A compound Danshen bionic synthesis engineering bacterium and its application

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a bionic synthesis engineering bacterium of compound Danshen and its application. The engineering bacterium is obtained by introducing cytochrome P450 reductase, tyrosine hydroxylase and D-lactate dehydrogenase of Lactobacillus pentosus on the basis of a Saccharomyces cerevisiae starting bacterium that produces protopanaxadiol, cryptotanshinone diene and borneol. The obtained Saccharomyces cerevisiae genetic engineering bacterium can simultaneously synthesize the active ingredients of compound Danshen preparations, namely protopanaxadiol, cryptotanshinone diene, danshensu and borneol, and has application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a compound Danshen bionic synthesis engineering bacterium and its application. Background Art

[0002] With the development of molecular pharmacognosy and artificial herbal medicine science, heterologous bionic synthetic biology of traditional Chinese medicine active ingredients has become a new driving force for promoting the green and sustainable development of traditional Chinese medicine resources. The synthetic pathways of many important traditional Chinese medicine active ingredients have been analyzed and heterologously produced by microorganisms. Compound Danshen preparations are traditional Chinese medicine compound preparations made by mixing Danshen, Panax notoginseng, and borneol in a certain proportion. Clinically, they are widely used to treat various diseases such as coronary heart disease and angina pectoris with symptoms of chest tightness, and have the characteristics of good effect, significant action, and small toxic and side effects. Modern pharmacology has proved that active substances such as danshensu, tanshinone, ginsenoside, and borneol are the main components for the pharmacological activity of compound Danshen preparations. At present, the heterologous synthesis of borneol, the precursor of tanshinone, miltiradiene, various ginsenosides, and danshensu has been achieved by microorganisms. (Literature: Ma R., et al., Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae. Synthetic and Systems Biotechnology 2022;7(1):490-497; Dai, Z. et al., Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae. Biotechnology and Bioengineering 2012, 109 (11), 2845-2853; Yan, X.; et al., Production of bioactive ginsenoside compound K in metabolically engineered yeast. Cell Research 2014, 24 (6), 770-773.).

[0003] However, most current studies are limited to the heterologous synthesis of single-component or single-source traditional Chinese medicine compound ingredients, and the bionic synthesis research of active ingredients of cross-species and multi-component compound preparations is still challenging.

[0004] The method for the biomimetic synthesis of the multi-component compound preparation proposed by the present invention is of great significance for expanding the production and application modes of the Compound Danshen Preparation. Summary of the Invention

[0005] Based on synthetic biology and molecular pharmacognosy, the present invention uses Saccharomyces cerevisiae as the chassis strain, and constructs an artificial herbal cell of Compound Danshen Preparation that can simultaneously synthesize the active ingredient precursors of Compound Danshen Preparation, namely danshensu, tanshinone diene, protopanaxadiol, and borneol, through heterologous pathway reconstruction, metabolic pathway optimization, and the construction of a co-production strain platform.

[0006] First, the present invention modifies the Saccharomyces cerevisiae chassis strain that can simultaneously synthesize cryptotanshinone diene, protopanaxadiol, and borneol to express the cytochrome P450 reductase VvCPR from grapes, the D-lactate dehydrogenase LpD-LDH of Lactobacillus pentosus, and the tyrosine hydroxylase BvCYP76AD from sugar beet, to obtain a Compound Danshen yeast strain that can simultaneously synthesize danshensu, tanshinone diene, protopanaxadiol, and borneol.

