CN103484375A - Fusarium oxysporum SC1301 capable of biologically synthesizing testolactone, and synthesis method - Google Patents

Abstract

The invention discloses Fusarium oxysporum SC1301 strain Fusarium oxysporum SC1301 and a method for producing testolactone by fermenting and transforming androstane or pregnane steroid compounds with the strain. The strain of the invention uses androsta-1,4-diene-3,17-dione as the sole carbon source, and is obtained by screening from soil collected from farm orchards in Xiqing District, Tianjin. The strain Fusarium oxysporum SC1301 is now preserved in the China General Biological Culture Collection and Management Center, and the preservation number is CGMCC No.5962. The production method is as follows: Fusarium oxysporum SC1301 is used as the strain, after primary seed cultivation and secondary fermentation expansion cultivation, and crushed or dissolved androstane or pregnane steroids are put into the bacterial fermentation broth The compound undergoes a conversion reaction, is extracted with an organic solvent, and is separated by silica gel column chromatography to obtain the product testolactone, and the yields are all greater than 78%. The operation in the reaction process is simple and the energy consumption is low.

Description

A strain of Fusarium oxysporum capable of biosynthesizing testolactone and its synthetic method

Technical field:

The invention relates to the field of bioengineering, and the invention relates to a strain and a synthesis method for biosynthesizing testolactone. the

Background technique:

Testolactone is a derivative of testosterone. Its English name is Testolactone or 1,2-dehydrotestololactone, etc. It has a chemical formula of C ₁₉ H ₂₄ O ₆ , and a formula weight of 300.39. It is a white or off-white crystalline powder with a melting point of 218-219°C. Optical rotation [α] D23+45.6° (chloroform), CAS968-93-4. Testolactone is an aromatase inhibitor that can prevent the conversion of testosterone into estradiol and androstenedione into estrone. Testolide and its derivatives are clinically used as cytotoxic anticancer drugs and for the treatment of male infertility and other diseases (Dunkel L. Use of aromatase inhibitors to increase final height. Molecular and Cellular Endocrinology. 2006, 254: 207).

The ring-opening and oxygenation process of testrolactone from androstane or pregnane steroids mainly involves the Baeyer-Villiger reaction (BVMO). This reaction was reported by Baeyer and Villiger in 1899 under the action of an oxidizing agent (usually peracid) to generate esters from acyclic ketones, and to generate lactones from cyclic ketones (Leisch H, Morler K, Lau P C K . Baeyer-Villiger Monooxygenases: More Than Just Green Chemistry. Chemical Reviews, 2011, 111(7): 4165-4222). Peroxidants are mainly used in organic chemical synthesis, but most of the traditional oxidants are toxic and/or explosive, and almost all products are racemates. In order to obtain suitable enantioselectivity, some transition metal oxidants and noble metal ligands have appeared, and low-temperature reactions are required. This method has expensive reagents and harsh reaction conditions, so it only has theoretical research significance. the

In 1948, when Turfitt was studying the biotransformation of a series of steroids, he discovered for the first time that microorganisms could catalyze the BVMO reaction (Turfitt, G E. The microbiological degradation of steroids: 4. Fission of the steroid molecule. The Biochemical Journal, 1948, 42( 3): 376-383). Subsequent literature reports have shown that BVMO reactions can be produced by a large number of bacteria (such as Arthrobacter, Noica, Rhodococcus, Streptomyces, etc.) Phytophthora, etc.) catalysis (Kolek T, Szpineter A, S'wizdor A. Baeyer-Villiger oxidation of DHEA, pregnenolone, and Androsten-edione by Penicillium lilacinum AM111. Steroids, 2008, 73: 1441-1445). the

So far, the microorganisms that can catalyze androstane or pregnane steroids to produce testolactone are mainly Penicillium and Aspergillus, such as Penicillium chrysogenum (Liu H M, Li H P, Shan L H. Synthesis of steroidal lactone by penicillium citreo-viride. Steroids, 2006, 71 (11-12): 931-934), Aspergillus terreus (Kudret Y, Ahmet U, Yasemin G E. Biotransformation of some steroids by Aspergillus terreus MRC 200365. Collection of Czechoslovak Chemical Communications. 2010, 75(6):665-673), Aspergillus (Kudret Y, Ahmet U, Yasemin G E.Baeyer-Villiger oxidation of some steroids by Aspergillus tamarii MRC 72400.Collection of Czechoslovak Chemical Communications.2011, 76(6) : 743-754), soil mold (Agnieszka B, Jadwiga D G. Transformation of steroids by Trichoderma hamatum. Enzyme and Microbial Technology. 2007, 40 (6): 1615-1621), etc., but there is a long transformation time or the product is not single issue. the

