Hqt enzyme mutant and its application in chlorogenic acid synthesis

Abstract

This invention belongs to the fields of biotechnology and enzyme engineering technology, specifically relating to an HQT enzyme mutant and its application in chlorogenic acid synthesis. Specifically, it relates to tobacco (…). Nicotiana tabacum Using HQT enzymes from [source] as the research subject, in order to improve [the effectiveness of] [the study]. Nt With the goals of improving the catalytic efficiency and substrate specificity of HQT enzymes, this study conducted computer-aided design-based experiments to systematically investigate the effects of key amino acid sites on enzyme activity and elucidate its molecular catalytic mechanism. Simultaneously, three rational design schemes were employed to achieve… Nt HQT enzyme molecule modification was used to screen for superior mutants with higher catalytic activity. Finally, the superior mutants were applied to the construction of chlorogenic acid engineered strains, which ultimately increased the yield of chlorogenic acid to the highest reported level to date. This laid a theoretical foundation for the technological advancement of microbial synthesis of chlorogenic acid and therefore has good practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and enzyme engineering technology, specifically relating to an HQT enzyme mutant and its application in chlorogenic acid synthesis. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Chlorogenic acid (CGA) is an important polyphenolic compound found in plants, primarily in medicinal plants such as honeysuckle, eucommia, and coffee. CGA possesses various pharmacological functions, including anti-inflammatory, antibacterial, antiviral, lipid-lowering, and hypoglycemic effects; it also exhibits certain therapeutic effects in tumor prevention, type II diabetes, and depression-related diseases. As an important pharmaceutical intermediate, CGA has enormous market potential in the pharmaceutical, chemical, and cosmetic industries.

[0004] In recent years, synthetic biology has made continuous progress, and the synthesis mechanism of chlorogenic acid (CGA) has been increasingly elucidated. Utilizing microorganisms with clear genetic backgrounds and high biosafety to synthesize CGA has become a developing trend in microbial fermentation for CGA synthesis. The CGA biosynthetic pathway has been successfully constructed and characterized in relatively safe strains such as *Escherichia coli*, *Saccharomyces cerevisiae*, *Streptomyces*, and *Corynebacterium glutamicum*. The CGA biosynthetic pathway exhibits bifurcation-convergence characteristics, making the assembly of biosynthetic modules extremely challenging.

[0005] Although current biosynthetic strategies have improved CGA yields at various levels, research on the modification of HQT enzymes, which play a key role in the CGA biosynthetic pathway, is weak. However, heterologous expression of this enzyme exhibits low catalytic activity and poor binding specificity to QA substrates, failing to meet the demands of industrial CGA production. Improving enzyme catalytic performance through enzyme engineering is a crucial strategy for increasing the yield of the target product. HQT, a key enzyme in CGA synthesis, belongs to the BAHD family of plant acyltransferases. This family possesses two conserved sequences, "HXXXD" and "DFGWG," and uses various acyl-CoA groups (cinnamyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, etc.) as acyl donors to catalyze the formation of esters or amides from various substrates (shikimic acid, QA, 4-hydroxyphenyllactic acid, gentianic acid, etc.). Currently, the structures of three enzymes from this family have been resolved. Cathie et al. identified two HQTs in artichokes and performed homology modeling and docking analysis based on two known structures in the BAHD family (VS and Dm3MaT3), predicting the structural features of HQTs in artichokes that may affect catalytic specificity. However, HQTs and the two previously reported BAHD superfamily structures belong to different branches, and the BAHD superfamily has evolved different substrate specificities through convergent evolution. Therefore, it is difficult to predict the true substrate preference of HQTs based solely on bioinformatics analysis. The structure of the HQT catalytic center has a double loop region, and the flexibility of the loop reduces the accuracy of homology modeling structures, failing to provide accurate references. This poses a significant obstacle to understanding the catalytic mechanism and its application and modification. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides an HQT enzyme mutant and its application in chlorogenic acid synthesis. Specifically, this invention uses tobacco ( Nicotiana tabacum Using HQT enzymes from [source] as the research subject, in order to improve [the effectiveness of] [the study]. Nt With the goals of improving the catalytic efficiency and substrate specificity of HQT enzymes, this study conducted computer-aided design-based experiments to systematically investigate the effects of key amino acid sites on enzyme activity and elucidate its molecular catalytic mechanism. Simultaneously, three rational design schemes were employed to achieve… Nt HQT enzyme molecule modification was conducted to screen for superior mutants with higher catalytic activity. These superior mutants were then applied to the construction of CGA engineered strains, ultimately increasing CGA yield to the highest reported level to date, laying a theoretical foundation for the technological advancement of CGA synthesis via microbial methods. Based on these research findings, this invention was completed.

[0007] Specifically, the present invention relates to the following technical solutions:

[0008] In a first aspect, the present invention provides an HQT enzyme mutant, which is obtained by mutating one or more amino acid sites from the 38th and 304th amino acids of the HQT enzyme, wherein the amino acid sequence of the HQT enzyme is shown in SEQ ID NO.2.

[0009] Furthermore, the HQT enzyme mutant is a mutation based on the above-mentioned HQT enzyme, and the HQT enzyme mutant is selected from any one or more mutants in the following group: G38A, V304E, G38A / V304E.

[0010] In a second aspect, the present invention provides a deoxyribonucleic acid molecule that encodes the above-mentioned HQT enzyme mutant.

[0011] A third aspect of the present invention provides a recombinant expression vector containing the deoxyribonucleic acid molecule described in the second aspect above, or a DNA sequence mutated from the deoxyribonucleic acid molecule as the parent.

[0012] Furthermore, the recombinant expression vector also contains At The gene encoding 4CL.

[0013] In a fourth aspect, the present invention provides a host cell capable of expressing the recombinant expression vector described in the third aspect or a chromosome integrated with the deoxyribonucleic acid molecule described in the second aspect.

