ECB Deacylase Mutants and Their Application in the Preparation of Anidulafungin Intermediates

Abstract

The present invention relates to an echinocandin B deacylase mutant with significantly improved catalytic performance, a coding gene, a vector containing the coding gene, a genetically engineered bacterium, and the application of the mutant in the catalytic synthesis of the echinocandin B core, an intermediate for anidulafungin. The echinocandin B deacylase mutant is obtained by single-point mutation or multi-point combined mutation of the amino acids shown in SEQ ID NO.1. Based on expanding the catalytic substrate pocket, the present invention conducts rational design by combining three-dimensional structure simulation and enzymatic property analysis, provides an ECB deacylase mutant with better catalytic performance, which can be used for the preparation of anidulafungin intermediates, and provides a new way for the synthesis of anidulafungin.

Description

(1) Technical Field

[0001] The present invention relates to an echinocandin B deacylase mutant, a coding gene, a vector containing the coding gene, a genetically engineered bacterium, and their application in the microbial catalysis for preparing an intermediate of anidulafungin. (2) Background Art

[0002] In recent years, the number of deaths caused by fungal infections has been increasing, which is related to the widespread use of corticosteroids, cytotoxic drugs, broad-spectrum antibiotics, immunosuppressants and the like in clinical practice. Echinocandins are the first class of antifungal drugs targeting the fungal cell wall, which can affect fungal synthesis by inhibiting the activity of glucan synthase. At present, the echinocandin drugs that have completed clinical trials and been successfully developed and marketed mainly include caspofungin, micafungin and anidulafungin, which are first-line antifungal drugs for the treatment of invasive infections.

[0003] As a third-generation echinocandin drug, anidulafungin has broad application prospects. The biocatalytic preparation of the key synthetic intermediate of anidulafungin - the echinocandin B core has become a research hotspot for antifungal drugs at home and abroad. The deacylation of echinocandin B (ECB) by ECB deacylase is often the rate-limiting step in the production process. ECB deacylase can specifically recognize and act on the amide bond of ECB to obtain the key intermediate echinocandin B core (ECBN).

[0004] At present, the activity of wild-type ECB deacylase is very low, which is difficult to meet the requirements of industrial production. With the rapid development of molecular biology and protein engineering, more and more protein structures have been resolved, and molecular modification has evolved from directed evolution to semi-rational or rational design. Rational design is based on biochemical data, and specific mutations are made at key sites after evaluating protein structure and molecular modeling data. In the absence of efficient and available high-throughput assay methods, rational design greatly reduces the time and effort required for screening mutant libraries. After rational design and modification, wild-type ECB deacylase has been able to obtain better enzymatic properties, which plays a crucial role in improving the catalytic activity of existing ECB deacylase. (3) Summary of the Invention

[0005] The object of the present invention is to overcome the deficiency of the low activity of ECB deacylase in the prior art, and provide an echinocandin B deacylase mutant with significantly improved catalytic performance, a coding gene, a vector containing the coding gene, a genetically engineered bacterium, and their application in the catalytic production of the echinocandin B core, an intermediate of anidulafungin.

[0006] The technical solution adopted by the present invention is:

[0007] An echinocandin B (ECB) deacylase mutant is obtained by single-point mutation or multi-point combined mutation at positions 53, 55, 57, 154, and 661 of the amino acid sequence shown in SEQ ID NO.1.