In the process of the present invention, a Fusarium oxysporum SC1301 was discovered for the first time through screening of a large number of microorganisms, which can efficiently transform several steroid substances to obtain the product testolactone. The product is single and the rate is fast, and there is no need to add a coenzyme system during the conversion process, which solves the problems of coenzyme addition and electron transfer during the conversion process. The screened Fusarium oxysporum SC1301 has a fast growth rate and is easy to cultivate, with stable catalytic performance and strong environmental adaptability. The catalytic reaction condition is mild, the reaction process is simple, easy to operate, the yield is high, the by-products are few, and the energy consumption is low. the

Contents of the invention

The object of the present invention is to provide a Fusarium oxysporum capable of efficiently transforming androstane or pregnane steroid compounds into testrolactone. the

Another object of the present invention is to provide a new method for synthesizing testolactone, which is characterized in that, using androstane or pregnane steroid compound, i.e., the compound of formula (1) as a substrate, utilizes Fusarium oxysporum biological Conversion obtains formula (2) testolactone. the

The reaction formula is as follows:

R ₁ is carbonyl or β-hydroxyl;

R ₂ is carbonyl or β-acetyl;

1,2 positions, 4,5 positions, 6,7 positions are single bonds or double bonds. the

Fusarium oxysporum SC1301 for biosynthesizing testolactone provided by the present invention is a fungus isolated and screened from soil collected from farm orchards in Xiqing District, Tianjin. The bacterial strain has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on March 29, 2012, and the classification is named Fusarium oxysporum (Fusarium oxysporum), and the preservation number is CGMCC No.5962. the

The morphological characteristics of the Fusarium oxysporum SC1301 are as follows: the strain grows rapidly on the PDA plate, the colony is round, white hyphae, creeping and spreading, and the mycelium is in the form of cotton wool and grows densely. The surface of the colony is flat, the color of the back of the colony is light yellow, and the colony is closely combined with the medium. Under the microscope, the mycelium is transparent, the branches have no septum, and white conidia are produced at the top of the mycelium. The spores are sickle-shaped and have a septum. the

The characteristics of the rDNA-ITS gene sequence of the strain: the rDNA-ITS has the nucleotide sequence shown in the sequence table, and the sequence length is 480bp. the

According to its morphological characteristics and rDNA-ITS gene sequence, the strain was identified as Fusarium oxysporum. the

Use Fusarium oxysporum of the present invention to transform androstane or pregnane steroid compound into the method for testolactone, comprising the steps:

(1) Seed and fermentation culture: Inoculate the strain of Fusarium oxysporum SC1301 in the primary seed culture medium, culture at 30-35°C, 200r/min for 48h-72h, and obtain the seed culture solution. Inoculate 5%-10% of the inoculum into a fresh fermentation medium, carry out secondary fermentation at 30-35°C, 200r/min, and cultivate for 24-30 hours to obtain bacterial fermentation broth. the

(2) Transformation reaction: the steroidal compound (1) is pulverized or completely or partially dissolved in a solvent and put into the bacterial fermentation broth, 30-35°C, 150-200r/min transformation for 9-24h to terminate the reaction. The solvent is selected from but not limited to acetone, ethyl acetate, methanol, ethanol, DMSO (dimethyl sulfoxide). When the above pulverization is fed, a solubilizer is added to the fermentation broth, and the solubilizer is Tween 80. the

(3) Extraction and refining of the product: after the conversion reaction in step (2), the fermentation broth was extracted several times with ethyl acetate, the extracts were combined, dried, concentrated under reduced pressure, and separated by silica gel chromatography column after concentration to obtain testrolactone. the

The used culture medium of described biotransformation adopts following proportioning:

Seed medium: glucose 10-15g/L, glycerol 12.63g/L, peptone 5g/L, yeast extract 5g/L, NaCl 2g/L, KH ₂ PO ₄ 5g/L, pH natural.

