WO2023103365A1 - Four engineering yeasts capable of efficiently synthesizing intermediate product or end product in synthetic route of ginsenoside ro and method - Google Patents

Abstract

Provided are four engineering yeasts, which are respectively engineering yeast CE-P1 capable of synthesizing calenduloside E, with a deposit number of CGMCC No. 23731, engineering yeast IVA-P1 capable of synthesizing chikusetsusaponin IVa, with a deposit number of CGMCC No. 23732, engineering yeast R1-P1 capable of synthesizing zingibroside R1, with a deposit number of CGMCC No. 23733, and engineering yeast Ro-P1 capable of synthesizing ginsenoside Ro, with a deposit number of CGMCC No. 23734. The de novo synthesis of a target compound is achieved by means of the above-mentioned engineering yeasts.

Description

4 strains of engineering yeast capable of efficiently synthesizing intermediate or final products of ginsenoside Ro synthesis pathway and methods thereof technical field

The invention belongs to the field of synthetic biology and metabolic engineering, and specifically relates to 4 strains of engineering yeast capable of efficiently synthesizing intermediate products or final products of ginsenoside Ro synthesis pathways and a method thereof.

Background technique

Ginsenoside is a sterol compound, also known as triterpene saponin. It is mainly found in ginseng medicinal materials. Ginsenoside is regarded as the main active ingredient in ginseng, which has a variety of pharmacological and physiological activities, including: anti-tumor, exciting central nervous system, anti-fatigue, improving memory and learning ability, promoting DNA and RNA synthesis, relieving fatigue, delaying 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., so various ginsenosides are widely used in health care and tumors, Treatment of myocardial systolic failure, liver disease, etc. caused by various reasons.

Ginsenosides all have a similar basic structure, both containing a sterane steroid core of 30 carbon atoms. They are divided into two groups according to the structure of the glycosidic group: dammarane type and oleanane type. Among them, the dammarane type is the main configuration of ginsenosides, which has been a research hotspot in recent years, and there are more and more microbial synthesis of dammarane type ginsenosides. And oleanane type ginsenosides exist less under natural conditions, such as rare ginsenoside Ro, which has a lower content (about 0.4%) in plants, so there are fewer studies, and ginsenoside Ro has anti-inflammatory, detoxification, and antithrombotic properties. , inhibition of acid system platelet aggregation, anti-hepatitis, and activation of macrophages have high research value, but its natural synthesis route is unknown (the last two steps of glycosyl modification enzymes have just been reported this year), and the glycosyl modification is complex (two kinds, a total of three) make it difficult to synthesize from scratch. No de novo synthesis of ginsenoside Ro and its precursor compounds, bamboo ginseng saponin Ⅳa and ginger-like notoginsenoside R1, has yet been reported by microorganisms.

Contents of the invention

In order to fill the above-mentioned gaps in this field, the present invention provides 4 strains of engineered yeasts that can efficiently participate in the ginsenoside synthesis pathway, and achieve high-efficiency ginsenoside Ro, de novo synthesis by culturing microorganisms without adding heterologous precursors or substrates. Three kinds of rare oleanane-type ginsenosides, including bamboo ginseng saponin IVa, ginger-like notoginseng glycoside R1, and calendulaside E.

The technical scheme adopted by the patent of the present invention to solve the technical problems is:

An engineered yeast strain CE-P1 capable of efficiently synthesizing calendulaside E is characterized in that its preservation number is CGMCC No.23731.

A method for efficiently synthesizing calendulaside E, characterized in that, cultivating the engineering yeast CE-P1 whose preservation number is CGMCC No.23731; preferably, said culturing refers to inoculating the engineering yeast CE-P1 into the YPD medium Cultivate for 3-7 days, preferably 5 days; preferably, the inoculation refers to inoculating the seed liquid of engineering yeast CE-P1 with an _OD600 of 2-5 into the YPD medium by volume ratio of 5%-15%, preferably 10% ; Preferably, the culture condition is 28-30°C.

An engineering yeast strain IVA-P1 capable of efficiently synthesizing bamboo ginseng saponin IVa is characterized in that its preservation number is CGMCC No.23732.

A method for efficiently synthesizing bamboo ginseng saponin IVa, characterized in that the engineering yeast IVA-P1 with the preservation number of CGMCC No.23732 is cultivated; preferably, the cultivation refers to inoculating the engineering yeast IVA-P1 into YPD culture 3-7 days, preferably 5 days; preferably, the inoculation refers to inoculating the seed liquid of the engineered yeast IVA-P1 with an _OD600 of 2-5 in a volume ratio of 5%-15%, preferably 10%, into the YPD medium; Preferably, the culture condition is 28-30°C.

