Engineered Escherichia coli and method for generating apocynin through whole-cell transformation thereof

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a method for generating apocynin through whole-cell transformation of engineered escherichia coli. Comprising the following steps: transforming a substrate 3, 4-dihydroxyacetophenone by using engineered escherichia coli carrying a plasmid pETM6-MsCOMT I319A-pmtn-plxS, so as to obtain apocynin; msCOMT refers to caffeic acid-O-methyltransferase from medicago sativa, mtn and luxS refer to two enzymes in SAM (S-adenosylmethionine) regeneration cycle, and SAM refers to S-adenosylmethionine; the MsCOMT I319A means that an amino acid residue I319 of the MsCOMT has been subjected to alanine scanning mutagenesis, and a mutant MsCOMT I319A is obtained; the invention also provides a novel engineered escherichia coli. Biotransformation conditions used in the method are normal temperature and normal pressure, various reagents for escherichia coli culture are cheap and environment-friendly, and the yield of apocynin obtained through biotransformation is considerable.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and particularly relates to an engineered Escherichia coli and a method for converting whole cells thereof into apocynin. Background Art

[0002] Apocynin (Ap), also known as Acetovanillone, is a small aromatic molecule compound with the chemical structure shown in Figure 1 . This molecule has a weak vanilla smell and is a light yellow or white crystalline powder. It is slightly soluble in water and easily soluble in organic solvents. It is one of the main active ingredients of the traditional Chinese medicine Picrorhizoma Coptidis and is widely present in other medicinal plants such as Sonneratia ovata, Lycopodiella vulgaris, Cyperus rotundus, Radix Isatidis, and Cyperus rotundus. At present, Lobumanine is mainly used in two fields: pharmaceutical and health care products and beauty and skin care products. In terms of pharmaceutical and health care products, Lobumanine is an important organic synthesis raw material with great development and utilization value. For example, the psychotropic drug iloperidone and a variety of new antimalarial drugs can be derived from it. At the same time, Lobumanine is a specific inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which can act on the respiratory, nervous, blood, immune and other systems, and has therapeutic effects on diseases such as asthma, atherosclerosis, arthritis, diabetes, hypertension, cancer, and myocardial and cerebral ischemia. In terms of beauty and skin care products, Lobumanine has the potential to be developed into anti-aging products. In 2019, researchers from Tokyo Dental University published a study in Nature, finding that apocynin can induce the expression of type XVII collagen (COL17A1) genes in the skin, thereby promoting benign competition among epidermal stem cells to maintain the youthful state of the skin. This research result was further confirmed in subsequent related studies.

[0003] Currently, traditional methods for obtaining apocynin include chemical synthesis and natural extraction. Chemical synthesis is the primary method, but it suffers from complex processes, demanding production conditions, difficulty separating reaction products, numerous reaction byproducts, and toxic reagents. For example, Zhou Yonghong et al. reported using nitrobenzene compounds as oxidants to oxidize lignin compounds to produce apocynin. This method not only requires the flammable and hazardous oxidants p-nitrobenzoic acid or 3-nitrobenzoic acid, but also suffers from high reaction temperatures (160°C to 200°C), cumbersome post-reaction processing, and low product yields. Huang Weibin et al. reported using guaiacol as a starting material, which reacts with n-propane bromide under alkaline conditions to produce 1-n-propoxy-2-methoxybenzene. This was then synthesized through non-regioselective Friedel-Crafts acetylation and selective depropylation. This method is complex, utilizes acetic anhydride, a controlled raw material for drug production, and also produces the byproduct isovanillyl acetone. Natural extraction is the most direct method to obtain apocynin. The extraction target is mainly the medicinal plant Coptis chinensis that contains this substance. However, due to the limitation of natural resources of wild Coptis chinensis, as well as the complicated extraction steps and low yield, this method is not suitable for large-scale acquisition of apocynin.

[0004] The biosynthesis of active molecules from traditional Chinese medicines (TCMs) is a key area of ​​green biomanufacturing. Biosynthesis of many important active molecules from TCMs has been achieved, including reports on apocynum venetum. Prasad et al. studied the microbial conversion of ferulic acid to apocynum venetum using Rhizopus oryzae cells. Their results showed that, in addition to the primary metabolite apocynum venetum, secondary metabolites such as dihydroferulic acid, coniferyl alcohol, and dihydroconiferyl alcohol, as well as trace metabolites such as vanillin, vanillyl alcohol, vanillic acid, and phenylethanol, were also formed. Furthermore, Negrel et al. isolated a soluble enzyme from tobacco suspension cultures stimulated with methyl jasmonate that converts feruloyl CoA to apocynum venetum in the presence of NAD. While both studies achieved the goal of biosynthesizing apocynum venetum, they each presented their own challenges. In Prasad's case, the conversion process produced numerous secondary and trace metabolites, significantly hindering the subsequent purification of apocynum venetum. Although there is no subsequent purification problem in the Negrel study, the soluble enzyme obtained has not been identified, and the substrates catalyzed by the enzyme, feruloyl CoA and NAD, are expensive and difficult to obtain, which brings difficulties to the large-scale production of Apocynin.

[0005] Based on the above analysis, whether traditional chemical synthesis and natural extraction or emerging biosynthesis in obtaining apocynin has many shortcomings, so it is necessary to explore new methods to obtain apocynin and provide effective solutions for efficient and green acquisition. In other words, this field needs a new method for biosynthesizing apocynin. Summary of the Invention

[0006] The present invention first provides a method for transforming the whole cells of engineered Escherichia coli into apocynin, wherein the substrate in the method is 3,4-dihydroxyacetophenone, and the plasmid pETM6-MsCOMT is used to transform the whole cells of engineered Escherichia coli into apocynin. I319A The engineered Escherichia coli transformed the substrate of -pmtn-pluxS to obtain apocynin.

