Echinocandin deacylase and uses thereof

Abstract

The application belongs to the field of bioengineering, bio-chemical industry and bio-medicine, and discloses aechinocandin deacylase and application. The application is a construction method of a novel echinocandin deacylase recombinant engineering bacterium and application of the echinocandin deacylase in biological catalysis of echinocandin compound hydrolysis and removal of fatty acid side chain to generate echinocandin cyclic peptide mother nucleus. The echinocandin deacylase engineering bacterium gene eca is an artificial synthetic DNA sequence, the gene is cloned into an integrative plasmid pSET152 and is recombined into a Streptomyces host chromosome DNA through conjugation transfer to obtain the application. The engineering bacterium constructed by the application can be successfully applied to the deacylation reaction of echinocandin compounds. The reaction is one of key technical links in the research and development process of echinocandin antifungal drugs. The conversion rates of the recombinant strain TK24pSET-pKasOECA to new structure echinocandin E within 12 hours are 78.38% respectively; the conversion rates of the recombinant strain TK24pSET-pKasOECA to micafungin precursor FR901379 in the deacylation reaction within 3 hours are 77.00% respectively.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering, biochemical engineering, and biomedicine, and more specifically, to an echinocandin deacylate (ECA) and its applications. This enzyme catalyzes the hydrolysis of fatty acid side chains of echinocandin drug precursors to generate deacylated echinocandin drug precursor compounds. Recombinant ECA engineered bacteria constructed by heterologously expressing the gene encoding ECA can efficiently catalyze deacylation reactions, enabling the deacylation of various compounds, including echinocandin compounds such as novel echinocandin compounds echinocandin E, FR901379, and echinocandin B. Background Technology

[0002] In recent years, the incidence of fungal infections has been increasing year by year due to factors such as cancer chemotherapy, organ transplantation, surgical treatment, pneumonia, and AIDS, posing a significant challenge to healthcare professionals. Unlike mammalian cells, fungi possess cell walls, making them ideal targets for treating fungal infections. Echinocandins primarily work by non-competitively inhibiting β-1,3-glucan synthase, preventing fungi from forming cell walls and thus achieving the goal of treating fungal infections. They have become first-line drugs for treating Candida, Aspergillus, and other invasive Candida infections.

[0003] The drug development process of echinocandins generally requires first removing the acyl side chain that is hemolytically toxic to human cells, and then adding a new, non-toxic side chain through chemical modification. Taking the currently marketed echinocandin antifungal drug anidulafungin as an example, the production process of anidulafungin first requires enzymatic removal of the hemolytically toxic acyl side chain of echinocandin B (ECB), obtaining the echinocandin B nucleus (ECBN) intermediate, and finally, the new side chain is added through an acylation reaction. Figure 1 This process yields the antifungal drug anidoxuridine. Therefore, deacylation is one of the key steps in the industrial production of echinocandins-based antifungal drugs.

[0004] Echinocandin E (ECE) is a novel tetradeoxyechinocandin drug intermediate obtained by modifying the ECB biosynthetic pathway (Org. Biomol. Chem., 2023, 21, 3552-3556). Figure 2As shown, compared to ECB, the azahemiacetal structure, which can cause chemical instability of anidoxurine, is structurally eliminated, thus giving ECE better chemical stability while possessing antifungal activity equivalent to ECB. Similar to ECB, removing the fatty acid side chain from ECE to obtain the echinocandin E core (ECEN) is a key step in developing ECE into a novel echinocandin drug. Figure 3 However, no known deacylase can catalyze the deacylation reaction of ECE. Therefore, the development of novel echinocandin acylase (ECA) for the deacylation reaction of functional compounds such as ECE has significant application value in multiple fields. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides an echinocandin deacylase and its applications.

[0006] This invention provides a protein that is a deacylase, the amino acid sequence of which is shown in ECASY1965AA. Figure 4 It can hydrolyze amide bonds and remove the acyl side chains of compounds.

[0007] The present invention provides a nucleic acid molecule that encodes the protein.

[0008] The present invention provides a biological material, wherein the biological material is any one of a1)-a4): a1) an expression cassette containing the nucleic acid molecule; a2) a recombinant vector containing the nucleic acid molecule; a3) a recombinant microorganism containing the nucleic acid molecule; a4) a transgenic cell line containing the nucleic acid molecule.

