Steroid c1,2 dehydrogenase mutants and their use in the synthesis of tetranic acid

Abstract

The application discloses a sterone C1,2 position dehydrogenase mutant and application thereof in synthesis of acetic acid tetraen. The application mines and modifies the sterone C1,2 position dehydrogenase by molecular biology technology and protein engineering technology, obtains a mutant with excellent properties, and the amino acid sequences of the sterone C1,2 position dehydrogenase mutant are shown as SEQ ID NO:5-SEQ ID NO:12. Under the participation of hydrogen donor and oxygen, the sterone C1,2 position dehydrogenase mutant can catalyze the oxidation of 60g / L acetic acid trien to acetic acid tetraen, the conversion rate reaches 99.1% within 10h, and the space-time yield reaches 5.94g / L / h. The application has the advantages of green safety, short reaction cycle, simple purification, low cost and the like, and has an excellent application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme catalysis and pharmaceutical engineering, and particularly relates to a steroid ketone C1,2 dehydrogenase mutant and application thereof in synthesis of tetraene acetate. BACKGROUND

[0002] Tetraene acetate (3TR), Chinese alias: 21-hydroxypregn-1, 4, 9(11), 16-tetraene-3, 20-dione-21-acetate, molecular formula is C 23 H 26 O4, the structural formula is as follows:

[0003]

[0004] 3TR is a synthetic steroid, which has a wide range of applications in in vitro studies and may have pharmacological effects such as anti-inflammatory, immunosuppressive and anti-tumor. At the same time, 3TR is also an important intermediate for synthesizing adrenocortical hormone drugs such as dexamethasone, betamethasone, triamcinolone acetonide and budesonide, and has a large market demand.

[0005] Initially, 3TR was mainly synthesized by chemical method. Patent CN102603843A uses 1, 4, 9(11)-triene-androst-3, 17-dione as a starting material, and sequentially undergoes alkyne, esterification, oxidation and rearrangement steps to finally synthesize 3TR with a purity of 98% and a yield of 50-70%. Patent CN105622699A uses 4, 9(11)-dien-3, 17-dione as a starting material, and through a series of reactions such as etherification, addition, hydrolysis, elimination, substitution, rearrangement and dehydrogenation, 3TR with a purity of 98.3% is finally prepared, and the yield is only 47%. Patent CN107814824A uses 1, 4, 9(11)-triene-androst-3, 17-dione as a starting material, and through a series of reaction steps including alkyne, esterification, bromine removal and displacement elimination, 3TR is synthesized. The alkyne step of this route uses unstable and flammable potassium acetylide as a raw material, which has a great risk in the production process. The chemical preparation of 3TR has poor atom economy, and the process needs strict condition control, even involves toxic chemical catalysts or raw materials, and has a high synthesis cost.

[0006] With the development of molecular biology technology, metabolic engineering and protein engineering, biosynthesis method began to be applied to the synthesis of 3TR. Biosynthesis method includes microbial transformation method and enzyme catalysis method, which are essentially enzyme-catalyzed reactions. Fokina et al. used Nocardioides simplex VKM Ac-2033D strain to biotransform acetoxytrienolide (2TR). Due to the complexity of enzyme system in the bacteria, in addition to the Δ1-dehydrogenation reaction, there is also a deacetylation reaction, resulting in the presence of 3TR, trienolide and tetraenolide in the fermentation broth, which seriously affects the production efficiency of 3TR.

[0007] Enzyme catalysis method is a process of using enzymes or enzyme-containing cells to specifically convert and synthesize target products under mild conditions, which has obvious advantages in the synthesis of 3TR. This method mainly uses acetoxytrienolide (2TR) as the substrate and utilizes steroidal ketone C1,2 dehydrogenase (KstD1) for catalysis to synthesize single 3TR, and the reaction process is as follows:

[0008]

[0009] Patent CN 116987754 A reports a process for synthesizing 3TR by enzyme catalysis. The resting cells TQ-0007 of steroidal ketone C1,2 dehydrogenase derived from Pseudomonas resinovorans are used to oxidize 15 g / L of 2TR at 28℃ for 24 h, and the conversion rate of 3TR reaches 96.1%. Although the substrate load has met the requirements of industrialization, the cycle of this method is long, and the space-time yield is only 0.625 g / L / h, so there is still a lot of room for development of this method. SUMMARY

[0010] To solve the above technical problems, an enzyme catalysis method for efficiently preparing acetoxytetraenolide (3TR) is provided to improve the substrate load and space-time yield of 3TR and reduce the synthesis cost of 3TR.