[0007] The present invention provides a genetically engineered Saccharomyces cerevisiae strain for the biomimetic synthesis of Compound Danshen, which, based on the starting Saccharomyces cerevisiae strain that produces protopanaxadiol, cryptotanshinone diene, and borneol, introduces the cytochrome P450 reductase VvCPR from grapes, the D-lactate dehydrogenase LpD-LDH of Lactobacillus pentosus, and the tyrosine hydroxylase BvCYP76AD1 from sugar beet;

[0008] Specifically, the D-lactate dehydrogenase LpD-LDH of Lactobacillus pentosus is obtained by gene synthesis and ligated with the GAL1p promoter to construct a plasmid; the tyrosine hydroxylase BvCYP76D1 from sugar beet is obtained by gene synthesis and ligated with the GAL10p promoter to construct a plasmid; the LpD-LDH ligated with the GAL1p promoter, the BvCYP76AD1 ligated with the GAL10p promoter, and the VvCPR ligated with the GAL7p promoter are transferred into the NDT80 locus of the strain SM-PPD-BOL to obtain the strain CDSY2.0.

[0009] The amino acid sequence of the D-lactate dehydrogenase LpD-LDH is as shown in SEQ ID NO:1, the amino acid sequence of the tyrosine hydroxylase BvCYP76AD1 is as shown in SEQ ID NO:2, and the coding genes of the D-lactate dehydrogenase LpD-LDH and the tyrosine hydroxylase BvCYP76AD1 are codon-optimized according to yeast expression preferences.

[0010] The present invention further provides the application of the above-mentioned engineered strain in the production of protopanaxadiol, cryptotanshinone diene, danshensu, and borneol.

[0011] The present invention also provides a method for preparing protopanaxadiol, cryptotanshinone diene, danshensu, and borneol using the engineered bacterium, characterized by the steps of culturing the engineered bacterium to produce protopanaxadiol, cryptotanshinone diene, danshensu, and borneol, and optionally including the further step of separating the produced protopanaxadiol, cryptotanshinone diene, danshensu, and borneol.

[0012] Specifically, the culture medium is an SD-Trp-Ura liquid selection medium with glucose and / or galactose and / or ethanol as the carbon source, and the culture conditions are culturing at 30 °C and 250 rpm.

[0013] Based on the guidance of molecular pharmacognosy and artificial herbal medicine science theories, the present invention regards the active ingredients of compound danshen preparations as a whole to achieve their microbial bionic synthesis. Through the reconstruction of heterologous pathways, the optimization of metabolic pathways, and the construction of a co-production strain platform, a compound danshen artificial herbal cell that simultaneously synthesizes the active ingredient precursors of compound danshen preparations, namely danshensu, tanshinone diene, protopanaxadiol, and borneol, has been constructed.

[0014] Experiments show that the engineered bacterium constructed in the present invention can produce 237.11 ± 4.65 mg / L of protopanaxadiol, 84.23 ± 3.30 mg / L of danshensu, 19.77 ± 1.20 mg / L of cryptotanshinone diene, and 1.25 ± 0.09 mg / L of borneol. Therefore, the simultaneous synthesis of the main active ingredients of compound danshen preparations in the same engineered cell has been achieved. That is, the innovation of the present invention lies in regarding the active ingredients of compound danshen preparations as a whole and constructing a compound danshen artificial herbal cell that simultaneously synthesizes multiple active components, providing a reference for the application of synthetic biology in traditional Chinese medicine compounds. At the same time, the present invention utilizes the reconstruction of heterologous pathways, the optimization of metabolic pathways, and the construction of a co-production strain platform to achieve the efficient de novo microbial synthesis of traditional Chinese medicine active ingredients, protopanaxadiol, cryptotanshinone diene, danshensu, and borneol, providing technical support for the green synthesis of traditional Chinese medicine active ingredients. Description of the Drawings

[0015] The drawings represent the results of the CDSY2.0 strain simultaneously producing protopanaxadiol, cryptotanshinone diene, danshensu, and borneol.

[0016] Figure 1 It is the analysis result of protopanaxadiol in the fermentation product of the CDSY 2.0 strain.

[0017] Figure 2 It is the analysis result of cryptotanshinone diene in the fermentation product of the CDSY 2.0 strain.

[0018] Figure 3 It is the analysis result of danshensu in the fermentation product of the CDSY 2.0 strain.