[0008] SEQ ID NO.1:

[0009] MKIKTGARILALSALTTMMFSASALASNAYGLGAQATVNGSGMVLANPHFPWQGAARFYRMHLKVPGRYDVEGAALIGDPIIGIGHNRTVAWSHTVSTARRFVWHRLSLVPGDPTSYYVDGRPERMRARTVTVQTGSGPVSRTFHDTRYGPVAVMPGTFDWTPATAYAITDVNAGNNRAFDGWLRMGQAKDVRALKAVLDRHQFLPWVNVIAADARGEALYGDHSVVPRVTGALAAACIPAPFQPLYASSGQAVLDGSRSDCALGADPDAAVPGILGPASLPVRFRDDYVTNSNDSHWLASPAAPLEGFPRILGNERTPRSLRTRLGLDQIQQRLAGTDGLPGKGFTTARLWQVMFGNRMHGAELARDDLVALCRRQPTATASNGAIVDLTAACTALSRFDERADLDSRGAHLFTEFALAGGIRFADTFEVTDPVRTPRRLNTTDPRVRTALADAVQRLAGIPLDAKLGDIHTDSRGERRIPIHGGRGEAGTFNVITNPLVPGVGYPQVVHGTSFVMAVELGPHGPSGRQILTYAQSTNPNSPWYADQTVLYSRKGWDTIKYTEAQIAADPNLRVYRVAQRGRGGSGGGSGHDGGYAALIRRASYGVPHITADDFGSLGFGVGYVQAEDNICVIAESVVTANGERSRWFGATGPDDADVRSDLFHRKAIDDRVAERLLEGPRDGVRAPSDDVRDQMRGFVAGYNHFLRRTGVHRLTDPACRGKAWVRPLSEIDLWRTSWDSMVRAGSGALLDGIVAATPPTAAGPASAPEAPDA

[0010] Preferably, the mutation is one of the following or a combination of two or more thereof: (1) the aspartic acid at position 53 is mutated to alanine; (2) the isoleucine at position 55 is mutated to phenylalanine; (3) the glycine at position 57 is replaced by methionine; (4) the phenylalanine at position 154 is mutated to leucine; (5) the glutamine at position 661 is mutated to leucine.

[0011] More preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO.3 (the coding gene sequence is as shown in SEQ ID NO.4).

[0012] SEQ ID NO.3:

[0013] MKIKTGARILALSALTTMMFSASALASNAYGLGAQATVNGSGMVLANPHFPWQGAARFYRMHLKVPGRYDVEGAALIGAPFIMIGHNRTVAWSHTVSTARRFVWHRLSLVPGDPTSYYVDGRPERMRARTVTVQTGSGPVSRTFHDTRYGPVAVMPGTFDWTPATAYAITDVNAGNNRALDGWLRMGQAKDVRALKAVLDRHQFLPWVNVIAADARGEALYGDHSVVPRVTGALAAACIPAPFQPLYASSGQAVLDGSRSDCALGADPDAAVPGILGPASLPVRFRDDYVTNSNDSHWLASPAAPLEGFPRILGNERTPRSLRTRLGLDQIQQRLAGTDGLPGKGFTTARLWQVMFGNRMHGAELARDDLVALCRRQPTATASNGAIVDLTAACTALSRFDERADLDSRGAHLFTEFALAGGIRFADTFEVTDPVRTPRRLNTTDPRVRTALADAVQRLAGIPLDAKLGDIHTDSRGERRIPIHGGRGEAGTFNVITNPLVPGVGYPQVVHGTSFVMAVELGPHGPSGRQILTYAQSTNPNSPWYADQTVLYSRKGWDTIKYTEAQIAADPNLRVYRVAQRGRGGSGGGSGHDGGYAALIRRASYGVPHITADDFGSLGFGVGYVQAEDNICVIAESVVTANGERSRWFGATGPDDADVRSDLFHRKAIDDRVAERLLEGPRDGVRAPSDDVRDLMRGFVAGYNHFLRRTGVHRLTDPACRGKAWVRPLSEIDLWRTSWDSMVRAGSGALLDGIVAATPPTAAGPASAPEAPDA

[0014] The present invention also relates to a gene encoding the echinocandin B deacylase mutant described above.

[0015] Preferably, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.4.

[0016] The present invention also relates to a recombinant vector and a genetically engineered bacterium containing the gene of the echinocandin B deacylase mutant described above.