[0011] To solve the above technical problems, the technical scheme adopted by the present application is that the steroidal ketone C1,2 dehydrogenase mutant is obtained by replacing any one of the amino acid residues in the amino acid sequence shown in SEQ ID No. 4 as follows:

[0012] 1) I343L;

[0013] 2) I343M;

[0014] 3) I343T;

[0015] 4) I343T / Y408F;

[0016] 5) I343T / Y408F / F116S;

[0017] 6) I343T / Y408F / F116D;

[0018] 7) I343T / Y408F / F116T;

[0019] 8) I343T / Y408F / F116V.

[0020] Wherein, the mutant format XnY represents that the n th amino acid residue X is replaced by the amino acid residue Y; I343T / Y408F represents that I343T and Y408F two kinds of amino acid residue replacement in the amino acid sequence occur simultaneously.

[0021] Further, the amino acid sequences of the steroid C1,2 dehydrogenase mutants are respectively shown in SEQ ID NO: 5 to SEQ ID NO: 12.

[0022] Further, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 4 is shown in SEQ ID No. 16; the nucleotide sequences encoding the steroid C1,2 dehydrogenase mutants are respectively shown in SEQ ID NO: 17 to SEQ ID NO: 24.

[0023] The above-mentioned steroid C1,2 dehydrogenase mutant can be applied in the synthesis of acetate tetraene.

[0024] The application provides a kind of expression plasmid of steroid C1,2 dehydrogenase, it is characterized in that, comprising promoter, RBS sequence, any one of nucleotide sequences in SEQ ID No.17~24 and terminator.

[0025] The application also provides a kind of engineering strain of expressing steroid C1,2 dehydrogenase, it is the E. coli comprising above-mentioned expression plasmid.Steroid C1,2 dehydrogenase E. coli engineering cell preparation process, with glycerol or glucose as carbon source, with lactose as inducing agent, to the engineering strain of expressing above-mentioned steroid C1,2 dehydrogenase is cultivated and induced, centrifugal collection E. coli wet bacteria.

[0026] The application further provides a kind of efficient preparation of enzyme catalysis method of acetate tetraene: with acetate triene as raw material, oxygen is hydrogen acceptor, under the catalysis of hydrogen transfer body and above-mentioned steroid C1,2 dehydrogenase mutant synthesis acetate tetraene.

[0027] As a possible implementation mode, further, the hydrogen transfer body is selected from PMS.

[0028] As a possible implementation, further, the steroid C1,2 dehydrogenase mutant is from a recombinant E. coli whole cell or cell broken liquid which co-expresses the steroid C1,2 dehydrogenase mutant by genetic engineering means.

[0029] As a preferred implementation, preferably, 18 g of acetic acid triene (2TR), 0.18 g of PMS, 15 g of E. coli wet cells (MTKstD-I343T / Y408F / F116S mutant) or cell broken liquid, and 300 mL of water are added in a 1L reactor. The aeration amount is set to 0.3 L / min, the stirring speed is 500 rpm, and the reaction temperature is 37℃. During the reaction, the pH of the reaction solution is adjusted to about 8 by dropwise adding 2M Na2CO3 or NaOH, and the conversion rate reaches 99.1% after 10 h of reaction.

