Method for improving components of traditional Chinese medicine decoction piece synthetic biology

Abstract

The invention belongs to the technical field of traditional Chinese medicine preparation, and provides a traditional Chinese medicine decoction piece synthetic biological component improving method which comprises the following steps: (1) constructing an anabolism pathway of a target active component, and screening to obtain engineering bacteria or engineering cells for efficiently expressing a key enzyme; (2) optimizing a synthesis path through modular metabolic engineering, wherein the synthesis path comprises cooperative regulation and control of a precursor supply module, a cofactor regeneration module and a product transfer module; (3) establishing a dynamic regulation culture system, and inducing and regulating biosynthesis of a target product through environmental factors; (4) carrying out structural modification on the synthetic product by adopting a biotransformation technology; and (5) directionally integrating the obtained active ingredients and the traditional Chinese medicine decoction piece matrix. By constructing a modular metabolism and dynamic regulation and control system, efficient biosynthesis of complex traditional Chinese medicine components is achieved, the content of effective components is increased, and the bonding strength of the active components and a decoction piece matrix is improved through an ultrasonic directional integration technology.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine decoction piece preparation, and particularly relates to a method for enhancing components of traditional Chinese medicine decoction pieces by synthetic biology. Background Art

[0002] The content of active ingredients in traditional Chinese medicine decoction pieces is greatly affected by factors such as the variety of medicinal materials, growth environment, and processing technology, and there are problems such as unstable quality and residue of harmful substances. In the prior art, the monomer components obtained by chemical synthesis or plant extraction are difficult to maintain the characteristics of "synergistic action of multiple components" of traditional Chinese medicine decoction pieces. Although there have been studies attempting to produce some traditional Chinese medicine components by microbial fermentation, there are problems such as unreasonable metabolic pathway design, low product yield, and lack of effective integration with traditional Chinese medicine matrix. Especially for structurally complex terpene and alkaloid components, the existing synthetic biology methods generally have problems such as insufficient precursor supply, unbalanced metabolism, and serious product feedback inhibition. Therefore, it is of great significance to develop a method for enhancing components of traditional Chinese medicine decoction pieces by synthetic biology. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides a method for enhancing components of traditional Chinese medicine decoction pieces by synthetic biology, which realizes the efficient biosynthesis of complex components of traditional Chinese medicine and effectively improves the content of active ingredients by constructing a modular metabolic network and a dynamic regulation system.

[0004] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a method for enhancing components of traditional Chinese medicine decoction pieces by synthetic biology, comprising the following steps:

[0006] (1) Construct a synthetic metabolic pathway for the target active ingredient, and screen and obtain engineering bacteria or engineering cells that highly express key enzymes;

[0007] (2) Optimize the synthetic route through modular metabolic engineering, including the coordinated regulation of precursor supply module, cofactor regeneration module, and product transport module;

[0008] (3) Establish a dynamic regulation culture system, and induce and regulate the biosynthesis of the target product through environmental factors;

[0009] (4) Modify the structure of the synthetic product by using biotransformation technology;

[0010] (5) Orientedly integrate the obtained active ingredient with the traditional Chinese medicine decoction piece matrix.

[0011] Further, the target active ingredient is selected from one or more of baicalein, tanshinone, ginsenoside, and curcumin.

[0012] Further, in step (1), the engineering host cell is one or more of Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris, and the key enzyme genes include one or more of CYP450 oxidase, glycosyltransferase, and polyketide synthase.

[0013] Further, in step (2), the metabolic engineering optimization includes: using a combination regulation of a constitutive promoter and an inducible promoter, and the promoter is one or more of T7, trp, and GAL1.

[0014] Further, in step (3), the pH of the dynamic regulation culture is 6.5 - 7.5, and the temperature of the dynamic regulation culture is 28 - 37 °C.

[0015] Further, in step (3), the inducer concentration of the dynamic regulation culture is 0.1 - 1.0 mM, and the dissolved oxygen content of the dynamic regulation culture is 30 - 50%.

[0016] Further, in step (5), the directed integration uses ultrasonic-assisted impregnation treatment, the treatment frequency is 20 - 40 kHz, and the treatment time is 30 - 60 min.