[0017] The recombinant expression vector can be constructed by ligating the nucleic acid encoding the ECB deacylase mutant gene of the present invention to various suitable vectors by conventional methods in the art. The vectors can be various conventional vectors in the art, such as commercially available plasmids, cosmids, phages or viral vectors, etc., as long as the recombinant expression vector can replicate normally in the corresponding expression host and express the ECB deacylase mutant. The ECB deacylase mutant gene can be operably linked downstream of a suitable regulatory sequence in the vector to achieve constitutive or inducible expression of the ECB deacylase mutant. The vector is preferably a plasmid, more preferably plasmid pET28a.

[0018] The recombinant expression transformant can be prepared by transforming the constructed recombinant expression vector into a host cell. The host cell is various conventional host cells in the art, as long as the recombinant expression vector can stably replicate itself and can effectively express the target protein after induction by an inducer. Escherichia coli is preferably used as the host cell in the present invention, and more preferably Escherichia coli E.coli BL21(DE3) is used for highly expressing the ECB deacylase mutant of the present invention.

[0019] The present invention also relates to the application of the echinocandin B deacylase mutant in the microbial catalytic preparation of anidulafungin intermediate.

[0020] Specifically, the application is as follows: using echinocandin B (ECB) as the reaction substrate, using the wet cells containing the echinocandin B deacylase mutant as the biocatalyst, using urea and choline chloride as co-solvents, and using a pH 7-8 potassium phosphate buffer as the reaction medium to form a reaction system, and performing a biocatalytic reaction at a temperature of 35-45°C and a stirring speed of 500-800 r / min to obtain the echinocandin B mother nucleus (ECBN).

[0021] The reaction involved is as follows:

[0022]

[0023] The biocatalyst shown can be any one of the following forms:

[0024] (1) Culturing the recombinant expression transformant and separating the transformed cells containing the ECB deacylase mutant;

[0025] (2) Culturing the recombinant expression transformant, separating the transformed cells containing the ECB deacylase mutant, and disrupting the transformed cells containing the ECB deacylase mutant to obtain a cell lysate.

[0026] The cultivation of the recombinant expression transformant is a conventional method and cultivation condition in the art. Specifically, the following steps can be selected: For recombinant Escherichia coli, the preferred medium is TB medium: 12 g / L of tryptone, 24 g / L of yeast powder, 5 g / L of glycerol, 2.31 g / L of potassium dihydrogen phosphate, 12.54 g / L of dipotassium hydrogen phosphate, pH 7.0. The preferred cultivation method is: inoculate the recombinant Escherichia coli constructed as described above into a medium containing kanamycin, and culture it overnight at 37°C with shaking at 180 rpm. Inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of TB medium (containing kanamycin) at an inoculation amount of 1-2% (v / v), and culture it with shaking at 37°C and 180 rpm. When the OD600 of the culture solution reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mmol / L as an inducer, and induce it at 16°C for 16 h. Then centrifuge the culture solution to collect the precipitate, and wash it twice with PBS to obtain recombinant expression transformant cells. Freeze-dry the harvested recombinant cells to obtain freeze-dried cells containing the ECB deacylase mutant. Suspend the harvested recombinant cells in 5-10 times the volume (v / w) of buffer, ultrasonically disrupt them, and centrifuge to collect the supernatant to obtain the cell lysate of the recombinant ECB deacylase mutant.

[0027] ECB deacylase enzyme activity assay method: At 40°C, the echinocandin B deacylase activity is measured in a total of 1 mL of reaction mixture, which consists of 0.1 M potassium phosphate buffer at pH 7.0, 10% cosolvent [urea][choline chloride], and 0.2 g / L of ECB. The reaction is carried out at 800 rpm for 30 minutes. After the reaction is completed, centrifuge to terminate the reaction. Subsequently, a high-performance liquid chromatograph is used to detect the product formation. One unit of echinocandin B deacylase activity (U) is defined as the amount of enzyme required to produce 1 μg of the echinocandin B nucleus per minute under standard conditions of 40°C and pH 7.0. The specific enzyme activity (U / g) is defined as the activity per gram of dry cells.

