Recombinant bacteria for synthesizing wogonin WGX-50 from glucose as carbon source, construction method and application thereof

Abstract

This invention relates to a recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source, its construction method, and its applications. The recombinant bacterial cell uses *Escherichia coli* as the host strain and integrates the following three heterologous biosynthetic modules: a 3,4-dimethoxyphenylethylamine de novo synthesis module, which contains an enzyme system catalyzing the generation of dopamine from L-tyrosine and further stepwise methylation of dopamine to 3,4-dimethoxyphenylethylamine; a cinnamoyl-CoA synthesis module, which contains an enzyme system catalyzing the generation of cinnamoyl-CoA from phenylalanine or glucose metabolic intermediates; and an amidation module, which contains an acyltransferase capable of catalyzing the condensation of 3,4-dimethoxyphenylethylamine and cinnamoyl-CoA to generate xanthocyanin WGX-50. This invention achieves, for the first time, the de novo biosynthesis of xanthocyanin in microorganisms, completely eliminating dependence on the expensive exogenous precursor 3,4-dimethoxyphenylethylamine and significantly reducing raw material costs.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic biology and metabolic engineering technology, specifically relating to a recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source, its construction method, and its application. Background Technology

[0002] Zanthoxylumin (WGX-50) is a natural active compound with an amide structure extracted from plants of the Zanthoxylum genus. It has been proven to have a variety of pharmacological activities, including anti-aging, anti-inflammatory, neuroprotective, and anti-tumor effects, and has broad application prospects in the fields of medicine and cosmetics. At present, the production of zanthoxylumin mainly relies on plant extraction and chemical synthesis. Plant extraction is limited by the source of raw materials, seasonality, and extremely low extraction rate, resulting in high costs and difficulty in scaling up. Chemical synthesis has problems such as complicated steps, use of toxic reagents, generation of harmful byproducts, and environmental pollution.

[0003] In recent years, heterologous synthesis of plant natural products using microbial cell factories has become a green and sustainable alternative strategy. Studies have reported the successful synthesis of various hydroxycinnamic acid compounds in Escherichia coli by adding exogenous precursors (such as tyramine, ferulic acid, etc.). For example, in the prior patent application (application number: 202411571876.1), our team has successfully constructed an engineered strain that can synthesize xanthocyanin by combining exogenously added 3,4-dimethoxyphenylethylamine as a precursor with endogenously generated cinnamoyl-CoA.

[0004] However, the reliance on the expensive precursor 3,4-dimethoxyphenylethylamine limits the economic viability and industrialization potential of this technology. Achieving "de novo synthesis" from inexpensive carbon sources (such as glucose) is key to reducing production costs and achieving true industrial production. Currently, there are no publicly available technical reports on a biosynthetic pathway that can achieve the complete synthesis of xanthocyanin from a basic carbon source in Escherichia coli or any other microorganism. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source, as well as its construction method and application, in order to solve the above-mentioned problems.

[0006] The present invention achieves the above objectives through the following technical solutions: This invention provides a recombinant bacterial cell that synthesizes xanthocyanin WGX-50 de novo using glucose as a carbon source. The recombinant bacterial cell uses Escherichia coli as a host strain and integrates the following three heterologous biosynthetic modules: (1) 3,4-Dimethoxyphenylethylamine de novo synthesis module, which contains an enzyme system that catalyzes the generation of dopamine from L-tyrosine and further steps-wise methylates dopamine to generate 3,4-dimethoxyphenylethylamine; (2) Cinnamyl-CoA synthesis module, which contains an enzyme system that catalyzes the production of cinnamyl-CoA from phenylalanine or glucose metabolic intermediates; (3) Amide module, which contains an acyltransferase that can catalyze the condensation of 3,4-dimethoxyphenethylamine with cinnamyl-CoA to generate xanthocyanin WGX-50.

