furthermore, in the enzymatic preparation method of scopolamine provided by the present invention, the sequence of the upstream primer F6'H1-BamHI-F is shown in SEQ ID NO.5; and the sequence of the downstream primer F6'H1-EcoRI-R is shown in SEQ ID NO.6.

[0016] Furthermore, in the enzymatic preparation method of scopolamine provided by the present invention, the sequence of the upstream primer FCS-EcoRI-rbs-F is shown in SEQ ID NO.9; and the sequence of the downstream primer FCS-XhoI-R is shown in SEQ ID NO.10.

[0017] Furthermore, in the enzymatic preparation method of scopolamine lactone provided by the present invention, the fermentation in step B specifically involves: inoculating the activated recombinant genetically engineered bacteria into LB medium at a ratio of 1:20-70, and culturing at 37°C and 250 rpm until the OD600 reaches 0.6-0.8; adding IPTG to a final concentration of 0.8-1.2 mM, and continuing to culture at 37°C and 250 rpm for 4-12 hours.

[0018] Furthermore, in the enzymatic preparation method of scopolamine provided by the present invention, the conversion process further includes adding an equal volume of ethyl acetate to the fermentation broth for extraction to obtain scopolamine.

[0019] 2. The present invention also provides an F6'H1 gene for preparing scopolamine lactone, the sequence of which is shown in SEQ ID NO.1.

[0020] 3. The present invention also provides a feruloyl-CoA synthase gene for preparing scopolamine lactone, the sequence of which is shown in SEQ ID NO.3.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. To address the issue of poor functional expression of the key enzyme F6'H1 for scopolamine synthesis derived from plants in Escherichia coli, we conducted rigorous technical screening and finally selected the F6'H1 enzyme derived from Arabidopsis thaliana after experimental verification. Furthermore, we optimized its codons based on the codon preference of E. coli, enabling it to be highly expressed in the recombinant host and exhibit good enzyme activity.

[0023] 2. A feruloyl-CoA synthase (FCS) derived from Streptomyces was used to replace the plant-derived 4-coumaryl-CoA ligase (4CL), and codon optimization was performed based on the codon preference of *E. coli*, resulting in high expression and good enzyme activity in the recombinant host. The feruloyl-CoA synthase expressed after sequence optimization in this invention exhibits higher precision and more efficient catalytic activity towards the substrate ferulic acid than the 4-coumaryl-CoA ligase. Its bacterial origin is more conducive to achieving efficient functional expression in *E. coli*, and this invention is the first to simultaneously construct feruloyl-CoA synthase and F6'H1 enzyme in *E. coli* and achieve efficient co-expression of both.

[0024] 3. We selected ferulic acid, which is widely available, inexpensive, safe, low-toxicity, and sustainable, as a substrate, and synthesized scopolamine lactone rapidly, efficiently, and conveniently through a "two-enzyme, two-step catalysis, one-pot synthesis" method. As ferulic acid is a major source of agricultural waste, the successful research of this invention is of great significance and value for the sustainable and value-added utilization of agricultural waste.

[0025] 4. We adjusted the gene expression order, placing the F6'H1 gene before the FCS gene and co-expressing it in a polycistronic form to reduce the toxic effects of intermediate product accumulation on cells and accelerate substrate transformation.

[0026] The method for producing scopolamine according to embodiments of the present invention, after catalysis with 1 mM ferulic acid as a substrate for 12 h, achieves a molar yield of 86.5%. By utilizing the recombinant Escherichia coli of the present invention for biotransformation of bio-based ferulic acid to synthesize scopolamine, the highest reported yield of scopolamine produced using ferulic acid as a substrate of 166.28 mg / L was obtained. The catalytic process is simple and environmentally friendly, and has broad prospects for industrial application.

[0027] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned by practice of the invention. Attached Figure Description

[0028] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0029] Figure 1 This invention relates to the pathway and strategy for the synthesis of scopolamine by two enzymes, FCS and F6'H1, according to embodiments of the present invention.

[0030] Figure 2 This is an electrophoresis image of the enzyme digestion identification of pGEX-F6'H1-rbs-FCS of the present invention.

[0031] Figure 3 This is an electrophoresis image of the pGEX-F6'H1-rbs-FCS bacterial culture for PCR identification in this invention.

[0032] Figure 4 This is the standard curve of scopolamine lactone in this invention.

[0033] Figure 5 The figures are chromatograms and mass spectra, where A is the chromatogram of the fermentation product of the recombinant bacteria of the present invention, 1 represents ferulic acid standard, 2 represents the product after 4 hours of fermentation, 3 represents the product after 12 hours of fermentation, and 4 represents scopolamine standard; B is the mass spectrum of the fermentation product.