[0007] In a specific embodiment, the engineered Escherichia coli in the method is named S23 strain, and the engineered Escherichia coli is BL21 (DE3) strain; preferably, the Escherichia coli strain DH5α is used as the host strain for plasmid construction and amplification, while the Escherichia coli strain BL21 (DE3) is used as the strain for protein expression and biotransformation.

[0008] In a specific embodiment, the method comprises firstly connecting MsCOMT, mtn and luxS respectively between the NdeI and XhoI sites of the pETM6 plasmid by a seamless cloning method.

[0009] In a specific embodiment, the method comprises using an inducer isopropyl-β-D-thiogalactopyranoside, i.e., IPTG, when culturing the engineered E. coli.

[0010] In a specific embodiment, in order to make the three genes MsCOMT, mtn, and luxS exist in a pseudooperon configuration, pETM6-mtn and pETM6-luxS are first cut with AvrII and SalI, and the resulting fragments are connected to the pETM6-MsCOMT vector cut with SpeI and SalI to obtain pETM6-MsCOMT-pmtn, pETM6-MsCOMT-pluxS, and pETM6-MsCOMT-pmtn-pluxS.

[0011] In a specific embodiment, alanine scanning mutagenesis of MsCOMT was achieved by whole plasmid PCR using site-directed mutagenesis primers and pETM6-MsCOMT as a template to obtain the mutant MsCOMT. I319A .

[0012] In a specific embodiment, a TB culture medium containing a certain concentration of substrate 3,4-dihydroxyacetophenone is used for biotransformation, preferably the composition of the TB culture medium includes 12 g·L -1 Tryptone, 24 g L -1 Yeast extract, 4 mL·L -1 Glycerol, 2.31 g·L -1 KH2PO4, 2.54 g·L -1 K2HPO4.

[0013] In a specific embodiment, the conversion temperature for whole-cell conversion to apocynin is 30-40° C., and the reaction time is more than 24 h, preferably 48 h to 72 h.

[0014] The present invention also provides an engineered Escherichia coli, wherein the engineered Escherichia coli carries the plasmid pETM6-MsCOMT I319A -pmtn-pluxS, the plasmid includes genes encoding three enzymes: MsCOMT, mtn and luxS, wherein MsCOMT refers to caffeic acid-O-methyltransferase from alfalfa, mtn and luxS are two enzymes in the SAM regeneration cycle, SAM is S-adenosylmethionine; the MsCOMT I319A The amino acid residue I319 of MsCOMT has been subjected to alanine scanning mutagenesis to obtain a mutant MsCOMT in which isoleucine I is mutated to alanine A. I319A ; pmtn and pluxS refer to the genes encoding mtn and luxS existing in a pseudooperon configuration, and the codon sequence of MsCOMT, the gene sequence of mtn and luxS are shown in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively.

[0015] In a specific embodiment, the engineered Escherichia coli is strain BL21(DE3).

[0016] The present invention has at least the following beneficial effects: It provides a novel engineered Escherichia coli and a method for transforming whole cells thereof to produce apocynin. The biotransformation conditions used in the present invention are normal temperature and pressure, the various reagents used for E. coli culture are not only inexpensive and environmentally friendly, and the biotransformation yield of apocynin is considerable. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the synthesis of apocynin by Escherichia coli using 3,4-dihydroxyacetophenone.

[0018] Figure 2 Figure 1 is an analysis diagram of the effects of three COMTs on Ap production, where Figure A is the HPLC test result and Figure B is the mass spectrometry identification result.

[0019] Figure 3 Figure A shows the effect of different culture media on Ap production, where Figure A shows the chromatographic analysis results and Figure B shows the comparison of Ap concentrations.

[0020] Figure 4 Figure 1 shows the effect of different SAM concentrations on Ap production. Figure A shows the chromatographic analysis results, and Figure B shows the comparison of Ap concentrations.

[0021] Figure 5This is the effect of the SAM regeneration system on Ap production.

[0022] Figure 6 Analysis of key amino acid residues in MsCOMT that interact with 3,4-DHAP. Figure A shows the molecular docking diagram, and Figure B compares the activities of the mutants.

[0023] Figure 7 This is the effect of biotransformation time on AP production.

[0024] Figure 8 Figures 1 and 2 show double enzyme digestion and enzyme protein expression of the pETM6-COMTs plasmid. Figure A shows double enzyme digestion of the pETM6-LcCOMT plasmid. Figure B shows double enzyme digestion of the pETM6-AtCOMT plasmid. Figure C shows double enzyme digestion of the pETM6-MsCOMT plasmid. Figure D shows prokaryotic expression of the pETM6-COMTs plasmid.

[0025] Figure 9 Figure 1 is a mass spectrometry analysis of whole-cell biotransformation. Figure A is the standard mass spectrum of AP; Figure B is the mass spectrum of the substances produced by whole-cell biotransformation.

[0026] Figure 10 Schematic diagram of pseudooperon construction. Panel A shows the assembly of MsCOMT and luxS as a pseudooperon. Panel B shows the assembly of MsCOMT and mtn as a pseudooperon. Panel C shows the assembly of MsCOMT, mtn, and luxS as a pseudooperon.

[0027] Figure 11 Schematic diagram of operon construction. Panel A shows MsCOMT and luxS assembled as an operon. Panel B shows MsCOMT and mtn assembled as an operon. Panel C shows MsCOMT, mtn, and luxS assembled as an operon.

