CN115232843A - Biological synthesis method of white ketone and iso white ketone - Google Patents

Abstract

The invention discloses a method for heterogeneously synthesizing white ketone and heterogenous white ketone in saccharomyces cerevisiae by utilizing synthetic biology technology, isopentenyl transferase SfG6DT and LaPT1 are respectively used as expression elements of white ketone and heterogenous white ketone, and a gene expression module is constructed in a saccharomyces cerevisiae cell factory to obtain active compounds white ketone and heterogenous white ketone. The synthesis method is economic, efficient, green and environment-friendly, and the synthesized white ketone and the synthesized iso-white ketone have high purity and important application value.

Description

A kind of biosynthesis method of white ketone and isovaterone

technical field

The present invention belongs to the field of biomedicine, and specifically, the present invention relates to a method for biosynthesizing wight ketone and isowert ketone in microorganism Saccharomyces cerevisiae.

Background technique

White ketones and isoflavones belong to the class of prenylated isoflavones, and the addition of isoprenoid moieties to isoflavones can enhance their biological activity and bioavailability relative to their non-prenylated parent compounds. Studies have shown that white ketone has good anti-cancer and anti-fungal activities, and isovaterone has antibacterial and anti-plasma parasite effects. At present, the methods for obtaining white ketone and isowaiter ketone are mainly direct extraction from plants or chemical synthesis. Due to the low content of white ketone and isovaterone in plants, it is more expensive to directly extract from plants, and will consume a large amount of the original plant, resulting in a serious shortage of plant resources; and there are fewer methods for chemical synthesis of white ketone and isovaterone. , and there are problems such as cumbersome steps and unfriendly environment. At present, the market price of white ketone and isovaterone is about 1,000 yuan/mg, which is relatively expensive. Therefore, it is urgent to find a new method for obtaining white ketone and isovaterone with high efficiency.

In recent years, synthetic biology has developed rapidly. The use of microbial yeast cell factories to synthesize rare and high-value medicinal components has become one of the important ways to produce drugs and develop new drugs. The microbial growth cycle is short, and it can be produced on a large scale with a variety of sugar raw materials. Compared with plant cultivation, extraction and chemical synthesis, microbial fermentation to produce white ketone and isovaterone is a cost-effective way. It is a new type of green Biomanufacturing mode. Therefore, the use of synthetic biology methods to construct the Saccharomyces cerevisiae microbial cell factory of white ketone and isovaterone will effectively solve the problem of sustainable resource utilization of white ketone and isovaterone.

The present invention utilizes bioinformatics analysis, mining, in vitro and in vivo experimental identification, and respectively verifies the key enzyme genes related to the biosynthesis of white ketone and isovaterone - isopentenyl transferase SfG6DT (Sophora flavescensgenistein 6-dimethylallyltransferase) and isopentenyl transferase. Alkenyl transferase LaPT1 (Lupinus albusisoflavonoid prenyltransferase), the present invention uses SfG6DT and LaPT1 as the expression elements of white ketone and isovaterone respectively, and constructs Saccharomyces cerevisiae cell factory for heterologous synthesis of white ketone and isovaterone.

SUMMARY OF THE INVENTION

The purpose of the present invention is, on the basis of the prior art, to screen out a cost-effective, green and environment-friendly method, that is, using Saccharomyces cerevisiae cell factory to synthesize compounds with anti-cancer and anti-fungal activities--white ketone and isotope White ketone.