An engineering yeast strain R1-P1 capable of efficiently synthesizing notoginseng glycoside R1 is characterized in that its preservation number is CGMCC No.23733.

A method for efficiently synthesizing notoginseng glycoside R1, characterized in that the engineering yeast R1-P1 with a preservation number of CGMCC No.23733 is cultivated; preferably, the cultivation refers to the engineering yeast R1-P1 and the bottom At the same time, the YPD medium is added to the YPD medium for 3-7 days, preferably 5 days; preferably, the culture condition is 28-30 ° C, and the inoculum size of the engineering yeast R1-P1 in the YPD medium is 5%-15% by volume, preferably 10 %; said inoculation refers to the inoculation of the engineering yeast seed solution with _OD600 of 2-5 into the liquid medium by volume ratio.

An engineering yeast strain Ro-P1 capable of efficiently synthesizing ginsenoside Ro is characterized in that its preservation number is CGMCC No.23734.

A method for efficiently synthesizing ginsenoside Ro, characterized in that the engineering yeast Ro-P1 whose preservation number is CGMCC No.23734 is cultivated; preferably, the culturing refers to inoculating the engineering yeast Ro-P1 into YPD medium for 3 - 7 days, preferably 5 days; preferably, the inoculation refers to inoculating the seed liquid of engineering yeast Ro-P1 with OD ₆₀₀ of 2-5 by volume ratio of 5%-15%, preferably 10% into the liquid medium; preferably Preferably, the culture conditions are 28-30°C.

Use of the enzyme shown in SEQ ID NO.1 in efficiently synthesizing calendulaside E.

A gene of an enzyme for efficiently synthesizing calendulaside E is characterized in that it has a nucleotide sequence as shown in SEQ ID NO.2.

Use of the enzyme shown in SEQ ID NO.3 in efficiently synthesizing notoginseng glycoside R1 and/or ginseng Ro.

A kind of high-efficiency gene for synthesizing the enzyme of notoginseng glycoside R1 and/or ginsenoside Ro, is characterized in that, has the nucleotide sequence as shown in SEQ ID NO.4.

Use of the enzyme shown in SEQ ID NO.5 in efficiently synthesizing bamboo ginsenoside IVa and/or ginsenoside Ro.

A kind of high-efficiency synthetic bamboo ginseng saponin IVa and/or the gene of the enzyme of ginsenoside Ro, is characterized in that, has the nucleotide sequence as shown in SEQ ID NO.6.

One aspect of 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. The engineered yeast CE-P1 provided by the present invention catalyzes the production of calendulaside E from oleanolic acid through the cellulose-like synthase derived from licorice and soybean compared with the reported existing methods, but the activity is low, resulting in the synthesis of The present situation of the bottleneck of the pathway, the bacterial strain CE-P1 of the present invention solves the synthesis bottleneck of oleanolic acid to calendulaside E, and can efficiently synthesize calendulaside E.

The second aspect of the present invention provides an engineered yeast strain IVA-P1, which realizes the synthesis of bamboo ginseng saponin IVa.

The third aspect of the present invention provides an engineered yeast strain R1-P1, which realizes the high-efficiency synthesis of notoginseng glycoside R1 by using the engineered yeast.

The fourth aspect of the present invention provides an engineered yeast strain Ro-P1, which solves the third problem of low glycosyl modification efficiency and realizes the efficient synthesis of ginsenoside Ro by using engineered yeast.

The effect achieved by the present invention is: without heterologous addition of substrates or precursors required for the synthesis of any link in the ginsenoside Ro synthesis pathway, only common YPD (glucose is used as carbon source input) medium is used to treat the above-mentioned engineered yeasts After culturing, after 5-7 days, the culture medium is separated and purified, and 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 bamboo Oleanane-type rare ginsenosides such as ginsenoside IVa and ginger-like notoginseng glycoside R1. Because the host used in the present invention is stable, the growth period is short, the culture conditions are mild, and the culture cost is low, the efficient de novo synthesis of the target compound is realized.