[0034] Figure 6 This is a graph showing the changes in the concentration of scopolamine and the growth of recombinant bacteria over time in the production of scopolamine from ferulic acid using recombinant bacteria, as described in this invention. Detailed Implementation

[0035] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0036] The test materials used in this invention are all commercially available products and can be purchased on the market; experimental methods that do not specify specific conditions in the examples are generally performed under conventional conditions or according to the conditions recommended by the manufacturer.

[0037] Materials used: *E. coli* strain BL21(DE3) and DH5α were commercially available. *E. coli* strain BL21(DE3) was used for the expression of all genes in this invention, and DH5α was used for vector construction. The *E. coli* expression vector pGEX-6p-1 was obtained from Novagen. DNA polymerase and restriction endonucleases were also purchased from Novagen. Plasmid extraction kits, DNA purification kits, and gel extraction kits were purchased from Tiangen.

[0038] The culture medium formulations in the following examples are:

[0039] LB liquid medium: 10 g / L NaCl, 10 g / L tryptone, and 5 g / L yeast extract, with the remainder being double-distilled water. Sterilize at 121°C for 20 min under 0.1 MPa pressure. A solid medium can be prepared by adding 1.5 g / 100 mL agar powder. When used for scopolamine production, add 1× trace element solution.

[0040] 1000× Trace Element Solution: 0.03g / L H3BO3, 1g / L Thiamine, 0.94g / L ZnCl2, 0.5g / L CoCl2, 0.38g / L CuCl2, 1.6g / L MnCl2, 3.6g / L FeCl2.

[0041] The primers used in the following examples are shown in Table 1:

[0042] Table 1 Primer sequences

[0043]

[0044] Note: Underlined lines represent restriction enzyme sites, and lowercase letters represent rbs sequences.

[0045] Example 1

[0046] Construction of expression vector pGEX-F6'H1 and recombinant strain

[0047] 1. Primer sequence and gene sequence

[0048] The primers for F6'H1 gene amplification are detailed in Table 1. The final optimized nucleotide sequence of feruloyl-CoA-6'-hydroxylase (F6'H1) is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0049] Arabidopsis thaliana ferulic yl-CoA hydroxylase (F6'H1), SEQ ID NO.1:

