Engineering yeast, enzyme, gene and method for efficiently synthesizing ginsenoside Ro
By developing engineered yeasts and optimizing culture conditions, the efficient synthesis of rare oleanoalkane ginseng saponins was achieved, which solved the problems of difficulty and cost in the existing technology, and achieved efficient de novo synthesis of the target compounds.
Patent Information
- Application Number
- CN202311172355.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-12
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art is difficult to efficiently synthesize rare oleanoane ginsenosides, such as ginsenoside Ro, whose natural synthesis pathways are unknown and the glycosyl modification process is complex, making de novo synthesis difficult.
By developing and optimizing engineered yeasts, such as Ro-P1, IVA-P1, R1-P1 and CE-P1, these yeasts are used to efficiently synthesize compounds such as ginseng saponin Ro, ginseng saponin IVa, ginger-like trinaganoside R1 and calendulanin E under specific culture conditions.
It is achieved that the target compounds can be synthesized efficiently by culturing microorganisms without adding heterologous precursors or substrates, which solves the problem of inefficient glycosyl modification, reduces synthesis costs, and increases yield.
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Figure CN119614405A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of synthetic biology and metabolic engineering, and specifically relates to an engineered yeast, enzyme, gene and method for efficiently synthesizing ginsenoside Ro. Background Art
[0002] Ginsenoside is a steroid compound, also known as triterpenoid saponin. It is mainly found in ginseng herbs. Ginsenoside is considered to be the main active ingredient in ginseng, and has a variety of pharmacological and physiological activities, including: anti-tumor, central nervous system excitation, anti-fatigue, improvement of memory and learning ability, promotion of DNA and RNA synthesis, fatigue relief, anti-aging, inhibition of platelet aggregation, anti-shock effect, improvement of myocardial ischemia and hypoxia, treatment and prevention of coronary heart disease, enhancement of myocardial function, protection of the body's own immune system, etc. Therefore, various ginsenosides are widely used in health care and the treatment of tumors, myocardial contractile failure caused by various reasons, liver disease, etc.
[0003] Ginsenosides all have similar basic structures, all containing a sterane steroid nucleus of 30 carbon atoms. They are divided into two groups according to the different glycoside base structures: dammarane type and oleanane type. Among them, dammarane type is the main configuration of ginsenosides, which has been a hot topic of research in recent years, and there are more and more reports on the microbial synthesis of dammarane type ginsenosides. Oleanane type ginsenosides are less common under natural conditions. For example, the rare ginsenoside Ro has a low content in plants (about 0.4%), so it has been less studied. Ginsenoside Ro has anti-inflammatory, detoxifying, anti-thrombotic, inhibition of acidic platelet aggregation, anti-hepatitis, and activation of macrophages, and has high research value, but its natural synthesis pathway is unknown (the glycosyl modification enzymes in the last two steps have just been reported this year), and the glycosyl modification is complex (two types, a total of three), which makes it difficult to synthesize it from scratch. There have been no reports on the de novo synthesis of ginsenoside Ro and its precursor compounds, bamboo ginsenoside IVa and ginger-shaped notoginseng glycoside R1, using microorganisms. Summary of the invention
[0004] In order to fill the above-mentioned gaps in the field, the engineered yeast, enzymes, genes and methods provided by the present invention can efficiently participate in the ginsenoside synthesis pathway, and achieve the de novo synthesis of three rare oleanane-type ginsenosides, including ginsenoside Ro, ginsenoside IVa, and ginger-like notoginseng glycoside R1, as well as calendulaside E, without adding heterologous precursors or substrates, simply by culturing microorganisms.
[0005] The technical solution adopted by the present invention to solve the technical problem is:
[0006] An engineered yeast strain Ro-P1 capable of efficiently synthesizing ginsenoside Ro, characterized in that its preservation number is CGMCC No.23734.
[0007] A method for efficiently synthesizing ginsenoside Ro, characterized in that the engineered yeast Ro-P1 with a preservation number of CGMCC No.23734 is cultured; preferably, the culture refers to inoculating the engineered yeast Ro-P1 with a YPD medium and culturing it for 3-7 days, preferably 5 days; preferably, the inoculation refers to culturing OD 600 The seed liquid of the engineered yeast Ro-P1 with a volume ratio of 2-5 is inoculated into the liquid culture medium at a volume ratio of 5%-15%, preferably 10%; preferably, the culture condition is 28-30°C.
[0008] An engineered yeast strain IVA-P1 capable of efficiently synthesizing panax ginsenoside IVa is characterized in that its preservation number is CGMCC No.23732.
