Compound salvia miltiorrhiza biomimetic synthetic engineering bacterium and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to compound salvia miltiorrhiza biomimetic synthesis engineering bacteria and application thereof. The engineering bacterium is obtained by introducing cytochrome P450 reductase, tyrosine hydroxylase and D-lactic dehydrogenase of lactobacillus pentosus on the basis of starting bacteria of saccharomyces cerevisiae for producing protopanoxadiol, hypotanshinone diene and borneol. The obtained saccharomyces cerevisiae genetically engineered bacterium can be used for simultaneously synthesizing active ingredients of protopanoxadiol, hypotanshinone diene, tanshinol and borneol of a compound salvia miltiorrhiza preparation, and has application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a compound salvia miltiorrhiza bionic synthetic engineering bacteria and application thereof. Background Art

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

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

[0004] The method for bionic synthesis of a multi-component compound preparation proposed in the present invention is of great significance for expanding the production and application of compound salvia miltiorrhiza preparations. Summary of the invention

[0005] The present invention is based on the theories of synthetic biology and molecular pharmacognosy, uses Saccharomyces cerevisiae as the base strain, and through heterologous pathway reconstruction, metabolic pathway optimization and construction of a co-production strain platform, constructs a compound Danshen artificial herbal cell that simultaneously synthesizes the active ingredient precursors of the compound Danshen preparation, tanshinone, tanshinone diene, protopanaxadiol and borneol.

[0006] Firstly, the present invention transforms the Saccharomyces cerevisiae chassis strain capable of simultaneously synthesizing tanshinone diene, protopanaxadiol and borneol to express grape-derived cytochrome P450 reductase VvCPR, D-lactate dehydrogenase LpD-LDH of Lactobacillus pentosus and beet-derived tyrosine hydroxylase BvCYP76AD, thereby obtaining a compound Salvia miltiorrhiza yeast strain capable of simultaneously synthesizing tanshinone diene, protopanaxadiol and borneol.

[0007] The present invention provides a biomimetic synthesis of compound salvia miltiorrhiza yeast genetic engineering bacteria, which is based on the brewer's yeast starting bacteria that produces protopanaxadiol, tanshinone diene and borneol, and introduces grape-derived cytochrome P450 reductase VvCPR, Lactobacillus pentosus D-lactate dehydrogenase LpD-LDH and beet-derived tyrosine hydroxylase BvCYP76AD1; Specifically, the D-lactate dehydrogenase LpD-LDH of Lactobacillus pentosus was obtained by gene synthesis, and it was connected to the GAL1p promoter to construct a plasmid; the tyrosine hydroxylase BvCYP76D1 derived from beet was obtained by gene synthesis, and it was connected to the GAL10p promoter to construct a plasmid; LpD-LDH connected to the GAL1p promoter, BvCYP76AD1 connected to the GAL10p promoter, and VvCPR connected to the GAL7p promoter were transferred into the NDT80 site of the strain SM-PPD-BOL to obtain the strain CDSY2.0.

[0008] The amino acid sequence of the D-lactate dehydrogenase LpD-LDH is shown in SEQ ID NO: 1, the amino acid sequence of the tyrosine hydroxylase BvCYP76AD1 is 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 preference.

[0009] The present invention further provides the use of the engineered bacteria in the production of protopanaxadiol, tanshinone diene, tanshinone and borneol.

[0010] The present invention also provides a method for preparing protopanaxadiol, tanshinone diene, tanshinone and borneol using the engineered bacteria, characterized in that it comprises the step of culturing the engineered bacteria to produce protopanaxadiol, tanshinone diene, tanshinone and borneol, and optionally, further separating the produced protopanaxadiol, tanshinone diene, tanshinone and borneol.

[0011] Specifically, the culture medium is a SD-Trp-Ura liquid selection medium with glucose and / or galactose and / or ethanol as carbon sources, and the culture conditions are 30° C. and 250 rpm.