[0007] As a further optimization of the present invention, the 3,4-dimethoxyphenethylamine de novo synthesis module comprises the following enzymes or their encoding genes: (1) 4-hydroxyphenylacetic acid-3-monooxygenase HpaBC, which catalyzes the hydroxylation of L-tyrosine to L-DOPA, has the nucleotide sequence of its encoding gene as shown in SEQ ID NO.1; (2) The DODC enzyme that catalyzes the decarboxylation of L-DOPA to produce dopamine has the nucleotide sequence of its encoding gene as shown in SEQ ID NO.2; (3) The first oxygen methyltransferase OMT1, which catalyzes the methylation of the phenolic hydroxyl group at the 3-position of dopamine to generate 3-methoxytyramine; (4) The second oxygen methyltransferase OMT2 catalyzes the para-phenolic hydroxymethylation of 3-methoxytyramine to generate 3,4-dimethoxyphenylethylamine.

[0008] As a further optimization of the present invention, the first oxygen methyltransferase OMT1 is AtOMT derived from Arabidopsis thaliana, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.3. The second oxygen methyltransferase OMT2 is 4-O-oxymethyltransferase MOMT derived from Clarkia breweri, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4.

[0009] As a further optimization of the present invention, the cinnamyl-CoA synthesis module comprises the following enzymes or their encoding genes: (1) Phenylalanine ammonia-lyase AtPAL, which catalyzes the deamination of phenylalanine to trans-cinnamic acid; (2) Catalyzes the activation of trans-cinnamic acid to cinnamic acid-co-A ligase HcCNL.

[0010] As a further optimization of the present invention, the phenylalanine ammonia-lyase PAL is AtPAL from Arabidopsis thaliana, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.5; the cinnamic acid coenzyme A ligase is HcCNL from Hypericum perforatum, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.6.

[0011] As a further optimization of the present invention, the amidation module comprises tyramine N-hydroxycinnamoyltransferase CaTHT or its N-terminal truncated variant trCaTHT derived from chili pepper, and the nucleotide sequence of the gene encoding tyramine N-hydroxycinnamoyltransferase CaTHT derived from chili pepper is shown in SEQ ID NO.7.

[0012] As a further optimization of the present invention, the Escherichia coli is Escherichia coli BL21 (DE3), Origami 2 (DE3), Rosetta-gami 2 (DE3) or MG1655 (DE3).

[0013] This invention also provides a method for constructing recombinant bacterial cells that de novo synthesize xanthocyanin WGX-50 using glucose as a carbon source as described above, comprising the following steps: (1) Construct expression vectors containing the coding genes of the 3,4-dimethoxyphenethylamine de novo synthesis module, the cinnamoyl-CoA synthesis module and the amidation module, respectively; (2) The expression vector obtained in step (1) is co-transformed or sequentially introduced into the Escherichia coli host strain to prepare the recombinant bacterial cells.

[0014] The present invention also provides the application of recombinant bacterial cells that synthesize xanthocyanin WGX-50 de novo using glucose as a carbon source in the production of xanthocyanin WGX-50.

[0015] As a further optimization of the present invention, the method for producing xanthocyanin using the recombinant bacterial cells as described above is as follows: the recombinant bacterial cells are inoculated into a fermentation medium for fermentation culture, the expression of enzyme proteins in each module is induced, and the product is synthesized. After fermentation, xanthocyanin WGX-50 is isolated and extracted from the culture. The fermentation medium uses glucose as the sole or primary carbon source.

[0016] The beneficial effects of this invention are as follows: 1) This invention successfully constructed and verified a complete new microbial synthesis pathway for 3,4-dimethoxyphenethylamine. In particular, the screening and combination of two-step specific methylation reaction enzyme systems has made outstanding contributions. For the first time, de novo biosynthesis of xanthocyanin in microorganisms has been achieved, completely eliminating the dependence on the expensive exogenous precursor 3,4-dimethoxyphenethylamine and significantly reducing the cost of raw materials. 2) This invention uses renewable glucose as raw material, and the fermentation process is mild and environmentally friendly, which is in line with the concept of green manufacturing. It lays a solid foundation for the low-cost and large-scale industrial production of xanthocyanin. At the same time, the engineered strain and modular strategy also provide a reference for the microbial synthesis of other complex phenolic amide natural products.