[0028] Figure 12 Figure 2 shows the 2D interaction of Ap docking. Figure A shows the docking of Ap with MsCOMT. Figure B shows the docking of Ap with mutant I319A. DETAILED DESCRIPTION

[0029] This study proposes to use genetic engineering technology to introduce caffeic acid O-methyl transferase (COMT) genes from three different plants into Escherichia coli. The whole-cell biotransformation method is then used to compare their ability to convert the inexpensive substrate 3,4-dihydroxyacetophenone to produce Ap. At the same time, key residue analysis and conversion conditions are explored for the COMT with the highest conversion activity, thereby laying the foundation for further realizing the de novo biosynthesis of Ap.

[0030] Figure 1 Schematic diagram of the synthesis of apocynin by Escherichia coli using 3,4-dihydroxyacetophenone. Figure 1 In the human body, Mtn and luxS are two important enzymes in the S-adenosylmethionine (SAM) regeneration cycle. SAH, SRH, Hcys, and Met represent S-adenosyl-L-homocysteine, S-ribosyl-L-homocysteine, L-homocysteine, and L-methionine, respectively.

[0031] Example 1

[0032] This example describes the materials and methods for synthesizing apocynin using 3,4-dihydroxyacetophenone in Escherichia coli.

[0033] 1. Culture medium, strains, and genes

[0034] LB medium (5 g·L -1 Yeast extract, 10 g L -1 Tryptone and 10 g·L -1 NaCl) is used for plasmid amplification, inoculum preparation and protein induction expression. Contains 4mmol·L -1 LB medium, TB medium, and M9Y medium were used for biotransformation of 3,4-dihydroxyacetophenone. The composition of M9Y medium was as follows: 17.1 g·L -1 Na2HPO4·12H2O,3g·L -1 KH2PO4, 1.5 g·L -1 NaCl, 1 g L -1 NH4Cl, 20 g·L -1 Glucose, 2 g·L -1 Tryptone, 7 g·L -1 Yeast extract, 1 mmol·L -1 MgSO4, 0.1mmol·L -1 CaCl2. The composition of TB culture medium is 12g·L -1 Tryptone, 24 g L -1 Yeast extract, 4 mL·L -1 Glycerol, 2.31 g·L -1 KH2PO4, 2.54 g·L -1 If necessary, add 100 μg·mL K2HPO4 to the culture medium -1ampicillin. Escherichia coli strain DH5α was used as the host for plasmid construction and amplification, while E. coli strain BL21(DE3) was used for protein expression and biotransformation. The codons of caffeic acid-O-methyltransferase (COMT) from Medicago sativa (gene accession number: GU066087.1), Arabidopsis thaliana (gene accession number: U70424.1), and Ligusticum chuanxiong (gene accession number: KR106206.1) were optimized for E. coli and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The Mtn and luxS genes were cloned from genomic DNA of BL21(DE3) using primers.

[0035] The codon sequence of MsCOMT, and the gene sequences of mtn and luxS are shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively.

[0036] SEQ ID NO 1:

[0037]

[0038]

[0039] SEQ ID NO 2:

[0040]

[0041] SEQ ID NO 3:

[0042]

[0043] 2. Plasmid construction

[0044] Different plasmids were constructed using different primers. The ePathBrick plasmid pETM6, derived from pETDuet-1, was used for gene assembly. Using seamless cloning, MsCOMT (Medicago truncatula), AtCOMT (Arabidopsis thaliana), LcCOMT (Ligusticum chuanxiong), mtn, and luxS were ligated between the NdeI and XhoI sites of the pETM6 plasmid. The pETM6 vector contains four isoenzyme sites (AvrII, XbaI, SpeI, and NheI), allowing for the ligation of multiple genes in different configurations. To create a pseudooperon configuration for the MsCOMT, mtn, and luxS genes, pETM6-mtn and pETM6-luxS were digested with AvrII and SalI. The resulting fragments were then ligated into the pETM6-MsCOMT vector digested with SpeI and SalI to generate pETM6-MsCOMT-pmtn, pETM6-MsCOMT-pluxS, and pETM6-MsCOMT-pmtn-pluxS. Alanine scanning mutagenesis of MsCOMT was performed by whole-plasmid PCR using site-directed mutagenesis primers and pETM6-MsCOMT as a template. The resulting PCR product was digested with DpnI. These plasmids were transformed into E. coli DH5α competent cells, and positive clones were identified by double restriction digestion and sequencing.

[0045] 3. Molecular docking of substrate 3,4-dihydroxyacetophenone with MsCOMT

[0046] The molecular structure of 3,4-dihydroxyacetophenone (Compound ID: 14530) was obtained from NCBIPubchem (http: / / pubchem.ncbi.nlm.nih.gov / ) and further docked with the crystal structure of the SAM-MsCOMT (PDB: 1KYW) binary complex using Autodock vina. PyMOL was used to view the structural model generated in this invention and to analyze the structure of MsCOMT surrounding 3,4-dihydroxyacetophenone. amino acid residues within the range.

[0047] 4. Alanine scanning mutagenesis

[0048] Following the methods described for plasmid construction, alanine scanning mutagenesis was performed on the aforementioned amino acid residues to identify key residues interacting with 3,4-dihydroxyacetophenone. Mutants with increased activity were selected for subsequent investigation of optimal biotransformation conditions.