[0050] 1 ATGGCGCCGA CCCTGTTGAC CACCCAGTTT AGCAATCCGG CGGAAGTGAC CGATTTTGTG

[0051] 61 GTGTATAAAG GCAATGGCGT GAAAGGCCTG AGCGAAAACCG GCATTAAAGC GCTGCCGGAA

[0052] 121 CAGTATATTC AGCCGCTGGA AGAACGCCTG ATTAATAAAT TTGTGAACGA AACCGATGAG

[0053] 181 GCGATTCCGG TGATTGATAT GAGCAATCCG GATGAAGATC GCGTGGCGGA AGCGGTGTGT

[0054] 241 GATGCGGCGG AAAAATGGGG CTTTTTTCAG GTGATTAATC ACGGCGTGCC GCTGGAAGTG

[0055] 301 CTGGATGATG TTAAAGCGGC GACCCATAAA TTTTTTAACC TGCCGGTGGA AGAAAAGCGC

[0056] 361 AAATTTACCA AAGAAAATAG CCTGAGCACC ACCGTGCGCT TTGGCACCAG CTTTAGCCCG

[0057] 421 TTAGCGGAAC AGGCGCTGGA ATGGAAAGAT TATCTGAGCC TGTTTTTTGT GAGCGAGGCG

[0058] 481 GAAGCGGAAC AGTTTTGGCC GGATATTTGC CGCAATGAAA CCCTGGAATA TATTAACAAA

[0059] 541 ACAAAAAGA TGGTGCGCCG CCTGCTGGAA TATCTGGGCA AAAATCTGAA TGTGAAAGAG

[0060] 601 CTGGATGAAA CCAAAGAAAG CCTGTTTATG GGCAGCATTC GCGTGAATCT GATTATTAT

[0061] 661 CCGATTTGCC CGAATCCGGA TCTGACCGTG GGCGTGGGTC GTCATAGCGA TGTTAGCAGC

[0062] 721 TTGACCATTC TGCTGCAGGA TCAGATTGGC GGCCTGCATG TGCGCAGCTT AGCGAGCGGT

[0063] 781 AATTGGGTTC ATGTGCCGCC GGTTGCGGGC AGCTTTGTTA TTAATATTGG CGATGCGATG

[0064] 841 CAGATCATGA GCAATGGCCT GTATAAAAAGC GTGGAACATC GCGTGCTGGC GAATGGCTAT

[0065] 901 AATAATCGCA TTAGCGTGCC GATTTTTGTG AATCCGAAAC CGGAAAGCGT GATTGGCCCG

[0066] 961 CTGCCAGAAG TTATTGCGAA TGGCGAAGAA CCGATTTATC GCGATGTGCT GTATAGCGAT

[0067] 1021 TATGTGAAT ATTTCTTCCG CAAGGCGCAC GATGGCAAGA AGACCGTGGATTATGCGAAA

[0068] 1081 HOME

[0069] Amino acid sequence, SEQ ID NO.2:

[0070] 1 MAPTLLTTQF SNPAEVTDFV VYKGNGVKGL SETGIKALPE QYIQPLEERL INKFVNETDE

[0071] 61 AIPVIDMSNP DEDRVAEAVC DAAEKWGFFQ VINHGVPLEV LDDVKAATHK FFNLPVEEKR

[0072] 121 KFTKENSLST TVRFGTSFSP LAEQALEWKD YLSLFFVSEA EAEQFWPDIC RNETLEYINK

[0073] 181 SKKMVRRLLE YLGKNLNVKE LDETKESLFM GSIRVNLNYY PICPNPDLTV GVGRHSDVSS

[0074] 241 LTILLQDQIG GLHVRSLASG NWVHVPPVAG SFVINIGDAM QIMSNGLYKS VEHRVLANGY

[0075] 301 NNRISVPIFV NPKPESVIGP LPEVIANGEE PIYRDVLYSD YVKYFFRKAH DGKKTVDYAK

[0076] 361 I*

[0077] 2 Experimental Methods

[0078] 2.1 Obtaining the gene encoding feruloyl-CoA hydroxylase F6'H1

[0079] Based on the codon preference of E. coli, the gene corresponding to feruloyl-CoA hydroxylase (F6'H1) (GenBank ID At3g13610) from Arabidopsis thaliana was codon optimized and then synthesized by Beijing BGI Biotechnology Co., Ltd., to obtain plasmid pUC57-F6'H1 containing the target gene.

[0080] 2.2 Amplification of the F6'H1 gene containing double restriction enzyme sites

[0081] The upstream primer F6'H1-BamHI-F (containing the Bam HI restriction site) and the downstream primer F6'H1-EcoRI-R (containing the Eco RI restriction site) were designed. Using pUC57-F6'H1 as a template, the target gene F6'H1:(BamHI) was amplified by PCR. ggatcc +F6'H1+ gaattc (EcoRI), the reaction system is shown in Table 2.

[0082] Table 2. Amplification system of F6'H1 gene containing double restriction sites.

[0083]

[0084] 2.3 Construction and transformation of pGEX-F6'H1

[0085] 2.3.1 Double enzyme digestion reaction

[0086] The F6'H1 gene fragment amplified above, containing BamHI and EcoRI restriction sites at both ends, was digested with the vector pGEX-6p-1 by double enzyme digestion with BamHI and EcoRI, respectively, and the digestion reaction was carried out at 37℃ for 1 h. The reaction system is shown in Table 3.

[0087] Table 3. Double enzyme digestion reaction system

[0088]

[0089] 2.3.2 Connect and convert

[0090] (1) The target fragment from 2.3.1, which had been double-digested with the same enzyme, was ligated to the vector at a ratio of 3:1 using T4 ligase and incubated overnight at 16°C. The reaction system is shown in Table 4 below:

[0091] Table 4. Target Fragment-Vector Connection System

[0092]

[0093] (2) After the ligation reaction was completed, the ligation solution was completely transferred to 100 μL of E.coli BL21(DE3) competent cells, and the solution was slowly shaken and placed in an ice bath for 30 min.

[0094] (3) The above-mentioned conversion mixture was heated in a water bath at 42°C for 90 seconds and then rapidly cooled in an ice bath for 2 minutes.

[0095] (4) Under aseptic conditions, add 900 μL of LB medium to the centrifuge tubes and then incubate at 37°C, 200 rpm for 1 h.

[0096] (5) Stir the above-cultured bacterial solution at 4000 rpm for 2 min, remove 800 μL of supernatant, mix the rest and add 100 μL to an LB plate containing Amp antibiotic, and spread it evenly with a spreader.

[0097] (6) After coating, seal the plate (using sealing film), let it stand at 37°C for 15 minutes (to completely dry the bacterial solution), and finally invert it for 12 hours.

