A histone deacetylase from Chaetomium oliveri and its encoding gene and application

Abstract

The present invention belongs to the field of microorganisms and biomedicine technology, and specifically relates to an olive-colored chaetomium histone deacetylase and its encoding gene and application. The present invention performs gene knockout on HDAC in the fungal strain olive-colored chaetomium (Chaetomium olivaceum) SD-80A, realizes the activation of the recessive biosynthetic gene cluster and increases the yield of the compound cochlidinol. Studies have found that the compound shows good antimicrobial, anti-inflammatory, anticoagulant and anticancer activities, has the potential to be developed as a drug lead compound, and therefore has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and biomedicine, and particularly relates to a Chaetomium olivineum histone deacetylase and a coding gene and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Chaetomium olivaceum belongs to the kingdom Fungi, class Ascomycetes, order Ascomycetes, family Chaetomaceae, and Chaetomium. Chaetomium strains have broad development and application prospects, and their biological information, active ingredients, and biosynthesis are all research hotspots, and they have important applications in biological control.

[0004] Cochliodinol is a benzoquinone compound, purple solid powder, with a molecular formula of C 32 H 30 N2O4, with a relative molecular mass of 506.22 and a CAS number of 11051-88-0, has an indole group and an isoprenyl group in its structure, and has multiple biological activities. It was isolated from Chaetomium species of the genus Chaetomium. Coccomycin can significantly inhibit the growth of a variety of microorganisms, and has good antimicrobial, anti-inflammatory, anticoagulant and anticancer activities, and has great potential for development as a drug lead compound.

[0005] Genomic information shows that microorganisms have abundant secondary metabolite synthesis gene clusters, but the compounds mined from them are very limited. Most secondary metabolite gene clusters are not expressed or expressed at a low level, which are called "silent gene clusters". Even the strains that have been widely studied have yet to explore their potential for producing secondary metabolites. Epigenetic modification is an effective means to mine silent gene clusters in microorganisms, which has a regulatory effect on the biosynthesis of secondary metabolites of various filamentous fungi. DNA methylation and histone modification are the main epigenetic regulation forms of filamentous fungi, which can affect the structure of chromatin and the transcription level of genes, thereby affecting the biosynthesis process of secondary metabolites. These two methods can increase the yield of a variety of known secondary metabolites, and can also induce filamentous fungi to produce new unknown metabolites by activating silent biosynthetic gene clusters. Among them, histone acetylation is conducive to the binding of transcription factors to DNA, thereby activating gene transcription, while histone deacetylation has the opposite effect. Histone deacetylase HDAC is an important class of epigenetic regulatory factors that can modify the lysine residues of histones, thereby controlling gene expression and silencing. HOS2 is an important member of class I HDAC and a regulatory factor for secondary metabolites. Summary of the invention

[0006] In view of the above-mentioned prior art, the present invention provides a histone deacetylase of Chaetomium olivaceum and its encoding gene and application. The present invention isolates the HDAC in the fungal strain Chaetomium olivaceum SD-80A from the excrement of Boselaphus tragocamelus and performs gene knockout, thereby activating the recessive biosynthetic gene cluster and increasing the yield of the compound cocamycin. Studies have found that the compound shows good antimicrobial, anti-inflammatory, anticoagulant and anticancer activities, and has the potential to be developed as a drug lead compound. Based on the above research results, the present invention is completed.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides an olive-colored chaete histone deacetylase, wherein the amino acid sequence of the olive-colored chaete histone deacetylase is any one of the following (a1)-(a2):

[0009] (a1) the amino acid sequence shown in SEQ ID NO.1;

[0010] (a2) The amino acid sequence of a protein derived from (b1) having the same biological activity after substitution and / or deletion and / or addition of one or several (such as 1, 2 or 3, etc.) conservative amino acid residues.

[0011] The second aspect of the present invention provides the gene CoHOS2 encoding the olive chaete histone deacetylase described in the first aspect, the nucleotide sequence of which is any one of the following (b1)-(b2):

[0012] (b1) the nucleotide sequence shown in SEQ ID NO.2;

[0013] (b2) A nucleotide sequence complementary to (a1).

