A process for the preparation of atorvastatin and its structural analogs

Abstract

The application belongs to the technical field of medicine production, and discloses a preparation method of aspitin and structural analogues, specifically discloses a kind of chalcone synthase ScCHS derived from Notholaena officinalis L. and oxidation-reduction enzyme sERED derived from anaerobic Eubacterium eligens, carbon glycosyltransferase AbCGT derived from aloe vera, and nucleic acid molecules encoding the above proteins, expression vectors of the nucleic acid molecules and engineered microorganisms containing corresponding vectors, and further contains biologically active dihydrochalcone carbon glycoside derivatives synthesized by the engineered microorganisms and a production method thereof.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical production technology, specifically involving the application of creating microbial cell factories using genetic engineering methods and biosynthesizing dihydrochalcone glycosides, represented by aspartame. Background Technology

[0002] Dihydrochalcone glycosides are an important class of plant polyphenols. For example, aspartame is a dihydrochalcone glycoside derived from the fermented leaves of Aspalathus Linearis, a plant endemic to South Africa. It has pharmacological activities such as antidiabetic and antioxidant effects. However, Aspalathus Linearis plant resources are limited and the aspartame content in plants is low. At the same time, the chemical synthesis involves problems such as poor selectivity and environmental pollution. Therefore, developing an efficient method for synthesizing dihydrochalcone glycosides, represented by aspartame, has important practical significance and application value.

[0003] In recent years, with the rapid development of synthetic biology, many important natural drugs or their precursors and intermediates (such as artemisinin and paclitaxel precursors) have been mass-produced by constructing microbial cell factories. Based on the concept of synthetic biology, creating artificial cell factories to produce bioactive natural products has become a new model for drug resource acquisition. Therefore, applying synthetic biology techniques to construct microbial cell factories to synthesize diverse natural or non-natural dihydrochalcone glycosides with drug development potential from inexpensive and readily available compounds is one viable approach.

[0004] The natural biosynthetic pathway of dihydrochalcone glycosides, represented by aspartame, involves phenylalanine being catalyzed by phenylalanine ammonia-lyase to cinnamic acid, followed by cinnamic acid hydroxylation at the 4-position to p-coumaric acid, then catalyzed by 4-coumaric acid:coenzyme A ligase to p-coumaryl-CoA, subsequently catalyzed by NADPH and oxidoreductase to dihydro-p-coumaryl-CoA, and then condensed with malonyl-CoA by chalcone synthase to form the dihydrochalcone nucleus. Literature reports that the pCDFDuet-At4CL-MatB::MatC plasmid can enhance the supply of malonyl-CoA, a precursor for chalcone synthesis, in recombinant strains. Chalcone synthase has been identified in plants such as maize and rice. Subsequently, CYP-P450 catalyzes the hydroxylation of the B-ring of the dihydrochalcone nucleus at the 3-position, and finally, carbonyl transferase completes the carbonylation to yield aspartame. However, it currently faces the problems of poor selectivity of chalcone synthase and low activity of CYP-P450 enzyme in the natural biosynthetic pathway, making it difficult to achieve efficient heterologous reconstruction of the natural biosynthetic pathway of aspartame and limiting its application in efficient biosynthesis.

[0005] In summary, current research on dihydrochalcone glycosides such as aspartame has the following limitations and shortcomings:

[0006] 1. Dihydrochalcone glycosides are rare in natural resources, difficult to synthesize chemically, and their sources are limited;

[0007] 2. Because the natural biosynthetic pathway of aspartame involves chalcone synthase with poor selectivity and cytochrome P450 enzyme with low activity, it is difficult to achieve efficient biosynthesis.

[0008] Therefore, there is an urgent need to establish a non-natural biosynthetic route for dihydrochalcone glycosides to achieve the efficient biosynthesis of aspartame. This patent innovatively constructs an artificial biosynthetic pathway for dihydrochalcone glycosides in *E. coli* by screening for highly efficient chalcone synthases and combining them with oxidoreductases and C-glycosyltransferases with broad substrate applicability. This method enables the preparation of structurally diverse and bioactive dihydrochalcone glycosides, represented by aspartame, through engineered microbial cell factories, providing a new method for obtaining these compounds and offering new compound entities for drug discovery. Summary of the Invention

[0009] To overcome the shortcomings of the prior art, the present invention aims to provide a method for the biosynthetic preparation of dihydrochalcone glycosides, characterized by:

[0010] 1. Construction of a high-yield engineered strain for chalcone backbone compound synthesis: The chalcone synthase gene ScCHS (amino acid sequence as shown in SEQ ID No. 1, nucleotide sequence as shown in SEQ ID No. 2) from *Stenolomachusanum* was constructed in the pET-28a expression vector; malonyl-CoA synthase MatB (AAP03025.1) (Chen, H., Kim, HU, Weng, H., Browse, J. Malonyl-CoA Synthetase, Encoded by Acyl Activating Enzyme 13, is Essential for Growth and Development of Arabidopsis. The Plant Cell, 2011, 23(6): 2247-2262.), malonic acid carrier protein MatC (KF765784.1) (An, JH, Kim, YSA Gene Cluster Encoding Malonyl-CoA Decarboxylase (MatA), Malonyl-CoA Synthetase (MatB) and a Putative Dicarboxylate Carrier Protein (MatC) in Rhizobiumtrifolii: Cloning, Sequencing, and Expression of the Enzymes in Escherichiacoli. European Journal of Biochemistry, 1998, 257(2):395-402.) was recombinantly ligated using a flexible linker and then sequentially constructed at two multiple cloning sites on the pCDFDuet-1 expression vector with 4-coumarate:coenzyme A ligase At4CL (AY376729.1) (Ehlting, J., Büttner, D., Wang, Q., Douglas, CJ, Somssich, IE, Kombrink, E. Three 4-Coumarate:Coenzyme A Ligases in Arabidopsis thaliana Represent Two Evolutionarily Divergent Classes in Angiosperms. The Plant Journal, 1999, 19(1):9-20.)The two recombinant expression plasmids were co-transformed into Escherichia coli host cells BL21(DE3), and strains containing both plasmids were screened to obtain high-yield engineered strains of chalcone backbone compound synthesis module.

[0011] 2. Construction of Chalcone Backbone Reduction Module Engineered Strains: The oxidoreductase sERED can catalyze the production of phlorizin from naringenin chalcone. The oxidoreductase gene sERED (amino acid sequence as shown in SEQ ID No. 3, nucleotide sequence as shown in SEQ ID No. 4) from the intestinal anaerobic eubacterium E. ramulus DSM 16296 was constructed in the pET-28a expression vector. The obtained recombinant plasmid was transformed into E. coli host cells BL21(DE3) to obtain a strain containing the above recombinant plasmid, namely the chalcone backbone reduction modular engineered strain.

[0012] 3. Construction of engineered strains with dihydrochalcone backbone glycosylation: The glycosyltransferase gene AbCGT (amino acid sequence as shown in SEQ ID No. 5, nucleotide sequence as shown in SEQ ID NO. 6) from the plant Aloe barbadensis was constructed in the pET-28a expression vector. The obtained recombinant plasmid was transformed into Escherichia coli host cells BL21(DE3) to obtain strains containing the above recombinant plasmids, namely, modular engineered strains with dihydrochalcone backbone glycosylation.

[0013] 4. Synthesis of Chalcone Skeleton Compounds: The engineered bacteria used in the chalcone skeleton synthesis module were cultured in M9Y medium. After induction of expression, phenylpropionic acid compounds such as malonic acid and caffeic acid were introduced for whole-cell reaction. The reaction conditions were 30℃, 200 rpm, and 24 h to synthesize the chalcone skeleton compounds. The reaction solution was extracted with ethyl acetate and the product was obtained by rotary evaporation and high-performance liquid chromatography (HPLC). UV-MS was used for further analysis. 1 H NMR, 13 The structure of the product was analyzed and identified using spectroscopic techniques such as C NMR.

[0014] 5. Reduction of Chalcone Skeleton Intermediates: Engineered bacteria for chalcone skeleton synthesis and chalcone skeleton reduction were cultured separately in M9Y medium. After induction of expression, malonic acid and caffeic acid compounds were added to the bacterial culture of the chalcone skeleton synthesis module to initiate a whole-cell reaction, synthesizing chalcone skeleton compounds. Then, the bacterial culture of the chalcone skeleton reduction module was added to the aforementioned bacterial culture for co-culture to synthesize dihydrochalcone compounds. All whole-cell reaction conditions were 30℃, 200 rpm, and 24 h. The reaction solution was extracted with ethyl acetate and the products were obtained by rotary evaporation and high-performance liquid chromatography (HPLC). UV, MS, and other analytical techniques were used to analyze the products. 1 H NMR, 13The structure of the product was analyzed and identified using spectroscopic techniques such as C NMR.