[0014] A fifth aspect of the present invention provides a method for preparing the above-mentioned HQT enzyme mutant, comprising: culturing the host cells described in the fourth aspect above to express the HQT enzyme mutant protein; and isolating and purifying the HQT enzyme mutant to obtain a pure HQT enzyme.

[0015] A sixth aspect of the present invention provides the application of the HQT enzyme mutant described in the first aspect, the deoxyribonucleic acid molecule described in the second aspect, the recombinant expression vector described in the third aspect, and the host cell described in the fourth aspect in the field of chlorogenic acid synthesis.

[0016] A seventh aspect of the present invention provides a method for synthesizing chlorogenic acid, the method comprising fermenting the aforementioned host cells to obtain chlorogenic acid.

[0017] Furthermore, the chlorogenic acid synthesis substrates include caffeoyl coenzyme A (Caffeoyl CoA) and quinic acid (QA).

[0018] The beneficial technical effects of one or more of the above technical solutions: The above technical solution utilizes computer-aided rational design, combined with NtThree-dimensional crystal structure analysis and substrate binding specificity analysis of HQT enzymes precisely identified two key mutation sites, G38 and V304. Through site-directed saturation mutagenesis and iterative combined mutagenesis, the desired HQT enzyme was obtained. Nt The HQT enzyme mutant G38A&V304E has significantly enhanced binding specificity to caffeoyl-CoA and quinic acid, and its catalytic efficiency is greatly improved compared to the wild type. The above technical solution will Nt HQT enzyme mutants and At A recombinant expression vector was constructed by tandemly constructing the 4CL gene and transformed into Escherichia coli BL21 (DE3) to obtain an engineered strain. This engineered strain can efficiently express functional mutant enzymes, and the yield of chlorogenic acid by single-strain fermentation reaches 100 mg / L. After combining with a bimicrobial synergistic fermentation system, the yield of shake-flask fermentation is further increased to 150 mg / L, breaking through the key rate-limiting bottleneck of microbial synthesis of chlorogenic acid. The microbial synthesis method for chlorogenic acid provided by the above technical solution has mild fermentation conditions, readily available raw materials, and is environmentally friendly. It also has a high yield of chlorogenic acid and is suitable for industrial-scale production. It provides a feasible technical solution for the large-scale biological preparation of chlorogenic acid, reduces the dependence of chlorogenic acid production on plant extraction, and has significant economic and social benefits. The HQT enzyme modification strategy established by the above technical solution provides a reference for the molecular modification of other enzymes in the acyltransferase family, enriches the technical means of enzyme engineering modification, and has important reference value for the directed evolution of enzyme molecules in the field of synthetic biology. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1 Electrophoretic band verification was performed on the saturation mutations G38 and V304.

[0021] Figure 2 Evaluation of the three-dimensional structural model of the target protein NtHQT.

[0022] Figure 3 The three-dimensional structure of the NtHQT protein constructed using the protein with PDB ID 4G22 as a template.

[0023] Figure 4 Laplace plot of the three-dimensional structural model of the target protein NtHQT.

[0024] Figure 5 : Nt Verify_3D evaluation results of HQT's 3D structural model.

[0025] Figure 6 : Nt Evaluation results of ERRAT, PROVE, and WHATCHECK for the three-dimensional structural model of HQT.

[0026] Figure 7 Target protein Nt The evaluation results of the three-dimensional structural model of HQT.

[0027] Figure 8 Target protein Nt HQT combined with Caffeoyl CoA and QA.

[0028] Figure 9 Caffeoyl CoA and QA with target proteins Nt 2D interaction diagram of HQT binding modes.

[0029] Figure 10 : The active site of the template protein HCT.

[0030] Figure 11 Fermentation diagram of V304 mutant.

[0031] Figure 12 Fermentation diagram of G38 mutant.

[0032] Figure 13 :BL21(DE3):pET28a- At4CL - NtHQT G38A&V304E CGA shake-flask fermentation yield.

[0033] Figure 14 Dual-strain fermentation (including) NtHQT G38A&V304E CGA shake flask fermentation yield detection.

[0034] Figure 15 CGA production and OD600 in fermenters.

[0035] Figure 16 : Glucose (Glu) and xylose (Xyl) consumption curves during fermentation. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0038] In a typical embodiment of the present invention, an HQT enzyme mutant is provided, which is obtained by mutating one or more amino acid sites of the 38th and 304th amino acids based on the HQT enzyme, wherein the amino acid sequence of the HQT enzyme is shown in SEQ ID NO.2.

[0039] In another specific embodiment of the present invention, the amino acid sequence of the HQT enzyme mutant has at least 80% homology with SEQ ID NO.2; more preferably, it has at least 90% homology; most preferably, it has at least 95% homology; such as having at least 95%, 96%, 97%, 98%, or 99% homology.

[0040] In another specific embodiment of the present invention, the HQT enzyme mutant is a mutation based on the above-mentioned HQT enzyme, and the HQT enzyme mutant is selected from any one or more mutants in the following group: G38A, V304E, G38A / V304E.

[0041] In another specific embodiment of the present invention, a deoxyribonucleic acid molecule is provided, wherein the deoxyribonucleic acid molecule encodes the above-mentioned HQT enzyme mutant.

[0042] In another specific embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector contains the above-mentioned deoxyribonucleic acid molecule or a DNA sequence mutated from the deoxyribonucleic acid molecule as the parent.

[0043] In another specific embodiment of the present invention, the recombinant expression vector is obtained by effectively linking the above-mentioned deoxyribonucleic acid molecules to the expression vector. The expression vector is any one or more of a viral vector, plasmid, bacteriophage, phage particle, granule, or artificial chromosome. The viral vector may include adenovirus vector, retrovirus vector, or adeno-associated virus vector. The artificial chromosome includes bacterial artificial chromosome, phage P1-derived vector, yeast artificial chromosome, or mammalian artificial chromosome. Preferably, the expression vector is a plasmid. In one specific embodiment of the present invention, the plasmid is PET28m.

[0044] In another specific embodiment of the present invention, the recombinant expression vector further comprises At The gene encoding the 4CL protein.