[0014] The third aspect of the present invention provides amplification primers designed based on the olive chaete histone deacetylase encoding gene CoHOS2, wherein the amplification primers include the nucleotide sequences shown in SEQ ID NO.3-6.

[0015] The fourth aspect of the present invention provides a recombinant expression vector, which contains at least the gene CoHOS2 encoding the olive chaete histone deacetylase.

[0016] The recombinant expression vector is obtained by effectively connecting the above-mentioned encoding gene to an expression vector, and the expression vector can be any one or more of a viral vector, a plasmid, a bacteriophage, a cosmid or an artificial chromosome; the viral vector may include an adenoviral vector, a retroviral vector or an adeno-associated viral vector, and the artificial chromosome includes a bacterial artificial chromosome, a bacteriophage P1-derived vector, a yeast artificial chromosome or a mammalian artificial chromosome; further preferably, it is a fungal plasmid.

[0017] The fifth aspect of the present invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium is a strain that inhibits the expression and / or activity reduction of the olive chaete histone deacetylase encoding gene CoHOS2 and its expression product.

[0018] Specifically, the engineered bacteria can be obtained by knocking out the olive-colored chaetocystis histone deacetylase encoding gene CoHOS2 in the starting strain.

[0019] Among them, the starting strain includes but is not limited to bacteria and fungi. In a specific embodiment of the present invention, the starting strain is a fungus, specifically Chaetomium olivaceum SD-80A, which has been deposited with the China General Microbiological Culture Collection Center, with the deposit number CGMCC No.40420, the deposit date is November 10, 2022, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0020] More specifically, the specific construction method of the engineered bacteria is as follows: construct a gene knockout box, and transform the knockout box using a PEG-mediated olive-colored chaete protoplast transformation method to obtain a recombinant olive-colored chaete strain in which the olive-colored chaete histone deacetylase encoding gene CoHOS2 is knocked out.

[0021] The primers used to construct the CoHOS2 gene knockout cassette are shown in SEQ ID NO.3-6.

[0022] Wherein, the olive-colored chaetomium is the above-mentioned olive-colored chaetomium (Chaetomium olivaceum) SD-80A.

[0023] The sixth aspect of the present invention provides the use of the olive chaete histone deacetylase, the olive chaete histone deacetylase encoding gene CoHOS2, the recombinant expression vector and / or the genetically engineered bacteria in any one or more of the following:

[0024] (c1) increasing the richness of secondary metabolites of olive chaete;

[0025] (c2) improving the ability of olive chaete to produce caesalpinia;

[0026] (c3) Improving the application effect of olive chaete in the medical field.

[0027] Among them, the olive-colored Chaetomium can be the above-mentioned genetically engineered bacteria, specifically the olive-colored Chaetomium genetically engineered bacteria.

[0028] The beneficial technical effects of one or more of the above technical solutions are:

[0029] The above technical scheme identified for the first time a histone deacetylase encoding gene CoHOS2 in Chaetomium olivaceum SD-80A. By constructing an engineered bacterium of Chaetomium olivaceum, the histone deacetylase encoding gene CoHOS2 was knocked out, its silent gene cluster was activated, and the expression level of fungal metabolites, especially cocamycin, was increased, which is helpful to provide lead compounds for new drug development, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 Schematic diagram of the CoHOS2 knockout cassette of the present invention.

[0032] Figure 2 HPLC profiles of ΔCoHOS2 of the present invention and the original strain.

[0033] Figure 3 is the structural formula of cotylosin highly expressed in ΔCoHOS2 of the present invention.