The present application finds that the expression and optimization of heterologous biosynthetic pathways in Saccharomyces cerevisiae require the introduction of multiple (continuous) genes at multiple different sites, and the application of the CRISPR-Cas9 system can speed up the construction of Saccharomyces cerevisiae strains. The chassis strain used in the present invention is IMX581 (MATa ura3-52 can1::cas9-natNT2 TRP1 LEU2 HIS3), and the IMX581 strain is a yeast mutant strain CEN.PK 113-5D inserted into the Cas9 gene to establish a CRISPR-Cas9 gene editing system. strains. In addition, the plasmid used in this editing is pMEL10 stored in the laboratory, and the sgRNA sequence is GGTATGTGCAGTTGATTCAC. The pMEL10 plasmid and sgRNA were recombined with 2×Ezmax-Single Clone MixPlus (TOLOBIO) to construct the pMEL10-XII-1 recombinant plasmid. The gene module expression cassettes XII-1up-pPGK1-SfG6DT-tCPS1-XII-1down, XII-1up-pTDH3-LaPT1-tSPO1-XII-1down and pMEL10-XII-1 recombinant plasmids were transformed into Saccharomyces cerevisiae strain IMX581, respectively. Yeast strains DR-01 and DR-02 were obtained (see Table 1). The DR-01 bacteria were cultured, and the crude protein was extracted after the bacteria were broken. Using genistein as the substrate, the gene expression products were detected by ultra-high performance liquid chromatography (UPLC), and the production of white ketone was found. The DR-02 bacteria were cultured, and microsomes were obtained by ultracentrifugation after breaking the bacteria. Using genistein as the substrate, the gene expression products of DR-02 were detected by UPLC, and it was found that the production of isowaiterone was found.

A kind of biosynthesis method of white ketone, is characterized in that, comprises the following steps:

Construction of yeast strain DR-01: The gene module expression cassettes XII-1up-pPGK1-SfG6DT-tCPS1-XII-1down and pMEL10-XII-1 recombinant plasmids were transformed into Saccharomyces cerevisiae strain IMX581 using lithium acetate high-efficiency transformation method. Coat the plate on the YNB medium containing leucine, tryptophan and histidine, cultivate at 30°C for 3-5 days, after a single colony grows, extract the genome of Saccharomyces cerevisiae for sequencing, and name this strain DR-01; (see Table 1), genistein and dimethylallyl pyrophosphate (DMAPP) can obtain compound 1-white ketone under the action of the isopentenyl transferase SfG6DT heterologously expressed in this yeast strain , the structure is as follows:

A kind of biosynthesis method of isovaterone, is characterized in that, comprises the following steps:

Construction of yeast strain DR-02: using lithium acetate high-efficiency transformation method to transform gene module expression cassettes XII-1up-pTDH3-LaPT1-tSPO1-XII-1down and pMEL10-XII-1 recombinant plasmids into Saccharomyces cerevisiae strain IMX581, and the transformed products Spread it on a plate containing YNB medium containing leucine, tryptophan and histidine, and cultivate it at 30°C for 3-5 days. After a single colony grows, extract the genome of Saccharomyces cerevisiae for sequencing, and name this strain DR -02; (see Table 1), genistein and dimethylallyl pyrophosphate (DMAPP) can obtain compound 2-isowaiterone under the action of the isopentenyltransferase LaPT1 heterologously expressed in this yeast strain , the structure is as follows:

The construction method of saccharomyces cerevisiae strain related to the synthesis of white ketone and isowhite ketone provided by the invention is shown in the following table 1:

Table 1. Construction of Saccharomyces cerevisiae strains related to the synthesis of white ketone and isovaterone

Note: The genes described in the table are: SfG6DT (Sophora flavescens genistein 6-dimethylallyltransferase) genistein-6-prenyltransferase; LaPT1 (Lupinus albusisoflavonoid prenyltransferase) isoflavone isopentenyltransferase.

As a preferred version, the above-mentioned biosynthesis method of a kind of white ketone, comprises the following steps:

Genistein and dimethylallyl pyrophosphate (DMAPP) are catalyzed by the isopentenyltransferase SfG6DT to generate white ketone in yeast strain DR-01

The 300 μL enzyme reaction system is: 1 M/L Tris-HCl 60 μL, 10 mg/mL substrate genistein 1.7 μL, 20 mM/L DMAPP 9 μL, 1 M/L MgCl ₂ 3 μL, 10 mM/L reduced coenzyme II ( NADPH) 60 μL, SfG6DT crude enzyme solution 160 μL, ultrapure water to make up to 300 μL, mix well; after overnight reaction in 30℃ water bath, extract three times with 500 μL ethyl acetate, combine the extracts, centrifuge and concentrate to dryness, then add methanol Reconstituted, filtered through a 0.22μm microporous membrane, and obtained ketone;