[0030] As a possible implementation, further, the enzyme catalytic method for efficiently preparing acetic acid tetraene also includes acetic acid tetraene purification, and the steps are as follows:

[0031] After the enzyme catalytic reaction is completed, the reaction solution is extracted with ethyl acetate for multiple times, the ethyl acetate is evaporated, the acetic acid tetraene crude product is obtained, the crude product is washed with water until the filtrate is colorless, and then dried to obtain a white powder.

[0032] The present application has the following advantages:

[0033] The present application uses molecular biology technology and protein engineering technology to mine and modify steroid C1,2 dehydrogenase, and obtains a mutant with excellent properties. Under the participation of hydrogen donor and oxygen, the mutant can catalyze the oxidation of 60 g / L of acetic acid triene to synthesize acetic acid tetraene, and the conversion rate reaches 99.1% within 10 h, and the space-time yield reaches 5.94 g / L / h. The present application has the advantages of green safety, short reaction period, simple purification, low cost, etc. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The HPLC detection results of the whole cell catalytic reaction solution;

[0035] Figure 2 The standard curve of 3TR (A) and 2TR (B) ;

[0036] Figure 3 The HPLC detection results of the purified 3TR sample. DETAILED DESCRIPTION

[0037] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0038] Example 1. Mining of sterone C1,2-dehydrogenase

[0039] Four kinds of sterone C1,2-dehydrogenases were mined from NCBI, the information of which is shown in Table 1, the amino acid sequences of which are respectively described as SEQ ID No. 1-4, and the DNA sequences of which are respectively described as SEQ ID No. 13-16. Recombinant plasmids pET28a-mskstd, pET28a-mrkstd, pET28a-mikstd and pET28a-mtkstd were respectively constructed, and were introduced into E. coli BL21(DE3) competent cells by heat shock transformation.

[0040] Table 1. Information of sterone C1,2-dehydrogenase

[0041]

[0042]

[0043] Example 2. Molecular modification of sterone C1,2-dehydrogenase

[0044] The mutant plasmid was amplified by circular plasmid PCR using the plasmid pET28a-mtkstd as a template and Primer Star DNA polymerase. The PCR program was as follows: 95℃, 2min; (95℃, 15s; Tm-5℃, 15s; 72℃, 2min) x 30 cycles; 72℃, 5min. Dpn I 0.2μL and corresponding buffer 0.8μL were added to the PCR product, which was digested at 37℃ for 2h, and then the PCR product was transformed into E. coli BL21(DE3) competent cells by heat shock transformation. The primer information of the mutant is shown in Table 2.

[0045] Table 2. Primer information

[0046]

[0047] Example 3. Shake flask culture of sterone C1,2-dehydrogenase engineering bacteria

[0048] The engineering bacteria were streaked on LB plates containing the corresponding resistance and incubated in a 37 °C constant temperature incubator overnight. When single colonies were grown, they were inoculated into 25 mL of LB liquid medium containing the corresponding resistance and incubated at 37 °C, 200 rpm for about 8 h. The culture was transferred into LB liquid medium containing the same resistance at a 1% volume inoculation amount and incubated at 37 °C, 200 rpm. When the OD 600 reached 0.6-0.8, IPTG was added (final concentration 0.1 mM) and the culture was continued to be incubated at 25 °C, 200 rpm overnight. The culture was centrifuged (7000 rpm, room temperature, 5 min) and the bacterial slurry was collected and resuspended in Tris-HCl buffer (50 mM, pH 8.0) to a whole cell concentration of 100 g / L.

[0049] Example 4. Screening of sterone C1,2 dehydrogenase mutants

[0050] The whole cell catalytic reaction was carried out in a 500 μL system for screening of the mutants. The system contained 2TR, 10 mg / mL whole cell, PMS and Tris-HCl buffer (50 mM, pH 8.0). The substrate load was adjusted step by step as the catalytic activity increased. The reaction was carried out at 37 °C, 200 rpm for 1 h. After the reaction was completed, the reaction was terminated with 2 volumes of ethyl acetate and incubated at 37 °C, 200 rpm for 1 h. After centrifugation at 12000 rpm for 1 min, 100 μL of the supernatant was diluted with 300 μL of chromatographic grade methanol, filtered through a 0.22 μm organic membrane and subjected to high performance liquid chromatography detection. The chromatographic column was C18 (4.6 mm x 250 mm, 5 μm); the mobile phase was acetonitrile / water = 60 / 40 (v / v); the injection amount was 5 μL; the flow rate was 0.8 ml / min; the column temperature was 35 °C; and the detection wavelength was 254 nm. Under these conditions, the elution of each substance was as shown in Table 1. Figure 1