[0049] 5. Enzyme protein expression and shake flask batch fermentation test

[0050] The strain was inoculated into 5 mL of 100 μg mL -1Penicillin-containing LB liquid medium was incubated at 37°C and 225 r / min -1 The next day, the seed solution was inoculated into new 50 mL LB liquid medium at a ratio of 1:50 and incubated at 37°C and 225 r·min. -1 When the optical density at 600 nm (OD 600 ) reached 0.6, the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the bacterial solution to make the final concentration 0.5 mmol·L -1 , then at 16℃ and 225r·min -1 After 16 hours of induction, 1 mL of the bacterial solution was taken for SDS-PAGE electrophoresis to identify the expression of enzyme proteins in the strain.

[0051] After the expression and identification of the enzyme protein, the remaining bacterial solution was subjected to batch fermentation tests according to the following steps. First, the bacterial solution was heated at 5000 r·min. -1 After centrifugation for 10 min, the cells were collected. -1 Suspend the cells in the culture medium containing 3,4-dihydroxyacetophenone and adjust the OD 600 Adjusted to 1. For the study on the promotion of MsCOMT activity by S-adenosyl-L-methionine (SAM), in addition to adding substrate to the culture medium, different concentrations of SAM (1 g·L -1 ,1.5g·L -1 and 2.0 g·L -1 Finally, the bacterial solution was heated at 28°C and 225 r·min -1 The transformation was carried out under the same conditions for 48 h, and 5 mL of fermentation broth was taken from the culture medium every 24 h for detection and comparison of Ap production.

[0052] 6. Discussion on the optimal conditions for biotransformation

[0053] According to the factors and levels designed in Table 1, the plasmid pETM6-MsCOMT I319A -pmtn-pluxS strain S23 was used for biotransformation orthogonal experiments. 5 mL samples were collected from each group 48 hours after transformation for Ap concentration testing. Based on the orthogonal test results, the optimal levels of each factor were selected for biotransformation experiments. 5 mL samples were collected at 12, 24, 36, 48, 60, and 72 hours to explore the effect of transformation time on Ap yield.

[0054] Table 1 Factors and levels of orthogonal experiment

[0055]

[0056] 7. Sample processing and product testing

[0057] The sample was extracted at 3000 r·min -1 The supernatant was collected and freeze-dried after centrifugation under the appropriate conditions for 10 minutes. The freeze-dried product was then reconstituted with 2 mL of methanol and filtered through a 0.22 μm filter. A 20 μL sample of the filtrate was then used for qualitative and quantitative analysis by high-performance liquid chromatography (HPLC, Shimadzu Corporation, Japan). The molecular weight of the target product in the filtrate was analyzed by liquid chromatography-mass spectrometry.

[0058] HPLC conditions: chromatographic column: ZORBAX SB-C18 column (4.6×250 mm, 5 μm); mobile phase: methanol: ultrapure water = 25:75; flow rate: 1 ml / min; detection wavelength: 276 nm.

[0059] Standard curve drawing: 1-(4-hydroxyphenol)-ethanol standard was dissolved in methanol and prepared at 0.1, 1.0, 10, and 100 mmol·L -1 , 1000mmol·L -1 The standard solution was analyzed by HPLC and the data were used to draw a standard curve using software.

[0060] Waters Xevo G2-XS QTof high-resolution mass spectrometry conditions: positive ion mode, drying gas flow rate of 6 L / min; nebulizer pressure of 40 Psig; nebulizing gas temperature of 325°C; sheath gas temperature of 350°C, sheath gas flow rate of 12 L / min; capillary voltage of 4000 V; mass spectrometry acquisition range of 50-1000 m / z.

[0061] 8. Data processing

[0062] The experimental data were obtained from at least three replicates and are presented as mean ± standard deviation. SPSS v19.0 statistical software was used to analyze the experimental data, and the minimum significant difference between samples was set at P < 0.05.

[0063] Example 2

[0064] This example shows the results of E. coli synthesizing apocynin using 3,4-dihydroxyacetophenone.

[0065] 1. Comparison of COMT biotransformation activity from three plant sources

[0066] The COMT genes from Arabidopsis thaliana, Ligusticum chuanxiong, and Medicago truncatula were constructed into the pETM6 vector and introduced into Escherichia coli BL21(DE3) to obtain strains S1, S2, and S3 that successfully expressed the enzyme protein. Using strain S0 containing the pETM6 vector as a control, the ability of S1, S2, and S3 to bioconvert 3,4-dihydroxyacetophenone to Ap was compared. HPLC test results showed that S0 and S3 could not produce a new chromatographic peak, but S1 and S2 could, and the new chromatographic peak was consistent with the chromatographic peak of the Ap standard ( Figure 2 A). Further mass spectrometry identification results showed that the mass-to-charge ratio (m / z) of the new substance produced by S1 and S2 was consistent with that of the Ap standard ( Figure 2 B) above Figure 2 A and Figure 2 Both results B indicate that S1 and S2 can convert 3,4-dihydroxyacetophenone into Ap. In addition, after 24h and 48h of conversion, the concentration of Ap generated by MsCOMT and LcCOMT was 90.75 mg·L -1 , 97.04mg·L -1 , 27.3mg·L -1 and 30.15 mg·L -1 ( Figure 2 B). This indicates that MsCOMT has a stronger ability to convert 3,4-dihydroxyacetophenone to Ap than LcCOMT, so MsCOMT was selected for subsequent studies.