[0015] 6. C-glycosylation of dihydrochalcone skeleton compounds: Engineered bacteria for chalcone skeleton synthesis, chalcone skeleton reduction, and glycosylation were cultured separately in suitable culture media. After induction of expression, malonic acid and caffeic acid compounds were added to the bacterial culture of the chalcone skeleton synthesis module to initiate a whole-cell reaction, synthesizing chalcone skeleton compounds. Then, the bacterial culture of the chalcone skeleton reduction module was added to the aforementioned bacterial culture for co-culture to synthesize dihydrochalcone compounds. Finally, the bacterial culture of the glycosylation module was added to the aforementioned bacterial culture for co-culture. All whole-cell reaction conditions were 30℃, 200 rpm, and 24 h. The reaction solution was extracted with ethyl acetate and then concentrated by rotary evaporation and separated by high-performance liquid chromatography (HPLC). The products were analyzed by UV, MS, and microscopy. 1 H NMR, 13 The structure of the product was analyzed and identified using spectroscopic techniques such as C NMR.

[0016] This invention has the following technical advantages:

[0017] 1. This invention provides a modular engineered microorganism for the efficient synthesis of chalcone compounds, which is economical, efficient, and environmentally friendly. Compared with existing technologies, it avoids the disadvantages of chemical synthesis methods, such as functional group protection, deprotection, and environmental pollution, while also enabling the production of large quantities of target compounds in a short time. Furthermore, because the chalcone synthase involved has a broad substrate spectrum, it can be developed into a chassis cell for the synthesis of a series of chalcone derivatives.

[0018] 2. This invention provides a modular engineered bacterium that efficiently completes the reduction of olefins in the chalcone skeleton. By catalyzing the reduction of olefin double bonds in the chalcone skeleton with engineered bacteria, the efficient production of dihydrochalcone compounds is achieved. This method is economical and practical, effectively reducing the cost of synthesizing dihydrochalcone compounds as reaction substrates.

[0019] 3. This invention provides a modular engineered bacterium that efficiently completes the glycosylation of dihydrochalcone skeleton. The engineered bacterium synthesizes glycosyl donors and combines them with highly catalytic glycosyltransferases to complete the glycosylation reaction, achieving self-sufficiency of glycosyl donors within the bacterium. This is economical, practical, and effectively reduces the cost of synthesizing dihydrochalcone carbon glycosylation products.

[0020] 4. The modular engineered bacteria involved in this invention effectively avoid the shortcomings of heavy cell load and weak vitality caused by multiple genes in the same host cell through combined co-culture by artificially controlling the reaction sequence. At the same time, it controls the orderly occurrence of each step of the reaction and gives full play to the catalytic ability of each functional bacteria, providing a new method for the efficient synthesis of dihydrochalcone glycoside compounds. Attached Figure Description

[0021] Figure 1 Schematic diagram of the artificially reconstructed biosynthetic pathway of aspartame

[0022] Figure 2 Summary of structural formulas of dihydrochalcone glycosides, substrates and intermediates

[0023] Figure 3 HPLC-MS chromatogram of whole-cell synthesis of aspirin by modular engineered bacteria through combined culture.

[0024] (1) HPLC chromatograms of caffeic acid and senna reference standards; (2) HPLC chromatogram of whole-cell synthesis of chalcone skeleton synthesizing engineered bacteria when caffeic acid substrate is added; (3) HPLC chromatogram of chalcone skeleton reducing engineered bacteria after the substrate is added and catalyzed by chalcone skeleton synthesizing engineered bacteria; (4) HPLC chromatogram of dihydrochalcone skeleton glycosylation engineered bacteria after the substrate is added and catalyzed by chalcone skeleton synthesizing and chalcone skeleton reducing engineered bacteria.

[0025] Figure 4 HPLC-MS chromatograms of whole-cell catalysis using ferulic acid as a substrate.

[0026] (1) HPLC chromatogram of ferulic acid reference standard; (2) HPLC chromatogram of whole-cell synthesis of chalcone skeleton synthesizing engineered bacteria when the substrate ferulic acid is added; (3) HPLC chromatogram of chalcone skeleton reducing engineered bacteria after the substrate is added and catalyzed by chalcone skeleton synthesizing engineered bacteria; (4) HPLC chromatogram of dihydrochalcone skeleton glycosylation engineered bacteria after the substrate is added and catalyzed by chalcone skeleton synthesizing and chalcone skeleton reducing engineered bacteria.