[0051] The contents of 3TR and 2TR in the reaction solution were calculated according to the standard curves of 2TR and 3TR ( Figure 2 ), and the conversion rate was calculated, with the results shown in Table 3. Among them, the mutant MtKstD-I343T / Y408F / F116S showed the highest conversion capacity, with a conversion rate of 52.5% after 1 h of catalysis at a substrate load of 60 mM.

[0052] Table 3 Conversion rates of KstD and its mutants

[0053]

[0054]

[0055] Superscript a ,​b , c , d respectively represent different reaction conditions, which are as follows:

[0056] a 10 mM 2TR, 3 mM PMS, 10% (v / v) DMF, Tris-HCl buffer (50 mM, pH 8.0), 10 mg / mL wet bacteria, 200 rpm, 30°C, 1 h;

[0057] b 20 mM 2TR, 6 mM PMS, 10% (v / v) DMF, Tris-HCl buffer (50 mM, pH 8.0), 10 mg / mL wet bacteria, 200 rpm, 30°C, 1 h;

[0058] c 40 mM 2TR, 12 mM PMS, 10% (v / v) DMF, Tris-HCl buffer (50 mM, pH 8.0), 10 mg / mL wet bacteria, 200 rpm, 30°C, 1 h;

[0059] d 60 mM 2TR, 18 mM PMS, 10% (v / v) DMF, Tris-HCl buffer (50 mM, pH 8.0), 10 mg / mL wet bacteria, 200 rpm, 30°C, 1 h.

[0060] Example 5. Preparation of MtKstD-I343T / Y408F / F116S E. coli engineered cells

[0061] The optimal steroid C1,2 dehydrogenase mutant MtKstD-I343T / Y408F / F116S (amino acid sequence and DNA sequence are SEQ ID No. 9 and SEQ ID No. 21, respectively) was activated on a kanamycin-resistant LB plate and incubated at 37°C overnight. After single colonies grew, a single colony was picked and inoculated into 25 mL of LB medium, which was incubated at 37°C, 200 rpm for about 12 h to obtain a first-stage seed solution. The second-stage seed solution was prepared by inoculating the first-stage seed solution into a second-stage seed medium (yeast powder 7.5 g, glycerol 1.5 g, K2HPO4·3H2O 1.2 g, NaH2PO4·2H2O 0.67 g, NaCl 0.9 g, (NH4)2SO4 0.75 g, citric acid monohydrate 0.63 g, glucose 0.6 g, MgSO4·7H2O 0.15 g, FeSO4·7H2O 0.1 g, and water 300 mL) at a 2% inoculation amount, and then incubated at 37°C, 200 rpm for about 12 h.

[0062] The parameters of a 5L fermenter with 3L fermentation medium (yeast powder 45g, glycerol 30g, K2HPO4-3H2O 12g, NaH2PO4-2H2O 6.72g, NaCl 9g, (NH4)2SO4 7.5g, citric acid monohydrate 6.3g, glucose 6g, MgSO4-7H2O 1.47g, FeSO4-7H2O 0.9g, water 3L) were set. The temperature was 37°C, the initial stirring speed was 300rpm, the air flow rate was 3L / min, and the tank pressure was 0.05MPa. The secondary seed liquid was transferred into the fermenter, and the dissolved oxygen-rotation speed correlation mode was started, with the dissolved oxygen threshold set at 30%. After the feeding signal appeared, the feed (66g yeast powder, 360g glycerol, 200mL water) was fed at a flow rate of 30mL / h to maintain the growth of the bacteria. The wet weight of the bacteria was 204g / L after 27.5h of fermentation, and the bacteria were collected by centrifugation (7000rpm, 10°C, 10min) and stored at -20°C for later use.