Culture deposit name: CE-P1

Deposit number: CGMCC No.23731

Class name: Saccharomyces cerevisiae

Latin name: Saccharomyces cerevisiae

Preservation unit: General Microbiology Center of China Committee for Microorganism Culture Collection

Address of Preservation Unit: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: November 5, 2021

Culture deposit name: IVA-P1

Deposit number: CGMCC No.23732

Class name: Saccharomyces cerevisiae

Latin name: Saccharomyces cerevisiae

Preservation unit: General Microbiology Center of China Committee for Microorganism Culture Collection

Address of Preservation Unit: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: November 5, 2021

Culture deposit name: R1-P1

Deposit number: CGMCC No.23733

Class name: Saccharomyces cerevisiae

Latin name: Saccharomyces cerevisiae

Preservation unit: General Microbiology Center of China Committee for Microorganism Culture Collection

Address of Preservation Unit: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: November 5, 2021

Culture deposit name: Ro-P1

Deposit number: CGMCC No.23734

Class name: Saccharomyces cerevisiae

Latin name: Saccharomyces cerevisiae

Preservation unit: General Microbiology Center of China Committee for Microorganism Culture Collection

Address of Preservation Unit: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: November 5, 2021

Description of drawings

Figure 1 shows the detection results of calendulaside E synthesized by engineered yeast CE-P1 by liquid chromatography-mass spectrometry.

Figure 2 is a comparison of liquid chromatography detection results of engineered yeast CE-P1, human UGDH, and reported cellulose synthase for the synthesis of calendulaside E.

Figure 3 is the liquid chromatography-mass spectrometry detection results of the synthesis of bamboo ginseng saponin IVa by engineered yeast IVA-P1.

Fig. 4 is the liquid chromatography-mass spectrometry detection result of the synthesis of bamboo ginger-like notoginsenoside R1 by engineered yeast R1-P1.

Fig. 5 is the detection result of ginsenoside Ro synthesized by engineered yeast Ro-P1 by liquid chromatography-mass spectrometry.

Fig. 6 is a graph showing the output of each compound synthesized by engineered yeast CE-P1, IVA-P1, R1-P1 and Ro-P1.

Detailed ways

The present invention is further illustrated below by the examples, but the protection scope of the present invention is not limited with this.

Embodiment 1, engineering yeast CE-P1 of the present invention

This group of examples provides an engineered yeast strain CE-P1 capable of efficiently synthesizing calendulaside E. The preservation number of the engineering yeast CE-P1 is CGMCC No.23731.

In a specific embodiment, the gene expression system in the engineering yeast CE-P1 contains the nucleotide sequence shown in SEQ ID NO.2 or the engineering yeast CE-P1 can express Amino acid sequence shown.

The 2nd group of embodiment, the method for the synthesis of calendulaside E of the present invention

This example provides a method for efficiently synthesizing calendulaside E. The method refers to: cultivating engineering yeast CE-P1 with a preservation number of CGMCC No. 23731.

In the preferred embodiment of this group, the cultivation refers to inoculation of engineering yeast CE-P1 in YPD medium for 3-7 days, preferably 5 days;

Preferably, the inoculation refers to inoculating the seed liquid of engineering yeast CE-P1 with an _OD600 of 2-5 into the YPD medium at a volume ratio of 5%-15%, preferably 10%;

Preferably, the culture condition is 28-30°C.

The 3rd group embodiment, engineering yeast IVA-P1 of the present invention

The examples of this group provide an engineering yeast strain IVA-P1 that can efficiently synthesize bamboo ginseng saponin IVa. The preservation number of the engineering yeast IVA-P1 is CGMCC No.23732.

In a specific embodiment, the engineering 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.

The 4th group embodiment, the method for synthesizing bamboo ginseng saponin IVa of the present invention

This group of examples provides a method for efficiently synthesizing bamboo ginseng saponin IVa. The method refers to: cultivating engineering yeast IVA-P1 with a preservation number of CGMCC No.23732;

Engineering Yeast IVA-P1

Preferably, the cultivation refers to adding the engineered yeast IVA-P1 and the substrate to YPD for 3-7 days, preferably 5 days;

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%;

Preferably, the inoculation refers to inoculating the engineered yeast seed solution with an OD600 of 2-5 into the liquid medium by volume ratio.

The 5th group embodiment, engineering yeast R1-P1 of the present invention

The examples of this group provide an engineering yeast strain R1-P1 capable of efficiently synthesizing notoginseng glycoside R1. The preservation number of the engineering yeast R1-P1 is CGMCC No.23733.

In a specific embodiment, the gene expression system in the engineering yeast R1-P1 contains the nucleotide sequence shown in SEQ ID NO.4 or the engineering yeast R1-P1 can express Amino acid sequence shown.