As a preferred version, the above-mentioned biosynthesis method of a kind of isowaiter ketone, comprises the following steps:

Genistein and dimethylallyl pyrophosphate (DMAPP) are catalyzed by the isopentenyltransferase LaPT1 to generate isowaiterone in yeast strain DR-02

The 300 μL enzyme reaction system is: 0.3 μL of 1M/L ditasitol (DTT), 7.5 μL of 1M/L 3-morpholinopropanesulfonic acid (MOPS), 1.7 μL of 10 mg/mL substrate genistein, 20 mM/L 9 μL of L DMAPP, 3 μL of 1 M/L MgCl ₂ , 60 μL of 10 mM/L reduced coenzyme II (NADPH), 210 μL of LaPT1 microsomes, supplemented with ultrapure water to 300 μL, and mixed well; after overnight reaction in a 30°C water bath , extracted three times with 500 μL of ethyl acetate, combined the extracts, centrifuged and concentrated to dryness, then reconstituted with methanol, and filtered through a 0.22 μm microporous membrane to obtain isowhite ketone.

Beneficial effects:

The present invention screened out two key enzyme genes through a large number of experiments - isopentenyl transferase SfG6DT (Sophoraflavescens genistein 6-dimethylallyltransferase) and LaPT1 (Lupinus albusisoflavonoid prenyltransferase) as the expression elements of white ketone and isoflurane ketone respectively, and constructed Saccharomyces cerevisiae Heterologous synthesis of white ketones and isoflurane ketones in cell factories. The synthesis method of the invention is economical, efficient, green and environmentally friendly, and the synthetically obtained white ketone and isowhite ketone have high purity.

Description of drawings

Fig. 1 is the UPLC diagram of the conversion of genistein into white ketone in Example 1.

Figure 2 is the mass spectrum of the product of Example 1, white ketone.

FIG. 3 is a UPLC diagram of the conversion of genistein to isowaiterone in Example 2. FIG.

Fig. 4 is the mass spectrum of the product of Example 2 isovaterone.

Detailed ways

In the present invention, gene modules are constructed and introduced into yeast cells for heterologous expression, and it is found that the strain DR-01 can produce white ketone, and the strain DR-02 can produce isowhite ketone. The present invention will be further described below in conjunction with specific implementations, but these implementations should not be construed as limiting the present invention.

Example 1 Isopentenyltransferase SfG6DT in strain DR-01 catalyzes the synthesis of white ketone from substrate genistein

Prenyltransferase SfG6DT catalyzes the enzymatic reaction of the substrate genistein as follows:

In strain DR-01, genistein and dimethylallyl pyrophosphate (DMAPP) can generate white ketone under the catalysis of isopentenyl transferase SfG6DT, and the product can be detected by ultra-high performance liquid chromatography (UPLC) White ketone to identify the enzymatic activity of the isopentenyltransferase SfG6DT.

The test method is as follows: The 300 μL enzyme reaction system is: 60 μL of 1M/L Tris-HCl, 1.7 μL of 10 mg/mL substrate genistein, 9 μL of 20 mM/L DMAPP, 3 μL of 1 M/L MgCl ₂ , 10 mM/L of additional Prototype coenzyme II (NADPH) 60 μL, isopentenyl transferase SfG6DT crude enzyme solution 160 μL, ultrapure water to make up to 300 μL, mix well; after overnight reaction in a 30°C water bath, extract three times with 500 μL ethyl acetate, and combine and extract The liquid was then centrifuged and concentrated to dryness, then reconstituted with methanol, filtered through a 0.22 μm microporous membrane to obtain the white ketone, and the subsequent filtrate was taken into UPLC. %; and the molecular weight of the product was determined by LC-MS, the results are shown in Figure 2.