Fermentation medium: glucose 30g/L, corn steep liquor dry powder 10~15g/L, NaNO ₃ 2g/L, KCl 0.5g/L, K ₂ HPO ₄ 3H ₂ O 2g/L, KH ₂ PO ₄ 1g/L, MgSO ₄ ·7H ₂ O 0.5g/L, FeSO ₄ ·7H ₂ O 0.02g/L, pH 8.0.

Described steroidal compound is compound (1) any one in the preferred following compound:

(1) Androst-4-ene-3,17-dione

(2) Pregn-4-ene-3,20-dione

(3) 3β-Hydroxy-5-androsten-17-one

(4) Androsta-1,4-diene-3,17-dione

The feeding concentration of the compound (1) as the substrate is 0.1-2 g/L. the

Beneficial effect of the present invention: a strain Fusarium oxysporum SC1301 capable of transforming androstane or pregnane steroidal compounds into testolactone was found by sampling from nature, the product is single and the rate is fast, and no coenzyme needs to be added during the transformation process The system solves the problem of coenzyme addition and electron transfer in the conversion process. The theoretical yield is 100%, and the recovered yield is 78-93%. The screened Fusarium oxysporum SC1301 grew fast and was easy to cultivate. It entered the logarithmic phase 12 hours after inoculation, and reached the stable phase 30 hours after inoculation. Catalytic stability, strong environmental adaptability. The catalytic reaction condition is mild, the reaction process is simple, easy to operate, the yield is high, the by-products are few, and the energy consumption is low. the

Description of drawings

Shown in Fig. 1 is the colony morphology diagram of Fusarium oxysporum SC1301;

What Fig. 2 shows is the spore morphology figure of Fusarium oxysporum SC1301;

What Fig. 3 shows is based on rDNA-ITS sequence, the diagram of phylogenetic tree of Fusarium oxysporum SC1301 and related strains;

What Fig. 4 shows is the ¹ H-NMR nuclear magnetic resonance figure of the testolactone that embodiment makes;

What Fig. 5 shows is the crystal structure X-ray diffractogram of testolactone prepared in the embodiment. the

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. the

Example 1: Screening and Identification of Bacterial Strains

1.1 Medium

Inorganic salt medium: K ₂ HPO ₄ 3H ₂ O 1.0g, Na ₂ HPO ₄ 3H ₂ O 1.0g, (NH ₄ ) ₂ HPO ₄ 2.0g, NaNO ₃ 2.0g, MgSO ₄ 7H ₂ O 0.2g , CaCl ₂ ·2H ₂ O 10mg, FeSO ₄ ·7H ₂ O 1.0mg, ZnSO ₄ 0.1mg, distilled water 1000mL.

A certain concentration of androst-1,4-diene-3,17-dione was added to the inorganic salt medium to prepare a corresponding enrichment medium. the

1.2 Isolation and purification of strains

Put 5g of the collected sample into a 250mL shaker flask containing 50mL of sterile water, add glass beads, shake on a shaker at 250r/min for 2h, and let it stand for 3-5min. After the soil particles settle, take 2mL of the supernatant and add into 50mL liquid enrichment medium (containing androst-1,4-diene-3,17-dione 1g/L). Cultivate at 30°C for 2-3 days on a shaker at 200r/min, take the enriched culture medium and dilute it on a plate (solid enriched medium containing androsta-1,4-diene-3,17-dione 1g/L) , isolate, purify until a single colony is obtained by screening, and inoculate the pure colony on the slant. the

1.3 Determination of the ability of strains to convert androsta-1,4-diene-3,17-dione into testolactone

Inoculate the purified single strain into the liquid fermentation medium from the slant, use the culture medium without inoculation as the control, shake the culture medium at 30°C and 200r/min until the medium is obviously turbid, add 1g/L androster- 1,4-diene-3,17-dione started to react, and samples were taken for analysis after conversion of 12h, 24h, and 48h respectively. Take a certain volume of fermentation broth, extract twice with an equal volume of ethyl acetate, vortex each time for 10 minutes to fully extract, centrifuge at 12,000 r/min for 10 minutes, take the supernatant and combine the extracts for TLC analysis. the