[0009] A method for efficiently synthesizing ginsenoside IVa of Panax notoginseng, characterized in that the engineered yeast IVA-P1 with a preservation number of CGMCC No. 23732 is cultured; preferably, the culture refers to inoculating the engineered yeast IVA-P1 with YPD for 3-7 days, preferably 5 days; preferably, the inoculation refers to OD 600 The seed liquid of the engineered yeast IVA-P1 of 2-5 is inoculated into the YPD medium at a volume ratio of 5%-15%, preferably 10%; preferably, the culture condition is 28-30°C.
[0010] An engineered yeast strain R1-P1 capable of efficiently synthesizing ginger-like notoginseng glycoside R1 is characterized in that its preservation number is CGMCC No.23733.
[0011] A method for efficiently synthesizing ginger-like notoginseng glycoside R1, characterized in that the engineered yeast R1-P1 with a preservation number of CGMCC No. 23733 is cultured; preferably, the culture refers to adding the engineered yeast R1-P1 and a substrate to a YPD medium and culturing for 3-7 days, preferably 5 days; preferably, the culture condition is 28-30° C., and the inoculation amount of the engineered yeast R1-P1 in the YPD medium is 5%-15% by volume, preferably 10%; the inoculation refers to adding OD 600 The engineered yeast seed solution is inoculated into the liquid culture medium at a volume ratio of 2-5.
[0012] Use of the enzyme shown in SEQ ID NO.3 in the efficient synthesis of notoginseng gingeroside R1 and / or ginsenoside Ro.
[0013] A gene for an enzyme that efficiently synthesizes ginger-like notoginseng glycoside R1 and / or ginsenoside Ro, characterized by having a nucleotide sequence as shown in SEQ ID NO.4.
[0014] Use of the enzyme shown in SEQ ID NO.5 in the efficient synthesis of ginsenoside IVa and / or ginsenoside Ro.
[0015] A gene for an enzyme that efficiently synthesizes ginsenoside IVa and / or ginsenoside Ro, characterized by having a nucleotide sequence as shown in SEQ ID NO.6.
[0016] On the one hand, the present invention provides an engineered yeast strain CE-P1, which solves the problem of insufficient supply of UDP-GlcA (uridine diphosphate-glucuronic acid) by Saccharomyces cerevisiae itself. Compared with the reported existing method that oleanolic acid is catalyzed by cellulose synthase derived from licorice and soybean to produce acalendulaside E, but the activity is low, thus causing the bottleneck of the synthesis pathway, the strain CE-P1 of the present invention solves the bottleneck of the synthesis of acalendulaside E from oleanolic acid, and can efficiently synthesize acalendulaside E.
[0017] The second aspect of the present invention provides an engineered yeast strain IVA-P1, which can synthesize Panax notoginseng saponin IVa.
[0018] The third aspect of the present invention provides an engineered yeast strain R1-P1, which realizes the efficient synthesis of notoginseng gingeroside R1 by using the engineered yeast.
[0019] The fourth aspect of the present invention provides an engineered yeast strain Ro-P1, which solves the problem of low efficiency of the third glycosyl modification and realizes the efficient synthesis of ginsenoside Ro using engineered yeast.
[0020] The effect achieved by the present invention is: there is no need to heterologously add the substrate or precursor required for the synthesis of any link in the ginsenoside Ro synthesis pathway, and only the ordinary YPD (glucose as carbon source input) medium is used to culture the above-mentioned engineered yeasts, and the culture fluid is separated and purified after 5-7 days, so that the intermediates and final products of each link in the ginsenoside Ro synthesis pathway can be efficiently obtained: including calendulaside E, ginsenoside Ro and its precursor compound ginsenoside IVa, ginger-shaped notoginseng glycoside R1 and other oleanane-type rare ginsenosides. Because the host used in the present invention is stable, the growth cycle is short, the culture conditions are mild, and the culture cost is low, the efficient de novo synthesis of the target compound is achieved.