[0067] Figure 2 Figure 1 is an analysis diagram of the effects of three COMTs on Ap production, where Figure A is the HPLC test result and Figure B is the mass spectrometry identification result. Figure 2 In A, peak 1: 3,4-dihydroxyacetophenone (3,4-DHAP) standard, peak 2: Ap standard. The asterisk represents Ap generated by biotransformation. Figure 2 Panel B shows comparison of Ap concentration. Bars represent Ap concentration, and dots indicate absorbance at 600 nm. T7, RBS, and "T" represent the T7 promoter, ribosome binding site, and T7 terminator, respectively. All data are presented as the mean ± SD of three biologically independent samples.

[0068] 2. Effects of three culture media on MsCOMT transformation activity

[0069] The expression of heterologous genes in E. coli will affect its transformation activity, and the gene expression level is regulated by many factors. One way to effectively improve its expression level is to optimize the culture medium. The present invention investigated the effects of TB culture medium, LB culture medium, and M9Y culture medium on MsCOMT transformation activity while maintaining other transformation conditions consistent. The results showed that TB culture medium had the most significant effect on MsCOMT among the three culture media ( Figure 3 A), the yields of which were 158.52 mg·L after 24 h and 48 h, respectively. -1 and 174.75 mg·L -1 ( Figure 3 B), so TB medium was selected for subsequent research.

[0070] Figure 3 Figure 1 shows the effect of different culture media on Ap production. Panel A shows the chromatographic analysis results, and Panel B shows a comparison of Ap concentrations. In Panel A, Peak 1 represents a 3,4-dihydroxyacetophenone (3,4-DHAP) standard, and Peak 2 represents an Ap standard. Asterisks represent Ap produced by biotransformation. In Panel B, bars represent Ap concentrations, and dots represent absorbance at 600 nm. All data are presented as the mean ± SD of three biologically independent samples.

[0071] 3. Effect of S-adenosylmethionine on the transformation activity of MsCOMT

[0072] To identify whether methyl donors affect the synthesis of Ap by MsCOMT, different concentrations of S-adenosylmethionine were added to TB medium. Figure 4 As shown in A, the addition of S-adenosylmethionine can significantly promote the production of Ap synthesized by MsCOMT, and the methyl donor concentration is positively correlated with the Ap synthesis concentration within the 24h and 48h conversion time ( Figure 4 B) These results suggest that SAM deficiency is a key factor restricting MsCOMT's ability to methylate 3,4-dihydroxyacetophenone. This also suggests that future research could enhance MsCOMT's ability to synthesize Ap by increasing the intracellular SAM supply.

[0073] Figure 4 The effect of different SAM concentrations on Ap production. Panel A shows the chromatogram, and Panel B compares Ap concentrations. In Panel A, Peak 1 represents a 3,4-dihydroxyacetophenone (3,4-DHAP) standard, and Peak 2 represents an Ap standard. Asterisks represent Ap produced by biotransformation. In Panel B, bars represent Ap concentration, and dots represent absorbance at 600 nm. All data are presented as the mean ± SD of three biologically independent samples.

[0074] 4. Effect of SAM regeneration system on MsCOMT transformation activity

[0075] From the above exogenous addition experiments, it can be seen that SAM has a great influence on the transformation activity of MsCOMT. However, as an auxiliary factor, SAM is expensive and not suitable for large-scale exogenous addition to transform 3,4-dihydroxyacetophenone into apocynin. Therefore, we used the mtn gene and luxS gene to construct a SAM regeneration system in Escherichia coli, and explored the effects of different arrangement configurations of them and the MsCOMT gene on the Ap yield on the vector. The results showed that when the mtn gene, luxS gene and MsCOMT gene were arranged on the vector in a pseudo-operon configuration, the yield of Ap synthesized by MsCOMT was greater than that of other configurations ( Figure 5 ), but it is higher than that of exogenous addition of 1g·L -1 The SAM is lower, which indicates that the mtn gene and luxS gene play a regenerative role in E. coli and provide SAM supplement for MsCOMT, but the supplementation level is not as high as that of exogenous addition of 1 g·L -1 Therefore, SAM regeneration needs to be further strengthened in future research.

[0076] Figure 5 The effect of the SAM regeneration system on Ap production is shown. Specifically, a comparison of apocynin (Ap) production is shown under different arrangements of the MsCOMT, mtn, and luxS genes. T7, RBS, and "T" represent the T7 promoter, ribosome binding site, and T7 terminator, respectively. Bars represent Ap concentration, and dots represent absorbance at 600 nm. All data are presented as the mean ± SD of three biologically independent samples.

[0077] Figure 5 The bottom group of bars are strains with S1 gene arrangement on the vector and 1 g·L exogenously added -1 The yield of synthetic Ap based on SAM, the second group of bars from the bottom up is the strain with an S1 arrangement configuration of genes on the vector and the yield of synthetic Ap without exogenous addition of SAM; the top group of bars is the strain with an S9 arrangement configuration of genes on the vector (MsCOMT, mtn and luxS genes are all arranged in a pseudo-operon configuration on the vector) and the yield of synthetic Ap without exogenous addition of SAM; the fourth group of bars from the top down is the strain with an S6 arrangement configuration of genes on the vector (mtn and luxS genes are all arranged in an operon configuration on the vector) and the yield of synthetic Ap without exogenous addition of SAM.