[0017] 3) The recombinant bacterial cell fermentation method provided in this invention can produce 20 mg / L of xanthocyanin WGX-50 after 48 hours of fermentation. This method uses inexpensive raw materials, is environmentally friendly, and has simple steps. It provides a new technical route for the large-scale and sustainable production of xanthocyanin and has significant industrial application prospects. Attached Figure Description

[0018] Figure 1 The structural formula of xanthocyanin WGX-50 is given.

[0019] Figure 2 This is a schematic diagram of the de novo synthesis pathway of xanthocyanin (WGX-50) constructed in Escherichia coli using glucose as a carbon source.

[0020] Figure 3 For screening of para-phenolic hydroxyl O-methyltransferases.

[0021] Figure 4 HPLC analysis of fermentation extracts from recombinant Escherichia coli carrying the xanthocyanin synthesis gene.

[0022] Figure 5 LC-MS analysis of fermentation extracts from recombinant Escherichia coli carrying the xanthocyanin synthesis gene.

[0023] Figure 6 To determine the fermentation yield of xanthocyanin by recombinant Escherichia coli carrying the xanthocyanin synthesis gene. Detailed Implementation

[0024] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0025] I. Reagents and Materials 1. Escherichia coli BL21 (DE3) is commercially available. Escherichia coli BL21 (DE3) is used in this invention for gene protein expression and biotransformation to produce xanthocyanin WGX-50. Escherichia coli BL21 (DE3) competent cells are prepared according to conventional methods. For the experimental methods in the following examples where specific conditions are not specified, they are carried out according to conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual, or according to the conditions recommended by the manufacturer of the corresponding biological reagent.

[0026] 2. Culture medium formulation: 1) Seed culture and protein expression medium LB medium: 5 g / L yeast extract, 10 g / L NaCl, 10 g / L tryptone, the remainder is water, sterilized at 121°C for 20 min.

[0027] 2) Biotransformation medium G medium: 10g / L glycerol, 6.8g / L KH2PO4, 17.9g / L Na2HPO4, 0.71g / L Na2SO4, 2.67 g / L NH4Cl, 15g / L Peptone, 5g / L yeast extract, 2 mM MgSO4, and 0.1mM CaCl2.

[0028] M9 medium: 0.1 mM 3,4-dimethoxyphenethylamine, 4.78 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 0.5 g / L NH4Cl, 0.12 g / L MgSO4, 4.35 g / L yeast extract, 20 g / L glucose.

[0029] In actual culture, a certain concentration of antibiotics can be added to the culture medium to maintain the stability of the plasmid, such as 100 mg / L ampicillin, 100 mg / L streptomycin, 50 mg / L kanamycin and 25 mg / L chloramphenicol.

[0030] Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0031] II. Methods 1. Screening and functional analysis of para-phenolic hydroxyl O-methyltransferase EjOMT (from loquat) and MOMT (from the plant *Clarkia breweri*) were codon-optimized and gene-synthesized (the gene synthesis was commissioned to General Biotechnology (Anhui) Co., Ltd., the same below) and respectively constructed into plasmid pET28a to obtain recombinant plasmids pET28a-EjOMT and pET28a-MOMT. The recombinant plasmids were introduced into *E. coli* BL21 and named *E. coli* BL21 / pET28a-EjOMT and *E. coli* BL21 / pET28a-MOMT, respectively. They were cultured in LB medium at 16℃ for 12 hours to induce protein expression. 2 mM dopamine was added to the culture and cultured at 30℃ for another 24 hours. The target product was extracted with ethyl acetate and the enzyme function was analyzed by HPLC. Recombinant bacteria transformed with empty plasmids were used as negative controls, and 4-methoxydopamine was used as a positive control.