[0019] Figure 4Analysis results of borneol in the fermentation product of CDSY2.0 strain.

[0020] Figure 5 Quantitative analysis results of protopanaxadiol, cryptotanshinone diene, danshensu and borneol in the fermentation product of CDSY 2.0 strain: A is the yield results of the four products; B is the analysis result of the proportion of the yield content of the four products. Specific embodiments

[0021] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0022] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0023] Unless otherwise specified, the order of nucleotide sequences in the embodiments is from 5' to 3', and the order of amino acid sequences is from the N-terminus to the C-terminus.

[0024] In the following embodiments, SPSS11.5 statistical software is used to process the data. The experimental results are expressed as mean ± standard deviation, and One-way ANOVA test is used.

[0025] The culture media used in the following embodiments and their compositions are as follows:

[0026] The solutes and their concentrations of the SD-Trp liquid selection medium are 2% (m / v) glucose and 0.8% (m / v) yeast selection medium SD-Trp (Phaneno), and the solvent is water.

[0027] The solutes and their concentrations of the SD-Trp-Ura solid selection medium are 2% (m / v) glucose, 2% (m / v) agar powder and 0.8% (m / v) yeast selection medium SD-Trp-Ura (Phaneno), and the solvent is water.

[0028] The solutes and their concentrations of the SD-Trp-Ura liquid selection medium are 2% (m / v) glucose and 0.8% (m / v) yeast selection medium SD-Trp-Ura (Phaneno), and the solvent is water.

[0029] The solutes and their concentrations in the secondary liquid fermentation medium of SD-Trp-Ura are 1% (m / v) galactose, 2% (v / v) ethanol, and 0.8% (m / v) yeast selection medium SD-Trp-Ura (PhanBase), and the solvent is water.

[0030] Example 1. Construction of Recombinant Plasmids

[0031] I. Obtaining the Nucleotide Sequence Encoding the Protein

[0032] GenScript synthesized the LpD-LDH gene and the BvCYP76AD1 gene according to the codon preference of Saccharomyces cerevisiae (the sequence accession number of the LpD-LDH protein is: WP_003640741, and the sequence accession number of the BvCYP76AD1 protein is: AKH61456.1). Then, the LpD-LDH gene and the BvCYP76AD1 gene were respectively ligated to the pUC57 vector to obtain the recombinant plasmids pUC57-LpD-LDH and pUC57-BvCYP76AD1 in sequence.

[0033] II. Construction of Recombinant Plasmids

[0034] 1. Construction of the recombinant plasmid pEASY-GAL1p-LpD-LDH-PRM9t and the recombinant plasmid pEASY-GAL10p-BvCYP76AD1-CWP2t

[0035] The vector DNA fragment pGAL1-ZT containing the GAL1p promoter and the terminator PRM9t (described in the invention patent: Identification and Application of Cytochrome P450 from Plants, Publication No.: CN 119060968 A);

[0036] The vector DNA fragment pGAL10-ZT containing the GAL10p promoter and the terminator CWP2t (described in the invention patent: Identification and Application of Cytochrome P450 from Plants, Publication No.: CN 119060968 A);

[0037] (1) Using the plasmid pUC57-LpD-LDH as a template, PCR amplification was carried out with primers, and the DNA fragment Sc-LpD-LDH was recovered.

[0038] (2) Using the pUC57-BvCYP76AD1 plasmid as a template, PCR amplification was carried out with primers, and the DNA fragment Sc-BvCYP76AD1 was recovered.

[0039] (3)Use a seamless cloning kit to seamlessly ligate the enzyme-treated DNA fragment pGAL1-ZT and the DNA fragment Sc-LpD-LDH to obtain the recombinant plasmid pEASY-GAL1p-LpD-LDH-PRM9t.