The 6th group embodiment, the method for synthesizing notoginseng glycoside R1 of the present invention

This group of examples provides a method for efficiently synthesizing notoginseng glycoside R1. The method refers to: cultivating engineering yeast R1-P1 with a preservation number of CGMCC No.23733;

Preferably, the cultivation refers to adding the engineered yeast R1-P1 and the substrate to the YPD medium for 3-7 days, preferably 5 days;

Preferably, the culture condition is 28-30°C, and the inoculation amount of engineered yeast R1-P1 in YPD medium is 5%-15% by volume, preferably 10%;

Preferably, the inoculation refers to inoculating the engineered yeast seed solution with an OD600 of 2-5 into the liquid medium by volume ratio.

The 7th group embodiment, engineering yeast Ro-P1 of the present invention

This group of examples provides an engineering yeast strain Ro-P1 that can efficiently synthesize ginsenoside Ro. The preservation number of the engineering yeast Ro-P1 is CGMCC No.23734.

The 8th group embodiment, the method for the synthesis of ginsenoside Ro of the present invention

This group of examples provides a method for efficiently synthesizing ginsenoside Ro. The method refers to: cultivating engineering yeast Ro-P1 with a preservation number of CGMCC No.23734;

Preferably, the culturing refers to simultaneously adding the engineered yeast Ro-P1 and the substrate to the YPD medium for 3-7 days, preferably 5 days;

Preferably, the culture condition is 28-30°C, and the inoculation amount of engineered yeast Ro-P1 in YPD medium is 5%-15% by volume, preferably 10%;

Preferably, the inoculation refers to inoculating the engineered yeast seed solution with an OD600 of 2-5 into the liquid medium by volume ratio.

Those skilled in the art adopt engineering yeast CE-P1 described in the first group of embodiments of the present invention, engineering yeast IVA-P1 described in the third group of embodiments, engineering yeast R1 described in the fifth group of embodiments 1 strain or 2 strains or 3 strains or 4 strains of engineered yeast strains in the engineering yeast Ro-P1 described in P1, the 7th group of embodiments The behavior of the intermediate product or the final product of each link in the synthesis pathway of ginsenoside Ro is uniform. Fall into the protection scope of the present invention.

The 9th group embodiment, the enzyme and its gene of efficient synthesis calendulaside E

The examples of this group provide the purposes of the enzyme shown in SEQ ID NO.1 in efficiently synthesizing calendulaside E.

Some embodiments provide an enzyme gene for efficiently synthesizing calendulaside E, characterized in that it has a nucleotide sequence as shown in SEQ ID NO.2.

The 10th group embodiment, the enzyme and its gene of highly efficient synthesis of notoginseng glycoside R1 and/or ginseng saponin Ro

This group of examples provides the use of the enzyme shown in SEQ ID NO.3 in efficiently synthesizing notoginseng glycoside R1 and/or ginseng saponin Ro.

Other embodiments of this group provide an enzyme gene for efficiently synthesizing notoginseng glycoside R1 and/or ginsenoside Ro, characterized in that it has a nucleotide sequence as shown in SEQ ID NO.4.

Example 11, Enzymes and genes for efficiently synthesizing bamboo ginsenoside IVa and/or ginsenoside Ro

This group of examples provides the use of the enzyme shown in SEQ ID NO.5 in efficiently synthesizing bamboo ginseng saponin IVa and/or ginseng saponin Ro.

Other embodiments of this group provide an enzyme gene for efficiently synthesizing bamboo ginseng saponin IVa and/or ginsenoside Ro, which is characterized in that it has a nucleotide sequence as shown in SEQ ID NO.6.

Experimental example, experimental operation of the present invention

YPD medium: about 10g/L yeast extract, 20g/L glucose, 20g/L peptone, prepare solid medium and add 20g/L agar powder.

During cultivation, use a 100mL Erlenmeyer flask, use a constant temperature shaker, 30°C, 200rpm, for cultivation, and generally cultivate for about 5 days.

Using engineered yeast CE-P1, using YPD medium to cultivate it for 5 days, as shown in Figure 1 and 2, realized the de novo synthesis of calendulaside E (CE) using engineered yeast CE-P1, and compared It can be found that the effect of AtUGDH is better than that of human HsUGDH, and the activity of Pn022859 is much higher than GmSyCSl3 and other cellulose synthases from soybean, which solves the bottleneck problem of CE synthesis. The yield of CE was 17.7±0.8mg/L, which was 8 times that of the best strain (2.2±0.1mg/L) reported in the literature using GmSyCSl3.