[0021] Strain deposit name: CE-P1
[0022] Accession number: CGMCC No.23731
[0023] Classification and nomenclature: Saccharomyces cerevisiae
[0024] Latin name: Saccharomyces cerevisiae
[0025] Depository: China National Microbiological Culture Collection Administration General Microbiology Center
[0026] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing
[0027] Date of deposit: November 5, 2021
[0028] Strain deposit name: IVA-P1
[0029] Accession number: CGMCC No.23732
[0030] Classification and nomenclature: Saccharomyces cerevisiae
[0031] Latin name: Saccharomyces cerevisiae
[0032] Depository: China National Microbiological Culture Collection Administration General Microbiology Center
[0033] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing
[0034] Date of deposit: November 5, 2021
[0035] Strain deposit name: R1-P1
[0036] Accession number: CGMCC No.23733
[0037] Classification and nomenclature: Saccharomyces cerevisiae
[0038] Latin name: Saccharomyces cerevisiae
[0039] Depository: China National Microbiological Culture Collection Administration General Microbiology Center
[0040] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing
[0041] Date of deposit: November 5, 2021
[0042] Strain deposit name: Ro-P1
[0043] Accession number: CGMCC No.23734
[0044] Classification and nomenclature: Saccharomyces cerevisiae
[0045] Latin name: Saccharomyces cerevisiae
[0046] Depository: China National Microbiological Culture Collection Administration General Microbiology Center
[0047] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing
[0048] Date of deposit: November 5, 2021 BRIEF DESCRIPTION OF THE DRAWINGS
[0049] Figure 1 These are the results of liquid chromatography-mass spectrometry detection of calendula glycoside E synthesized by engineered yeast CE-P1.
[0050] Figure 2 The liquid chromatography detection results of the engineered yeast CE-P1, human UGDH and reported cellulose synthase on the synthesis of acalendula glycoside E were compared.
[0051] Figure 3 These are the results of liquid chromatography-mass spectrometry detection of the engineered yeast IVA-P1 synthesizing panax ginsenoside Ⅳa.
[0052] Figure 4 The results of liquid chromatography-mass spectrometry detection of the engineered yeast R1-P1 synthesizing ginger-like Panax notoginseng glycoside R1.
[0053] Figure 5 This is the result of liquid chromatography-mass spectrometry detection of ginsenoside Ro synthesized by engineered yeast Ro-P1.
[0054] Figure 6 The yield diagram of each compound synthesized by engineered yeast CE-P1, IVA-P1, R1-P1, and Ro-P1. DETAILED DESCRIPTION
[0055] The present invention is further illustrated by the following examples, but the protection scope of the present invention is not limited thereto.
[0056] The first group of examples, the engineered yeast Ro-P1 of the present invention
[0057] This group of embodiments provides an engineered yeast Ro-P1 that can efficiently synthesize ginsenoside Ro. The deposit number of the engineered yeast Ro-P1 is CGMCC No.23734.
[0058] The second group of examples, the method for synthesizing ginsenoside Ro of the present invention
[0059] This group of embodiments provides a method for efficiently synthesizing ginsenoside Ro. The method comprises: culturing the engineered yeast Ro-P1 with a preservation number of CGMCC No. 23734;
[0060] Preferably, the culturing refers to adding the engineered yeast Ro-P1 and the substrate to YPD medium at the same time and culturing for 3-7 days, preferably 5 days;
[0061] Preferably, the culture condition is 28-30°C, and the inoculation amount of the engineered yeast Ro-P1 in the YPD medium is 5%-15% by volume, preferably 10%;
[0062] Preferably, the inoculation refers to inoculating engineered yeast seed liquid with an OD600 of 2-5 into a liquid culture medium according to a volume ratio.
[0063] The actions of those skilled in the art to synthesize the intermediate products or final products of each link in the synthesis pathway of ginsenoside Ro by using one, two, three or four strains of the engineered yeast CE-P1 described in the first group of embodiments of the present invention, the engineered yeast IVA-P1 described in the third group of embodiments, the engineered yeast R1-P1 described in the fifth group of embodiments, and the engineered yeast Ro-P1 described in the seventh group of embodiments of the present invention fall within the scope of protection of the present invention.
[0064] The third group of examples, the engineered yeast IVA-P1 of the present invention
[0065] This group of examples provides an engineered yeast strain IVA-P1 that can efficiently synthesize Panax notoginseng saponin IVa. The deposit number of the engineered yeast strain IVA-P1 is CGMCC No.23732.
[0066] In a specific embodiment, the engineered yeast IVA-P1 can express the gene sequence shown in SEQ ID NO.6 or the amino acid sequence shown in SEQ ID NO.5.