[0078] 5. Analysis of key residues affecting MsCOMT transformation activity

[0079] Molecular docking results showed that 3,4-dihydroxyacetophenone was embedded in the active site of MsCOMT, and its surrounding There are 13 amino acid residues within the range, namely M130, L136, F163, H166, F176, M180, W266, H269, D270, I319, M320, H323, N324 ( Figure 6 A). These 13 amino acid residues were subjected to alanine scanning mutagenesis to obtain 13 recombinant E. coli containing the MsCOMT mutant gene. Whole-cell biotransformation of these bacteria showed that the transformation activity of 12 mutants was lower than that of the wild type, of which the M130A and W266A mutants showed the greatest reduction in transformation activity ( Figure 6 B) I319A was the only mutant among the 13 mutants with improved transformation activity, with its yields at 24 and 48 h, respectively, being 198.32 mg·L -1 and 228.37 mg·L -1 ( Figure 6 B), can be used as a subsequent research object for the optimization of whole-cell biotransformation conditions.

[0080] Figure 6 This is an analysis of the key amino acid residues that interact with 3,4-DHAP in MsCOMT. Figure A is a molecular docking diagram, and Figure B is a comparison of mutant activities. The left side of Figure A is a global molecular docking diagram, and the right side is a local molecular docking diagram. Green, yellow, blue, and purple represent MsCOMT, 3,4-DHAP, SAM, and 13 amino acid residues, respectively. Figure B compares the changes in MsCOMT activity after the 13 amino acids were mutated to alanine under the same whole-cell transformation conditions, using wild-type MsCOMT as a control. The bars represent Ap concentrations, and the dots represent absorbance values ​​at 600 nm. All data are presented as the mean ± standard deviation of three biologically independent samples.

[0081] 6. Discussion on the optimal conditions for whole-cell transformation

[0082] In order to optimize the whole-cell transformation conditions of the S23 strain, four factors, namely substrate concentration, inducer IPTG concentration, induction temperature and transformation temperature, were selected as independent variables, and the change in apocynum yield was used as the dependent variable to conduct a 4-factor 3-level orthogonal experiment. The orthogonal experiment results and range analysis are shown in Table 2, and the variance analysis results are shown in Table 3. The range R values ​​and variance analysis F values ​​in Tables 2 and 3 show that the order of influence of each factor on apocynum yield is A>C>D>B, that is, transformation temperature>induction temperature>substrate concentration>IPTG concentration. Size analysis showed that the best combination was A2B1C1D3, i.e., a conversion temperature of 35°C and an IPTG concentration of 0.1 mmol·L -1 , induction temperature 15°C and substrate concentration 8 mmol·L -1Based on the optimal combination conditions, the effect of conversion time on the concentration of apocynin was further explored. The results showed that within the selected time period, S23 could steadily convert 3,4-dihydroxyacetophenone into apocynin. After 72 hours of conversion, the apocynin yield was 544 mg·L -1 The yield was 40.9%, and the yield after 60 h of conversion was 537 mg·L -1 Compared with the yield of 40.4%, there is no significant difference ( Figure 7 ), which indicates that the conversion of substrate to product by S23 has gradually reached its maximum value since 60h.

[0083] Figure 7 Figure 2 shows the effect of bioconversion time on AP production. Bars represent AP concentration, and circles represent absorbance at 600 nm. All data points are presented as the mean ± standard deviation of three biologically independent samples.

[0084] The final preferred plasmid of the present invention is the pETM6 plasmid carrying COMT from alfalfa, mtn and LuxS from Escherichia coli. In the plasmid, MsCOMT, mtn and LuxS are arranged in a pseudooperon form, and the amino acid residue I319 of MsCOMT has been subjected to alanine scanning mutagenesis to obtain a mutant MsCOMT in which I is mutated to A. I319A .

[0085] Table 2 Orthogonal test results

[0086]

[0087] Table 3 Results of variance analysis

[0088]

[0089] Note: ** indicates significant difference (P<0.01).

[0090] Figure 8 Figure 1 shows the double enzyme digestion and protein expression of the pETM6-COMTs plasmid. Figure A shows the double enzyme digestion of the pETM6-LcCOMT plasmid. Figure B shows the double enzyme digestion of the pETM6-AtCOMT plasmid. Figure C shows the double enzyme digestion of the pETM6-MsCOMT plasmid, where M is a 5000bp DNA ladder and lane 1 shows the double enzyme digestion results. Figure D shows the prokaryotic expression of pETM6-COMTs, where M is a protein molecular weight standard and lanes 1, 3, 5, and 7 show pETM6, pETM6-LcCOMT, pETM6-AtCOMT, and pETM6-MsCOMT after 0.5 mmol / L -1After IPTG induction for 0 h, lanes 2, 4, 6, and 8 showed pETM6, pETM6-LcCOMT, pETM6-AtCOMT, and pETM6-MsCOMT were induced with 0.5 mmol·L -1 IPTG induction for 5 h.

[0091] Figure 9 Figure 1 shows the mass spectrometry analysis of whole-cell biotransformation. Figure A shows the standard mass spectrum of AP; Figure B shows the mass spectrum of the product produced by whole-cell biotransformation. 167.07 is the mass-to-charge ratio of AP after adding an H+.

[0092] Figure 10 Schematic diagram of pseudooperon construction. Figure A shows the assembly of MsCOMT and luxS as a pseudooperon. Figure B shows the assembly of MsCOMT and mtn as a pseudooperon. Figure C shows the assembly of MsCOMT, mtn, and luxS as a pseudooperon. The pseudooperon structure is generated by digesting the donor vector with the restriction enzymes AvrII (A) and SalI (L) and then ligating it with the recipient vector that has been digested with the enzymes SpeI (S) and SalI (L). Black boxes and arrows: T7 promoter and lacO terminator; orange boxes: ribosome binding site; green boxes with arrows: open reading frame; red boxes: T7 terminator; bla: ampicillin resistance gene; lacI: lactose repressor gene.