[0045] Therefore, specifically, the recombinant expression vector can be PET28m- At4CL-NtHQT .in, NtHQT This represents the aforementioned deoxyribonucleic acid molecules.

[0046] In another specific embodiment of the present invention, a host cell is provided, wherein the host cell contains the above-mentioned recombinant expression vector or the above-mentioned deoxyribonucleic acid molecule is integrated into the chromosome.

[0047] The host cell can be a prokaryotic cell or a eukaryotic cell.

[0048] In another specific embodiment of the present invention, the host cell is any one or more of bacterial cells and fungal cells; The bacterial cells mentioned therein are any species within the genera Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas, or Staphylococcus; In another specific embodiment of the present invention, the bacterial cells are Escherichia coli (such as Escherichia coli BL21(DE3)), Agrobacterium tumefaciens (such as GV3101), Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, or Pseudomonas fluorescens.

[0049] The fungal cells include yeasts (such as Pichia pastoris).

[0050] In another specific embodiment of the present invention, a method for preparing the above-mentioned HQT enzyme mutant is provided, comprising the steps of: culturing the host cells of the present invention to express the HQT enzyme mutant protein; and isolating and purifying the HQT enzyme mutant pure enzyme.

[0051] In another specific embodiment of the present invention, the application of the above-mentioned HQT enzyme mutant, deoxyribonucleic acid molecule, recombinant expression vector, and host cell in the field of chlorogenic acid synthesis is provided.

[0052] In another specific embodiment of the present invention, a method for synthesizing chlorogenic acid is provided, the method comprising fermenting the above-mentioned host cells to obtain chlorogenic acid.

[0053] Furthermore, the chlorogenic acid synthesis substrates include caffeoyl coenzyme A (Caffeoyl CoA) and quinic acid (QA).

[0054] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0055] Example I. Experimental Methods: 1. Obtain At4CL Gene 1.1 Experimental Procedure: 1. Take about 100 mg of fresh Arabidopsis thaliana tissue or about 30 mg of dry tissue, add liquid nitrogen and grind thoroughly.

[0056] 2. Quickly transfer the ground powder into a centrifuge tube pre-filled with 700 µl of GP1 preheated buffer at 65°C (add mercaptoethanol to the preheated GP1 before the experiment to make the final concentration 0.1%), quickly invert and mix, then place the centrifuge tube in a 65°C water bath for 20 min, inverting the centrifuge tube several times during the water bath to mix the sample.

[0057] 3. Add 700 µl of chloroform, mix thoroughly, and centrifuge at 12,000 rpm (~13,400×g) for 5 min.

[0058] 4. Carefully transfer the upper aqueous phase obtained in the previous step into a new centrifuge tube, add 700 µl of buffer GP2, and mix thoroughly.

[0059] 5. Transfer the mixed liquid into the adsorption column CB3, centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, and discard the waste liquid.

[0060] 6. Add 500 µl of buffer GD to the adsorption column CB3 (please check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0061] 7. Add 600 µl of wash buffer PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0062] 8. Repeat step 7.

[0063] 9. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400×g) for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0064] The concentration of the extracted genome was determined, diluted to a certain concentration, and then amplified using the primers listed in the table below. At The 4CL gene and the 50μL reaction system are shown below: Components volume 2X Primestar Max Mix 25 μL Primer F 1 μL Primer R 1 μL Template 1 μL <![CDATA[ddH2O]]> 22 μL Primer name sequence At4CL-NdeI-F-up-pck CATATGACCACCCAGGAC At4CL-NotI-R-up-pck GCGGCCGCATAAGGTACC After amplification, the product was validated by gel electrophoresis, and the correct bands were recovered. The reaction is shown below: 1. Once the desired DNA fragment is completely separated on the agarose gel, carefully cut off the fragment using a clean, sharp scalpel, removing as much excess gel as possible; 2. Transfer the gel block containing the target fragment to a 1.5 mL centrifuge tube and weigh it to obtain the weight of the gel block.

[0065] 3. Add an equal volume of XP2 Binding Buffer to the centrifuge tube, equal to the volume of the colloid; 4. Incubate at 50-60℃ for 7 minutes or until the gel is completely melted, shaking or vortexing the mixture every 2-3 minutes. If the gel block is large or the gel concentration is high, the incubation time can be extended appropriately until the gel is completely melted. After the gel is completely melted, pay attention to the pH value of the gel-XP2 binding buffer mixture. If the pH value is greater than 8, the DNA yield will be greatly reduced. Observe the color of the mixture. If it is orange-red, add 5uL of 5M sodium sulfate (pH 5.2) to lower its pH value. After this adjustment, the color of the mixture will return to the normal yellow / light yellow.

[0066] 5. Place the HiBind® DNA Mini Column into a 2mL collection tube; 6. Transfer no more than 700 μL of DNA sol to a HiBind® DNA Mini Column, centrifuge at 10,000 g for 1 minute at room temperature, discard the filtrate, and reattach the column to a 2 mL collection tube; 7. If the volume of the DNA bath solution exceeds 700 μL, repeat step 6; 8. Discard the filtrate and reattach the HiBind® DNA Mini Column to the 2 mL collection tube. Add 300 μL of XP2 Binding Buffer to the binding column and centrifuge at the maximum speed (≥13,000) for 1 minute at room temperature. Discard the filtrate. 9. Replace the HiBind® DNA Mini Column into the 2 mL collection tube. Add 700 μL of SPW buffer (diluted with anhydrous ethanol) to the binding column. Centrifuge at 10,000 xg for 1 minute at room temperature and discard the filtrate; 10. Place the HiBind® DNA Mini Column into a clean 1.5 mL centrifuge tube, add 15-30 μL to the binding column matrix, incubate at room temperature for 2 minutes, and centrifuge at 13000 x g for 1 minute to elute the DNA.

[0067] After obtaining the target gene, the PET28m and cDNA templates were processed using a double enzyme digestion method, as shown in the reaction system below: Components volume PET28m 6μL I enzyme 1 μL I enzyme 1 μL Buffer(10X) 5μL <![CDATA[ddH2O]]> 37μL PCR procedure: react at 37℃ for 5 hours, then determine the DNA concentration.