[0032] The results are as follows Figure 2As shown, MOMT derived from the fairy fan can catalyze the synthesis of 4-methoxydopamine.

[0033] 2. Construction of recombinant Escherichia coli strains for de novo synthesis of xanthocyanin Genes encoding the following enzymes (as shown in SEQ ID NO. 1-7) were synthesized and codon-optimized based on GenBank accession numbers: AtPAL (NP_190894.1; SEQ ID NO. 5), HcCNL (AFS60176.1, SEQ ID NO. 6), CaTHT (NP_001311493.1; SEQ ID NO. 7), HpaBC (derived from Escherichia coli K12; SEQ ID NO. 1), LbDODC (derived from Lactobacillus brevis; SEQ ID NO. 2), AtOMT (NP_850118.2; SEQ ID NO. 3), MOMT (derived from the plant Clarkiabreweri; SEQ ID NO. 7). NO.4), the AtPAL and HcCNL genes were cloned into the pRSFDuet-1 vector to form the plasmid pRSF-PAL-CNL (cinnamoyl-CoA module), the CaTHT gene was cloned into the pACYCDuet-1 vector to form the plasmid pACC-THT (amidation module), and the HpaBC, LbDODC, AtOMT and MOMT genes were cloned into the pETDuet-1 and pCDFDuet-1 vectors respectively to form the plasmids pET-HpaBC-DODC and pCDF-OMT1-OMT2 (3,4-dimethoxyphenylethylamine (DMPEA) de novo synthesis module).

[0034] The three recombinant plasmids were electroporated into Escherichia coli BL21(DE3) competent cells. The recombinant engineered strain carrying three functional modules was obtained by screening with antibiotic plates (kanamycin, chloramphenicol, ampicillin, and streptomycin) and named E. coli W-Core.

[0035] 3. Fermentation verification of de novo synthesis of xanthocyanin by recombinant Escherichia coli 1) Pick a single colony of E. coli W-Core, culture it overnight in LB seed culture, and transfer it to G medium (or optimized M9Y medium) with 2% inoculum at 20 g / L glucose as the sole carbon source. Incubate at 37°C until OD600 is about 0.6-0.8, add 0.5 mM IPTG, lower the temperature to 25°C, and induce expression for 24-72 hours.

[0036] 2) Take the fermentation broth, centrifuge to remove the bacterial cells, and extract the supernatant with an equal volume of ethyl acetate. After concentration and redissolution, perform HPLC and LC-MS analysis. Compare the retention time, characteristic UV absorption peak (277 nm), and molecular ion peak (m / z 312.16 [M+H]) with the standard. + The product was confirmed to be xanthoside (WGX-50), and the synthesis process is as follows: Figure 3 As shown.

[0037] Experimental results are as follows Figure 4-5 As shown, both HPLC and LC-MS analyses detected the characteristic peak of xanthocyanin in the fermentation broth. Quantitative calculations showed that the engineered strain E. coli W-Core produced 15 mg / L of xanthocyanin after 48 hours of fermentation. The LC-MS spectral data were completely consistent with the standard, further confirming the product structure.

[0038] 4. Optimization of different fermentation conditions Based on step 3 above, we attempted to optimize fermentation conditions to increase yield. For example, we added glucose after 24 hours of fermentation; added 2 mM L-tyrosine and 2 mM L-phenylalanine to enhance precursor supply; and added L-methionine (10 mM) to enhance the supply of the methyl donor S-adenosylmethionine (SAM). Under these optimized conditions, the yield of xanthocyanin was further increased, as shown by quantitative calculations (e.g., ...). Figure 6 As shown in the figure, the engineered strain E. coli W-Core achieved a xanthocyanin yield of 20 mg / L after 48 hours of fermentation.