[0028] The beneficial effects of the present invention are mainly reflected in: Based on rational design by expanding the catalytic substrate pocket, and using three-dimensional structure simulation combined with enzymatic property analysis, the present invention provides an ECB deacylase mutant with better catalytic performance, which can be used for the preparation of anidulafungin intermediates, providing a new way for the synthesis of anidulafungin. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the reaction process of wild-type echinocandin B deacylase and its mutant to transform into ECBN.

[0030] Figure 2 It is the influence of different cosolvents on the catalytic reaction.

[0031] Figure 3 Effect of different cosolvent concentrations on the catalytic reaction. (V) Specific implementation manners

[0032] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments:

[0033] The culture media involved in the following embodiments are as follows:

[0034] LB medium (g / L): Tryptone 10, yeast extract 5, NaCl 10, pH 7.0;

[0035] TB medium (g / L): Tryptone 12, yeast extract 24, glycerol 5, potassium dihydrogen phosphate 2.31, dipotassium hydrogen phosphate 12.54, pH 7.0. Kanamycin was added to a final concentration of 50 μg / mL before inoculation.

[0036] Method for determining the enzyme activity of echinocandin B deacylase:

[0037] At 40 °C, the echinocandin B deacylase activity was determined in a total of 1 mL of reaction mixture, which consisted of pH 7.0, 0.1 M potassium phosphate buffer and 0.2 g / L ECB. The reaction was carried out at 800 rpm for 30 minutes. After the reaction, centrifugation was used to terminate the reaction. Subsequently, high performance liquid chromatography was used to detect the product formation. One unit of echinocandin B deacylase activity (U) was defined as the amount of enzyme required to produce 1 μg of the echinocandin B parent nucleus per minute under standard conditions of 40 °C and pH 7.0. The specific enzyme activity (U / g) was defined as the activity per gram of dry cells.

[0038] Note: For the molecular biology experimental methods not specifically described in the following embodiments, they were all carried out with reference to the specific methods listed in the third edition of "Molecular Cloning: A Laboratory Manual", or according to the kits and product manuals.

[0039] Example 1: Construction of an expression plasmid for an ECB deacylase mutant

[0040] The wild-type echinocandin B deacylase was obtained from the laboratory-preserved plasmid, and the nucleotide sequence was as shown in SEQ ID No. 2. Using the preserved plasmid as a template, mutations were introduced at specific sites to obtain a mutant expression vector.

[0041] The amplification conditions (50 μL system) were as follows: 25 μL of 2×Phanta Max buffer, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of dNTPs, 1 μL of DNA polymerase (Vanzyme, Nanjing, China), 1 μL of plasmid template, and the reaction volume was adjusted to 50 μL with sterile water.

[0042] The amplification conditions were as follows: pre-denaturation at 95°C for 10 minutes, 30 cycles: denaturation at 95°C for 30 seconds, annealing at 59°C for 30 seconds, extension at 72°C for 5 minutes, and finally a final extension at 72°C for 10 minutes.

[0043] The amplified products were verified by agarose gel electrophoresis. 1 μL of DpnI enzyme was added to the PCR products that were positive in the electrophoresis detection and digested, and the original template was removed by incubation at 37°C for 30 minutes.