[0009] This invention also provides applications of the protein, specifically for hydrolyzing the amide bonds of echinocandins, cephalosporins, penicillin, and capsaicin to remove acyl side chains. Further, it is used to hydrolyze the amide bonds of echinocandins, removing acyl side chains to generate a cyclic peptide core. Still further, it is used to hydrolyze the amide bonds of echinocandins FR901379, echinocandin E, and echinocandin B compounds, removing acyl side chains to generate a cyclic peptide core.

[0010] This invention also provides applications of the aforementioned nucleic acid molecules and biomaterials for producing the proteins described herein, catalyzing the hydrolysis of amide bonds. Further, for producing the proteins described herein, the amide bonds of echinocandin compounds, cephalosporin compounds, penicillin, and capsaicin are hydrolyzed, and their acyl side chains are removed. Still further, for producing the proteins described herein, the acyl side chains of echinocandin compounds are hydrolyzed to generate a cyclic peptide core. Even further, for producing the proteins described herein, the amide bonds of echinocandin compounds FR901379, echinocandin E, and echinocandin B compounds are hydrolyzed, and the acyl side chains are removed to generate a cyclic peptide core.

[0011] This invention provides an expression cassette for heterologous expression of the novel echinocandin deacylase gene eca, comprising a promoter nucleic acid sequence, an RBS nucleic acid sequence, an ECA coding sequence nucleic acid sequence, and a terminator nucleic acid sequence connected in sequence. The core is the ECA coding sequence, which can be used for intracellular expression based on either a targeting N-terminal signal peptide sequence or the removal of the signal peptide sequence. A separation tag, such as 6×His, can be added to the ECA coding sequence to facilitate affinity chromatography separation.

[0012] This invention relates to the construction of recombinant vectors and ECA expression strains. ECA can be heterologously expressed to construct recombinant expression strains for the production, isolation, and purification of ECA proteins and their application in deacylation reactions; the recombinant strains can also be directly applied to biotransformation deacylation reactions. The heterologous expression engineered strains described in this invention are not limited to different expression hosts and expression vectors. Taking Streptomyces expression systems as an example, they include, but are not limited to, *Streptomyces coelicolor*, *Streptomyces lividans*, and *Streptomyces albus*. Taking the integrative expression vector pSET152 (containing the promoter pKasO) as an example, the assembled deacylation enzyme expression cassette is ligated to the recombinant vector pSET152 to obtain, as shown... Figure 5 The recombinant plasmid is shown. The recombinant plasmid was transformed into *Escherichia coli* DH5α, and the correct transformed strain was obtained through antibiotic selection. The plasmid was extracted and sequenced to ensure the gene sequence was accurate. The correct recombinant plasmid was transformed into *E. coli* ET12567 (PUZ8002) to obtain an *E. coli* vector for conjugation transfer. This vector was then conjugated into a *Streptomyces* host, such as *S. lividans* TK24. The plasmid recombined into the host's genomic DNA, resulting in an engineered strain that highly expresses recombinant echinocandin deacylase.

[0013] This invention provides a method for purifying the recombinant echinocandin deacylase from engineered bacteria. The specific method involves: fermenting the recombinant engineered bacteria for 72 hours, then collecting the supernatant. The supernatant is then filtered through a 0.45 μm NY microporous membrane. Protein affinity chromatography was performed using a protein purifier and a Ni-NTA column. Gradient elution was performed with imidazole buffers at concentrations of 20 mM, 50 mM, 100 mM, 300 mM, and 500 mM. The purification effect of each gradient fraction was analyzed by SDS-PAGE protein gel and Coomassie brilliant blue staining, and the purified target protein was collected.

[0014] This invention provides a method for deacylation reaction catalyzed by echinocandin deacylase ECA. Specifically, purified ECA protein undergoes a deacylation reaction in a buffer system. Echinocandins as substrates include, but are not limited to, echinocandin E, FR901379, and echinocandin B. For example, the ECA is converted to a final concentration of 2 μM in 50 mM Tris-HCl buffer at pH 6.8 at 28°C for 2 h. The production of the corresponding deacylation products is detected by HPLC or LCMS.