[0012] The present invention is based on the guidance of molecular pharmacognosy and artificial herbal science theory, and regards the active ingredients of compound Danshen preparation as a whole to realize its microbial biomimetic 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 is constructed that simultaneously synthesizes the active ingredient precursors of compound Danshen preparation, Danshensu, Tanshinone Diene, Protopanaxadiol and Borneol.

[0013] Experiments show that the engineered bacteria constructed by the present invention can produce 237.11±4.65 mg / L protopanaxadiol, 84.23±3.30 mg / L danshinone, 19.77±1.20 mg / L tanshinone diene and 1.25±0.09 mg / L borneol. Therefore, the main active ingredients of the compound danshen preparation are synthesized simultaneously in the same engineered cell. That is, the innovation of the present invention is to regard the active ingredients of the compound danshen preparation as a whole, and to construct a compound danshen artificial herbal cell that synthesizes multiple active components simultaneously, which provides a reference for the application of synthetic biology in traditional Chinese medicine compound. At the same time, the present invention realizes the efficient microbial de novo synthesis of the active ingredients of traditional Chinese medicine protopanaxadiol, tanshinone diene, danshinone and borneol by reconstruction of heterologous pathways, optimization of metabolic pathways and construction of a co-production strain platform, providing technical support for the green synthesis of active ingredients of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The attached figure represents the results of CDSY2.0 strain producing protopanaxadiol, tanshinone diene, tanshinone and borneol simultaneously.

[0015] Figure 1 These are the analysis results of protopanaxadiol in the fermentation products of CDSY 2.0 strain.

[0016] Figure 2 The results show the analysis of tanshinone diene in the fermentation product of CDSY 2.0 strain.

[0017] Figure 3 The results show the analysis of tanshinone in the fermentation product of CDSY 2.0 strain.

[0018] Figure 4The results show the analysis of borneol in the fermentation product of CDSY2.0 strain.

[0019] Figure 5 These are the quantitative analysis results of protopanaxadiol, tanshinone diene, tanshinone and borneol in the fermentation products of CDSY 2.0 strain: A is the yield result of the four products; B is the yield content ratio analysis result of the four products. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0021] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0022] Unless otherwise specified, the order of nucleotide sequences in the examples is from 5' to 3', and the order of amino acid sequences is from N-terminus to C-terminus.

[0023] In the following examples, SPSS11.5 statistical software was used to process the data, and the experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA.

[0024] The culture medium and its composition used in the following examples are as follows: The solutes and their concentrations of the SD-Trp liquid selection medium were 2% (m / v) glucose and 0.8% (m / v) yeast selection medium SD-Trp (Panquinone), and the solvent was water.

[0025] The solutes and their concentrations of SD-Trp-Ura solid selection medium were 2% (m / v) glucose, 2% (m / v) agar powder, and 0.8% (m / v) yeast selection medium SD-Trp-Ura (Panquino), and the solvent was water.

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

[0027] The solutes and their concentrations of the SD-Trp-Ura secondary liquid fermentation medium were 1% (m / v) galactose, 2% (v / v) ethanol and 0.8% (m / v) yeast selection medium SD-Trp-Ura (Panquino), and the solvent was water.

[0028] Example 1. Construction of recombinant plasmid 1. Obtaining the nucleotide sequence encoding the protein The LpD-LDH gene and BvCYP76AD1 gene were synthesized by GenScript after optimizing 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). The LpD-LDH gene and the BvCYP76AD1 gene were then connected to the pUC57 vector to obtain the recombinant plasmids pUC57-LpD-LDH and pUC57-BvCYP76AD1, respectively.