[0067] The fourth group of examples, the method for synthesizing Panax notoginseng saponin IVa of the present invention
[0068] This group of embodiments provides a method for efficiently synthesizing Panax notoginseng saponin IVa. The method comprises: culturing the engineered yeast IVA-P1 with a preservation number of CGMCC No. 23732;
[0069] Engineered yeast IVA-P1
[0070] Preferably, the culturing refers to adding the engineered yeast IVA-P1 and the substrate to YPD at the same time and culturing for 3-7 days, preferably 5 days;
[0071] Preferably, the culture condition is 28-30°C, and the inoculation amount of the engineered yeast IVA-P1 in the YPD medium is 5%-15% by volume, preferably 10%;
[0072] Preferably, the inoculation refers to inoculating engineered yeast seed liquid with an OD600 of 2-5 into a liquid culture medium according to a volume ratio.
[0073] The fifth group of examples, the engineered yeast R1-P1 of the present invention
[0074] This group of embodiments provides an engineered yeast strain R1-P1 that can efficiently synthesize ginger-like notoginseng glycoside R1. The deposit number of the engineered yeast strain R1-P1 is CGMCC No.23733.
[0075] In a specific embodiment, the gene expression system in the engineered yeast R1-P1 contains the nucleotide sequence shown in SEQ ID NO.4 or the engineered yeast R1-P1 can express the amino acid sequence shown in SEQ ID NO.3.
[0076] Group 6 Example: Method for synthesizing ginger-like notoginseng glycoside R1 of the present invention
[0077] This group of embodiments provides a method for efficiently synthesizing ginger-like notoginseng glycoside R1. The method comprises: culturing the engineered yeast R1-P1 with a preservation number of CGMCC No. 23733;
[0078] Preferably, the culturing refers to adding the engineered yeast R1-P1 and the substrate to YPD medium at the same time and culturing for 3-7 days, preferably 5 days;
[0079] Preferably, the culture condition is 28-30°C, and the inoculation amount of the engineered yeast R1-P1 in the YPD medium is 5%-15% by volume, preferably 10%;
[0080] Preferably, the inoculation refers to inoculating engineered yeast seed liquid with an OD600 of 2-5 into a liquid culture medium according to a volume ratio.
[0081] The seventh group of examples, the engineered yeast CE-P1 of the present invention
[0082] This group of examples provides an engineered yeast strain CE-P1 that can efficiently synthesize calendulaside E. The deposit number of the engineered yeast strain CE-P1 is CGMCC No.23731.
[0083] In a specific embodiment, the gene expression system in the engineered yeast CE-P1 contains the nucleotide sequence shown in SEQ ID NO.2 or the engineered yeast CE-P1 can express the amino acid sequence shown in SEQ ID NO.1.
[0084] Group 8 Example: Method for synthesizing calendulaside E of the present invention
[0085] This embodiment provides a method for efficiently synthesizing calendulaside E. The method comprises: culturing the engineered yeast CE-P1 with a preservation number of CGMCC No. 23731.
[0086] In the preferred embodiments of this group, the culturing refers to inoculating the engineered yeast CE-P1 into YPD medium and culturing for 3-7 days, preferably 5 days;
[0087] Preferably, the inoculation refers to the OD 600 The seed solution of the engineered yeast CE-P1 of 2-5 is inoculated into the YPD medium at a volume ratio of 5%-15%, preferably 10%;
[0088] Preferably, the culture condition is 28-30°C.
[0089] Example 9: Enzymes and genes for efficiently synthesizing calendulaside E
[0090] This group of embodiments provides the use of the enzyme shown in SEQ ID NO.1 in the efficient synthesis of calendulaside E.
[0091] Some embodiments provide a gene for an enzyme that efficiently synthesizes calendulaside E, characterized by having a nucleotide sequence as shown in SEQ ID NO.2.
[0092] Example 10: Enzymes and genes for efficiently synthesizing ginger-like notoginseng glycoside R1 and / or ginsenoside Ro
[0093] This group of embodiments provides the use of the enzyme shown in SEQ ID NO.3 in the efficient synthesis of notoginseng gingeroside R1 and / or ginsenoside Ro.
[0094] Other embodiments of this group provide a gene for an enzyme that efficiently synthesizes ginger-like notoginseng glycoside R1 and / or ginsenoside Ro, characterized in that it has a nucleotide sequence as shown in SEQ ID NO.4.
[0095] Example 11: Enzymes and genes for efficiently synthesizing ginsenoside IVa and / or ginsenoside Ro
[0096] This group of embodiments provides the use of the enzyme shown in SEQ ID NO.5 in the efficient synthesis of ginsenoside IVa and / or ginsenoside Ro.
[0097] Other embodiments of this group provide a gene for an enzyme that efficiently synthesizes ginsenoside IVa and / or ginsenoside Ro, characterized in that it has a nucleotide sequence as shown in SEQ ID NO.6.