[0027] Figure 5 HPLC-MS chromatograms of whole-cell catalysis using 4-hydroxycinnamic acid as a substrate.

[0028] (1) HPLC chromatogram of 4-hydroxycinnamic acid reference standard; (2) HPLC chromatogram of whole-cell synthesis of chalcone skeleton synthesizing engineered bacteria when 4-hydroxycinnamic acid substrate is added; (3) HPLC chromatogram of chalcone skeleton reducing engineered bacteria after the substrate is added and catalyzed by chalcone skeleton synthesizing engineered bacteria; (4) HPLC chromatogram of dihydrochalcone skeleton glycosylation engineered bacteria after the substrate is added and catalyzed by chalcone skeleton synthesizing and chalcone skeleton reducing engineered bacteria.

[0029] Figure 6 Compound 2 1 1H NMR spectrum (DMSO-d6, 700MHz)

[0030] Figure 7 Compound 2 13C10 NMR spectrum (DMSO-d6, 176MHz)

[0031] Figure 8 Compound 3 1 1H NMR spectrum (DMSO-d6, 700MHz)

[0032] Figure 9 Compound 3 13 C10 NMR spectrum (DMSO-d6, 176MHz)

[0033] Figure 10 Compound 4 1 1H NMR spectrum (DMSO-d6, 700MHz)

[0034] Figure 11 Compound 4 13 C10 NMR spectrum (DMSO-d6, 176MHz) Detailed Implementation

[0035] To further understand the present invention, the following embodiments are only used to further illustrate the present invention, but do not imply any limitation on the present invention.

[0036] Example 1: Construction of pET-28a-ScCHS recombinant expression vector

[0037] Based on the gene sequences flanking the site on the expression vector pET-28a-NdeⅠ-SalⅠ, primers containing homologous arms of the vector were designed. Homologous arms were introduced to both sides of the target gene ScCHS (codon optimized for E. coli) using PCR technology, and the PCR products were recovered via gel electrophoresis. Homologous recombination was used to ligate the target gene with the linearized pET-28a vector. Transformed into Trans1-T1 competent cells, and sequencing confirmed that the candidate gene was correctly ligated to pET-28a. The recombinant plasmid pET-28a-ScCHS was obtained by extracting plasmids from the transformants.

[0038] 1.1. PCR reaction system:

[0039]

[0040]

[0041] 1.2. PCR cycling:

[0042] 1) 94℃ for 2 min; 2) 98℃ for 10 sec; 3) 58℃ for 30 sec; 4) 68℃ for 1 min; 5) Cycle 2 to 4 for 35 times; 6) 68℃ for 10 min; 7) Hold at 10℃.

[0043] Example 2: Construction of pCDFDuet-1-At4CL-MatB::MatC recombinant expression vector

[0044] The 4-coumaric acid:coenzyme A ligase gene At4CL, optimized for E. coli codons, was inserted into the NotI single restriction site at the pCDFDuet cloning site. The genes encoding malonyl-CoA synthase MatB and malonate carrier protein MatC were linked using a flexible linker and then inserted into the XhoI single restriction site at the pCDFDuet cloning site. The recombinant plasmid pCDFDuet-At4CL-MatB::MatC was constructed (specific method as in Example 1). This plasmid was transformed into Trans1-T1 competent cells. Sequencing confirmed that the candidate genes were correctly linked to pET-28a. The recombinant plasmid pCDFDuet-1-At4CL-MatB::MatC was obtained by extracting the plasmid from the transformant.

[0045] Example 3: Construction of pET-28a-sERED recombinant expression vector

[0046] Based on the gene sequences flanking the expression vector pET-28a-NdeⅠ-SalⅠ site, primers containing homologous arms were designed. Homologous arms were introduced flanking the target gene sERED (codon optimized for E. coli) using PCR (specific method as in Example 1). The PCR products were then recovered via gel electrophoresis. Homologous recombination was used to ligate the target gene with the linearized pET-28a vector. Transformed into Trans1-T1 competent cells, and sequencing confirmed that the candidate gene was correctly ligated to pET-28a. The resulting transformants were then used to extract the recombinant plasmid pET-28a-sERED.

[0047] Example 4: Construction of the pET-28a-AbCGT recombinant expression vector

[0048] Based on the gene sequences flanking the expression vector pET-28a-NdeⅠ-SalⅠ site, primers containing homologous arms were designed. Homologous arms were introduced flanking the target gene AbCGT (codon optimized by *E. coli*) using PCR (specific method as in Example 1). The PCR products were then recovered via gel electrophoresis. Homologous recombination was used to ligate the target gene with the linearized pET-28a vector. Transformed into Trans1-T1 competent cells, and sequencing confirmed that the candidate gene was correctly ligated to pET-28a. The resulting transformants were then used to extract the recombinant plasmid pET-28a-AbCGT.