[0029] 2. Construction of recombinant plasmid 1. Construction of recombinant plasmids pEASY-GAL1p-LpD-LDH-PRM9t and pEASY-GAL10p-BvCYP76AD1-CWP2t The vector DNA fragment pGAL1-ZT containing the GAL1p promoter and terminator PRM9t (recorded in the invention patent: Identification and application of plant-derived cytochrome P450, publication number: CN 119060968 A); A vector DNA fragment pGAL10-ZT containing the GAL10p promoter and the terminator CWP2t (described in the invention patent: Identification and application of plant-derived cytochrome P450, publication number: CN 119060968 A); (1) Using plasmid pUC57-LpD-LDH as a template, PCR amplification was performed using primers to recover the DNA fragment Sc-LpD-LDH.

[0030] (2) Using the pUC57-BvCYP76AD1 plasmid as a template and primers, PCR amplification was performed to recover the DNA fragment Sc-BvCYP76AD1.

[0031] (3) The enzyme-treated DNA fragment pGAL1-ZT and the DNA fragment Sc-LpD-LDH were seamlessly connected using a seamless cloning kit to obtain the recombinant plasmid pEASY-GAL1p-LpD-LDH-PRM9t.

[0032] (4) The enzyme-treated DNA fragment pGAL10-ZT and the DNA fragment Sc-BvCYP76AD1 were seamlessly connected using a seamless cloning kit to obtain the recombinant plasmid pEASY-GAL10p-BvCYP76AD1-CWP2t.

[0033] Example 2: Construction of recombinant strains 1. Acquisition of CDSY 2.0 strain 1. Obtaining modules (1) Using the recombinant plasmid pEASY-GAL1p-LpD-LDH-PRM9t as a template, homologous recombination primers containing homologous arms were used for PCR amplification to recover the M1 module.

[0034] (2) Using the recombinant plasmid pEASY-GAL10p-BvCYP76AD1-CWP2t as a template, homologous recombination primers containing homologous arms were used for PCR amplification to recover the M2 module.

[0035] (3) Using the recombinant plasmid pEASY-GAL7p-VvCPR-SPG5t (described in the invention document: Identification and Application of Plant-derived Cytochrome P450, Publication No.: CN 119060968 A) as a template, PCR amplification was performed using homologous recombination primers containing homologous arms to recover the M3 module.

[0036] 2. Acquisition of CDSY 2.0 strain (1) Activation of the SM-PPD-BOL strain as an example of the present invention in SD-Trp liquid selection medium (described in the literature: Li R, Wang J, Han Y, Dai Z. Compound Danshen Yeast 1.0. Sci Tradition Chin Med 2024;2(4):303–311, which was preserved and provided by the present applicant), and then the SM-PPD-BOL strain competent cells were prepared by the lithium acetate method.

[0037] (2) Add 200 ng of plasmid NDT80-gRNA (recorded in the literature: Yang Tingting, Wang Dong, Li Wenhao, et al. Creation of a Saccharomyces cerevisiae cell factory for fermentation 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 the SM-PPD-BOL strain, transform, and culture at 30°C for 48 h to obtain transformants.

[0038] (3) The transformants were plated and cultured in SD-Trp-Ura solid selective medium, and then colony verification was performed to obtain positive transformants.

[0039] The genomic DNA of the transformant was extracted and used as a template to design primers for PCR amplification. The PCR amplification product was sequenced and confirmed to be a positive transformant.

[0040] In the CDSY 2.0 strain, the GAL7p-VvCPR-SPG5t, GAL1p-LpD-LDH-PRM9t expression cassettes, and GAL10p-BvCYP76AD1-CWP2t expression cassettes were integrated into the NDT80 site of the chromosome of the SM-PPD-BOL strain.

[0041] Example 3: Application of the recombinant strain obtained in Example 1 in the production of protopanaxadiol, tanshinone diene, danshensu and borneol 1. Activate CDSY 2.0 strain on SD-Trp-Ura solid selection medium, then inoculate the activated test bacteria into SD-Trp-Ura liquid selection medium, culture at 30°C and 250rpm for 16h to obtain seed solution.