[0068] 2. Expression vector ligation and amplification Using T4 DNA ligase to ligate the recovered DNA At4CL The gene fragment and the PET28m plasmid fragment were ligated into the complete recombinant plasmid PET28m- At4CL The reaction system is shown below: Components volume PET28m 4 μL 4 μL T4 DNA Ligation Buffer 1 μL T4 DNA ligase 1 μL Transfer the ligation product into E. coli Amplification of recombinant plasmids in DH5α competent cells: 1) Take 10 μL of the ligation product and add 100 μL of the product. E. coli Mix well in DH5α competent cells and incubate on ice for 20 min; 2) After the ice bath, place it in a metal bath at 42 ℃ for 45 s for heat shock; 3) After the heat shock, immediately place the container on an ice bath for 2 min, then immediately add 900 μL of LB medium, mix well, and incubate at 37 ℃ and 180 rpm for 1 h with shaking. 4) After the recovery culture is completed, spread the bacterial culture on LB agar plates containing 50 mg / L Kan and incubate upside down at 37 °C overnight.

[0069] After overnight incubation, single colonies were selected for colony PCR to verify whether the recombinant plasmid was successfully constructed.

[0070] 3. Obtain NtHQT Gene Experimental procedure: Similar to the at4cl gene acquisition, design the corresponding RNA primers, and add the corresponding restriction enzyme sites to the primers: Hin dIII and Bam The HI restriction site and the nucleotide sequence of the primer are shown below.

[0071] Primer name sequence HQT-BamHI-F-up-pck GGATCCatgggaagtgaaaaa HQT-HindIII-R-up-pck AAGCTTtcaaaattcatacaa RNA reverse transcription into cDNA is also related to At 4 CL The reverse transcription of the gene was identical, and the PET28m- was constructed. At4CL-NtHQT Plasmid vectors.

[0072]

[0073] 4. Computer-based rational design NtHQT Mutation screening 4.1 Site-directed mutation 4.1.1 Single mutant NtHQT G38 , NtHQT V304 Build Site-directed mutagenesis PCR for PET28m- At4CL-NtHQT In NtHQT In this embodiment, saturation mutations are performed on G38 and V304 of the gene. The mutant library is constructed by computer simulation and the mutants with improved catalytic activity are screened. The single-point mutants obtained by screening are iteratively combined to construct and screen mutant libraries, and finally the superior mutants with improved catalytic efficiency are obtained, so as to realize the directed evolution of enzyme molecules.

[0074] Based on the results of protein crystal structure analysis and computer-aided design, the amino acids at key sites were determined, and site-directed saturation mutagenesis was performed on the key amino acids G38 and V304. Site-directed saturation mutagenesis primers were designed, using pET28a- At4CL-NtHQT Using plasmids as templates, amplification NtHQT Gene fragments. The purified PCR product was digested with the appropriate restriction endonucleases. The target fragment was then recovered and recombined with the pET-28a(+) expression vector. The recombinant product was then transformed into the expression host cell using a heat shock method. E. coli BL21(DE3) competent cells were plated on LB plates containing 50 μg / mL Kan and cultured overnight at 37°C for 12–16 h to obtain mutant libraries.

[0075] 1) Using PET28m- At4CL-NtHQT Using plasmids as templates, saturation mutations were performed on G38 and V304, respectively. Design corresponding primers, the nucleotide sequences of which are shown below.