[0040] 4)Use a seamless cloning kit to seamlessly ligate the enzyme-treated DNA fragment pGAL10-ZT and the DNA fragment Sc-BvCYP76AD1 to obtain the recombinant plasmid pEASY-GAL10p-BvCYP76AD1-CWP2t.

[0041] Example 2. Construction of Recombinant Strains

[0042] I. Obtaining of CDSY 2.0 Strain

[0043] 1. Obtaining of Modules

[0044] (1)Using the recombinant plasmid pEASY-GAL1p-LpD-LDH-PRM9t as a template, perform PCR amplification with homologous recombination primers containing homologous arms, and recover the M1 module.

[0045] (2)Using the recombinant plasmid pEASY-GAL10p-BvCYP76AD1-CWP2t as a template, perform PCR amplification with homologous recombination primers containing homologous arms, and recover the M2 module.

[0046] (3)Using the recombinant plasmid pEASY-GAL7p-VvCPR-SPG5t (recorded in the invention document: Identification and Application of Plant-Derived Cytochrome P450, Publication No.: CN 119060968 A) as a template, perform PCR amplification with homologous recombination primers containing homologous arms, and recover the M3 module; the protein sequence of cytochrome P450 reductase is encoded by positions 601 - 2715 of the sequence shown in SEQ ID NO.8 in the patent document with Publication No.: CN 119060968 A.

[0047] 2. Obtaining of CDSY 2.0 Strain

[0048] (1)Activate the SM-PPD-BOL strain (recorded in the literature: Li R, Wang J, Han Y, Dai Z. Compound Danshen Yeast 1.0. Sci Tradit Chin Med 2024;2(4):303–311, preserved and provided by the applicant) in SD-Trp liquid selection medium, and then prepare competent cells of the SM-PPD-BOL strain by the lithium acetate method.

[0049] (2)Add 200 ng of plasmid NDT80-gRNA (described in the literature: Yang Tingting, Wang Dong, Li Wenhao, et al. Creating a Saccharomyces cerevisiae cell factory for fermentative production of the aromatic essential oil valencene [J]. Acta Pharmaceutica Sinica, 2023, 58(06): 1619-1628), 200 ng of M1 module, 200 ng of M2 module, and 200 ng of M3 module to the competent cells of SM-PPD-BOL strain, transform, and culture at 30 °C for 48 h to obtain transformants.

[0050] (3)Plate and culture the transformants on SD-Trp-Ura solid selection medium respectively, and then perform colony verification to obtain positive transformants.

[0051] Extract the genomic DNA of the transformants and use it as a template, design primers for PCR amplification. After the PCR amplification product is verified by sequencing, it is determined as a positive transformant.

[0052] In the CDSY 2.0 strain, the GAL7p-VvCPR-SPG5t, GAL1p-LpD-LDH-PRM9t expression cassettes, and GAL10p-BvCYP76AD1-CWP2t expression cassettes are integrated into the NDT80 locus of the chromosome of the SM-PPD-BOL strain.

[0053] Application of the recombinant strains obtained in Example 3 and Example 1 in the production of protopanaxadiol, cryptotanshinone, danshensu, and borneol

[0054] 1. Activate the CDSY 2.0 strain on SD-Trp-Ura solid selection medium, and then inoculate the activated test bacteria into SD-Trp-Ura liquid selection medium, culture at 30 °C and 250 rpm for 16 h to obtain a seed solution.

[0055] 2. Inoculate 1% (v / v) of the seed solution into a triangular flask (specification: 100 ml) containing 15 ml of SD-Trp-Ura liquid selection medium with 2% (m / v) glucose as the carbon source, culture at 30 °C and 250 rpm for 48 h to obtain a culture solution.

[0056] 3. Place the culture solution obtained in step 2 into a sterile centrifuge tube (specification: 50 ml), centrifuge at 6000 rpm for 5 min, and collect the precipitate. Add 15 ml of SD-Trp-Ura secondary liquid fermentation medium with 1% (m / v) galactose and 2% (v / v) ethanol as the carbon source to the precipitate, mix well and transfer it to a new triangular flask (specification: 100 ml), add 1.5 ml (v / v = 1:10) of dodecane as an extractant, and ferment and culture at 30 °C and 250 rpm for 96 h to obtain a fermentation broth.