[0042] 2. Inoculate 1% (v / v) seed solution in a conical flask (100 ml) containing 15 ml of SD-Trp-Ura liquid selection medium with 2% (m / v) glucose as the carbon source, and culture at 30°C and 250 rpm for 48 h to obtain the culture solution.

[0043] 3. Place the culture solution obtained in step 2 in a sterile centrifuge tube (50 ml), centrifuge at 6000 rpm for 5 min, and collect the precipitate. Add 15 ml of SD-Trp-Ura secondary liquid fermentation medium containing 1% (m / v) galactose and 2% (v / v) ethanol as carbon sources to the precipitate, mix well and transfer to a new Erlenmeyer flask (100 ml), add 1.5 ml (v / v=1:10) dodecane as an extractant, and ferment at 30°C and 250 rpm for 96 h to obtain a fermentation solution.

[0044] 4. Product testing: Protopanaxadiol detection: 2 ml of fermentation broth was placed in a crushing tube, centrifuged at 13000 rpm for 1 min, the supernatant was removed, and the bacteria were collected. Then 1 ml of mixed solution (mixed by 1 volume of methanol and 1 volume of acetone) and appropriate glass beads were added to the bacteria, and the mixture was crushed by a oscillating crusher for 10 min and ultrasonicated for 30 min. After that, the supernatant was centrifuged at 13000 rpm for 1 min and the supernatant was collected. Finally, the supernatant was filtered with a Jinlong organic nylon membrane (pore size of 0.22 μm), the filtrate was collected, and protopanaxadiol was detected by HPLC (Agilent1200). Protopanaxadiol standard was used for quantification (product of Shanghai Yuanye Biotechnology Co., Ltd., item number B21619). HPLC determination conditions: UV detector: 203 nm; chromatographic column: Waters Symmetry C18 column (250 mm×4.6 mm, 5 μm); chromatographic conditions: gradient elution system consisting of 10% methanol-water solution (A) and 100% acetonitrile (B). The column was first equilibrated with 10% A and 90% B, then injected and maintained at a ratio of 10% A and 90% B for 20 minutes. The mobile phase flow rate was 1.0 mL / min, and the column temperature was maintained at 30°C during the separation process.

[0045] Danshensu detection: 1 ml of fermentation broth was placed in a 1.5 ml centrifuge tube, centrifuged at 13000 rpm for 1 min, and the aqueous phase in the supernatant was collected. Finally, the aqueous phase in the supernatant was filtered with a Jinlong water system nylon membrane (pore size 0.22 μm), the filtrate was collected, and Danshensu was detected by HPLC (Agilent 1200). Danshensu sodium standard was used for quantification (product of Shanghai Aladdin Biochemical Technology Co., Ltd., item number S110197). HPLC determination conditions: UV detector: 281 nm; chromatographic column: Waters Symmetry C18 column (250 mm×4.6 mm, 5 μm); chromatographic conditions: gradient elution system composed of water (A) and 100% acetonitrile (B). The chromatographic column was first equilibrated with 90% A and 10% B, then injected, and maintained at a ratio of 90% A and 10% B for 20 minutes. The mobile phase flow rate was 1.0 mL / min, and the column temperature was maintained at 30 °C during the separation.

[0046] Detection of tanshinone diene and borneol: 10 ml of fermentation broth was placed in a 15 ml centrifuge tube, centrifuged at 6000 rpm for 5 min, 100 μl of dodecane in the supernatant was aspirated, and 900 μl of n-hexane was added for dilution. Finally, the filtrate was filtered with a Jinlong organic nylon membrane (pore size 0.22 μm), and the filtrate was collected and detected by GC-MS (Agilent GC-MS Agilent 7890A / 5975C). The borneol and tanshinone diene standards were used for quantification (the borneol standard grade was a product of Shanghai Yuanye Biotechnology Co., Ltd., with a product number of B25349, and the tanshinone diene standard was stored in the laboratory). GC-MS measurement conditions: injection port temperature 250 ℃, injection volume 1μL, no splitting, solvent delay 3 min; chromatographic column: DB-35 ms (30m*0.25 mm); chromatographic conditions: initial temperature 45℃, hold for 1 min, increase the temperature to 130℃ at 5℃ / min, hold for 1 min, continue to increase the temperature to 300℃ at 10°C / min, hold for 1 min; MS conditions: Full Scan: 40-600 amu.