[0076] Primer name sequence HQT-G38F-F-up-pck tctaacttagatttaatagtgttcaga HQT-G38F-R-up-pck tactgttaaaagatgaattctgaacac HQT-G38L-F-up-pck tctaacttagatttaatagtgctaaga HQT-G38L-R-up-pck tactgttaaaagatgaattcttagcac HQT-G38I-F-up-pck tctaacttagatttaatagtgttaaga HQT-G38I-R-up-pck tactgttaaaagatgaattcttaacac HQT-G38M-F-up-pck tctaacttagatttaatagtgatgaga HQT-G38M-R-up-pck tactgttaaaagatgaattctctacac HQT-G38V-F-up-pck tctaacttagatttaatagtggtaaga HQT-G38V-R-up-pck tactgttaaaagatgaattcttaccac HQT-G38S-F-up-pck tctaacttagatttaatagtgtcaaga HQT-G38S-R-up-pck tactgttaaaagatgaattcttgacac HQT-G38P-F-up-pck aacttagatttaatagtgccaaga HQT-G38P-R-up-pck tactgttaaaagatgaattcttggcac HQT-G38T-F-up-pck tctaacttagatttaatagtgacaaga HQT-G38T-R-up-pck tactgttaaaagatgaattcttgtcac HQT-G38A-F-up-pck tctaacttagatttaatagtggcaaga HQT-G38A-R-up-pck tactgttaaaagatgaattcttgccac HQT-G38Y-F-up-pck tctaacttagatttaatagtgtataga HQT-G38Y-R-up-pck tactgttaaaagatgaattctatacac HQT-G38H-F-up-pck tctaacttagatttaatagtgcataga HQT-G38H-R-up-pck tactgttaaaagatgaattctatgcac HQT-G38Q-F-up-pck tctaacttagatttaatagtgcaaaga HQT-G38Q-R-up-pck tactgttaaaagatgaattctttgcac HQT-G38N-F-up-pck tctaacttagatttaatagtgaataga HQT-G38N-R-up-pck tactgttaaaagatgaattctattcac HQT-G38K-F-up-pck tctaacttagatttaatagtgaaaaga HQT-G38K-R-up-pck tactgttaaaagatgaattcttttcac HQT-G38D-F-up-pck tctaacttagatttaatagtggataga HQT-G38D-R-up-pck tactgttaaaagatgaattctagccac HQT-G38E-F-up-pck tctaacttagatttaatagtggaaaga HQT-G38E-R-up-pck tactgttaaaagatgaattctttccac HQT-G38C-F-up-pck tctaacttagatttaatagtgtgtaga HQT-G38C-R-up-pck tactgttaaaagatgaattctacacac HQT-G38W-F-up-pck tctaacttagatttaatagtgtggaga HQT-G38W-R-up-pck tactgttaaaagatgaattctccacac HQT-G38R-F-up-pck tctaacttagatttaatagtgcgaaga HQT-G38R-R-up-pck tactgttaaaagatgaattcttcgcac HQT-V304F-F-up-pck ccaggttacttaggaaatgttttcttc HQT-V304F-R-up-pck tgccataggtgtgcctgtgaagaaaac HQT-V304L-F-up-pck ccaggttacttaggaaatgttctgttc HQT-V304L-R-up-pck tgccataggtgtgcctgtgaacagaac HQT-V304I-F-up-pck ccaggttacttaggaaatgttatcttc HQT-V304I-R-up-pck tgccataggtgtgcctgtgaagataac HQT-V304M-F-up-pck ccaggttacttaggaaatgttatgttc HQT-V304M-R-up-pck tgccataggtgtgcctgtgaacataac HQT-V304G-F-up-pck ccaggttacttaggaaatgttgggttc HQT-V304G-R-up-pck tgccataggtgtgcctgtgaacccaac HQT-V304S-F-up-pck ccaggttacttaggaaatgtttcgttc HQT-V304S-R-up-pck tgccataggtgtgcctgtgaacgaaac HQT-V304P-F-up-pck ccaggttacttaggaaatgttccgttc HQT-V304P-R-up-pck gccataggtgtgcctgtgaacggaac HQT-V304T-F-up-pck ccaggttacttaggaaatgttacgttc HQT-V304T-R-up-pck tgccataggtgtgcctgtgaacgtaac HQT-V304A-F-up-pck ccaggttacttaggaaatgttgcgttc HQT-V304A-R-up-pck tgccataggtgtgcctgtgaacgcaac HQT-V304Y-F-up-pck ccaggttacttaggaaatgtttagttc HQT-V304Y-R-up-pck tgccataggtgtgcctgtgaactaaac HQT-V304H-F-up-pck ccaggttacttaggaaatgttcacttc HQT-V304H-R-up-pck tgccataggtgtgcctgtgaagtgaac HQT-V304Q-F-up-pck ccaggttacttaggaaatgttcagttc HQT-V304Q-R-up-pck tgccataggtgtgcctgtgaactgaac HQT-V304N-F-up-pck ccaggttacttaggaaatgttaacttc HQT-V304N-R-up-pck tgccataggtgtgcctgtgaagttaac HQT-V304K-F-up-pck ccaggttacttaggaaatgttaagttc HQT-V304K-R-up-pck tgccataggtgtgcctgtgaacttaac HQT-V304D-F-up-pck ccaggttacttaggaaatgttgacttc HQT-V304D-R-up-pck tgccataggtgtgcctgtgaagtcaac HQT-V304E-F-up-pck ccaggttacttaggaaatgttgagttc HQT-V304E-R-up-pck tgccataggtgtgcctgtgaactcaac HQT-V304C-F-up-pck ccaggttacttaggaaatgtttgtttc HQT-V304C-R-up-pck tgccataggtgtgcctgtgaaacaaac HQT-V304W-F-up-pck ccaggttacttaggaaatgtttggttc HQT-V304W-R-up-pck tgccataggtgtgcctgtgaaccaaac HQT-V304R-F-up-pck ccaggttacttaggaaatgttcggttc HQT-V304R-R-up-pck tgccataggtgtgcctgtgaaccgaac 2) Prepare a 20 μL system in an RNase-free centrifuge tube according to the table below.

[0077] 2x Primestar Max Mix 10μL Primer F 2μL Primer R 2uL Template 1ul <![CDATA[ddH2O]]> 5ul 3) Run the PCR instrument at 98℃ for 3 minutes, and then perform 30 cycles. The specific annealing and extension temperatures need to be changed according to different primers. After the reaction, maintain at 4℃.

[0078] 4) Dpn I. Digestive Template System PCR products 20μL 10x cutsmart 2.5μL Dpnl 1uL <![CDATA[ddH2O]]> 1.5ul PCR procedure: 37°C water bath for 1 hour.

[0079] 5) Transformation Remove competent cells and thaw them on ice. Then add 10 μL of the digested solution described above, mix gently, and incubate on ice for 20 min. Incubate at 42°C for 90 s, then immediately incubate on ice for 3 min. Add 1 mL of blank LB and incubate at 37°C with gentle shaking for 1 h. Centrifuge and plate all cells. Observe colony formation overnight and perform sequencing verification.

[0080] 4.1.2 Site-directed mutagenesis screening The obtained mutant library was screened by fermentation. Single colonies were selected from the resistance plate and inoculated into a 96-well screening plate, and cultured at 37℃ and 200 r / min for 12 h. 100 μL of the cultured seed culture was transferred to a 48-well plate containing 1 mL of M9 fermentation medium. CA and QA substrate were added to the fermentation medium to a final concentration of 150 mg / L and 1 g / L, respectively, for fermentation. OD was collected. 600 To achieve a concentration of 0.6-0.8, IPTG at a final concentration of 100 μM was added to induce expression at 30 ℃ for at least 16 h. The supernatant from the fermentation broth was collected by centrifugation, and the synthesis of CGA was detected by HPLC. Nt HQT wild-type was used as a negative control to screen for mutants with the highest CGA yield.

[0081] 4.2 Iterative Combinatorial Mutation and Screening The selected single-site mutants were subjected to iterative combined mutations to obtain two-site combined mutants. The combined mutant plasmids were then transformed into the expression host using a heat shock method. E. coliBL21(DE3) competent cells were plated on LB agar plates containing 50 μg / mL Kan and cultured overnight at 37°C for 12–16 h to obtain a hybrid mutant strain. Positive colonies were selected and inoculated into 96-well selection plates containing 500 μL of LB medium per well for overnight culture. 100 μL of seed culture was transferred to 48-well deep-dip plates containing 1 mL of M9 fermentation medium (50 μg / mL Kan) for fermentation. The fermentation medium was supplemented with 150 mg / L caffeic acid and 1 g / L QA substrate. OD was collected. 600 When the concentration reached 0.6-0.8, IPTG at a final concentration of 100 μM was added to induce expression at 30℃ for more than 16 h; the supernatant was collected by centrifugation of the fermentation broth and the synthesis of CGA was detected by HPLC.