[0057] 4. Product detection:

[0058] Detection of protopanaxadiol: Place 2 ml of the fermentation broth into a crushing tube, centrifuge at 13,000 rpm for 1 min, discard the supernatant, and collect the cells. Then add 1 ml of a mixed solution (composed of 1 volume part of methanol and 1 volume part of acetone) and appropriate glass beads to the cells, and use a shaking crusher to crush for 10 min and sonicate for 30 min. Then centrifuge at 13,000 rpm for 1 min and collect the supernatant. Finally, filter the supernatant through a Jinlong organic nylon membrane (pore size 0.22 μm), collect the filtrate, and detect protopanaxadiol by HPLC (Agilent 1200). Quantify with a protopanaxadiol standard product (product of Shanghai Yuanye Bio-Technology Co., Ltd., catalog number B21619). HPLC determination conditions: UV detector: 203 nm; chromatographic column: Waters Symmetry C18 column (250 mm×4.6 mm, 5 μm); chromatographic conditions: The gradient elution system consists of 10% methanol-aqueous solution (A) and 100% acetonitrile (B). The chromatographic column is first equilibrated with 10% A and 90% B, then injected, and maintained at a ratio of 10% A and 90% B for 20 min. The mobile phase flow rate is 1.0 mL / min, and the column temperature is maintained at 30°C during the separation process.

[0059] Detection of danshensu: Place 1 ml of the fermentation broth into a 1.5 ml centrifuge tube, centrifuge at 13,000 rpm for 1 min, and collect the aqueous phase in the supernatant. Finally, filter the aqueous phase in the supernatant through a Jinlong aqueous nylon membrane (pore size 0.22 μm), collect the filtrate, and detect danshensu by HPLC (Agilent 1200). Quantify with a danshensu sodium standard product (product of Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number S110197). HPLC determination conditions: UV detector: 281 nm; chromatographic column: Waters Symmetry C18 column (250 mm×4.6 mm, 5 μm); chromatographic conditions: The gradient elution system consists of water (A) and 100% acetonitrile (B). The chromatographic column is first equilibrated with 90% A and 10% B, then injected, and maintained at a ratio of 90% A and 10% B for 20 min. The mobile phase flow rate is 1.0 mL / min, and the column temperature is maintained at 30°C during the separation process.

[0060] Detection of cryptotanshinone diene and borneol: Place 10 ml of the fermentation broth in a 15 ml centrifuge tube, centrifuge at 6000 rpm for 5 min, aspirate 100 μl of dodecane in the supernatant, and dilute it with 900 μl of n - hexane. Finally, filter it through a Jinlong organic nylon membrane (pore size 0.22 μm), collect the filtrate, and detect cryptotanshinone diene and borneol by GC - MS (Agilent 7890A / 5975C gas chromatograph - mass spectrometer). Quantify with borneol and cryptotanshinone diene standards (the borneol standard is a product of Shanghai Yuanye Bio - Technology Co., Ltd., product number B25349, and the cryptotanshinone diene standard is stored in the laboratory). GC - MS determination conditions: injection port temperature 250 °C, injection volume 1 μL, splitless, solvent delay 3 min; chromatographic column: DB - 35 ms (30 m * 0.25 mm); chromatographic conditions: initial temperature 45 °C, hold for 1 min, increase the temperature at 5 °C / min to 130 °C, hold for 1 min, continue to increase the temperature at 10 °C / min to 300 °C, hold for 1 min; MS conditions: Full Scan: 40 - 600 amu.