[0098] 2.3.3 PCR identification of bacterial culture

[0099] (1) Pick a single colony and inoculate it into 1 mL of LB liquid medium containing 50 mg / L ampicillin. Incubate at 37°C and 200 rpm for 4-6 h. Amplify the colony using primers F6'H1-F and F6'H1-R. The reaction system is shown in Table 5 below:

[0100] Table 5. PCR reaction system for bacterial detection

[0101]

[0102] (2) After PCR amplification is terminated, a small amount of PCR product is taken and detected by 1% gel electrophoresis. The results are analyzed to preliminarily screen out positive recombinant bacteria.

[0103] 2.3.4 Extraction of recombinant plasmids

[0104] The recombinant bacteria that were correctly sequenced in the previous step were inoculated into fresh LB liquid medium (Amp) at a ratio of 1:50. + The plasmid was incubated overnight for 12 hours at 37°C and 200 rpm in a solution of 50 mg / L. Then, the recombinant plasmid was extracted according to the procedure outlined in the Tiangen plasmid mini-prep kit. The specific steps are as follows:

[0105] (1) Take 2 mL of bacterial culture, centrifuge at 12000 rpm for 30 s and remove as much of the supernatant as possible;

[0106] (2) Slowly add 250 μL of solution P1 to the centrifuge tube above, and repeatedly mix the bacterial cells with a pipette.

[0107] (3) Slowly add 250 μL of solution P2 to the above solution, and slowly invert the solution 6-8 times until the bacteria are completely broken down and lysed into a transparent solution.

[0108] (4) Take 350 μL of solution P3 and add it to the centrifuge tube above. Quickly invert the tube 6-8 times and centrifuge at 12000 rpm for 10 min.

[0109] (5) Slowly draw the supernatant obtained by centrifugation into the adsorption column AC, then centrifuge at 12000 rpm for 1 min and discard the filtrate;

[0110] (6) Pipette 500 μL of solution WB into the adsorption column, then centrifuge at 12000 rpm for 1 min and discard the filtrate;

[0111] (7) Centrifuge the adsorption column again at 12,000 rpm for 1 min to remove the residual solution to the greatest extent possible;

[0112] (8) Transfer the adsorption column to an enzyme-free centrifuge tube, slowly add 50 μL of ddH2O from the center of the column, let stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and store the product at -20℃.

[0113] 2.3.5 Enzyme digestion identification

[0114] The pGEX-F6'H1 plasmid extracted in the previous step was identified by double digestion with BamHI and EcoRI. The double digestion reaction system is shown in Table 3.

[0115] 2.3.6 Sequencing Identification

[0116] The bacterial culture that initially tested positive was sent to a sequencing company for sequencing. The sequencing results showed that the expression vector had been successfully constructed.

[0117] 3 Results

[0118] The bacterial culture PCR identification results were detected by agarose gel electrophoresis, and the amplified fragment size was approximately 1086 bp. Plasmids from strains that tested positive by bacterial culture PCR were extracted and subjected to double digestion with BamHI and EcoRI. The digestion results, detected by agarose gel electrophoresis, showed fragment sizes of approximately 5400 bp and 1086 bp, respectively. The former matched the fragment obtained from the empty pGEX-6p-1 vector, while the latter matched the size of the target fragment F6'H1, further validating the successful construction of the prokaryotic expression vector. Finally, the bacterial culture initially identified as positive was sent to a sequencing company for sequencing. The results showed that F6'H1 had been successfully ligated into the pGEX-6p-1 prokaryotic expression vector via BamHI and EcoRI restriction sites.

[0119] Example 2

[0120] Construction of expression vector pGEX-F6'H1-rbs-FCS and recombinant strain

[0121] 1 primer

[0122] The primers for FCS gene amplification are detailed in Table 1. The final optimized nucleotide sequence of feruloyl-CoA synthase FCS is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0123] Streptomyces sp.strain V-1 Functional Chemical Conjugation (FCS),SEQ ID NO.3:

[0124] 1 ATGCGCAATC AGGGCTTGGG TAGCTGGCCA GTTCGTCGCG CGCGTATGAG CCCACATGCG

[0125] 61 ACCCGGTTC GTCATGGTGG TACCGCGTTG ACCTATGCGG AATTAAGCCG TCGCGTGGCG

[0126] 121 CGTCTGGCGC ATGGTTTGCG CGAAGCGGGT GTTCGTCCAG GTGATCGTGT TGCGTATTTG

[0127] 181 GGCCCGAATC ATCCGGCGTA TCTGGAAACC CTGTTTGCGT GCGGCCAGGC GGGTGCGGTT

[0128] 241 TTTGTTCCAT TAAATTTTCG CCTGGGCGTG CCGGAACTGG ATCATGCGCT GGCGGATAGC

[0129] 301 GGTGCGAGCG TTTTAATTCA TACCCCGGAA CATGCGGAAA CCGTGGCGGC GTTAGCGGGT

[0130] 361 GATCGTTTAT TGCGCGTTCC AGCGGGTGAA TTGGAAGCGG CGGATGATGA ACCACTGGAT

[0131] 421 CTGCCGGTTG GCTTGGATGA TGTTTGCCTG CTGATGTATA CCAGCGGCAG CACCGGCCGT

[0132] 481 CCAAAAGGCG CGATGTTAAC CCATGGTAAT CTGACCTGGA ATTGCGTGAA TGTGCTGGTG

[0133] 541 GAAACCGATC TGGCGAGCGA TGAACGCGCG TTAGTTGCGG CGCCATTATT TCATGCGGCG

[0134] 601 GCGTTAGGCA TGGTGTGTCT GCCGACTTTA TTGAAAGGCG GCACCGTTAT TTTGCATAGC

[0135] 661 GCGTTTGATC CGGGCGCGGT GTTAAGCGCG GTTGAACAAG AACGCGTTAC CCTGGTGTTT

[0136] 721 GGCGTGCCGA CTATGTATCA GGCGATTGCG GCGCATCCGC GCTGGCGTAG CACTGATTTG

[0137] 781 AGCAGCTTAC GTACCCTGCT GTGCGGTGGC GCGCCAGTTC CAGCGGATTT AGCGGGTCGT

[0138] 841 TATCTGGATC GTGGCCTGGC GTTTGTGCAA GGCTATGGCA TGACCGAAGC GGCGCCAGGT

[0139] 901 GTTCTGGTTT TAGATCGCGC GCATGTTGCG GAAAAAATTG GCAGCGCGGG CGTGCCGAGC

[0140] 961 TTTTTTACCG ATGTGCGCGT GGCGGGCCCG AGTGGCGAAC CAGTTCCACC AGGTGAAAAA

[0141] 1021 GGCGAAATTG TGGTGAGCGG CCCGAATGTG ATGAAAGGCT ATTGGGGCCGCCCGGAAGCG

[0142] 1081 ACTGCGGAAG TTTTAAGAGA TGGTTGGTTT CGCAGCGGCG ATGTGGCGACTGTTGATGGT

[0143] 1141 GATGGTTATT TTCATGTGGT GGATCGCCTG AAAGATATGA TTATTAGCGGCGGCGAAAAT

[0144] 1201 ATCTATCCGG CGGAAGTGGA AAATGAGCTG TATGGCTATC CGGGCGTGGAAGCGTGTGCG

[0145] 1261 GTTATTGGCG TTCCAGATCC GCGCTGGGGT GAAGTTGGTA AAGCGGTGGTTGTTCCAGCG

[0146] 1321 GCGGGTAGTC GAATTGATGG TGCGGAATTA TTGGCGTGGT TGCGCACCCGTCTGGCGGGT

[0147] 1381 TATAAAGTGC CAAAAAGCGT GGAATTTACC GACCGCCTGC CGACCACCGGCAGCGGCAAA

[0148] 1441 ATTTTAAAAG GCGAAGTGCG CCGCCGCTTT GGCTAA

[0149] Amino acid sequence, SEQ ID NO.4:

[0150] 1 MRNQGLGSWP VRRARMSPHA TAVRHGGTAL TYAELSRRVA RLAHGLREAG VRPGDRVAYL

[0151] 61 GPNHPAYLET LFACGQAGAV FVPLNFRLGV PELDHALADS GASVLIHTPE HAETVAALAG

[0152] 121 DRLLRVPAGE LEAADDEPLD LPVGLDDVCL LMYTSGSTGR PKGAMLTHGN LTWNCVNVLV

[0153] 181 ETDLASDERA LVAAPLFHAA ALGMVCLPTL LKGGTVILHS AFDPGAVLSA VEQERVTLVF

[0154] 241 GVPTMYQAIA AHPRWRSTDL SSLRTLLCGG APVPADLAGR YLDRGLAFVQ GYGMTEAAPG

[0155] 301 VLVLDRAHVA EKIGSAGVPS FFTDVRVAGP SGEPVPPGEK GEIVVSGPNV MKGYWGRPEA

[0156] 361 TAEVLRDGWF RSGDVATVDG DGYFHVVDRL KDMIISGGEN IYPAEVENEL YGYPGVEACA

[0157] 421 VIGVPDPRWG EVGKAVVVPA AGSRIDGAEL LAWLRTRLAG YKVPKSVEFT DRLPTTGSGK

[0158] 481 ILKGEVRRRF G*

[0159] 2 Experimental Methods

[0160] 2.1 Obtaining the gene encoding feruloyl-CoA synthase FCS

[0161] Based on the codon preference of E. coli, the gene corresponding to feruloyl-CoA synthase (FCS) from Streptomyces sp. strain V-1 was codon optimized and then synthesized by Beijing BGI Biotechnology Co., Ltd., to obtain plasmid pUC57-FCS containing the target gene.