A strain capable of efficiently transforming androsta-1,4-diene-3,17-dione into testolactone was screened by the above method. The colony of this strain is round, with white hyphae, creeping and spreading, and the mycelium is in the form of cotton wool and grows densely. The surface of the colony is flat, the color of the back of the colony is light yellow, and the colony is closely combined with the medium, as shown in Figure 1. Under the microscope, the mycelium was transparent, with no septum in the branches, and white conidia were produced at the top of the mycelium. The spores were sickle-shaped and had a septum, as shown in Figure 2. Further, the total DNA of the bacterial strain is used as a template, and the universal primers of the rDNA-ITS gene are used for PCR amplification to obtain amplified fragments for recovery and sequencing. After sequencing, its sequence is shown in the sequence table. The sequence was compared with the known sequence in the GenBank database by BLAST analysis, and the rDNA-ITS sequence of the relevant species was obtained from the database, and a phylogenetic tree was constructed, as shown in Figure 3. According to the morphological characteristics and rDNA-ITS gene sequence, the strain was identified as Fusarium oxysporum. the

Embodiment 2: take androst-4-ene-3,17-dione as the method for substrate synthetic testolactone

1 Preparation of seed solution

The strain of Fusarium oxysporum SC1301 was inoculated in the first-grade seed culture medium, cultured at 30°C for 48 hours, and the shaker speed was 200r/min to obtain the seed culture solution. the

The seed medium is configured according to the following proportions: glucose 10g/L, glycerol 12.63g/L, peptone 5g/L, yeast extract 5g/L, NaCl 2g/L, KH ₂ PO ₄ 5g/L, pH natural.

2 secondary fermentation culture

The seed culture solution obtained in step 1 was transferred to fresh fermentation medium according to the inoculation amount of 5%, the culture conditions were: 30°C, the rotating speed of the shaker was 200r/min, and the fermentation time was 30h, and the bacterial cell fermentation solution was obtained. the

The composition of the fermentation medium is: glucose 30g/L, corn steep liquor dry powder 10g/L, NaNO ₃ 2g/L, KCl 0.5g/L, K ₂ HPO ₄ 3H ₂ O 2g/L, KH ₂ PO ₄ 1g /L, MgSO ₄ ·7H ₂ O 0.5g/L, FeSO ₄ ·7H ₂ O 0.02g/L, pH 8.0.

3 Transformation reactions

Androst-4-ene-3,17-dione prepared by dissolving in DMSO was added to the bacterial fermentation broth, the substrate concentration was 1 g/L, 30° C., 200 r/min for 15 h, and then the reaction was terminated. the

4 Extraction and identification of products

After the reaction, the fermentation liquid was extracted several times with ethyl acetate, the extracts _were combined, dried over anhydrous _Na2SO4 , concentrated under reduced pressure, and the product was separated by silica gel chromatography after concentration, and the eluent was ethyl acetate:petroleum ether= 7:3, the yield was 82.9%.

Embodiment 3: the method for synthesizing testolactone with pregnan-4-ene-3,20-dione as substrate

As in Example 1, Fusarium oxysporum SC1301 was subjected to primary culture and secondary culture, and pregna-4-ene-3,20-dione prepared by dissolving in DMSO was added to the bacterial fermentation broth, and the substrate The concentration is 1g/L, 30°C, 200r/min conversion for 15h, and the reaction is terminated. After the reaction, the fermentation liquid was extracted several times with ethyl acetate, the extracts _were combined, dried over anhydrous _Na2SO4 , concentrated under reduced pressure, and the product was separated by silica gel chromatography after concentration, and the eluent was ethyl acetate:petroleum ether= 7:3, the yield was 93%.

Embodiment 4: take 3β-hydroxyl-5-androsten-17-ketone as substrate synthetic testolactone method

As in Example 1, Fusarium oxysporum SC1301 was subjected to primary and secondary culture, and 3β-hydroxy-5-androsten-17-one prepared by dissolving in DMSO was added to the bacterial fermentation broth, and the substrate concentration was 1g/L, 30°C, 200r/min conversion for 24h and then stop the reaction. After the reaction, the fermentation liquid was extracted several times with ethyl acetate, the extracts _were combined, dried over anhydrous _Na2SO4 , concentrated under reduced pressure, and the product was separated by silica gel chromatography after concentration, and the eluent was ethyl acetate:petroleum ether= 5:5, the yield was 78%.