[0082] 5. Fermentation culture for the production of chlorogenic acid 5.1 Culture medium and fermentation method: 1) LB medium (1L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride (NaCl), bring the volume to 1L with double-distilled water, sterilize at 121 ℃ for 15 min. If preparing solid medium, add 15 g agar.

[0083] 2) M9 basal medium: Contains 0.5 g / L sodium chloride (NaCl), 17.1 g / L sodium dihydrogen phosphate dodecahydrate (Na2HPO4·12H2O), 1.0 g / L ammonium chloride (NH4Cl), 3.0 g / L dipotassium hydrogen phosphate (KH2PO4), 5 mM magnesium sulfate (MgSO4), and 0.1 mM calcium chloride (CaCl2), supplemented with the required proportions of glucose and xylose. Trace elements are added to the medium to achieve a final concentration of 0.5 mg / L cobalt chloride (CoCl2), 1 mg / L thiamine, 0.03 mg / L boric acid (H3BO3), 0.38 mg / L copper chloride (CuCl2), 0.4 mg / L disodium ethylenediaminetetraacetate (Na2EDTA), 1.6 mg / L manganese chloride (MnCl2), 0.94 mg / L zinc chloride (ZnCl2), and 3.6 mg / L... Ferrous chloride (FeCl2). At the start of fermentation, add 100 μg / mL kanamycin to the culture medium. If necessary, supplement with 1 mM isopropyl-β-D-thiogalactoside (IPTG) and supplement the culture with 50 mg / L phenylalanine or 50 mg / L tyrosine.

[0084] 3) Fermentation medium: glucose 20 g / L, xylose 10 g / L, (Na2HPO4·12H2O) 17.10 g, potassium dihydrogen phosphate (KH2PO4) 3.00 g, sodium chloride 0.50 g, ammonium chloride 1.00 g, yeast extract 5.00 g, tryptone 10 g, magnesium sulfate (MgSO4) 246.00 mg, calcium chloride dihydrate (CaCl2·2H2O) 14.70 mg, and 1 ml of trace element solution. Each liter of the trace element solution contains: 20.00 g of ferric chloride hexahydrate (FeCl3·6H2O), 10.00 g of calcium chloride monohydrate (CaCl2·H2O), 0.03 g of copper sulfate pentahydrate (CuSO4·5H2O), 0.05 g of manganese chloride tetrahydrate (MnCl2·4H2O), and 0.10 g of zinc sulfate heptahydrate (ZnSO4·7H2O).

[0085] 5.2 Shake flask fermentation and fermenter fermentation 5.2.1 Shake-flask fermentation Take either the culture tube or a piece of the two constructed fermentation strains, CA production strain MG09 (MG1655: ) ptsG : pykA : pykF : tyrR : pheA :Δ manZ :pETDuet-1- tyrA fbr - aroG fbr - tktA -P trc aroL :pET28m- R g tal - hpaBC ) and CGA producing strain BD07 (BW25113 Δ xylA :Δ tyrA :Δ ydiI :pACYCDuet-1- aroG fbr - aroB - tktA- P trc -qutB2 :pET28m-A t4cl - NthqtInoculate each culture into a shake flask containing 20 mL of LB medium and incubate overnight at 37°C and 180 rpm. Add 2% seed culture to a shake flask containing 50 mL of fermentation medium, add the appropriate antibiotic, and incubate at the appropriate temperature and 200 rpm for cell OD. 600 When the concentration reaches approximately 0.6, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1.0 mmol / L and glucose and xylose at a concentration of 5 g / L.

[0086] 5.2.2 Fermentation in a fermenter High-density CGA fermentation was conducted using a 5L microbial reactor with 2L of fermentation medium. CGA fermentation strains MG09 and BD07 were transferred from preservation tubes or picked from plates to 20mL of LB medium and cultured overnight at 37℃ and 180rpm in a shaker. Then, at a 1% ratio, they were transferred to 200mL of LB medium and cultured at 37℃ and 180rpm in a shaker until the cell OD600 reached approximately 0.6–0.8. The medium containing MG09 and BD07 was then used as a seed culture and transferred to a fermenter containing 2L of fermentation medium to simultaneously culture both strains for synergistic fermentation. The microbial reactor could monitor pH changes in real time, and ammonia was automatically added to maintain the pH at 7.00. The system could be coupled with the agitator speed; by controlling the agitation speed at 300–600 rpm, dissolved oxygen (DO) was maintained at >20%, and the fermenter aeration rate was set at 1.5–2.0 vvm. When the bacterial OD600 reached 10, the temperature was adjusted to 28℃, and IPTG was added to a final concentration of 1 mmol. Samples were taken every 2 hours, and the carbon source and CGA in the fermentation broth were determined by liquid chromatography, as well as the bacterial OD600. When the glucose concentration was below 10 g / L, glucose was added to bring the glucose concentration back to at least 10 g / L. When the xylose concentration was below 5 g / L, xylose was added to bring the xylose concentration back to at least 5 g / L.

[0087] II. Experimental Results: 1 Nt Exploring the Structure-Function Relationship of HQT Protein 1.1 Nt Analysis of the three-dimensional crystal structure of HQT protein 1.1.1 Establishment of target protein homology model Based on the HQT protein sequence information (SEQ ID NO.2), sequence alignment was performed in the PDB database to identify templates with high sequence identity for homology modeling. X-ray analysis revealed that the crystal structure with PDB ID 4G22 showed the highest sequence identity with the target protein, and also had the highest resolution of 1.70 Å. Considering all factors, we believe that PDB ID 4G22 is more suitable as the template protein.