[0162] 2.2 Amplification of the FCS gene containing double restriction enzyme sites and ribosome binding sites

[0163] The upstream primer FCS-EcoRI-rbs-F (containing the EcoRI restriction site and the ribosome binding site rbs) and the downstream primer FCS-XhoI-R (containing the XhoI restriction site) were designed. Using pUC57-FCS as a template, the FCS gene fragment containing both restriction sites and the ribosome binding site was amplified by PCR: (BamHI)ggatcc+(rbs)aaggagatatacca+FCS+ctcgag(XhoI). The reaction system is shown in Table 2.

[0164] 2.3 Construction and transformation of pGEX-F6'H1-rbs-FCS

[0165] 2.3.1 Double enzyme digestion reaction

[0166] The FCS gene fragment amplified above, containing EcoRI and XhoI restriction sites at both ends, was digested with the vector pGEX-F6'H1 using EcoRI and XhoI, respectively. The reaction system is shown in Table 3.

[0167] 2.3.2 Connect and convert

[0168] Following the procedure in Example 1, the recombinant strain BL21(DE3) was obtained, carrying the recombinant plasmid pGEX-F6'H1-rbs-FCS, and named ySL88.

[0169] 2.3.3 PCR identification of bacterial culture

[0170] Operate according to the method in Example 1

[0171] 2.3.4 Extraction of recombinant plasmids

[0172] Following the procedure in Example 1, the pGEX-F6'H1-rbs-FCS plasmid was obtained.

[0173] 2.3.5 Enzyme digestion identification

[0174] The pGEX-F6'H1-rbs-FCS plasmid extracted in the previous step was identified by double digestion with BamHI and EcoRI and double digestion with EcoRI and XhoI, respectively. Figure 2 This is an electrophoresis image of the enzyme digestion identification of pGEX-F6'H1-rbs-FCS of the present invention.

[0175] 2.3.6 Sequencing Identification

[0176] The bacterial culture that initially tested positive was sent to a sequencing company for sequencing. The sequencing results showed that the expression vector had been successfully constructed.

[0177] 3 Results

[0178] Figure 3 The image shows the electrophoresis diagram of the pGEX-F6'H1-rbs-FCS bacterial culture PCR identification. The PCR identification results, detected by agarose gel electrophoresis, showed an amplified fragment size of approximately 1476 bp. Plasmids from strains identified as positive by PCR were extracted and subjected to double digestion with BamHI and EcoRI. The digestion results, detected by agarose gel electrophoresis, showed a double-digested fragment size of approximately 1086 bp, consistent with the size of the F6'H1 fragment. Double digestion with EcoRI and XhoI, detected by agarose gel electrophoresis, showed double-digested fragments of approximately 1476 bp each, consistent with the size of the FCS plus rbs fragment. Finally, the bacterial culture initially identified as positive was sent to a sequencing company for sequencing, indicating that the pGEX-F6'H1-rbs-FCS prokaryotic expression vector was successfully constructed.

[0179] Example 3

[0180] Method for preparing scopolamine by biotransformation of ferulic acid using recombinant strains

[0181] The strain used was the recombinant strain ySL88 constructed in Example 2.

[0182] 1. Establishment of the standard curve of scopolamine lactone

[0183] Accurately weigh 10 mg of scopolamine standard and dissolve it in chromatographic methanol, then dilute to 1 mL with chromatographic methanol to obtain a 1000 mg / L standard solution. This solution is then mixed with chromatographic methanol in specific proportions to prepare standard solutions with concentrations of 1, 5, 10, 50, 100, and 200 mg / L. The solutions are then analyzed by high-performance liquid chromatography (HPLC). Finally, based on the experimental results, a linear regression equation was established between the peak area and concentration of scopolamine standard, as shown below. Figure 4 As shown, this provides a theoretical basis for the quantitative analysis of scopolamine concentration in subsequent fermentation experiments.