[0063] Example 6. Enzymatic synthesis of 3TR

[0064] In a 1L reactor, 18g of 2TR, 0.18g of PMS, 15g of MtKstD-I343T / Y408F / F116S resting cells or cell lysate, and 300mL of water were added. The aeration amount was set at 0.3L / min, the stirring speed was 500rpm, and the reaction temperature was 37°C. During the reaction, the pH of the reaction solution was adjusted to about 8 by dropwise addition of 2M Na2CO3 or NaOH. After 10h of reaction, the conversion rate reached 99.1%.

[0065] Example 7. Purification of 3TR

[0066] The 300mL reaction solution of Example 6 was filtered to remove insoluble impurities, and extracted with 200mL of ethyl acetate multiple times until no solid residue was observed in the lower layer. The upper organic phase was collected, and the ethyl acetate was recovered by evaporation under reduced pressure, during which yellow solids precipitated. The yellow solids were collected, slurried with 200mL of water, and filtered to obtain white solids. The white solids were dried at 60°C to obtain white powder, which was the finished product of 3TR, with a purity of more than 99%. Figure 3 )。

[0067] The above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A sterol C1,2 dehydrogenase mutant, characterized in that: The sterone C1,2 dehydrogenase mutant is obtained by performing any of the following amino acid residue substitution mutations on the amino acid sequence shown in SEQ ID No. 4: 1)I343L; 2)I343M; 3)I343T; 4)I343T / Y408F; 5)I343T / Y408F / F116S; 6)I343T / Y408F / F116D; 7)I343T / Y408F / F116T; 8)I343T / Y408F / F116V.

2. The sterone C1,2 dehydrogenase mutant according to claim 1, characterized in that The amino acid sequences of the sterone C1,2 dehydrogenase mutants are shown in SEQ ID NO: 5 to SEQ ID NO: 12, respectively.

3. The sterone C1,2 dehydrogenase mutant according to claim 1, characterized in that The nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 4 is shown in SEQ ID No. 16; The nucleotide sequences encoding the sterone C1,2 dehydrogenase mutants are shown in SEQ ID NO: 17 to SEQ ID NO: 24, respectively.

4. Use of the sterone C1,2 dehydrogenase mutant according to any one of claims 1 to 3 in the synthesis of acetate tetraenes.

5. An expression plasmid for sterol C1,2 dehydrogenase, characterized in that: It comprises a promoter, an RBS sequence, a nucleotide sequence of any one of SEQ ID No. 17 to 24, and a terminator.

6. An engineered strain expressing sterol C1,2 dehydrogenase, characterized in that: The engineered strain is Escherichia coli comprising the expression plasmid according to claim 5.

7. An efficient enzymatic method for preparing tetraene acetate, characterized in that: Acetate tetraene is synthesized by using acetate triene as raw material and oxygen as hydrogen acceptor under the catalysis of hydrogen donor and the sterone C1,2 dehydrogenase mutant according to any one of claims 1 to 3.

8. The enzymatic method for efficiently preparing tetraene acetate according to claim 7, characterized in that: The hydrogen donor is PMS.

9. The enzymatic method for efficiently preparing tetraene acetate according to claim 7, characterized in that: The sterone C1,2 dehydrogenase mutant is derived from whole cells or cell fragments of recombinant Escherichia coli that co-express the sterone C1,2 dehydrogenase mutant by genetic engineering.

10. The enzymatic method for efficiently preparing tetraene acetate according to claim 7, characterized in that: It also includes the purification of tetraene acetate, the steps are as follows: After the enzyme-catalyzed reaction is completed, the reaction solution is extracted with ethyl acetate several times, and the ethyl acetate is evaporated to obtain a crude tetraene acetate. The crude product is washed with water until the filtrate is colorless, and then dried to obtain a white powder.

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