Embodiment 5: take androst-1,4-diene-3,17-dione as the method for substrate synthesis testolactone

As in Example 1, the Fusarium oxysporum SC1301 was subjected to primary and secondary culture, and androst-1,4-diene-3,17-dione prepared by dissolving in DMSO was added to the bacterial fermentation broth , the substrate concentration is 1g/L, 30°C, 200r/min conversion for 15h, and the reaction is terminated. After the reaction, the fermentation liquid was extracted several times with ethyl acetate, the extracts _were combined, dried over anhydrous _Na2SO4 , concentrated under reduced pressure, and the product was separated by silica gel chromatography after concentration, and the eluent was ethyl acetate:petroleum ether= 7:3, the yield was 82.3%.

The product testolactone structure detection data obtained in the above example are as follows:

¹ H NMR: δ(ppm): 7.02(d, J=10.2Hz, 1H), 6.26(dd, J=10.2Hz, 1H), 6.09(s, 1H), 2.72-2.55(m, 2H), 2.50 -2.40(m, 2H), 2.16-1.96(m, 4H), 1.70-1.65(m, 2H), 1.62-1.50(m, 2H), 1.48-1.42(m, 1H), 1.39(s, 3H) , 1.30-1.26 (m, 1H), 1.22 (s, 3H), 1.16-1.09 (m, 1H). (see accompanying drawing 4); crystal structure X-ray diffraction (see accompanying drawing 5).

Claims (6)

1. Fusarium oxysporum Fusarium oxysporum SC1301, depositary institution is Chinese common micro-organisms DSMZ, deposit number is CGMCC No.5962.

2. the fermentation culture method of the described bacterial strain of claim 1, it comprises step:

1) seed culture

By in the seed liquid nutrient medium after Fusarium oxysporum Fusarium oxysporum SC1301 access sterilizing, in 30～35 ℃, 200r/min cultivates 48～72h, obtains seed liquor,

Being formulated as follows of liquid nutrient medium: glucose 10～15g/L, glycerine 12.63g/L, peptone 5g/L, yeast extract 5g/L, NaCl 2g/L, KH ₂pO ₄5g/L, the pH nature;

2) fermentation culture

The seed liquid that step (1) is obtained is inoculated in fermention medium and carries out fermentation culture by inoculum size 5～10%, culture condition: 30～35 ℃, and shaking speed 200r/min, fermentation time 24～30h, obtain the fermented liquid of thalline,

The composition of described fermention medium is: glucose 30g/L, Dried Corn Steep Liquor Powder 10～15g/L, NaNO ₃2g/L, KCl 0.5g/L, K ₂hPO ₄3H ₂o 2g/L, KH ₂pO ₄1g/L, MgSO ₄7H ₂o 0.5g/L, FeSO ₄7H ₂o 0.02g/L, pH 8.0.

3. a biosynthetic means for preparing testolactone, is characterized in that, take etioallocholane or pregnane steroidal compounds is that formula (1) compound is substrate, adopts the described bacterial strain bio-transformation of claim 1 to obtain formula (2) testolactone,

Reaction formula is as follows:

R ₁for carbonyl or beta-hydroxy;

R ₂for carbonyl or β-ethanoyl;

1,2,4,5,6,7 is singly-bound or two key.

Comprise the steps:

1) conversion reaction: fermentation culture bacterial strain claimed in claim 1, formula (1) compound is pulverized or, with dissolution with solvents, dropped in cultured thalline fermented liquid and carry out bio-transformation, 30～35 ℃, 150～200r/min transforms 9～24h termination reaction;

2) extraction of product is with refining: after step (1) conversion reaction finishes, be extracted with ethyl acetate fermented liquid for several times, and combining extraction liquid, drying, concentrating under reduced pressure, adopt silica gel chromatographic column to separate after concentrating and obtain testolactone.

4. biosynthetic means as claimed in claim 3, it is characterized in that: the solvent of described dissolving substrate is selected from but is not limited only to a kind of of acetone, ethyl acetate, methyl alcohol, ethanol, dimethyl sulfoxide (DMSO), preferred dimethyl sulfoxide (DMSO), add-on is 1～4% (v/v).

5. biosynthetic means as claimed in claim 3 is characterized in that a kind of of the preferably following several compounds of the described compound as substrate (1):

4-AD;

DELTA4-pregn-3,20-dione;

3 beta-hydroxies-5-androstene-17-ketone;

Androsta-1,4-diene-3,17-dione.

6. those skilled in the art can make according to the present invention various changes or distortion, only otherwise break away from technological thought of the present invention, all belong to the defined scope of the claims in the present invention.

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