[0044] The primers were designed as follows:

[0045] Forward primer F for mutation site D53A:

[0046] 5′-TGATCGGCgcgCCGATCATTGGCATTGG-3′

[0047] Reverse primer R for mutation site D53A:

[0048] 5′-ATGATCGGcgcGCCGATCAGCGCC-3′

[0049] Forward primer F for mutation site I55F:

[0050] 5′-GACCCGTTCATTTGGATTGGTCAC-3′

[0051] Reverse primer R for mutation site I55F:

[0052] 5′-TCCAAATGAACGGGTCGCCGATCA-3′

[0053] Forward primer F for mutation site G57M:

[0054] 5′-ATCATTATGATTGGTCACAACCGTACCG-3′

[0055] Reverse primer R for mutation site G57M:

[0056] 5′-CCAATCATAATGATCGGGTCGCCG-3′

[0057] Forward primer F for mutation site F154L:

[0058] 5′-CGTGCGCTGGATGGTTGGCTGCGT-3′

[0059] Reverse primer R for mutation site F154L:

[0060] 5′-CCAACCATCCAGCGCACGGTTGTTA-3′

[0061] Forward primer F for the mutation site Q661L:

[0062] 5′-GTTCGTGATCTGATGCGTGGTTTCG-3′

[0063] Reverse primer R for the mutation site Q661L:

[0064] 5′-CCACGCATCAGATCACGAACATCATC-3′

[0065] Example 2: Preparation of ECB deacylase mutant strains and expression of recombinant proteins

[0066] The recombinant expression plasmid constructed in Example 1 was transformed into competent cells of Escherichia coli E. coli BL21(DE3) (purchased from Invitrogen). The transformed competent cells were spread on an LB agar plate containing 50 μg / mL kanamycin resistance (a plate medium containing LB solid medium) and cultured overnight at 37°C (for the preparation and transformation of E. coli competent cells, refer to the Molecular Cloning Laboratory Manual). After the appearance of transformants, single colonies were picked for verification, and positive transformants were selected for sequencing. The transformants with correct sequences were used for large-scale preparation of recombinant plasmids.

[0067] An activated colony was picked into 100 mL of TB medium and cultured at 37°C and 180 rpm for 2 h until the OD600 reached 0.8. The composition of the medium was: 12 g / L of tryptone, 24 g / L of yeast extract, 5 g / L of glycerol, 2.31 g / L of potassium dihydrogen phosphate, and 12.54 g / L of dipotassium hydrogen phosphate.

[0068] An inducer IPTG with a final concentration of 0.1 mM was added to the fermentation broth, and the mixture was transferred to a shaker at 16°C and 180 rpm for enzyme induction.

[0069] The fermentation broth was placed in a 50 mL centrifuge tube and centrifuged at 4°C and 5000 rpm for 5 min. The supernatant was discarded, and the bacterial cells were collected. The centrifuged bacterial cells were resuspended with an equal volume of 0.1 mM PBS. The bacterial suspension was diluted by an appropriate multiple and then the cells were broken using an ultrasonic cell disruptor. The broken cell suspension was centrifuged at 4°C and 8000 rpm for 10 min, and the centrifuged supernatant (the supernatant part of the broken cells) was collected. The activities of wild echinocandin B deacylase and its mutants were measured.

[0070] Example 3: Enzyme activity assay of ECB deacylase mutants

[0071] Method for determining the enzyme activity of echinocandin B deacylase: At 40 °C, the echinocandin B deacylase activity was determined in a total reaction mixture of 1 mL, which consisted of 0.1 M potassium phosphate buffer at pH 7.0, 10% co-solvent [urea][choline chloride] (choline chloride:urea molar ratio = 1:2), and 0.2 g / L ECB. The reaction was carried out at 800 rpm for 30 minutes. After the reaction, centrifugation was used to terminate the reaction. Subsequently, high performance liquid chromatography was used to detect the product formation. One unit of echinocandin B deacylase activity (U) was defined as the amount of enzyme required to produce 1 μg of the echinocandin B parent nucleus per minute under the standard conditions of 40 °C and pH 7.0. The specific enzyme activity (U / g) was defined as the activity per gram of dry cells.

[0072] The best mutant of ECB deacylase, D53A / I55F / G57M / F154L / Q661L (MT), after mutation, the volume of the catalytic pocket effectively increased, which was beneficial for the better entry of the substrate ECB into the interior of the catalytic pocket. The enzyme activity of the ECB deacylase mutant increased by 296% and reached 286.5 U / g.