[0015] This invention provides an application of the recombinant engineered bacteria using echinocandin deacylase ECA in the biotransformation of echinocandin E (ECE) to generate echinocandin E nucleus (ECEN), such as... Figure 3 As shown. The specific method is as follows: The fermentation broth of the recombinant echinocandin deacylase engineered bacteria, after fermentation for 24 h, was mixed with substrate ECE to a final concentration of ~0.1 mg / mL (0.1 mM). The mixture was converted at 28℃ and 200 rpm for 12 h. An equal volume of methanol was added to quench the reaction, and the mixture was centrifuged at 13000 rpm for 30 min. The supernatant was then used as the sample. The deacylation product ECEN from ECE was detected by HPLC and LC-TOFMS, and the conversion rate was determined by HPLC. HPLC and LC-TOFMS conditions: YMC-5μC18 column, 250×4.60 mm, 5 μm; mobile phase A was 0.5‰ TFA water, and B was acetonitrile; elution program: 0-15 min, 15%-40% (v / v, B in A) gradient elution; 10-40 min, 40%-100% gradient elution; 40-50 min, 100% isocratic elution. Flow rate: 0.6 mL / min; injection volume: 20 μL; detection wavelength: 222 nm.

[0016] This invention provides the application of the recombinant echinocandin deacylase engineered bacteria in the biocatalytic generation of the micaflavin intermediate FR179642 from FR901379, such as... Figure 6As shown. The specific method is as follows: FR901379 was added to the fermentation broth of recombinant echinocandin deacylate engineered bacteria after 24 hours of fermentation to a final concentration of 0.2 mg / mL (0.17 mM). The mixture was converted at 28℃ and 200 rpm for 3 hours. An equal volume of methanol was added to quench the reaction. After centrifugation at 13000 rpm for 30 minutes, the supernatant was used as the sample. The deacylated product FR179642 of FR901379 was detected by HPLC, and the conversion rate was determined by HPLC. HPLC conditions: YMC-5μC18 column, 250×4.60 mm, 5 μm; mobile phase A was 0.5‰ TFA water, and B was acetonitrile; elution program: 0-5 min, 5% (v / v, B in A) isocratic elution; 5-10 min, 5%-55% gradient elution; 10-25 min, 55%-100% gradient elution; 25-35 min, 100% isocratic elution. Flow rate: 1 mL / min; injection volume: 20 μL; detection wavelength: 210 nm.

[0017] Advantages of this invention:

[0018] The echinocandin deacylate or its recombinant engineered strain provided by this invention possesses a novel amino acid sequence, can efficiently catalyze deacylation reactions, and exhibits good substrate generalization, capable of catalyzing the deacylation reactions of various substrates including multiple echinocandin compounds, cephalosporin compounds, penicillin compounds, and capsaicin. It shows promising application prospects for the development of new drugs, various functional products, and their intermediates. Attached Figure Description

[0019] Figure 1 The flowchart shows the process of generating ECBN intermediate by deacylation of ECB, and finally obtaining the antifungal drug anidoxane by reacylation. The arrows in the diagram indicate the azahemiacetal sites that can cause poor stability of ECB and anidoxane.

[0020] Figure 2 Chemical structure diagrams of echinocandin B (ECB) and echinocandin E (ECE), where ECE is a tetradeoxy derivative of ECB, the black arrows indicate the four deoxy sites of ECB; the hollow arrows indicate the azahemiacetal structure.

[0021] Figure 3 A schematic diagram of the reaction in which echinocandin E is hydrolyzed by echinocandin E deacylase (ECA) or ECA recombinant engineered bacteria to remove fatty acid side chains and generate ECE nucleus (ECEN);

[0022] Figure 4 ECA amino acid sequence;

[0023] Figure 5 A schematic diagram of a recombinant plasmid containing an ECA gene expression cassette;

[0024] Figure 6 A schematic diagram of the reaction in which the echinocandin deacylase (ECA) or a recombinant engineered strain of ECA catalyzes the formation of the intermediate FR179642 of the antifungal drug micafungin from the echinocandin natural product FR901379.

[0025] Figure 7 DNA sequence encoding ECA;

[0026] Figure 8 A schematic diagram of the integration of the recombinant plasmid into the heterologous host S. lividans TK24 chromosome and the amplification site used for genotype verification;

[0027] Figure 9 Agarose gel electrophoresis image of the genotype verification results of the recombinant strain.

[0028] In the figure, A: upstream integration site of pSET-pKasOECA; B: downstream integration site of pSET-pKasOECA;

[0029] Figure 10 SDS-PAGE electrophoresis image of echinocandin deacylase ECA.