[0049] Example 5: Construction of the engineered chalcone backbone synthesizing strain BL21(DE3):pCDFDuet-1-At4CL-MatB::MatC-pET-28a-ScCHS

[0050] The recombinant plasmids pCDFDuet-1-At4CL-MatB::MatC,pET-28a-ScCHS from Examples 1-2 were co-transformed into Escherichia coli BL21(DE3) competent cells. Colony PCR screening was performed, and the positive transformants, which are engineered bacteria containing both of the above recombinant plasmids, were named S01.

[0051] Example 6: Construction of chalcone skeleton-reducing engineered bacteria BL21(DE3):pET-28a-sERED

[0052] The pET-28a-sERED recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells. By screening for positive transformants, engineered bacteria carrying the recombinant plasmid were obtained and named S02.

[0053] Example 7: Construction of chalcone skeleton-reducing engineered bacteria BL21(DE3):pET-28a-AbCGT

[0054] The pET-28a-AbCGT recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells. By screening for positive transformants, engineered bacteria carrying the recombinant plasmid were obtained and named S03.

[0055] Example 8: Synthesis of Chalcone Skeleton Compounds

[0056] 1) The engineered bacteria S01 with the correct sequence was inoculated into LB medium containing both kanamycin (resistance carried by pET-28a vector) and streptomycin (resistance carried by pCDFDuet-1 vector) and cultured at 37°C with shaking at 200 rpm for 12 h to obtain seed culture;

[0057] 2) Inoculate the activated seed culture into fresh M9Y medium containing the same concentration of antibiotics (use 250mL Erlenmeyer flasks, 100mL per flask), until the bacterial concentration reaches OD200. 600 =0.1;

[0058] 3) Incubate at 37℃ and 200 rpm until OD 600 =0.6, add IPTG to each culture bottle to a final concentration of 0.1mM; induce the expression of the target protein at 16℃ and 200rpm;

[0059] 4) After 16 h of induction culture, add substrate malonic acid and phenylpropionic acid compounds to final concentrations of 18 mM and 3 mM, respectively; culture at 30 °C and 200 rpm for 24 h with shaking.

[0060] 5) Add 200 μL of culture to 400 μL of methanol, centrifuge at 15,000 rpm for 30 min, collect the supernatant, and detect the chalcone product by HPLC (chromatographic column: Shiseido Capcellpak C). 18 MGIII column (250 mm × 4.6 mm I.D., 5 μm, Shiseido Co., Ltd., Tokyo, Japan); mobile phase: A (0.1% formic acid aqueous solution) / B (methanol), gradient elution: 15% B, 5 min; 15-100% B, 30 min; 100% B, 5 min; flow rate: 1 mL / min; column temperature: 30 °C; λ max =290nm.

[0061] 6) Extract 800 mL of culture with 1.6 L of ethyl acetate. Collect the ethyl acetate phase and dry it under reduced pressure by rotary evaporation to obtain a concentrated solution. Redissolve the concentrated solution in methanol, centrifuge at 15,000 rpm for 30 min, and collect the supernatant. Prepare the chalcone product using HPLC (chromatographic column: Shiseido Capcellpak C10). 18 MG II column (250mm × 10mm ID, 5μm, Shiseido Co., Ltd., Tokyo, Japan); mobile phase: chromatographic methanol (B) / pure water (A), gradient elution: 40%-65% B, 20 min; 65%-100% B, 0.1 min; 100% B, 2.9 min; flow rate: 3 mL / min; column temperature: 30℃; λ max =290nm).

[0062] When caffeic acid was used as a phenylpropionic acid substrate, the sennaol chalcone obtained by spontaneous cyclization showed a retention time of 18.5 min according to HPLC-UV detection. Further analysis by MS / MS and NMR (with attached...) confirmed the presence of sennaol. Figure 3 , 6 The structure identified as sennaol (2a) was 131.4 mg / L. Spectroscopic data are as follows:

[0063] Sacred herbol (2a): C 15 H 12 O6: ESI-MSm / z 287.0561[MH] - ; 1 HNMR (DMSO-d6, 700MHz) δ H12.14(s,5-OH),10.79(s,7-OH),9.06(s,3'-OH),9.02(s,4'-OH),6.87(t,J=1.3Hz,H-5'),6.74(s,H-2'),6.74(s,H-6') ,5.87(q,J=2.1Hz,H-6,8),5.37(dd,J=12.5,3.1Hz,H-2),3.17(dd,J=13.1,4.0Hz,H-3α),2.67(dd,J=17.1,3.2Hz,H-3β).