[0073] Example 4: Catalytic generation of the echinocandin B parent nucleus by the ECB deacylase mutant

[0074] The 10 mL reaction system contained 0.5 mM KCl, 200 mg / L ECB, 0.1 M PBS buffer, crude enzyme solution, and 10% co-solvent [urea][choline chloride] (choline chloride:urea molar ratio = 1:2). The reaction was carried out at 40 °C for 0.5, 2, 4, 6, 8, 10, and 12 h respectively. Hydrochloric acid was added to terminate the reaction, and the supernatant was taken by centrifugation. The yield of the product echinocandin B parent nucleus was detected by high performance liquid chromatography.

[0075] The determination of the reaction process of the conversion of ECB deacylase (WT) and ECB deacylase mutant (MT) to generate ECBN was carried out, and the experimental results were as Figure 1 shown. The yield and reaction rate of the mutant were higher than those of the wild-type enzyme, and the results further verified that the mutant with a larger effective cavity volume had a higher catalytic efficiency than the wild-type enzyme.

[0076] Example 5: Optimization of different co-solvents and co-solvent ratios in the catalytic reaction

[0077] Different co-solvents were added to the catalytic reaction respectively: dimethyl sulfoxide (DMSO), dimethylacetamide (DMF), methanol, deep eutectic solvent [choline chloride][urea] (DES) (choline chloride:urea molar ratio = 1:2), and isopropanol. The reaction system was 0.5 mM KCl, different types of co-solvents, 0.2 g / L ECB, 0.1 M PBS buffer, crude enzyme solution, and pH 7.0. The enzyme activity was determined at 40 °C. For the specific method of enzyme activity determination, see Example 3.

[0078] The enzymatic reactions were carried out under DES at proportions of 10%, 20%, 40%, 60%, and 80% respectively. The reaction system was 0.5 mM KCl, different proportions of DES, 0.2 g / L ECB, 0.1 M PBS buffer, crude enzyme solution, and pH 7.0. The enzyme activity was measured at 40°C. For the specific method of enzyme activity measurement, see Example 3.

[0079] The results are as Figure 2 and Figure 3 shown. The addition of the cosolvent [choline chloride][urea] has the best effect. It and the PBS buffer in the reaction system together form a two-phase catalytic reaction system, which helps the reaction proceed. Among them, the effect is the best when the addition proportion is 10%.

[0080] In summary, based on the wild-type echinocandin B deacylase, the present invention provides a mutant echinocandin B deacylase with improved catalytic performance. The present invention confirms that increasing the volume of the catalytic pocket plays an important role in improving catalytic activity, providing an important clue for studying the catalytic mechanism of echinocandin B deacylase.

[0081] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An echinocandin B deacylase mutant with an amino acid sequence as shown in SEQ ID NO.

3.

2. A gene encoding the echinocandin B deacylase mutant according to claim 1.

3. The gene according to claim 2, wherein The nucleotide sequence of the gene is as shown in SEQ ID NO.

4.

4. A recombinant vector containing the gene encoding the echinocandin B deacylase mutant according to claim 1.

5. A genetically engineered bacterium containing the gene encoding the echinocandin B deacylase mutant according to claim 1.

6. Use of the echinocandin B deacylase mutant according to claim 1 in the microbial catalysis for preparing the intermediate of anidulafungin - the echinocandin B core compound.

7. The application according to claim 6, wherein The use is as follows: Using echinocandin B as a reaction substrate, using the wet cells containing the echinocandin B deacylase mutant as a biocatalyst, using urea and choline chloride as cosolvents, and using a pH 7 - 8 potassium phosphate buffer as a reaction medium to form a reaction system, and performing a biocatalytic reaction under the conditions of a temperature of 35 - 45 °C and a stirring speed of 500 - 800 r / min to obtain the echinocandin B core compound.

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