[0034] Figure 4 The expression levels of the compound kinase inhibitor ΔCoHOS2 of the present invention and the original strain are compared. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0037] In a typical embodiment of the present invention, a histone deacetylase encoding gene was identified from Chaetomium olivaceum SD-80A. By conducting whole genome sequencing and bioinformatics analysis on SD-80A, detailed gene information of CoHOS2 was obtained, and its nucleotide sequence is shown in SEQ ID NO.2:

[0038]

[0039] Its amino acid sequence is shown in SEQ ID NO.1:

[0040] MDAAAYRYRPPKPNYLPHNIEADGPVVEEYALPLGKADDRENAIFFNKMKFAAQEYGIVRPKGYTVSYHATPEMEKHHFGQTHPMKPWRLTLTKSLVTSYGMPFAMDNYVTREATYEELNAFHSN DYLDYLSTAAPEDQPRDIENPDKEVKYNLGGSDCPLFHGLYNYCSMSSAGCSLDAARKITSNQSDIAIAWGGGLHHAKRYEASGFCYINDIVLAILQLLRTHPRVLYIDIDVHHGDGVEEAFFSTD RVMTVSFHKYHPETFFPGTGALEDNGPKSEHNPGAHHAINVPLQDGITDEQYETLFQSIIGSINERYRPSAIALQCGADSLAGDRLGRFNLRVQGHGACVRYCKSLGIPMIVFGGGGYTPRNVAR AWAYETSIAIGADTRIPETIPDHTPWREHFIHDTLFPSLEQSMNEPRHNRNSEKRLREIITHVQEQLRFVSHAPSVQSSIIPPDLGPIRDDVEARLKEENDERNGGLRRIQEEGLGRDMEF(SEQ ID NO.1).

[0041] In another specific embodiment of the present invention, an engineered bacterium of olive color with a knockout histone deacetylase encoding gene CoHOS2 is provided, and the construction method thereof comprises the following steps:

[0042] (1) Constructing a knockout cassette containing upstream and downstream fragments of CoHOS2: using the genome sequence as a template, using primer sequences SEQ ID NO.3-4 to amplify the upstream fragment of CoHOS2, and using primer sequences SEQ ID NO.5-6 to amplify the downstream fragment of CoHOS2; synthesizing the Hyg-R resistance fragment, and connecting the three fragments using Overlap PCR;

[0043] (2) The constructed knockout cassette was transferred into olive Chaetomium protoplasts using the PEG-CaCl2 method;

[0044] (3) Using the Hyg-R resistance of the vector, colony PCR was performed to screen positive mutants.

[0045] Wherein, in the step (2), the olive-colored chaetomium is the olive-colored chaetomium (Chaetomium olivaceum) SD-80A.

[0046] The olive-colored Chaetomium engineered bacteria (ΔCoHOS2) with the CoHOS2 gene knocked out obtained by the above construction method were detected to have the original CoHOS2 sequence replaced by a Hyg-R resistance fragment, thereby achieving the editing of the CoHOS2 gene.

[0047] It should be noted that, although the present invention provides a method for obtaining an olive-colored chaetomium engineered bacterium with high cocamycin production based on knocking out the histone deacetylase encoding gene CoHOS2, based on the inventive concept of the present invention, other methods for inhibiting the expression and / or reducing the activity of the olive-colored chaetomium histone deacetylase encoding gene CoHOS2 and its expression products to obtain the corresponding genetically engineered bacteria also fall within the scope of protection of the present invention.

[0048] In another specific embodiment of the present invention, the use of the olive-colored Chaetomium engineered bacteria in any one or more of the following is provided:

[0049] (c1) increasing the richness of secondary metabolites of Chaetomium olivei;

[0050] (c2) improving the ability of Chaetomium olivei to produce caesalpinioides;

[0051] (c3) Improving the application effect of clomiphene citrate in the medical field.

[0052] Among them, Chaetomium olivaceae can be the above-mentioned genetically engineered bacteria, specifically Chaetomium olivaceae genetically engineered bacteria.

[0053] In (c3), the application specifically includes providing a guide compound for the research and development of new drugs related to coquinomycin. Therefore, specifically, the application can be used to improve the application effect of coquinomycin in the preparation of coquinomycin-related drugs.

[0054] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0055] Sources of biological materials:

[0056] Chaetomium olivaceum SD-80A is a Chaetomium strain screened and identified by our laboratory. Its accession number in the China General Microbiological Culture Collection is CGMCC No.40420, and the deposit date is November 10, 2022. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Fungal genome extraction kit was purchased from Beijing Adlai Biotechnology Co., Ltd., high-fidelity DNA polymerase was purchased from Nanjing Novozymes, and PDA medium and PDB medium were purchased from Qingdao Haibo Company.