[0064] 13 C NMR (DMSO-d6, 176MHz)δ C 196.35(C-4),166.69(C-7),163.47(C-9),162.90(C-5),145.72(C-4'),145.19(C-3'),129.45(C-1'),117.9 4(C-6'),115.33(C-5'),114.34(C-2'),101.77(C-10),95.75(C-6),94.96(C-8),78.45(C-2),42.08(C-3).;

[0065] When ferulic acid is used as a phenylpropionic acid substrate, the obtained chalcone compounds are analyzed by MS / MS (with attached...). Figure 4 The structure was identified as 3-methoxy-2',4,4',6'-tetrahydroxychalcone (6).

[0066] When 4-hydroxycinnamic acid is used as a phenylpropionic acid substrate, the obtained chalcone compounds are analyzed by MS / MS (with attached...). Figure 5 The structure was identified as 4,2',4',6'-tetrahydroxychalcone (10).

[0067] Example 9: Reduction of chalcone skeleton compounds

[0068] 1) The engineered bacteria S01, whose sequencing verification was correct, was inoculated into LB medium containing both kanamycin (resistance carried by pET-28a vector) and streptomycin (resistance carried by pCDFDuet-1 vector); the engineered bacteria S02, whose sequencing verification was correct, was inoculated into LB medium containing kanamycin (resistance carried by pET-28a vector), and cultured at 37°C and 200 rpm for 12 h to obtain seed culture;

[0069] 2) Inoculate the activated seed culture into fresh M9Y medium containing the same concentration of antibiotics (use 250mL Erlenmeyer flasks, 100mL per flask), until the bacterial concentration reaches OD200. 600 =0.1;

[0070] 3) Incubate at 37℃ and 200 rpm until OD 600 =0.6, add IPTG to each culture bottle to a final concentration of 0.1mM; induce the expression of the target protein at 16℃ and 200rpm;

[0071] 4) After induction culture for 16 h, add substrate malonic acid and phenylpropionic acid compounds to the S01 culture to a final concentration of 18 mM and 3 mM, respectively; culture at 30℃ and 200 rpm for 24 h with shaking.

[0072] 6) Add an equal volume of SO2 bacterial solution to SO1 reaction solution and mix. Use 500mL Erlenmeyer flasks, with each flask containing no more than 200mL of solution. Incubate at 30℃ and 200rpm for 24h with shaking. The post-processing and detection steps for the bacterial solution are the same as in Example 8. Prepare the corresponding product using HPLC (chromatographic column: Shiseido Capcell Pak C18 MG II column (250mm×10mm ID, 5μm, Shiseido Co., Ltd., Tokyo, Japan); mobile phase: methanol (B) / pure water (A), gradient elution: 40%-65% B, 20min; 65-100% B, 0.1min; 100% B, 2.9min; flow rate: 3mL / min; column temperature: 30℃; λ max =290nm).

[0073] When caffeic acid was used as a phenylpropionic acid substrate, the dihydrochalcone compound obtained had a retention time of 18.8 min according to HPLC-UV detection. Further analysis by MS and NMR (with attached...) confirmed the presence of the chalcone compound. Figure 3 , 8 The structure identified by 9) is dihydroseriol (3). The spectral data are as follows:

[0074] Dihydrosuccinyl sorbitol (3): C 15 H 14 O6: ESI-MSm / z 289.0718[MH] - ; 1 HNMR (DMSO-d6, 700MHz) δ H 6.64–6.59(m,H-2',5'),6.46(dd,J=8.2,2.1Hz,H-6'),5.81(d,J=1.9Hz,H-3",5"),3.20(t,J=7.7Hz,H-α,α'),2.70(t,J=7.8Hz,H-β,β').

[0075] 13 C NMR (DMSO-d6, 176MHz)δ C204.25(C-1),164.53(C-2”,6”),164.17(C-1”),144.95(C-3’),143.22(C-4’),132.46(C-1’),118. 91(C-6'),115.75(C-2'),115.46(C-5'),103.73(C-4”),94.65(C-3”,5”),45.45(C-α),29.62(C-β).