[0093] Figure 11 Schematic diagram of operon construction. Panel A shows the assembly of MsCOMT and luxS as an operon. Panel B shows the assembly of MsCOMT and mtn as an operon. Panel C shows the assembly of MsCOMT, mtn, and luxS as an operon. The operon structure is generated by digesting the donor vector with the restriction enzyme pair XbaI (X) and SalI (L) and then ligating it with the recipient vector digested with the enzyme pair SpeI (S) and SalI (L). Black boxes and arrows: T7 promoter and lacO terminator; orange boxes: ribosome binding site; green boxes with arrows: open reading frame; red boxes: T7 terminator; bla: ampicillin resistance gene; lacI: lactose repressor gene.

[0094] Figure 12 Figure 2 shows the 2D interaction of the Ap molecule docking. Figure A shows the docking of Ap with MsCOMT. Figure B shows the docking of Ap with mutant I319A. Green dashed lines indicate hydrogen bonds between amino acid residues and Ap. The red underline indicates the amino acid residue corresponding to position 319 in the wild-type and mutant.

[0095] Analysis and Conclusion

[0096] As a valuable asset of traditional Chinese medicine, Chinese herbal medicines have been used to extract a variety of active ingredients with significant clinical effects, such as artemisinin, ginsenosides, and tanshinone. However, these ingredients are usually present in Chinese herbal medicines in the form of secondary metabolites, so their content in plant cells is usually low, making it difficult to obtain them in large quantities through traditional extraction methods. The biosynthesis of active substances in Chinese herbal medicines is an important branch of current green biomanufacturing, and many important active molecules have been synthesized through this method. Currently, microbial whole-cell transformation is one of the commonly used methods to achieve green biomanufacturing. It usually involves the precise modification, transformation, or expansion of microbial metabolic pathways, the construction of new metabolic pathways, and the change of the original metabolic characteristics of microorganisms. It is also combined with microbial genetic regulation, metabolic regulation, and biochemical engineering to improve the activity or yield of natural products. According to literature reports, people have successfully synthesized a variety of natural products, including terpenes, phenylpropanoids, alkaloids, etc., using microbial whole-cell transformation methods. Therefore, the present invention selects the model microorganism E. coli as the experimental strain and uses genetic engineering technology to genetically transform it to achieve the goal of whole-cell conversion of 3,4-dihydroxyacetophenone to produce apocynin.

[0097] MsCOMT, a caffeic acid-O-methyltransferase discovered by Gowri et al. from alfalfa (Medicago sativa L.), catalyzes the methylation of the 3- or 5-hydroxyl groups of phenolic substrates. Zubieta et al. conducted a structural study of MsCOMT, revealing that it consists of a large C-terminal domain and a smaller N-terminal domain: the former primarily involved in SAM / SAH binding and phenolic substrates, while the latter primarily mediates enzyme dimerization. Further analysis of the catalytic mechanism revealed that MsCOMT is similar to previously characterized plant O-methyltransferases and rat protein arginine N-methyltransferase 3, all of which utilize histidine as a key catalytic residue. In the structural structure of MsCOMT, His269 deprotonates the 3- or 5-hydroxyl group on the phenyl ring of the phenolic substrate, thereby facilitating the transfer of the reactive methyl group from SAM to the nascent phenolate anion. Furthermore, amino acid residues at other positions in MsCOMT also influence catalytic activity. On the one hand, D270 and E297 are adjacent to H269, which have a positive impact on the correct spatial orientation of H269 and the dissociation constant of the phenolic hydroxyl group; on the other hand, M130, M180, I316, I319, etc. surround the alkyl tail of the phenolic substrate and form a relatively hydrophobic binding pocket, which gives MsCOMT selectivity for the alkyl tail, that is, it prefers aldehydes, ketones and alcohols with neutral alkyl tails rather than negatively charged carboxylic acids. Alanine scanning mutagenesis of 13 amino acid residues surrounding 3,4-dihydroxyacetophenone revealed that, with the exception of mutant I319A, all mutants exhibited reduced activity compared to wild-type MsCOMT. Molecular docking of mutant I319A with 3,4-dihydroxyacetophenone and comparison of its interactions with the wild-type revealed that the I-to-A mutation at position 319 allowed D270, in addition to H269, to form hydrogen bonds with the phenolic hydroxyl group of 3,4-dihydroxyacetophenone. This suggests that mutant I319A facilitates the catalytic function of H269 with the assistance of D270. Furthermore, mutants I130A, L136A, F176A, and M180A exhibited significantly reduced catalytic activity. This is likely due to the disruption of the hydrophobic pocket on the alkyl tail of phenolic substrates, which prevents the substrates from binding properly to MsCOMT, leading to the significant reduction in MsCOMT activity.