Using engineered yeast IVA-P1, using YPD medium to cultivate it for 5 days, as shown in Figure 3, realized the de novo synthesis of bamboo ginseng saponin Ⅳa by using engineered yeast IVA-P1, and the yield was 44.2±7.3mg/L . There is no report on the synthesis of this compound by yeast yet.

Using engineered yeast R1-P1, using YPD medium to cultivate it for 5 days, as shown in Figure 4, realized the de novo synthesis of notoginseng glycoside R1 by using engineered yeast R1-P1, and the yield was 60.7±0.8mg/ L. There is no report on the synthesis of this compound by yeast yet.

The engineered yeast Ro-P1 was used to cultivate it in YPD medium for 5 days, as shown in Figure 5, the de novo synthesis of the trisaccharide compound ginsenoside Ro by the engineered yeast Ro-P1 was achieved, and the yield was 57.9±2.0mg/ L. There is no report on the synthesis of this compound by yeast yet.

Claims (14)

An engineered yeast strain CE-P1 capable of efficiently synthesizing calendulaside E is characterized in that its preservation number is CGMCC No.23731. A method for efficiently synthesizing calendulaside E, characterized in that the engineering yeast CE-P1 whose preservation number is CGMCC No.23731 is cultivated; and/or, the cultivation refers to inoculation of engineering yeast CE-P1 into YPD culture Medium culture for 3-7 days, preferably 5 days; and/or, the inoculation refers to inoculating the seed liquid of engineering yeast CE-P1 with an _OD600 of 2-5 in a volume ratio of 5%-15%, preferably 10% to YPD culture medium; and/or, the culture condition is 28-30°C. An engineering yeast strain IVA-P1 capable of efficiently synthesizing bamboo ginseng saponin IVa is characterized in that its preservation number is CGMCC No.23732. A method for efficiently synthesizing bamboo ginseng saponin IVa, characterized in that the engineering yeast IVA-P1 with the preservation number of CGMCC No.23732 is cultivated; and/or, the cultivation refers to inoculating the engineering yeast IVA-P1 into YPD Cultivate for 3-7 days, preferably 5 days; and/or, the inoculation refers to inoculating the seed liquid of engineering yeast IVA-P1 with OD ₆₀₀ of 2-5 to YPD medium by volume ratio of 5%-15%, preferably 10% Medium; and/or, the culture condition is 28-30°C. An engineering yeast strain R1-P1 capable of efficiently synthesizing notoginseng glycoside R1 is characterized in that its preservation number is CGMCC No.23733. A method for efficiently synthesizing notoginseng glycoside R1, characterized in that the engineering yeast R1-P1 with the preservation number of CGMCC No.23733 is cultivated; and/or, the cultivation refers to engineering yeast R1-P1 and The substrate is simultaneously added to the YPD medium for 3-7 days, preferably 5 days; and/or, 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 inoculating the seed liquid of engineered yeast with an _OD600 of 2-5 into the liquid medium by volume. An engineering yeast strain Ro-P1 capable of efficiently synthesizing ginsenoside Ro is characterized in that its preservation number is CGMCC No.23734. A method for efficiently synthesizing ginsenoside Ro, characterized in that the engineering yeast Ro-P1 whose preservation number is CGMCC No.23734 is cultivated; and/or, the cultivation refers to inoculating the engineering yeast Ro-P1 with YPD medium for cultivation 3-7 days, preferably 5 days; and/or, the inoculation refers to inoculating the seed liquid of engineering yeast Ro-P1 with an _OD600 of 2-5 in a volume ratio of 5%-15%, preferably 10%, into the liquid medium and/or, the culture condition is 28-30°C. Use of the enzyme shown in SEQ ID NO.1 in efficiently synthesizing calendulaside E. A gene of an enzyme for efficiently synthesizing calendulaside E is characterized in that it has a nucleotide sequence as shown in SEQ ID NO.2. Use of the enzyme shown in SEQ ID NO.3 in efficiently synthesizing notoginseng glycoside R1 and/or ginseng Ro. A kind of high-efficiency gene for synthesizing the enzyme of notoginseng glycoside R1 and/or ginsenoside Ro, is characterized in that, has the nucleotide sequence as shown in SEQ ID NO.4. Use of the enzyme shown in SEQ ID NO.5 in efficiently synthesizing bamboo ginsenoside IVa and/or ginsenoside Ro. A kind of high-efficiency synthetic bamboo ginseng saponin IVa and/or the gene of the enzyme of ginsenoside Ro, is characterized in that, has the nucleotide sequence as shown in SEQ ID NO.6.

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