[0030] In the figure, the arrows indicate the locations of the large and small subunits of the mature ECD. Lane 1 is the fermentation supernatant of the model strain S. lividans TK24; lane 2 is the fermentation supernatant of the engineered strain of echinocandin deacetylate; lane 3 is the fermentation supernatant of the empty plasmid control strain TK24pSET-pKasO; lane 4 is elution buffer of 20 mM imidazole; lane 5 is elution buffer of 50 mM imidazole; lane 6 is elution buffer of 100 mM imidazole; lane 7 is elution buffer of 300 mM imidazole; and lane 8 is elution buffer of 500 mM imidazole.

[0031] Figure 11 LC-MS chromatogram of echinocandin deacylase ECA catalyzing the formation of ECEN from ECE, where ECE and ECEN are standards for echinocandin E and its parent nucleus, respectively.

[0032] Figure 12 LC-MS chromatogram of the biotransformation of ECE to ECEN by recombinant engineered bacteria of echinocandin deacylase ECA. In the figure, TK24pSET-pKasOECA is the recombinant engineered bacteria of echinocandin deacylase ECA, Vehicle is the strain that has been conjugated and transferred with pSET152 empty plasmid, and ECE and ECEN are the standards of echinocandin E and echinocandin E nucleus, respectively.

[0033] Figure 13 HPLC chromatogram of FR901379 to FR179442 by biotransformation of echinocandin deacylase ECA recombinant engineered bacteria. In the figure, TK24pSET-pKasOECA is the echinocandin deacylase ECA recombinant engineered bacteria, Vehicle is the strain that has been conjugated and transferred with the pSET152 empty plasmid, and FR901379 and FR179442 are the substrate and product standards, respectively. Detailed Implementation

[0034] The present invention will be further described below with reference to the embodiments and accompanying drawings, but is not limited thereto.

[0035] The culture medium formulations involved in the following examples are as follows:

[0036] Escherichia coli was cultured using LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 5.0 g / L; solid medium was cultured with agar 20.0 g / L added.

[0037] The solid culture medium for Streptomyces was MS medium: soybean flour 20.0 g / L, mannitol 20.0 g / L, CaCO3 3.0 g / L, agar 20.0 g / L;

[0038] Streptomyces seed culture was performed using TSB medium: 30.0 g / L TSB medium powder;

[0039] Streptomyces fermentation was performed using SGGP medium: glucose 10.0 g / L, tryptone 4.0 g / L, yeast extract 4.0 g / L, casein amino acids 4.0 g / L, K2HPO3 1.36 g / L, and magnesium sulfate heptahydrate 0.5 g / L.

[0040] Example 1: Construction of recombinant engineered strains with echinocandin deacylate

[0041] 1) Constructing a gene expression cassette for echinocandin deacylate ECA

[0042] Synthesis such as Figure 7 The DNA sequence shown (ecaSY1965DNA) was assembled sequentially to the 3′ end of the ecaSY1965DNA gene fragment using overlap PCR, with the 6×His separator tag and lambda t0 terminator DNA fragment attached to both ends. This resulted in a linear DNA fragment with AflII and EcoRI restriction sites at both ends, representing the ECA gene expression cassette.

[0043] 2) Construct the pSET-pKasOECA recombinant plasmid

[0044] The ECA gene expression cassette DNA and plasmid pSET152 were double-digested with AflII and EcoRI, respectively. After separation by gel electrophoresis, the digested expression cassette insert and linearized plasmid backbone DNA fragments were recovered separately. The insert and plasmid backbone were ligated using T4 DNA ligase and transformed into E. coli DH5α for antibiotic selection (50 μg / mL apramycin). Positive transformants were selected. Single colonies were picked for colony PCR verification, amplifying the fragment containing the transcription unit. The plasmid was extracted and sequenced for verification. The recombinant plasmids with correct sequences were named pSET-pKasOECA. The plasmid map is shown below. Figure 5 As shown.

[0045] 3) Construction of recombinant engineered bacteria with echinocandin deacylate (ECA)

[0046] The correctly recombinant plasmid pSET-pKasOECA was transformed into E. coli ET12567 (PUZ8002), and then transferred into Streptomyces lividans TK24 via conjugation transfer plasmid. The Mg2+ concentration used during conjugation transfer was 10 mM, the pre-germination time of Streptomyces was 3 h, and the final concentrations of apramycin and nalidixic acid were 1.25 mg / ml and 0.5 mg / ml, respectively. After conjugation transfer, MS plates were incubated statically at 28°C, and single colonies were picked after 3-5 days. Figure 8 As shown, PCR primers pSL_F5 and pSL_R5, pSL_F7 and pSL_R7 were used to verify the genotype and ensure that the plasmid was integrated into the correct position of the S. lividans TK24 chromosome DNA.