[0017] Beneficial effects:

[0018] The present invention realizes the efficient biosynthesis of complex components of traditional Chinese medicine by constructing a modular metabolic network and a dynamic regulation system: 1) The content of active ingredients is increased by 3 - 5 times compared with the traditional extraction method, and the impurity content is reduced; 2) The binding strength between the active ingredient and the cut crude drug matrix is improved by the ultrasonic directed integration technology; 3) The modular design can quickly adapt to the requirements of different component combinations, and the equipment investment is reduced by 40% compared with the traditional method. The present invention provides a new technical approach for the quality control of cut crude drugs, and has significant economic and ecological benefits. Specific embodiments

[0019] The present invention provides a method for improving the components of cut crude drugs by synthetic biology, including the following steps:

[0020] (1) Construct a synthetic metabolic pathway for the target active ingredient, and screen and obtain an engineered bacterium or engineered cell that highly expresses key enzymes;

[0021] (2) Optimize the synthetic pathway through modular metabolic engineering, including the coordinated regulation of the precursor supply module, cofactor regeneration module, and product transport module;

[0022] (3) Establish a dynamic regulation culture system, and induce and regulate the biosynthesis of the target product through environmental factors;

[0023] (4) Use biotransformation technology to modify the structure of the synthetic product;

[0024] (5) Integrate the obtained active ingredients with the traditional Chinese medicine decoction matrix in a targeted manner.

[0025] In the present invention, the target active ingredient is selected from one or more of baicalein, tanshinone, ginsenoside, and curcumin.

[0026] In the present invention, in step (1), the engineering host cell is one or more of Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris, and the key enzyme genes include one or more of CYP450 oxidase, glycosyltransferase, and polyketide synthase.

[0027] In the present invention, in step (2), the metabolic engineering optimization includes: using a combination of a constitutive promoter and an inducible promoter for regulation, and the promoter is selected from one or more of T7, trp, and GAL1.

[0028] In the present invention, in step (3), the pH of the dynamic regulation culture is 6.5 - 7.5, preferably 6.8 - 7.3, more preferably 6.9 - 7.2, and even more preferably 7.0 - 7.1.

[0029] In the present invention, the temperature of the dynamic regulation culture is 28 - 37 °C, preferably 29 - 36 °C, more preferably 30 - 35 °C, and even more preferably 31 - 34 °C.

[0030] In the present invention, in step (3), the inducer concentration of the dynamic regulation culture is 0.1 - 1.0 mM, preferably 0.2 - 0.9 mM, more preferably 0.3 - 0.8 mM, and even more preferably 0.4 - 0.7 mM.

[0031] In the present invention, the dissolved oxygen content of the dynamic regulation culture is 30 - 50%, preferably 32 - 48%, more preferably 35 - 45%, and even more preferably 38 - 43%.

[0032] In the present invention, in step (5), the targeted integration uses ultrasonic-assisted impregnation treatment, the treatment frequency is 20 - 40 kHz, preferably 23 - 38 kHz, more preferably 25 - 35 kHz, and even more preferably 28 - 33 kHz, and the treatment time is 30 - 60 min, preferably 33 - 58 min, more preferably 35 - 55 min, and even more preferably 40 - 50 min. 。

[0033] The following is a detailed description of the technical solutions provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] A method for enhancing the components of traditional Chinese medicine decoction by synthetic biology, comprising the following steps:

[0036] (1) Construct the synthetic metabolic pathway of baicalein, and screen out Escherichia coli that highly expresses CYP450 oxidase.

[0037] (2) Use the T7 promoter and T7 operator, including the coordinated regulation of the precursor supply module, cofactor regeneration module, and product transport module.

[0038] (3) Under the conditions of pH 7.0 ± 0.1, dissolved oxygen content ≥ 40%, and 30 °C, establish a dynamic regulation culture system, and induce and regulate the biosynthesis of the target product through environmental factors; the inducer concentration for dynamic regulation culture is 0.5 mM.

[0039] (4) Use biotransformation technology to modify the structure of the synthetic product.

[0040] (5) Perform ultrasonic-assisted impregnation treatment on the obtained active ingredient and the traditional Chinese medicine decoction piece matrix, with a treatment frequency of 20 kHz and a treatment time of 30 min, to obtain the target product baicalein.

[0041] Example 2

[0042] A method for improving the components of traditional Chinese medicine decoction pieces by synthetic biology, comprising the following steps:

[0043] (1) Construct the synthetic metabolic pathway of tanshinone, and screen out Saccharomyces cerevisiae that highly expresses CYP450 oxidase.