[0088] Using this as a template, homology modeling was performed on the Swiss-model platform, and the resulting three-dimensional structural model of the target protein was evaluated as follows. Figure 2 As shown. The protein model was optimized using SPDB software to minimize energy, which was then used for model evaluation and molecular docking. The constructed protein model is shown below. Figure 3 As shown.

[0089] 1.1.2 Target Protein Nt Evaluation of HQT homology model The established homology model was evaluated using PROCHECK (https: / / servicesn.mbi.ucla.edu / PROCHECK / ). The Laplace plot analysis focused on the rationality of the non-glycine and non-proline carbon backbone in the model, providing the number and proportion of amino acids remaining in the most desirable regions. In the PROCHECK Laplace plot analysis of the model, black squares represent non-glycine and non-proline residues; red areas indicate optimal regions; bright yellow indicates reasonable regions; light yellow indicates generally reasonable regions; and white areas indicate unacceptable regions. A high-quality model requires that the proportion of amino acids in the red regions exceeds 90%, and the proportion in the white regions is less than 5%.

[0090] The overall quality factor of the established homology model was evaluated using SAVE 6.0 (http: / / services.mbi.ucla.edu / SAVES / ), primarily based on the following four metrics: Verify 3D, ERRAT, PROVE, and WHATCHECK. Verify 3D compares the model to the primary structure of amino acids, achieving a PASS rating. A 3D / 1D value greater than 0.2 for over 80% of residues indicates a satisfactory model quality; values ​​below 0.2 require further correction. ERRAT calculates the number of non-bonded interactions (side chains) between different atomic pairs within a 0.35 nm range. A higher Overall quality factor value in the ERRAT results is better; this value can reach 95% for high-resolution crystal structures, but only around 91% for medium-resolution structures. PROVE evaluates the difference between the model and a pre-calculated column of standard volumes, expressed as a Z-score. The Z-score, as a statistical value, shows the degree of matching between the template protein and the target protein; a low Z-score indicates no matching structure was found. WHATCHECK includes a large number of tests that can detect differences between the submitted protein structure and the normal structure. If the green color accounts for a large proportion, the test is considered successful.

[0091] target protein Nt The Laplace plot analysis results of the HQT 3D structural model are as follows: Figure 4 As shown, the modeled structure contains 369 N-Gly and N-Pro amino acid residues. Of these, 325 (88.1%) are located in the optimal regions marked A, B, and L; 41 (11.1%) are located in the reasonable regions marked a, b, l, and p; 2 (0.5%) are located in the maximum acceptable regions marked -a, -b, -l, and -p; and 1 (0.3%) is located in the disallowed region. These data indicate that the target protein... Nt The dihedral angles of the peptide chains obtained by HQT based on homology modeling of 4G22 are reasonable, and the overall structure of the model has good stability and stereochemical properties.

[0092] target protein Nt The HQT 3D structural model received a PASS rating in Verify_3D. Figure 5As shown, 91.14% of the residues have a 3D / 1D value greater than 0.2, indicating that the model quality is acceptable. The ERRAT results show an overall quality factor of 93.705; the average Z-score for PROVE evaluation is -0.555, meaning the searched structure is matched; the WHATCHECK results show a high proportion of green, indicating a pass, such as... Figure 6 As shown. The three-dimensional structural model of target protein 52 can be used for further analysis.

[0093] Molprobity is currently a relatively comprehensive protein structure detection tool in structural biology, targeting proteins. Nt HQT test results are as follows Figure 7 As shown in the prediction results, the constructed three-dimensional model of the target protein can basically pass the model detection and can be used for the next step of molecular docking.

[0094] 1.2 Analysis of the substrate-specific binding structure of caffeoyl coA and QA The structural information of Caffeoyl CoA and QA is shown in Table 1 below. (The two are then compared with...) Nt HQT target proteins were docked using Autodock Vina software to obtain preliminary docking phase structures. The phases with the highest docking energies were selected for structural extraction (Caffeoyl CoA had a highest docking energy of -8.5 kcal / mol, and QA -4.8 kcal / mol). Interaction analysis of the conformations revealed that Caffeoyl CoA and QA could dock well into the pre-defined binding pockets, such as... Figure 8 As shown, this demonstrates that the predicted binding pocket is reasonable. A 2D diagram of the binding patterns of Caffeoyl CoA and QA with the target protein is shown below. Figure 9As shown, Caffeoyl CoA forms hydrogen bonds with Ile36, Leu33, His159, Ala284, Asp286, Ser289, Arg290, Asn371, and Arg375 of the target protein, and hydrophobic interactions with Val304 and Leu332 of the target protein. QA forms hydrogen bonds with Gly38, Arg39, His41, and Arg358 of the target protein. The amino acids that form hydrogen bonds with the target protein, such as His159, Asn371, Gly38, Arg39, His41, and Arg358, may be key amino acids affecting the binding of small molecules Caffeoyl CoA and QA to the target protein. Further analysis of the key amino acids in the target protein influencing the binding of Caffeoyl CoA and QA, combined with the bioinformatics analysis results of the target protein's HQT structure, is needed to provide a theoretical basis for future mutation studies.

[0095] Table 1 Structural information of small molecule compounds

[0096] 2. Computer-Aided Rational Design Implementation Nt Preliminary modification of HQT enzyme molecules 2.1 Nt HQT Active Center Prediction Preliminary project research revealed that the catalytic center of the HCT protein's crystal structure has been identified. The plan is to use the catalytic center of the HCT protein as a reference to predict target proteins. Nt The active site of HQT protein, such as Figure 10 As shown. The predicted amino acids surrounding the active site for Caffeoyl CoA binding include: I40, T44, Y46, V157, D163, G164, L165, S167, I168, I171, K246, L259, V283, A284, D286, R290, V304, L332, N371, S372, W373, T374, R375, L376, and M391. The predicted amino acids surrounding the active site for QA binding include: G38, R39, I40, H41, L42, A284, V304, F363, N369, N371, W373, T401, and Y403.