[0076] When ferulic acid is used as a phenylpropionic acid substrate, the resulting dihydrochalcone compound is analyzed by MS / MS (with attached...). Figure 4 The structure was identified as 2',4,4',6'-tetrahydroxy-3-methoxydihydrochalcone (7).

[0077] When 4-hydroxycinnamic acid is used as a phenylpropionic acid substrate, the resulting dihydrochalcone compound is analyzed by MS / MS (with attached...). Figure 5 The structure was identified as 4,2',4',6'-tetrahydroxydihydrochalcone (11).

[0078] Example 10: Carbonylation of dihydrochalcone skeleton compounds

[0079] 1) The engineered bacteria S01, whose sequencing verification was correct, were inoculated into LB medium containing both kanamycin (resistance carried by the pET-28a vector) and streptomycin (resistance carried by the pCDFDuet-1 vector); the engineered bacteria S02 and S03, whose sequencing verification was correct, were inoculated into LB medium containing kanamycin (resistance carried by the pET-28a vector) and cultured at 37°C and 200 rpm for 12 h to obtain seed culture;

[0080] 2) Inoculate the activated seed culture into fresh M9Y medium containing the same concentration of antibiotics (use 1000mL Erlenmeyer flasks, 100mL per flask), until the bacterial concentration reaches OD200. 600 =0.1;

[0081] 3) Incubate at 37℃ and 200 rpm until OD 600 =0.6, add IPTG to each culture bottle to a final concentration of 0.1mM; induce the expression of the target protein at 16℃ and 200rpm;

[0082] 4) After induction culture for 16 h, add substrate malonic acid and phenylpropionic acid compounds to the S01 culture to a final concentration of 18 mM and 3 mM, respectively; culture at 30℃ and 200 rpm for 24 h with shaking.

[0083] 6) Add an equal volume of SO2 bacterial solution to SO1 reaction solution and mix. Use a 1000mL Erlenmeyer flask, with each flask containing no more than 400mL of liquid. Incubate at 30℃ and 200rpm for 24h with shaking.

[0084] 6) Add an equal volume of SO3 bacterial solution to SO1 and SO2 reaction solutions and mix. Use a 1000mL Erlenmeyer flask, with each flask containing no more than 400mL of liquid. Incubate at 30℃ and 200rpm for 24h with shaking. The post-processing and detection steps for the bacterial solution are the same as in Example 8. Prepare the corresponding product using HPLC (chromatographic column: Shiseido Capcell Pak C18 MG II column (250mm×10mm ID, 5μm, Shiseido Co., Ltd., Tokyo, Japan); mobile phase: chromatographic methanol (B) / pure water (A), gradient elution: 40%-65% B, 20min; 65-100% B, 0.1min; 100% B, 2.9min; flow rate: 3mL / min; column temperature: 30℃; λ max =290nm).

[0085] When caffeic acid was used as a phenylpropionic acid substrate, the dihydrochalcone glycoside obtained by HPLC-UV detection had a retention time of 13.6 min. Further analysis by MS and NMR (with attached...) confirmed the presence of the glycoside. Figure 3 , 10 The identified structures were aspartate (4), with a yield of 192.9 mg / L. The spectral data are as follows:

[0086] Aspartame (4): C 21 H 24 O 11 ESI-MSm / z 451.1237[MH] - ; 1 HNMR (DMSO-d6, 700MHz) δ H13.80(s,H-18),11.02(s,H-19),10.06(s,H-17),8.71(s,H-30),8.61(s,H-31),6.61(s,H-2),6.60(d,J=2.1H z,H-5),6.46(dd,J=8.0,2.1Hz,H-6),5.94(s,H-3'),4.83(d,J=4.6Hz,1H),4.79(d,J=4.5Hz,1H),4.50(d,J=5. 5Hz,H-1”),4.46(t,J=5.7Hz,1H),3.88(td,J=9.1,5.2Hz,H-2”),3.65(dd,J=12.0,4.2Hz,H-6”),3.40(dt,J=11 .3,5.4Hz,H-6”),3.22–3.20(m,H-α,α’),3.16–3.13(m,H-5”),3.11–3.10(m,H-4”),2.70(t,J=7.7Hz,H-β,β’).

[0087] 13 C NMR (DMSO-d6, 176MHz)δ C 204.46(C-10),164.83(C-4'),163.66(C-6'),161.62(C-2'),145.00(C-3), 143.27(C-4),132.38(C-1),118.86(C-6),115.73(C-10),115.45(C-5),104 .06(C-1'),103.60(C-5'),94.56(C-3'),81.35(C-5”),78.93(C-3”),73.55 (C-1”),70.63(C-4”),70.47(C-2”),61.31(C-6”),45.51(C-α),29.71(C-β).