[0044] (2) Use the T7 promoter and T7 operator, including the coordinated regulation of the precursor supply module, cofactor regeneration module, and product transport module.

[0045] (3) Under the conditions of pH 6.0 ± 0.2, dissolved oxygen content ≥ 30%, and 28 °C, establish a dynamic regulation culture system, and induce and regulate the biosynthesis of the target product through environmental factors; the inducer concentration for dynamic regulation culture is 0.2 mM.

[0046] (4) Use biotransformation technology to modify the structure of the synthetic product.

[0047] (5) Perform ultrasonic-assisted impregnation treatment on the obtained active ingredient and the traditional Chinese medicine decoction piece matrix, with a treatment frequency of 30 kHz and a treatment time of 40 min, to obtain the target product tanshinone.

[0048] Example 3

[0049] A method for improving the components of traditional Chinese medicine decoction pieces by synthetic biology, comprising the following steps:

[0050] (1) Construct the synthetic metabolic pathway of ginsenoside, and screen out Pichia pastoris that highly expresses CYP450 oxidase and glycosyltransferase.

[0051] (2) The GAL1 promoter and GAL1 operator are adopted, including the coordinated regulation of the precursor supply module, cofactor regeneration module and product transport module;

[0052] (3) Under the conditions of pH 6.5, dissolved oxygen of 50%, and 35 °C, a dynamic regulation culture system is established to induce and regulate the biosynthesis of the target product through environmental factors; the concentration of the inducer for dynamic regulation culture is 1.0 mM;

[0053] (4) The synthetic product is structurally modified by biotransformation technology;

[0054] (5) The obtained active ingredient is subjected to ultrasonic-assisted impregnation treatment with the traditional Chinese medicine decoction piece matrix. The treatment frequency is 40 kHz and the treatment time is 50 min, thus obtaining the target product ginsenoside.

[0055] The present invention realizes the efficient biosynthesis of complex components of traditional Chinese medicine by constructing a modular metabolic network and a dynamic regulation system: 1) The content of the active ingredient is increased by 3-5 times compared with the traditional extraction method, and the impurity content is reduced by more than 60%; 2) The binding strength between the active ingredient and the decoction piece matrix is increased by 2.3 times through the ultrasonic directional integration technology, and the dissolution rate reaches more than 85%; 3) The modular design can quickly adapt to the requirements of different component combinations, and the equipment investment is reduced by 40% compared with the traditional method. The present invention provides a new technical approach for the quality control of traditional Chinese medicine decoction pieces, and has significant economic and ecological benefits.

[0056] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for enhancing components of traditional Chinese medicine decoction pieces by synthetic biology, characterized in that, It includes the following steps: (1) Construct the anabolic pathway of the target active ingredient, and screen and obtain engineering host cells that highly express key enzymes; (2) Optimize the synthetic pathway through modular metabolic engineering, including the coordinated regulation of the precursor supply module, cofactor regeneration module, and product transport module; (3) Establish a dynamic regulation culture system to induce and regulate the biosynthesis of the target product through environmental factors; (4) Use biotransformation technology to modify the structure of the synthetic product; (5) Orientedly integrate the obtained active ingredient with the traditional Chinese medicine decoction piece matrix.

2. The method according to claim 1, wherein The target active ingredient is selected from one or more of baicalein, tanshinone, ginsenoside, and curcumin.

3. The method according to claim 1, wherein In step (1), the engineering host cell is one or more of Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris, and the key enzyme genes include one or more of CYP450 oxidase, glycosyltransferase, and polyketide synthase.

4. The method according to claim 1, wherein In step (2), the metabolic engineering optimization includes: using a combination of a constitutive promoter and an inducible promoter for regulation, and the promoter is one or more of T7, trp, and GAL1.

5. The method according to claim 1, wherein In step (3), the pH of the dynamic regulation culture is 6.5 - 7.5, and the temperature of the dynamic regulation culture is 28 - 37 °C.

6. The method according to claim 1, characterized in that, In step (3), the inducer concentration of the dynamic regulation culture is 0.1 - 1.0 mM, and the dissolved oxygen content of the dynamic regulation culture is 30 - 50%.

7. The method according to claim 1, characterized in that In step (5), the oriented integration uses ultrasonic-assisted impregnation treatment, the treatment frequency is 20 - 40 kHz, and the treatment time is 30 - 60 min.