[0057] Example 1: Construction of engineered bacteria with knockout of the CoHOS2 gene of Chaetomium olivineum

[0058] (1) Construction of gene knockout cassette

[0059] Olive chaete SD-80A was inoculated into PDB medium. After culturing for 3 days, the genome was extracted using a fungal genome extraction kit. The genome was used as a template to amplify the upstream fragment of the sequence using primers SEQ ID NO.3 (CAGGTGGTGGCTACCGTG) and SEQ ID NO.4 (TTATCAGGTCACGATGTGTCC). The downstream fragment of the sequence was amplified using primers SEQ ID NO.5 (GAGGAGGGGCTCGGGCG) and SEQ ID NO.6 (TCGAACGCCATCAACAGC). Hyg-R with the linker of the upstream and downstream fragments of the sequence was synthesized, and the three fragments were connected by Overlap PCR to form a gene knockout cassette ( Figure 1 ).

[0060] (2) Protoplast preparation

[0061] Olive-colored chaetocystis SD-80A was inoculated on a spore-forming plate and cultured in a dark incubator at 28°C for about 14 days to produce spores;

[0062] Use a spatula to scrape the spores on the spore-producing plate, put them into a 10 mL sterile centrifuge tube with glass beads, add 5 mL of PDB medium, and vortex thoroughly for 5 minutes to disperse the spores;

[0063] The spore suspension was added to 100 mL of PDB medium and placed in a constant temperature shaking incubator at 28°C and 200 rpm for overnight germination;

[0064] Prepare enzymatic solution: dissolve 0.05 g of lytic enzyme in 5 mL of OM solution (1.2 M MgSO4, 10 mM NaH2PO4, 10 mM Na2HPO4, pH 5.8), and sterilize by filtering with a 0.2 μM filter membrane;

[0065] The young mycelial balls germinated from the spores were collected into the filter, washed twice with OM solution, added to the pre-prepared enzymatic solution, and placed in a constant temperature shaking incubator at 30°C and 100 rpm for enzymatic hydrolysis for 4 hours;

[0066] After 4 hours of enzymatic hydrolysis, samples were taken for microscopic examination. When a large number of transparent protoplasts appeared in the field of vision, the enzymatic hydrolysis was stopped.

[0067] Filter the enzymatic solution into a 50 mL centrifuge tube with a filter, add 20 mL of refrigerated Trapping Buffer (0.6 M Sorbitol, 10 mM Tris-HCl, pH 7.0), centrifuge at 4 ° C, 5000 rpm for 10 min, discard the supernatant, add an appropriate amount of STC Buffer (1.2 M Sorbitol, 10 mM CaCl2, 10 mM Tris-HCl, pH 7.5) to resuspend the protoplasts and count them, and adjust the protoplast concentration to about 10 7 Pieces / mL.

[0068] (3) Protoplast transformation and mutant screening

[0069] Add 100 μL of protoplast suspension and 5 μg of knockout box fragment to a 1.5 mL sterile centrifuge tube, mix gently, and place on ice for 30 min; add 600 μL of PEG (60% PEG4000, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5) solution, mix gently, and place at room temperature for 15 min; spread on MYG-S agar plate containing hygromycin B (200 μg / mL), and culture at 28°C in the dark for 5-7 days;

[0070] After transformation, 10 positive transformants were obtained. The genomes of ΔCoHOS2 and the original strain were extracted as templates, and PCR verification was performed using primers SEQ ID NO.7 (GTTGCTGAGCGCCGAAG) and SEQ ID NO.8 (CGTGGTCCGTCTGCTGGT), and sequencing was used to verify the correctness.

