CN114315778B - Trichoderma harzianum acid A compound and trichoderma harzianum mutant strain for producing same - Google Patents
Trichoderma harzianum acid A compound and trichoderma harzianum mutant strain for producing same Download PDFInfo
- Publication number
- CN114315778B CN114315778B CN202011082163.0A CN202011082163A CN114315778B CN 114315778 B CN114315778 B CN 114315778B CN 202011082163 A CN202011082163 A CN 202011082163A CN 114315778 B CN114315778 B CN 114315778B
- Authority
- CN
- China
- Prior art keywords
- trichoderma harzianum
- compound
- acid
- mutant strain
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a trichoderma harzianum acid A compound, the molecular structure of which is shown as a formula (I), and the compound has low cytotoxicity and high anti-inflammatory activity. The invention also relates to a trichoderma harzianum mutant strain for producing the trichoderma harzianum acid A compound, which is trichoderma harzianum for knocking out histone deacetylase gene HOS2, and the trichoderma harzianum mutant strain is used for producing the trichoderma harzianum acid A compound, and has the advantages of simple operation, high yield and good repeatability.
Description
Technical Field
The invention belongs to the field of natural products of fungi, and relates to a trichoderma harzianum acid A compound and a trichoderma harzianum mutant strain for producing the compound.
Background
Secondary metabolites of fungi are an important source of new drugs and lead compounds, however, as more and more compounds are repeatedly discovered, the development of natural products of fungal origin gradually reaches a bottleneck. With the maturation of genome sequencing technology and the perfection of bioinformatics analysis methods, researchers find that the biosynthetic gene clusters (Biosynthetic gene clusters, BGCs) of fungi are quite abundant, however, more than 70% of the biosynthetic gene clusters are in a silent or undeveloped state due to the limitation of laboratory culture conditions, which indicates that there is a great room for the development of natural products of fungi. Therefore, how to activate these silent gene clusters is critical for research on fungal natural products.
There are four main strategies for the research of fungal natural product discovery: (1) The culture conditions are optimized using extreme environmental microorganisms (novel compounds found in closely related fungi from specific environments such as hot springs, volcanic, saline-alkali soil, etc.). The method is simple to operate, but has large workload and low efficiency, and only a very small amount of novel compounds are obtained in a large number of experiments; (2) heterologous expression. The method can obtain a new active compound and simultaneously clarify the function of silencing gene clusters, but the gene clusters are required to be assembled, and the experiment difficulty coefficient is large because the gene clusters are often larger; (3) transcriptional regulation. The secondary metabolism of fungi involves a complex regulatory network, and editing of regulatory genes facilitates the discovery of new compounds. Wherein, the epigenetic method modifies the histone and can play a role in overall regulation. Chemical apparent modification is commonly used, and small molecule epigenetic modifier inhibitors are suitable. The operation is relatively simple, but a transformant with hereditary property cannot be obtained, and the experimental repeatability is poor. Therefore, the searching of the natural product mining strategy with simple experimental operation and good repeatability has important significance.
Disclosure of Invention
It is an object of the present invention to address the deficiencies of the prior art and to provide a trichoderma harzianum acid a compound having low cytotoxicity and high anti-inflammatory activity.
The second object of the invention is to provide a trichoderma harzianum mutant strain for producing trichoderma harzianum acid A compound, which is simple to operate and high in yield when being used for producing trichoderma harzianum acid A compound.
To this end, in a first aspect, the present invention provides a trichoderma harzianum acid a compound (trichoharzianin A) having a molecular structure represented by formula (i):
according to the invention, the trichoderma harzianum acid a compound has low cytotoxicity; specifically, the antitumor activity of the trichoderma harzianum acid A compound is as follows: the inhibition rates of the Trichoderma harzianum acid A compound with the concentration of 10 mu M on tumor cells A549, HCT-8, hepG2 and MCF-7 are 4.84%, 11.82%, 5.30% and 2.47%, respectively.
According to the invention, the trichoderma harzianum acid a compound has high anti-inflammatory activity; specifically, the anti-inflammatory activity of the trichoderma harzianum acid a compound is: the inhibition rate of the trichoderma harzianum A compound with the concentration of 10 mu M on NO produced by the microglial cells BV2 induced by lipopolysaccharide and inflammatory factors IL-1, IL-6 and TNF-alpha is 41.11%, 75.00%, 16.67% and 28.57%, respectively.
In some embodiments of the present invention, the trichoderma harzianum acid a compound has the formula C 13 H 18 O 3 The molecular weight is 223.13.
In a second aspect, the invention provides a trichoderma harzianum mutant strain for producing a trichoderma harzianum acid A compound, which is trichoderma harzianum with the histone deacetylase gene HOS2 (M431 DRAGFT_ 478849) knocked out.
In the invention, the sequence of the trichoderma harzianum of the histone deacetylase gene HOS2 is shown as SEQ No. 3.
In some embodiments of the present invention, the trichoderma harzianum has a preservation number of CGMCC 3.9236.
The invention also provides an application of the trichoderma harzianum mutant strain in the production of trichoderma harzianum acid A compounds with the molecular structure shown in the formula (I).