Example 2 Isopentenyltransferase LaPT1 in strain DR-02 catalyzes the synthesis of isowaiterone from substrate genistein

The prenyltransferase LaPT1 catalyzes the enzymatic reaction of the substrate genistein as follows:

In strain DR-02, genistein and dimethylallyl pyrophosphate (DMAPP) can be catalyzed by isopentenyltransferase LaPT1 to generate isovaterone, which can be detected by ultra-high performance liquid chromatography (UPLC) The product white ketone was used to identify the enzymatic activity of the prenyltransferase LaPT1.

The test method is as follows: 300μL enzyme reaction system is: 1M/L ditauritol (DTT) 0.3μL, 1M/L 3-morpholinopropanesulfonic acid (MOPS) 7.5μL, 10mg/mL substrate genistein 1.7μL μL, 9 μL of 20 mM/L DMAPP, 3 μL of 1 M/L MgCl ₂ , 60 μL of 10 mM/L reduced coenzyme II (NADPH), 10 μL of isopentenyl transferase LaPT12, supplemented with ultrapure water to 300 μL, and mixed; After reacting in a water bath overnight, extract three times with 500 μL ethyl acetate, combine the extracts, centrifuge and concentrate to dryness, then add methanol to redissolve, and filter through a 0.22 μm microporous membrane to obtain isowhite ketone. UPLC, the liquid phase results are shown in Figure 3, the conversion rate is 56%, and the purity is about 97%; the molecular weight of the product is determined by LC-MS, and the results are shown in Figure 4.

Example 3 Industrial application of saccharomyces cerevisiae strains to produce white ketone and isovaterone

1. Application in the field of medicine

White ketone, a product obtained by fermentation in Saccharomyces cerevisiae cell factory, can be used in pharmaceutical production due to its anticancer activity.

2. Anti-Plasmodium

Isowaiterone, a product obtained by fermentation in a Saccharomyces cerevisiae cell factory, can be used as one of the active components of an anti-plasma parasite because of its anti-plasma parasite activity.

3. Antifungal application

White ketone and isowhite ketone have good antifungal activity and can be used as antifungal agents in agriculture and medical fields.

4. Biologics

White ketones and isowhite ketones, which are synthesized by heterologous biosynthesis of Saccharomyces cerevisiae, can be used as biological preparations for biochemical component standards or product raw materials.

The above descriptions are only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. For those skilled in the art, without departing from the principles of the present invention, various possible equivalent changes or modifications can be made according to the technical solutions of the present invention and the description of the preferred embodiments thereof. and retouching should also be regarded as the protection scope of the present invention.

Claims (8)

1. The method for synthesizing the white ketone is characterized by comprising the following steps:

constructing a yeast strain DR-01: the gene module expression cassette XII-1up-pPGK1-SfG6DT-tCPS1-XII-1down and pMEL10-XII-1 recombinant plasmids are transformed into the plasmid by using a lithium acetate high-efficiency transformation methodIn the wine yeast strain IMX581, the transformed product is coated on a plate of YNB medium containing leucine, tryptophan and histidine, and cultured for 3-5 days at 30 ℃, after a single strain grows out, the genome of the saccharomyces cerevisiae is extracted and sequenced, and the strain is named as DR-01; obtaining compound 1-white ketone with molecular formula C under the action of genistein and dimethylallyl pyrophosphate in isopentenyl transferase SfG6DT heterologously expressed in the yeast strain DR-01 ₂₀ H ₁₈ O ₅ The structural formula is as follows:

2. a method for the biosynthesis of a white ketone as claimed in claim 1, comprising the steps of:

in yeast strain DR-01, genistein and dimethylallyl pyrophosphate DMAPP generate white ketone under the catalysis of isopentenyl transferase SfG6DT

The enzyme reaction system is as follows: 1M/L Tris-HCl,10mg/mL genistein as substrate, 20mM/L dimethylallyl pyrophosphate, and 1M/L MgCl ₂ 10mM/L reduced coenzyme II, 160 mu L isoamylene transferase SfG6DT and ultrapure water are mixed evenly; reacting in a water bath kettle at 25-30 ℃ overnight, extracting with ethyl acetate, combining the extracts, centrifuging and concentrating to dryness, adding methanol for redissolution, and filtering through a 0.22 mu m microporous membrane to obtain the white ketone.