[0088] When ferulic acid is used as a phenylpropionic acid substrate, the resulting dihydrochalcone carbon glycoside compound is analyzed by MS / MS (with attached...). Figure 4 The structure was identified as 3-methoxy-northernfarin (8).

[0089] When 4-hydroxycinnamic acid is used as a phenylpropionic acid substrate, the resulting dihydrochalcone carbon glycoside compound is analyzed by MS / MS (with attached...). Figure 5 The structure was identified as the Norsefagen (12).

Claims

1. A method for preparing aspartame by biosynthesis of caffeic acid using multi-enzyme cascade recombinant Escherichia coli, characterized in that, It is prepared through engineered microbial "cell factories," and the specific steps are as follows: a. The engineered microorganisms that synthesize chalcone backbone containing the chalcone synthase gene ScCHS, the engineered microorganisms that reduce chalcone backbone containing the oxidoreductase gene sERED, and the engineered microorganisms that glycosyltransferase gene AbCGT were induced to express recombinant chalcone synthase ScCHS, oxidoreductase sERED, and glycosyltransferase AbCGT, respectively. b. The above engineered microorganisms were cultured and induced in M9Y medium, and then adjusted to an appropriate concentration and an appropriate amount of substrate (such as malonic acid and caffeic acid) was added. The reaction was carried out at 30℃ and 200rpm for 24h. c. Aspartame and its structural analogues were obtained by extracting the bacterial broth with ethyl acetate and then purifying it by rotary evaporation concentration and high performance liquid chromatography.

2. The preparation method according to claim 1, characterized in that, The recombinant chalcone synthase ScCHS is derived from the plant *Stenoloma chusanum*, and its amino acid sequence is shown in SEQ ID No. 1, or has more than 90% homology with the sequence shown in SEQ ID No.

1.

3. A nucleic acid molecule encoding the chalcone synthase ScCHS of claim 2, characterized in that, Its nucleic acid sequence is shown in SEQ ID NO.2, or has more than 90% homology with the sequence shown in SEQ ID NO.

2.

4. A recombinant expression vector comprising the nucleic acid molecule of claim 3 and a regulatory element operatively linked thereto.

5. An engineered microorganism comprising the gene of claim 3 or the vector of claim 4.

6. The use of the chalcone synthase of claim 2, the gene of claim 3, the vector of claim 4, or the engineered microorganism of claim 5 in the synthesis of dihydrochalcone glycoside intermediates.

7. The preparation method according to claim 1, characterized in that, The oxidoreductase sERED is derived from the intestinal anaerobic eubacterium ramulus, and its amino acid sequence is shown in SEQ ID No. 3, or has more than 90% homology with the sequence shown in SEQ ID No.

3.

8. A nucleic acid molecule encoding the oxidoreductase sERED of claim 7, characterized in that, Its nucleic acid sequence is shown in SEQ ID NO.4, or has more than 90% homology with the sequence shown in SEQ ID NO.

4.

9. A recombinant expression vector, characterized in that, It comprises the gene as described in claim 8 and a regulatory element operatively connected thereto.

10. An engineered microorganism, characterized in that, It contains the gene of claim 8 or the vector of claim 9.

11. The use of the oxidoreductase of claim 7, the gene of claim 8, the vector of claim 9, or the engineered microorganism of claim 10 in the synthesis of dihydrochalcone glycoside intermediates.

12. The preparation method according to claim 1, characterized in that, The glycosyltransferase AbCGT is derived from the plant Aloe barbadensis, and its amino acid sequence is shown in SEQ ID No. 5, or has more than 90% homology with the sequence shown in SEQ ID No.

5.

13. A nucleic acid molecule encoding the glycosyltransferase AbCGT of claim 12, characterized in that, Its nucleic acid sequence is shown in SEQ ID NO.6, or has more than 90% homology with the sequence shown in SEQ ID No.

6.

14. A recombinant expression vector, characterized in that, It comprises the gene as described in any one of claims 13 and a regulatory element operatively connected thereto.

15. An engineered microorganism, characterized in that, It comprises the gene of claim 13 or the vector of claim 14.

16. The use of the glycosyltransferase of claim 12, the gene of claim 13, the vector of claim 14, or the engineered microorganism of claim 15 in the preparation of glycosylated dihydrochalcone derivatives.

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