[0097] 2.2 Mutation screening using computer-predicted sites 2.2.1 Site-directed saturation mutagenesis and fermentation screening Based on predictions, G38 and V304 were identified as the active sites requiring modification. This study constructed a total of 38 mutants using site-directed saturation mutagenesis, and the obtained mutant library was then... NtUsing wild-type HQT as a negative control, fermentation screening was conducted to identify mutants with the highest CGA yield. Experimental results showed that the G38A and V304E mutants produced the highest CGA yields. Figure 11 and Figure 12 As shown, their concentrations are 80.6 mg / L and 89.2 mg / L, respectively.

[0098] 2.2.2 Iterative combination of mutations to screen for superior mutants Selected single-point mutants Nt HQT G38A , Nt HQT V304E Iterative combined mutations were performed to obtain two-site combined mutants. Nt HQT G38A&V304E ( Figure 12 ). Transform the combined mutant plasmid into E. coli The synthesis of CGA was detected by post-fermentation of BL21(DE3). The experimental results show that... Nt HQT G38A&V304E The mutant expression yielded the highest CGA production, such as Figure 13 As shown, the concentration reached 100 mg / L. Finally, the selected dominant mutants were... Nt HQT G38A&V304E The applicant previously designed and developed a "dual-microbe symbiotic" co-fermentation system, referencing the article "High-Level Biosynthesis of Chlorogenic Acid from Mixed Carbon Sources of Xylose and Glucose through a Rationally Refactored Pathway Network" by Yuhui Wang et al. J Agric Food Chem The techniques and conditions described in [reference needed](doci: 10.1021 / acs.jafc.3c08587) are described below. In this system, one strain is designed to metabolize xylose to synthesize caffeic acid; the other strain metabolizes glucose to synthesize quinic acid and the final product chlorogenic acid. Both strains are designed to metabolize a "glucose-xylose" dual carbon source, which effectively reduces growth competition between them. This engineered strain is genetically stable, can be scaled up, and the use of xylose reduces production costs, demonstrating potential for industrial production. Figure 14 As shown, the final CGA yield in shake flasks was 150 mg / L, a 1.5-fold increase compared to the yield in the authors' earlier studies. The final CGA yield in a 5L fermenter was 5916.82 mg / L. Figure 15 The amount of glucose and xylose consumed during this fermentation process is as follows: Figure 16 As shown, the final fermenter data for CGA are compared with those published by Wenjing He et al. De Novo Biosynthesis of Chlorogenic Acid in Yarrowia lipolytica throughCis-Acting Element Optimization and NADPH Regeneration Engineering( J Agric Food Chem The yield in (doi: 10.1021 / acs.jafc.4c12056) was 4837.32 mg / L, which is 1.22 times higher.

[0099] The amino acid sequence information involved in this invention is as follows: Arabidopsis thaliana-derived At 4CL enzyme (SEQ ID NO.1) Tobacco-derived Nt HQT enzyme MGSEKMMKINIKESTLVKPSKPTPTKRLWSSNDLIVGRIHLLTVYFYKPNGSSNFFDSKIMKEALSNVLVSFYPMAGRLARDEQGRIEINCNGEGVLFVEAESDAFVDD FGDFTPSLELRKLIPTVDTSGDISTFPLIIFQVTRFKCGGVSLGGGVFHTLSDGLSSIHFINTWSDIARGLSVAIPPFIDRTLLRARDPPTSSFEHVEYHPPPSLISSSK SLESTSPKPSTTTMLKFSSDQLGLLKSKSKHDGSTYEILAAHIWRCTCKARALSDDQLTKLHVATDGSRLCPPLPPGYLGNVVFTGTPMAKSSELLQEPLTNSAKRIHS ALSKMDDNYLRSALDYLELLPDLSALIRGPTYFASPNLNINSWTRLPVHDSDFGWGRPIHMGPACILYEGTVYILPSPNSKDRNLRLAVCLDADHMPLFEKYLYEF(SEQ ID NO.2) The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mutant HQT enzyme, characterized in that, It is obtained by mutating one or more amino acid sites in the 38th and 304th amino acid sites of the HQT enzyme, wherein the amino acid sequence of the HQT enzyme is shown in SEQ ID NO.

2.

2. The HQT enzyme mutant of claim 1, wherein The HQT enzyme mutant is a mutation based on the above-mentioned HQT enzyme, and the HQT enzyme mutant is selected from any one or more mutants in the following group: G38A, V304E, G38A / V304E.

3. A deoxyribonucleic acid molecule, characterized in that, The deoxyribonucleic acid molecule encodes the above-mentioned HQT enzyme mutant.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the deoxyribonucleic acid molecule of claim 3 or a DNA sequence mutated from the deoxyribonucleic acid molecule as the parent; Further, the recombinant expression vector further comprises At 4CL, the coding gene of which is shown as SEQ ID NO.

1. At The amino acid sequence of 4CL is shown as SEQ ID NO.

1.

5. A host cell, characterized in that, The host cell is capable of expressing the recombinant vector of claim 4 or the chromosome integrated with the deoxyribonucleic acid molecule of claim 3.

6. A method of preparing the HQT enzyme mutant according to claim 1, characterized in that, include: The host cells of claim 5 are cultured to express the HQT enzyme mutant protein; and the HQT enzyme mutant pure enzyme is isolated and purified.

7. The application of the HQT enzyme mutant of claim 1 or 2, the deoxyribonucleic acid molecule of claim 3, the recombinant expression vector of claim 4, and the host cell of claim 5 in the field of chlorogenic acid synthesis.

8. A method for synthesizing chlorogenic acid, the method comprising fermenting the host cell described in claim 5 to obtain chlorogenic acid.

9. The method of synthesis of claim 8, wherein, The method includes: the chlorogenic acid synthesis substrate includes caffeoyl-CoA and quinic acid.

10. The method of synthesis of claim 8, wherein, The fermentation is carried out using a dual-microbial synergistic fermentation system.

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