[0047] The primers for colony PCR are as follows:

[0048] pSL_F5: 5′-cacgttttcccaggtcagaagc-3′

[0049] pSL_R5: 5′-atgctcaactaaagtggggcg-3′

[0050] pSL_F7: 5′-gaccatcgtgatcggcgtgt-3′

[0051] pSL_R7: 5′-gcgtcgagccgcataatcagg-3′

[0052] PCR validation example and validation results are as follows: Figure 9 As shown, the recombinant plasmid has been correctly recombined into the correct position on the heterologous host S. lividans TK24 chromosome, and the resulting recombinant engineered bacterium is named TK24pSET-pKasOECA.

[0053] Example 2: Purification of Echinocandin Deacylase

[0054] Ferment the verified recombinant strain TK24pSET-pKasOECA for 72 hours. Use the supernatant. Protein affinity chromatography was performed using a protein purification instrument and a Ni-NTA column. The separation and purification results were analyzed using SDS-PAGE gel chromatography. Figure 10 As shown, compared with the control group *S. lividans* TK24 and the empty plasmid control group *TK24pSET-pKasO*, the experimental group showed obvious single purified bands in lanes 6 and 7. Both enzyme expression bands were located at 50-70 kDa, which is consistent with the size of ECB deacylases (a superfamily of hydrolases). These enzymes typically consist of an inactive precursor of 80-90 kDa that self-cleaves, resulting in a mature large subunit of approximately 63 kDa and a small subunit of 18-20 kDa. The observation of distinct large subunit and relatively weak small subunit bands indicates that the two deacylase genes were successfully secreted in *S. lividans* TK24. The purified echinocandin deacylase A was obtained by eluting with 300 mM imidazole elution buffer.

[0055] Example 3: Echinocandin deacylation enzyme ECA catalyzes the deacylation of ECE to generate ECEN.

[0056] The purified ECA protein was used to catalyze the deacylation of echinocandin E in a buffer system. The catalytic reaction was carried out in 50 mM Tris-HCl buffer at pH 6.8, with a final ECA concentration of 1.5 μM and a substrate ECE concentration of 0.11 mM. Incubation was performed at 28 °C for 30 min. The formation of the corresponding deacylated ECEN product was detected by LC-MS. Figure 11 ).

[0057] Example 4: Biotransformation of ECE to ECEN by recombinant engineered strain of echinocandin deacylate ECA

[0058] Fermentation broth from the engineered strain of recombinant echinocandin deacylase, after 24 hours of fermentation, was mixed with ECE to a final concentration of 0.1 mM. The mixture was then converted at 28°C and 200 rpm for 12 hours. The conversion results were detected by HPLC, and the conversion rate was determined. The results are as follows: Figure 12As shown, after 12 h of biotransformation, the consumption of substrate ECE and the formation of product ECEN were clearly observed in the reaction mixture. The formation of ECEN was further verified by LC-TOFMS. The quasi-molecular ion peaks of ECEN were m / z 734.3730 [M+H]+ and m / z 756.3568 [M+Na]+ (cacld.: C34H52N7O11+, m / z 734.3719, C34H51N7NaO11+ m / z 756.3579). This indicates that both heterologously expressed recombinant deacylates in the biotransformation system successfully catalyzed the deacylation of ECE and produced the target compound ECEN. The TK24pSET-pKasOECA recombinant engineered strain achieved a 78.38% conversion rate of ECE within 12 h.

[0059] Example 5: Biotransformation of FR901379 by recombinant engineered strain of echinocandin deacylase ECA to generate FR179642

[0060] Referring to the biotransformation method described in Example 4, the deacylation activity of the recombinant engineered strain of echinocandin deacylate ECA on the micafungin precursor FR901379 was further tested. The fermentation broth of the engineered strain after 24 hours of fermentation was added to a final concentration of 0.17 mM with FR901379, and the transformation was carried out at 28°C and 200 rpm for 3 hours. HPLC results are as follows. Figure 13 As shown, compared to the control strain, the recombinant strain successfully transformed FFR901379 to obtain the deacylated product FR179642. The TK24pSET-pKasOECA recombinant engineered strain achieved a transformation rate of 77.00% for FR901379 within 3 hours.

Claims

1. Application of deacylase with the amino acid sequence shown in SEQ ID NO:1 in the hydrolysis of echinocandin E to remove the acyl side chain and generate a cyclic peptide core.

Images