[0039] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source, characterized in that, The recombinant bacteria, using Escherichia coli as the host strain, integrates the following three heterologous biosynthetic modules: (1) 3,4-Dimethoxyphenylethylamine de novo synthesis module, which contains an enzyme system that catalyzes the generation of dopamine from L-tyrosine and further steps-wise methylates dopamine to generate 3,4-dimethoxyphenylethylamine; (2) Cinnamyl-CoA synthesis module, which contains an enzyme system that catalyzes the production of cinnamyl-CoA from phenylalanine or glucose metabolic intermediates; (3) Amide module, which contains an acyltransferase that can catalyze the condensation of 3,4-dimethoxyphenethylamine with cinnamyl-CoA to generate xanthocyanin WGX-50.

2. The recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source according to claim 1, characterized in that, The 3,4-dimethoxyphenethylamine de novo synthesis module contains the following enzymes or their encoding genes: (1) 4-hydroxyphenylacetic acid-3-monooxygenase HpaBC, which catalyzes the hydroxylation of L-tyrosine to L-DOPA, has the nucleotide sequence of its encoding gene as shown in SEQ ID NO.1; (2) The DODC enzyme that catalyzes the decarboxylation of L-DOPA to produce dopamine has the nucleotide sequence of its encoding gene as shown in SEQ ID NO.2; (3) The first oxygen methyltransferase OMT1, which catalyzes the methylation of the phenolic hydroxyl group at the 3-position of dopamine to generate 3-methoxytyramine; (4) The second oxygen methyltransferase OMT2 catalyzes the para-phenolic hydroxymethylation of 3-methoxytyramine to generate 3,4-dimethoxyphenylethylamine.

3. The recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source according to claim 2, characterized in that, The first oxygen methyltransferase OMT1 is AtOMT derived from Arabidopsis thaliana, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

3. The second oxygen methyltransferase OMT2 is 4-O-oxymethyltransferase MOMT derived from Angelica dahurica, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

4.

4. The recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source according to claim 1, characterized in that, The cinnamyl-CoA synthesis module contains the following enzymes or their encoding genes: (1) Phenylalanine ammonia-lyase PAL, which catalyzes the deamination of phenylalanine to trans-cinnamic acid; (2) Catalyzes the activation of trans-cinnamic acid to cinnamic acid-co-A ligase HcCNL.

5. The recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source according to claim 4, characterized in that, The phenylalanine ammonia-lyase PAL is AtPAL from Arabidopsis thaliana, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

5. The cinnamic acid coenzyme A ligase is HcCNL from Hypericum perforatum, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

6.

6. The recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source according to claim 1, characterized in that, The amidation module comprises tyramine N-hydroxycinnamoyltransferase CaTHT or its N-terminal truncated variant trCaTHT derived from chili pepper, and the nucleotide sequence of the gene encoding tyramine N-hydroxycinnamoyltransferase CaTHT derived from chili pepper is shown in SEQ ID NO.

7.

7. The recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source according to claim 1, characterized in that, The Escherichia coli is Escherichia coli BL21 (DE3), Origami 2 (DE3), Rosetta-gami 2 (DE3), or MG1655 (DE3).

8. A method for constructing recombinant bacterial cells for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source as described in any one of claims 1-7, characterized in that, Includes the following steps: (1) Construct expression vectors containing the coding genes of the 3,4-dimethoxyphenethylamine de novo synthesis module, the cinnamoyl-CoA synthesis module and the amidation module, respectively; (2) The expression vector obtained in step (1) is co-transformed or sequentially introduced into the Escherichia coli host strain to prepare the recombinant bacterial cells.

9. The application of a recombinant bacterial cell for de novo synthesis of xanthocyanin WGX-50 using glucose as a carbon source as described in any one of claims 1-7 in the production of xanthocyanin WGX-50.

10. The application according to claim 9, characterized in that, The method for producing xanthocyanin using the recombinant bacterial cells as described in any one of claims 1-7 is as follows: the recombinant bacterial cells are inoculated into a fermentation medium for fermentation culture, the expression of enzyme proteins in each module is induced, and the product is synthesized. After fermentation, xanthocyanin WGX-50 is isolated and extracted from the culture. The fermentation medium uses glucose as the sole or primary carbon source.

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