3. A method for the biosynthesis of white ketone according to claim 2, comprising the steps of:

in yeast strain DR-01, genistein and dimethylallyl pyrophosphate generate white ketone under the catalysis of isopentenyl transferase SfG6DT

The 300. Mu.L enzyme reaction system is: 1M/L Tris-HCl 60. Mu.L, 10mg/mL substrate genistein 1.7. Mu.L, 20mM/L dimethylallyl pyrophosphate 9. Mu.L, 1M/L MgCl ₂ mu.L, 60 mu.L of 10mM/L reduced coenzyme II, 160 mu.L of isoamylene transferase SfG6DT and 300 mu.L of ultrapure water, and uniformly mixing; reacting in a water bath kettle at 30 ℃ overnight, extracting with 500 mu L ethyl acetate for three times, combining the extracts, centrifuging and concentrating to dryness, adding methanol for redissolving, and filtering through a 0.22 mu m microporous membrane to obtain the white ketone.

4. A method for the biosynthesis of isophytanone, which comprises the following steps:

constructing a yeast strain DR-02: transforming gene module expression box XII-1up-pTDH3-LaPT1-tSPO1-XII-1down and pMEL10-XII-1 recombinant plasmid into Saccharomyces cerevisiae strain IMX581 by using a lithium acetate efficient transformation method, coating the transformed product on a plate of YNB culture medium containing leucine, tryptophan and histidine, culturing for 3-5 days at 30 ℃, extracting Saccharomyces cerevisiae genome sequencing after a single strain grows out, and naming the strain as DR-02; obtaining compound 2-isophytone with molecular formula C by the action of genistein and dimethylallyl pyrophosphate in isopentenyl transferase LaPT1 heterologously expressed in the yeast strain DR-02 ₂₀ H ₁₈ O ₅ The structural formula is as follows:

5. the method of claim 4, comprising the steps of:

in the yeast strain DR-02, genistein and dimethylallyl pyrophosphate generate the isophytone under the catalysis of isopentenyl transferase LaPT1

The enzyme reaction system is as follows: 1M/L of dithiothreitol, 1M/L of 3-morpholine propanesulfonic acid, 10mg/mL of substrate genistein, 20mM/L of dimethylallyl pyrophosphate, and 1M/L of MgCl ₂ Mixing 10mM/L reduced coenzyme II, isopentenyl transferase LaPT1 microsome and ultrapure water; reacting in a water bath kettle at 25-30 ℃ overnight, extracting with ethyl acetate, combining the extracts, centrifuging and concentrating to dryness, adding methanol for redissolution, and filtering through a 0.22 mu m microporous membrane to obtain the isophyton.

6. The method of claim 5, comprising the steps of:

in yeast strain DR-02, genistein and dimethylallyl pyrophosphate generate isophytone under the catalysis of isopentenyl transferase LaPT1

The 300. Mu.L enzyme reaction system is: 0.3. Mu.L of 1M/L of stachyose, 7.5. Mu.L of 1M/L of 3-morpholinopropanesulfonic acid, 1.7. Mu.L of 10mg/mL of substrate genistein, 9. Mu.L of 20mM/L of DMAPP, and 1M/L of MgCl ₂ mu.L of reduced coenzyme II (60 mu.L) with concentration of 10mM/L, 210 mu.L of LaPT1 microsome and 300 mu.L of ultrapure water are added and mixed uniformly; reacting in a water bath kettle at 30 ℃ overnight, extracting with 500 mu L ethyl acetate for three times, combining the extracts, centrifuging and concentrating to dryness, adding methanol for redissolving, and filtering through a 0.22 mu m microporous membrane to obtain the isophyton.

7. Use of the prepared white ketone of any one of claims 1 to 3 in the preparation of an anticancer drug and an antifungal agent.

8. Use of isophytone prepared according to claims 4-6 for the preparation of an antifungal agent and an plasmodium resistant agent.

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