[0071] One of the screened transformants had its CoHOS2 sequence replaced by the Hyr-R sequence, which was a correct transformant. Example 2: Analysis of secondary metabolites of Chaetomium olive and its engineered strains

[0072] Olive Chaetomium and its engineered strains were inoculated on PDA plates and grown for 3-5 days, inoculated into PDB medium, and grown in a shaker at 28°C and 200 rpm for 3-5 days, which were used as seed liquid;

[0073] The seed solution was inoculated into 30 bottles of 500 mL conical flasks with rice culture medium (40 g rice / bottle), and cultured in an incubator at 28° C. for 30 days;

[0074] The fermentation product was extracted 5 times with an equal volume of ethyl acetate, and the crude extract was obtained after vacuum concentration. 10 mg of the crude extract was weighed and dissolved in 1 mL of chromatographic methanol, and chromatographic analysis was performed using HPLC ( Figure 2 );

[0075] The crude extract was purified by gradient elution using silica gel column chromatography with petroleum ether:ethyl acetate (100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100, v / v) as the eluent, and a total of 11 fractions (Fr.1-11) were eluted; Fr.4 was further separated and purified using Sephadex LH-20 gel column (eluent dichloromethane / methanol = 2 / 1) to obtain 462 mg of clomiphene citrate ( Figure 3 ), and its yield reached 385 mg / kg rice culture medium, which was 12.03 times higher than that of the original strain ( Figure 4 ), which was 334.8 times higher than the yield of Chaetomium subaffine MFFC22 (1.15 mg / L).

[0076] Table 1 Comparison of cohosin expression in ΔCoHOS2 and other strains

[0077]

[0078] Example 3: Analysis of expression levels of genes for biosynthesis of secondary metabolites of Chaetomium olive and its engineered strains

[0079] RNA was extracted from olive-colored chaetocystis and its engineered strain after 30-day rice fermentation, and reverse transcribed into cDNA. Actin gene was used as the internal reference gene and the core gene of the biosynthesis of cyclosporin as the target gene. Fluorescence quantitative PCR was used to analyze the transcription level. The gene expression level of the engineered strain increased by 8 times.

[0080] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria is a strain in which the olive-colored chaete histone deacetylase encoding gene CoHOS2 is knocked out from the starting strain; The nucleotide sequence of the olive chaete histone deacetylase encoding gene CoHOS2 is shown in SEQ ID NO.2; Wherein, the starting strain is specifically olive-colored chaete ( Chaetomium olivaceum )SD-80A, which has been deposited by the China General Microbiological Culture Collection Center, with the deposit number CGMCC No.40420, the deposit date is November 10, 2022, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The genetically engineered bacterium according to claim 1, characterized in that The specific construction method of the genetically engineered bacteria is as follows: construct a gene knockout box, transform the knockout box using a PEG-mediated olive-colored Chaetomium protoplast transformation method, and obtain a recombinant olive-colored Chaetomium strain with the olive-colored Chaetomium histone deacetylase encoding gene CoHOS2 knocked out.

3. The genetically engineered bacterium according to claim 2, characterized in that: The primers used to construct the CoHOS2 gene knockout cassette are shown in SEQ ID NO.3-6.

4. The use of olive chaete histone deacetylase in improving the ability of olive chaete to produce cytomycin, characterized in that: The coding gene of olive-colored Chaetomium histone deacetylase is knocked out in the starting strain to improve the ability of olive-colored Chaetomium to produce caerulein. The amino acid sequence of olive-colored Chaetomium histone deacetylase is shown in SEQ ID NO.

1. The starting strain is specifically olive-colored Chaetomium ( Chaetomium olivaceum )SD-80A, which has been deposited by the China General Microbiological Culture Collection Center, with the deposit number CGMCC No.40420, the deposit date is November 10, 2022, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

5. The use of the olive chaete histone deacetylase encoding gene CoHOS2 in improving the ability of olive chaete to produce cocamycin, characterized in that: The olive-colored Chaetomium histone deacetylase encoding gene CoHOS2 was knocked out in the starting strain to improve the olive-colored Chaetomium's ability to produce cocamycin. The nucleotide sequence of the olive-colored Chaetomium histone deacetylase encoding gene CoHOS2 is shown in SEQ ID NO.

2. The starting strain is specifically olive-colored Chaetomium ( Chaetomium olivaceum )SD-80A, which has been deposited by the China General Microbiological Culture Collection Center, with the deposit number CGMCC No.40420, the deposit date is November 10, 2022, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

6. Use of the genetically engineered bacteria according to any one of claims 1 to 3 in improving the ability of Chaetomium olivei to produce caesalpinia.

Images