[0098] When whole-cell transformation of engineered E. coli is performed, the transformation conditions significantly influence product yield. First, the present invention compared the culture media used for transformation, and the results showed that TB medium performed better than the other two media. This may be due to its ability to stabilize pH and promote chaperone expression, thereby significantly improving protein solubility. Second, SAM is a key cofactor for COMT, providing reactive methyl groups for enzymatic reactions. Therefore, the present invention not only attempted in vitro SAM feeding but also explored the construction of an in vivo SAM regeneration system. The results showed that, while the in vivo SAM regeneration system was not as effective as in vitro feeding, it still improved yield compared to strains without a regeneration system. The yield-boosting effect was particularly pronounced when mtn, luxS, and MSCOMT were arranged in a pseudooperon configuration on the vector. These results demonstrate that SAM supply plays a key role in improving MsCOMT catalytic efficiency and product yield. Furthermore, they suggest that a gene cluster co-expression strategy, where each gene is controlled by a single promoter, can effectively overcome the strain's inherent SAM synthesis bottleneck. This provides valuable insights for further improving in vivo SAM regeneration systems, potentially achieving results that match or even exceed those achieved by direct in vitro supplementation. Finally, the present invention uses orthogonal experiments to explore the effects of four conversion factors (substrate concentration, inducer IPTG concentration, induction temperature and conversion temperature) on product yield. From the results, conversion temperature and induction temperature ranked first and second among the influencing factors, and their optimal temperatures were 37°C and 16°C, respectively. This shows that temperature has a key influence on whole-cell conversion, among which 37°C is an ideal temperature for rapid growth of E. coli and for most enzymes to exert their optimal catalytic effects, while 16°C can slow down the expression rate of enzyme genes and provide sufficient time for the correct folding of the spatial conformation of enzyme proteins. Substrate concentration and IPTG concentration ranked third and fourth among the influencing factors, among which substrate concentration is the maximum concentration, indicating that the substrate not only determines whether the enzymatic reaction occurs, but also that its content is linearly related to the amount of product generated. As an inducer, IPTG can promote the expression of enzyme proteins as the concentration increases, but its toxicity to cells will also increase accordingly, so the optimal level is determined to be 0.1mmol·L -1 It is relatively reasonable. This concentration can produce a good induction effect while not affecting the growth of bacteria due to high concentration.

[0099] The microbial whole-cell transformation method explored in this invention is a new approach to synthesizing apocynin, offering several unique advantages over traditional chemical synthesis methods. For example, the biotransformation conditions used in this method are normal temperature and pressure, and the various reagents used for E. coli cultivation are both inexpensive and environmentally friendly. However, the current yield of this method is lower than that of chemical synthesis, with some substrate accumulation. This may be primarily due to two factors: insufficient soluble expression of the MsCOMT gene in E. coli and insufficient amounts of the cofactor SAM required for the MsCOMT enzyme in E. coli. Therefore, in future research, we will implement measures to further promote soluble expression of the MsCOMT enzyme and enhance the ability of E. coli to regenerate SAM. Furthermore, we will also employ other, more effective fermentation processes to increase yield, such as fed-batch fermentation or continuous fermentation. It is hoped that through the implementation of the above methods and measures, the yield of the microbial whole-cell transformation method will be further increased, thereby enabling practical application of apocynin synthesis using this method.

[0100] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for producing apocynin by transforming whole-cell engineered Escherichia coli, characterized in that: The substrate in the method is 3,4-dihydroxyacetophenone, and the plasmid pETM6-MsCOMT is used. I319A The engineered Escherichia coli transformed the substrate of -pmtn-pluxS to obtain apocynin.

2. The method according to claim 1, characterized in that The engineered E. coli in the method is named S23 strain, and the engineered E. coli is BL21 (DE3) strain; preferably, E. coli strain DH5α is used as the host strain for plasmid construction and amplification, while E. coli strain BL21 (DE3) is used as the strain for protein expression and biotransformation.

3. The method according to claim 1, characterized in that The method comprises first connecting MsCOMT, mtn and luxS respectively between the NdeI and XhoI sites of the pETM6 plasmid by a seamless cloning method.

4. The method according to claim 1, characterized in that The method includes using an inducer, isopropyl-β-D-thiogalactopyranoside, i.e., IPTG, when culturing the engineered E. coli.

5. The method according to claim 1, characterized in that: In order to make the three genes MsCOMT, mtn, and luxS exist in a pseudooperon configuration, pETM6-mtn and pETM6-luxS were first digested with AvrII and SalI, and the resulting fragments were ligated with the pETM6-MsCOMT vector digested with SpeI and SalI to obtain pETM6-MsCOMT-pmtn, pETM6-MsCOMT-pluxS, and pETM6-MsCOMT-pmtn-pluxS.

6. The method according to claim 1, characterized in that Alanine scanning mutagenesis of MsCOMT was achieved by whole-plasmid PCR using site-directed mutagenesis primers and pETM6-MsCOMT as a template. I319A .

7. The method according to claim 1, characterized in that: TB culture medium containing a certain concentration of substrate 3,4-dihydroxyacetophenone is used for biotransformation, preferably the composition of the TB culture medium includes 12g·L -1 Tryptone, 24 g L -1 Yeast extract, 4 mL·L -1 Glycerol, 2.31 g·L -1 KH2PO4, 2.54 g·L -1 K2HPO4.

8. The method according to claim 1, characterized in that: The conversion temperature for whole cell transformation to produce apocynin is 30-40° C., and the reaction time is more than 24 hours, preferably 48 hours to 72 hours.

9. An engineered Escherichia coli, characterized in that The engineered E. coli carries the plasmid pETM6-MsCOMT I319A -pmtn-pluxS, the plasmid includes genes encoding three enzymes: MsCOMT, mtn and luxS, wherein MsCOMT refers to caffeic acid-O-methyltransferase from alfalfa, mtn and luxS are two enzymes in the SAM regeneration cycle, SAM is S-adenosylmethionine; the MsCOMT I319A The amino acid residue I319 of MsCOMT has been subjected to alanine scanning mutagenesis to obtain a mutant MsCOMT in which isoleucine I is mutated to alanine A. I319A ; pmtn and pluxS refer to the genes encoding mtn and luxS existing in a pseudooperon configuration, and the codon sequence of MsCOMT, the gene sequence of mtn and luxS are shown in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively.

10. The engineered Escherichia coli according to claim 9, characterized in that The engineered Escherichia coli is strain BL21 (DE3).

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