[0061] The results of the production of protopanaxadiol, cryptotanshinone diene, danshensu, and borneol by CDSY 2.0 strain are shown in the figure. Figure 1 It represents the HPLC chromatogram of protopanaxadiol analysis in the fermentation products of CDSY 2.0 strain, as well as the HPLC chromatogram of the protopanaxadiol standard; Figure 2 It represents the GC chromatogram of cryptotanshinone diene analysis in the fermentation products of CDSY 2.0 strain, as well as the GC chromatogram of the cryptotanshinone diene standard; Figure 3 It represents the HPLC chromatogram of danshensu analysis in the fermentation products of CDSY 2.0 strain, as well as the HPLC chromatogram of the danshensu standard; Figure 4 It represents the GC chromatogram of borneol analysis in the fermentation products of CDSY 2.0 strain, as well as the GC chromatogram of the borneol standard. Figure 5 In it, A represents the production data of protopanaxadiol, cryptotanshinone diene, danshensu, and borneol in the fermentation products of CDSY 2.0 strain; Figure 5 In it, B represents the content ratio analysis data of protopanaxadiol, cryptotanshinone diene, danshensu, and borneol in the fermentation products of CDSY 2.0 strain.

[0062] The results show that CDSY 2.0 strain can simultaneously produce 237.11 ± 4.65 mg / L of protopanaxadiol, 84.23 ± 3.30 mg / L of danshensu, 19.77 ± 1.20 mg / L of cryptotanshinone diene, and 1.25 ± 0.09 mg / L of borneol.

[0063] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, in accordance with the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. The application of some basic features can be made within the scope of the appended claims below.

Claims

1. An engineered bacterium for the biomimetic synthesis of compound Danshen, characterized in that, It is obtained by introducing the cytochrome P450 reductase gene, tyrosine hydroxylase, and the D-lactate dehydrogenase gene of Lactobacillus pentosus on the basis of the yeast starting strain SM-PPD-BOL strain that produces protopanaxadiol, cryptotanshinone diene, and borneol; the yeast starting strain SM-PPD-BOL strain is described in the literature: Li R, Wang J, Han Y, Dai Z. Compound Danshen Yeast 1.

0. Sci Tradit Chin Med 2024; 2(4): 303–311; The protein sequence accession number of the D-lactate dehydrogenase is: WP_003640741, and the protein sequence accession number of the tyrosine hydroxylase is: AKH61456.1; the protein sequence of cytochrome P450 reductase is encoded by positions 601-2715 of the sequence shown in SEQ ID NO.8 in the patent document with the publication number: CN 119060968A.

2. The engineered bacterium according to claim 1, wherein The P450 reductase gene is operably linked to the GAL7p promoter, the tyrosine hydroxylase gene is operably linked to the GAL10p promoter respectively, and the D-lactate dehydrogenase gene of Lactobacillus pentosus is operably linked to the GAL1p promoter.

3. The engineered bacteria according to claim 1 or 2, characterized in that, The introduced genes are transferred into the NDT80 locus of the yeast starting strain.

4. The engineered bacterium according to claim 1 or 2, characterized in that, The coding genes of the D-lactate dehydrogenase and tyrosine hydroxylase are codon-optimized according to yeast expression preferences.

5. Use of the engineered bacterium according to any one of claims 1 to 4 in the production of protopanaxadiol, cryptotanshinone diene, danshensu, and / or borneol.

6. A method for preparing protopanaxadiol, cryptotanshinone diene, danshensu and / or borneol using the engineered bacteria according to any one of claims 1 to 4, characterized in that, It comprises the step of culturing the engineered bacterium as described to produce protopanaxadiol, cryptotanshinone diene, danshensu, and / or borneol.

7. The method according to claim 6, wherein It further comprises the step of isolating the produced protopanaxadiol, cryptotanshinone diene, danshensu, and / or borneol.

8. The method according to claim 6, wherein The culture medium used during culturing is a medium with glucose and / or galactose and / or ethanol as the carbon source, and the culture conditions are culturing at 30 °C and 250 rpm.

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