[0184] 2. Recombinant bacteria fermentation synthesis of scopolamine lactone

[0185] 2.1 Activation of recombinant strains

[0186] Activation of recombinant bacteria involves re-inoculating cryopreserved glycerol-containing recombinant bacteria into fresh, preheated culture medium (containing the appropriate antibiotics) for expansion culture. After multiple expansion cultures and screenings, the desired recombinant bacteria with good growth, a single bacterial strain, and stable status are obtained. The activated recombinant bacteria can be identified as having undergone mutations through bacterial PCR, enzyme digestion, and sequencing. The specific activation procedures are as follows:

[0187] (1) Immediately place the glycerol bacteria stored at -80℃ into a water bath at 38-40℃ and shake it continuously until it is completely melted into liquid;

[0188] (2) Under aseptic conditions, use an inoculation loop to streak a small amount of thawed inoculum onto a plate containing ampicillin;

[0189] (3) After sealing the streaked plate, let it stand at 37°C for 15 minutes (to absorb the bacterial solution), and finally invert it in a constant temperature incubator at 37°C for 12 hours to observe the colonies.

[0190] (4) Under aseptic conditions, pick a single colony from the plate and transfer it to 1 mL of LB liquid medium (Amp). + (50 mg / L), incubated overnight at 37°C and 200 rpm.

[0191] The activated recombinant bacterial culture should be stored at 4°C in preparation for subsequent fermentation experiments. If the recombinant bacterial culture is stored for more than one week, it needs to be newly expanded, activated, and identified.

[0192] 2.2 Fermentation transformation and extraction of its products

[0193] (1) The activated recombinant bacteria were inoculated into 50 mL of fresh LB medium (Amp) at a ratio of 1:50. + In 50 mg / L solution, incubate at 37°C and 250 rpm until OD is reached. 600 Once the concentration reaches 0.6-0.8, add exogenous inducer IPTG to a final concentration of 1 mM, and continue culturing at 37℃ and 250 rpm for 4 h.

[0194] (2) Then add 1 mM ferulic acid to (1);

[0195] (3) Fermented in a constant temperature incubator at 37℃ and 250rpm for 12h;

[0196] (4) Shake the fermentation broth thoroughly. Under aseptic conditions, take 1 mL of the fermentation broth and place it in a 2 mL centrifuge tube. Add the same volume of ethyl acetate and repeat the extraction 3 times.

[0197] (5) After extraction, combine the ethyl acetate phases and place them in a fume hood to air dry naturally;

[0198] (6) Dissolve the obtained residual components in chromatographic methanol and store at 4°C before injection.

[0199] 3 Results and Analysis

[0200] 3.1 High-performance liquid chromatography (HPLC) detection

[0201] Figure 5 The chromatogram and mass spectrum are shown. Figure 5 In diagram A, the chromatograms show different substances. Curve 4 is the chromatogram of scopolamine standard, showing a distinct peak at approximately 7.9 min. Curve 1 is the chromatogram of ferulic acid standard, showing a distinct peak at approximately 9.6 min. Curves 2 and 3 are the chromatograms of recombinant bacterial fermentation products, showing a peak at 9.6 min that corresponds to the chromatogram of ferulic acid standard. Figure 1 The presence of this peak indicates that it represents ferulic acid, i.e., the unconverted substrate. Furthermore, in curves 2 and 3, there is another distinct chromatographic peak at an elution time of approximately 7.9 min, the elution time of which is consistent with that of the scopolamine standard. Figure 5 B represents the substance in curve 3 with an elution time of approximately 7.9 min, which was identified as scopolamine by mass spectrometry. The experimental results indicate that the recombinant strain ySL88 constructed in this study possesses excellent catalytic activity and can efficiently catalyze the conversion of ferulic acid to scopolamine.

[0202] 3.2 Yield and conversion rate of scopolamine lactone

[0203] 3.2.1 Yield of Scopolamine

[0204] Analysis of the chromatograms and mass spectra of the fermentation products confirmed the presence of scopolamine in the fermentation broth of the recombinant bacteria. The content of scopolamine in the fermentation products can be determined by analyzing the peak areas of the chromatographic peaks and using the established scopolamine standard curve. Figure 4 As shown, a working standard curve was obtained by plotting the peak area against the mass concentration. The linear regression equation for the scopolamine standard was Y = 24.863X + 1.591, with a correlation coefficient R0. 2 =0.998. Where Y is the concentration of scopolamine standard, in mg / L; X is the peak area, in mAU*s. The yield of scopolamine over time was calculated as follows: Figure 6 As shown, the highest yield was 166.28 mg / L, which is a molar concentration of 0.865 mM.