[0047] The results of CDSY 2.0 strain producing protopanaxadiol, tanshinone diene, tanshinone and borneol are shown in the figure. Figure 1 HPLC peak diagram representing the analysis of protopanaxadiol in the fermentation product of CDSY 2.0 strain, and HPLC peak diagram of protopanaxadiol standard; Figure 2 GC peaks representing the analysis of tanshinone dienes in the fermentation product of CDSY 2.0 strain, and GC peaks of tanshinone diene standards; Figure 3 HPLC peak diagram representing the analysis of danshensu in fermentation products of CDSY 2.0 strain, and HPLC peak diagram of danshensu standard; Figure 4 Represents the GC peak diagram of borneol analysis in the fermentation product of CDSY 2.0 strain, and the GC peak diagram of borneol standard. Figure 5 A in the middle represents the production data of protopanaxadiol, tanshinone diene, tanshinone and borneol in the fermentation products of CDSY 2.0 strain; Figure 5 B in the figure represents the content ratio analysis data of protopanaxadiol, tanshinone diene, tanshinone and borneol in the fermentation products of CDSY 2.0 strain.

[0048] The results showed that the CDSY 2.0 strain was able to simultaneously produce 237.11±4.65 mg / L protopanaxadiol, 84.23±3.30 mg / L tanshinone, 19.77±1.20 mg / L tanshinone diene, and 1.25±0.09 mg / L borneol.

[0049] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. An engineering bacterium synthesized by biomimetic synthesis of compound salvia miltiorrhiza, characterized in that: The method is obtained by introducing cytochrome P450 reductase gene, tyrosine hydroxylase and D-lactate dehydrogenase gene of Lactobacillus pentosus into a yeast starting strain producing protopanaxadiol, tanshinone diene and borneol.

2. The engineered bacteria according to claim 1, characterized in that The P450 reductase gene is operably linked to the GAL7p promoter, the tyrosine hydroxylase gene is operably linked to the GAL10p promoter, 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 yeast starting bacteria is a saccharomyces cerevisiae starting bacteria.

4. The engineered bacteria according to claim 3, characterized in that The brewer's yeast starter can simultaneously produce tanshinone diene, protopanaxadiol and borneol.

5. The engineered bacteria according to claim 4, characterized in that The P450 reductase gene comes from grapes; the tyrosine hydroxylase gene comes from sugar beets; the introduced genes are transferred into the NDT80 site of the yeast starter.

6. The engineered bacteria according to claim 4, characterized in that 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 coding genes of the D-lactate dehydrogenase and tyrosine hydroxylase are codon-optimized according to yeast expression preference.

7. Use of the engineered bacteria according to any one of claims 1 to 6 in the production of protopanaxadiol, tanshinone diene, tanshinone and / or borneol.

8. A method for preparing protopanaxadiol, tanshinone diene, danshensu and / or borneol using the engineered bacteria according to any one of claims 1 to 6, characterized in that: The method comprises the steps of culturing the engineering bacteria to produce protopanaxadiol, tanshinone diene, tanshinone and / or borneol.

9. The method according to claim 8, characterized in that The method also includes a step of separating the produced protopanaxadiol, tanshinone diene, tanshinone and / or borneol.

10. The method according to claim 8, characterized in that The culture medium used during the culture is a culture medium with glucose and / or galactose and / or ethanol as carbon sources, and the culture conditions are 30° C. and 250 rpm.

Images