[0098] Experimental Examples and Experimental Procedures of the Present Invention
[0099] YPD medium: about 10g / L yeast extract, 20g / L glucose, 20g / L peptone. Add 20g / L agar powder to prepare solid culture medium.
[0100] The culture was carried out in a 100 mL Erlenmeyer flask using a constant temperature shaker at 30°C and 200 rpm, and generally for about 5 days.
[0101] The engineered yeast CE-P1 was cultured in YPD medium for 5 days. Figure 1 , 2 As shown, the de novo synthesis of calendula glycoside E (CE) was achieved using the engineered yeast CE-P1. By comparison, it was found that AtUGDH was better than human HsUGDH, and the activity of Pn022859 was much higher than GmSyCSl3 from soybean and other cellulose synthases, which solved the bottleneck problem of CE synthesis.
[0102] The engineered yeast IVA-P1 was cultured in YPD medium for 5 days. Figure 3 As shown, the de novo synthesis of Panax notoginseng saponin IVa was achieved using the engineered yeast IVA-P1. There is currently no report on the synthesis of this compound using yeast.
[0103] The engineered yeast R1-P1 was cultured in YPD medium for 5 days. Figure 4 As shown, the de novo synthesis of ginger-like notoginseng glycoside R1 was achieved using engineered yeast R1-P1. Currently, there is no report on the synthesis of this compound using yeast.
[0104] The engineered yeast Ro-P1 was cultured in YPD medium for 5 days. Figure 5 As shown, the de novo synthesis of the trisaccharide compound ginsenoside Ro was achieved using the engineered yeast Ro-P1. There is currently no report on the synthesis of this compound using yeast.
Claims
1. An engineered yeast strain Ro-P1 capable of efficiently synthesizing ginsenoside Ro, characterized in that: Its accession number is CGMCC No.23734.
2. A method for efficiently synthesizing ginsenoside Ro, characterized in that: Cultivate the engineered yeast Ro-P1 with the accession number of CGMCC No.23734; preferably, the culturing refers to inoculating the engineered yeast Ro-P1 with YPD medium and culturing for 3-7 days, preferably 5 days; preferably, the inoculation refers to OD 600 The seed liquid of the engineered yeast Ro-P1 with a volume ratio of 2-5 is inoculated into the liquid culture medium at a volume ratio of 5%-15%, preferably 10%; preferably, the culture condition is 28-30°C.
3. An engineered yeast strain IVA-P1 that can efficiently synthesize Panax notoginseng saponin IVa, characterized in that: Its accession number is CGMCC No.23732.
4. A method for efficiently synthesizing Panax notoginseng saponin IVa, characterized in that: Cultivate the engineered yeast IVA-P1 with the accession number of CGMCC No.23732; preferably, the culturing refers to inoculating the engineered yeast IVA-P1 with YPD for 3-7 days, preferably 5 days; preferably, the inoculation refers to OD 600 The seed liquid of the engineered yeast IVA-P1 of 2-5 is inoculated into the YPD medium at a volume ratio of 5%-15%, preferably 10%; preferably, the culture condition is 28-30°C.
5. An engineered yeast strain R1-P1 capable of efficiently synthesizing ginger-like notoginseng glycoside R1, characterized in that: Its accession number is CGMCC No.23733.
6. A method for efficiently synthesizing ginger-like notoginseng glycoside R1, characterized in that: Cultivating the engineered yeast R1-P1 with a preservation number of CGMCC No.23733; preferably, the culturing refers to adding the engineered yeast R1-P1 and the substrate to the YPD medium and culturing for 3-7 days, preferably 5 days; preferably, the culture condition is 28-30°C, and the inoculation amount of the engineered yeast R1-P1 in the YPD medium is 5%-15% by volume, preferably 10%; the inoculation refers to adding the OD 600 The engineered yeast seed solution is inoculated into the liquid culture medium at a volume ratio of 2-5.
7. Use of the enzyme shown in SEQ ID NO.3 in the efficient synthesis of ginger-like notoginseng glycoside R1 and / or ginsenoside Ro.
8. A gene for an enzyme that efficiently synthesizes ginger-like notoginseng glycoside R1 and / or ginsenoside Ro, characterized in that: It has a nucleotide sequence as shown in SEQ ID NO.
4.
9. Use of the enzyme shown in SEQ ID NO.5 in the efficient synthesis of ginsenoside IVa and / or ginsenoside Ro.
10. A gene for an enzyme that efficiently synthesizes ginsenoside IVa and / or ginsenoside Ro, characterized in that: It has a nucleotide sequence as shown in SEQ ID NO.6.