[0205] 3.2.2 Molar conversion rate of scopolamine lactone

[0206] When using recombinant microorganisms to bioconvert substrates into target products, the utilization rate of the substrate needs to be calculated to measure the bioconversion capacity of the recombinant bacteria. The molar conversion rate of scopolamine refers to the number of moles of scopolamine produced per mole of ferulic acid after fermentation by recombinant bacteria during the biosynthesis of scopolamine. In this study, the substrate concentration used was 1 mM, while the molar concentration of scopolamine in the fermentation broth was 0.865 mM. Based on this definition, the molar conversion rate of scopolamine in this study can be calculated to be 86.5%.

[0207] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. An enzymatic method for preparing scopolamine lactone, characterized in that, Includes the following steps: A. Prepare recombinant genetically engineered bacteria containing the F6'H1 gene and the feruloyl-CoA synthase gene; B. After activating and fermenting the recombinant genetically engineered bacteria, scopolamine was obtained by further fermentation and transformation using ferulic acid as a substrate. The sequence of the F6'H1 gene is shown in SEQ ID NO.1, and the sequence of the feruloyl-CoA synthase gene is shown in SEQ ID NO.3; The expression strain for the recombinant genetically engineered bacteria prepared in step A is Escherichia coli strain BL21(DE3).

2. The enzymatic preparation method of scopolamine lactone according to claim 1, characterized in that, The specific preparation method for the recombinant genetically engineered bacteria in step A is as follows: 1) The artificially synthesized F6'H1 gene was constructed into plasmid pUC57 to obtain plasmid pUC57-F6'H1 containing the target gene; an upstream primer F6'H1-BamHI-F containing a BamHI restriction site and a downstream primer F6'H1-EcoRI-R containing an EcoRI restriction site were designed. Using pUC57-F6'H1 as a template, the F6'H1 gene containing double restriction sites was amplified by PCR: ggatcc+F6'H1+gaattc; the vector pGEX-6p-1 was also digested with BamHI and EcoRI respectively; the above target fragment and vector were ligated using T4 ligase; after ligation, the fragments were transformed into competent host cells; positive clones containing the F6'H1 gene were selected. 2) The artificially synthesized feruloyl-CoA synthase gene was constructed into plasmid pUC57 to obtain plasmid pUC57-FCS containing the target gene; upstream primer FCS-EcoRI-rbs-F containing EcoRI restriction site and ribosome binding site rbs and downstream primer FCS-XhoI-R containing XhoI restriction site were designed. Using pUC57-FCS as template, the FCS gene fragment containing double restriction site and ribosome binding site was amplified by PCR: ggatcc+aaggagatatacca+FCS+ctcgag; Similarly, the vector pGEX-F6'H1 was digested with BamHI and EcoRI respectively; the target fragment and the vector were ligated using T4 ligase; after ligation, it was transformed into competent host cells; finally, recombinant genetically engineered bacteria containing the F6'H1 gene and the feruloyl-CoA synthase gene were obtained.

3. The enzymatic preparation method of scopolamine lactone according to claim 2, characterized in that, The sequence of the upstream primer F6'H1-BamHI-F is shown in SEQ ID NO.5; the sequence of the downstream primer F6'H1-EcoRI-R is shown in SEQ ID NO.

6.

4. The enzymatic preparation method of scopolamine lactone according to claim 2, characterized in that, The sequence of the upstream primer FCS-EcoRI-rbs-F is shown in SEQ ID NO.9; the sequence of the downstream primer FCS-XhoI-R is shown in SEQ ID NO.

10.

5. The enzymatic preparation method of scopolamine lactone according to claim 1, characterized in that, The fermentation in step B is as follows: the activated recombinant genetically engineered bacteria are inoculated into LB medium at a ratio of 1:20-70, and cultured at 37℃ and 250 rpm until the OD600 reaches 0.6-0.

8. IPTG is then added to a final concentration of 0.8-1.2 mM, and the culture is continued at 37℃ and 250 rpm for 4-12 hours.

6. The enzymatic preparation method of scopolamine lactone according to claim 1, characterized in that, The conversion process also includes extracting scopolamine by adding an equal volume of ethyl acetate to the fermentation broth.

7. An F6'H1 gene for the preparation of scopolamine lactone, characterized in that, The sequence of the F6'H1 gene is shown in SEQ ID NO.

1.

8. A gene for synthetase of ferulic acid-CoA for the preparation of scopolamine, characterized in that, The sequence of the feruloyl-CoA synthase gene is shown in SEQ ID NO.3.

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