According to some embodiments of the invention, the application comprises inoculating the trichoderma harzianum mutant strain into a fermentation medium for fermentation culture, and then separating and purifying the obtained fermentation culture solution to obtain the trichoderma harzianum acid A compound with the molecular structure shown in the formula (I).
In the present invention, the fermentation medium includes one or more of PDA, PDB and rice medium.
In some embodiments of the invention, the fermentation temperature is 25-30 ℃; the fermentation time is 10-14 days.
In some embodiments of the invention, separating and purifying the obtained fermentation broth comprises performing UPLC detection by extracting the crude extract with ethyl acetate, separating the metabolite by ODS column, gradient eluting with 30% -100% methanol/water, concentrating the fraction, and separating by semi-preparative liquid chromatography.
The invention has the following advantages:
(1) The invention provides a novel compound with a molecular structure shown as a formula (I), which has low cytotoxicity and high anti-inflammatory activity.
(2) Histone deacetylase HOS2 was considered as a positive or negative regulator of secondary metabolism in the previous studies, but was first studied in trichoderma harzianum (Trichoderma harzianum), and trichoderma harzianum mutant strains producing trichoderma harzianum a compound were obtained by knocking out the histone deacetylase gene HOS2 from trichoderma harzianum.
(3) The trichoderma harzianum mutant strain for producing the trichoderma harzianum compound A is simple to operate and high in yield.
Drawings
The invention is described in further detail below with reference to the accompanying drawings:
FIG. 1 is a verification electrophoresis diagram of a transformant TMY3, wherein a is a PCR system, and primers used are RT-F and RT-R; b is a PCR system, and the used primers are 5F-F and 3F-R; WT is Trichoderma harzianum with the preservation number of CGMCC3.9236, namely the genome DNA of which the template used for PCR is WT; n is negative control, and the PCR template is ultrapure water; TMY3 is a transformant knocked out of HOS 2; 1.1, 1.2, 1.3: three transformants were in parallel, and the template used for PCR was the genomic DNA of the transformant.
FIG. 2 is a UPLC liquid phase analysis spectrum (rice culture medium).
Fig. 3 shows the molecular structure of trichoderma harzianum acid a compound.
FIG. 4 is a diagram of 1 H NMR(CDCl 3 ) A spectrogram.
FIG. 5 is a schematic view of a display 13 C NMR(CDCl 3 ) A spectrogram.
FIG. 6 is a diagram of 1 H- 1 H COSY(CDCl 3 ) A spectrogram.
FIG. 7 shows HSQC (CDCl) 3 ) A spectrogram.
FIG. 8 is a schematic diagram of HMBC (CDCl) 3 ) A spectrogram.
FIG. 9 is a schematic diagram of construction of transformant TMY3 by knocking out gene HOS 2.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail below with reference to the accompanying drawings. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I terminology
The term "transformant" as used herein refers to a transformed mutant strain, and refers to a bacterial cell or other recipient cell having a novel genetic marker obtained by knocking out a certain gene or introducing a foreign gene.
II. Embodiment
As previously mentioned, existing strategies for research of fungal natural product discovery are always unsatisfactory, and have problems such as (1) using extreme environmental microorganisms, working with great inefficiency, and obtaining only very small amounts of new compounds in a large number of experiments; (2) Heterologous expression is needed to be assembled by gene clusters, and the experiment difficulty coefficient is large because the gene clusters are often larger; (3) Transcription regulation and common chemical apparent modification cannot obtain a transformant with stable heredity, and experimental repeatability is poor. In order to find a transformant which is simple to operate, good in repeatability and stable in heredity and has high yield of functional natural compounds, the inventor conducts a great deal of research on a natural product mining strategy.
The inventor researches and discovers that a transformant with stable heredity for producing a novel compound can be obtained by knocking out a histone deacetylase gene HOS2 from trichoderma harzianum, the compound has low cytotoxicity and high anti-inflammatory activity, and the production of the compound by using the transformant is easy to operate and good in repeatability, so that the invention is obtained.
Accordingly, the trichoderma harzianum acid a compound according to the first aspect of the present invention has a molecular structure as shown in formula (i) (see fig. 3):
the compound is a novel natural functional compound with low cytotoxicity and high anti-inflammatory activity.
Activity analysis of the trichoderma harzianum acid a compound:
the inhibition rate of the compound on tumor cells A549, HCT-8, hepG2 and MCF-7 is detected by an MTT method to evaluate the anti-tumor activity; the anti-inflammatory activity of the compound is evaluated by detecting the inhibition rate of NO and inflammatory factors IL-1, IL-6 and TNF-alpha generated by lipopolysaccharide induced microglial cell BV2 through Griess method and ELISA kit, and the result shows (see table 8): the antitumor activity of the trichoderma harzianum acid A compound is as follows: the inhibition rate of the trichoderma harzianum acid A compound with the concentration of 10 mu M on tumor cells A549, HCT-8, hepG2 and MCF-7 is 4.84%, 11.82%, 5.30% and 2.47%, respectively; the anti-inflammatory activity of the trichoderma harzianum acid A compound is as follows: the inhibition rate of the trichoderma harzianum acid A compound with the concentration of 10 mu M on NO generated by lipopolysaccharide induced microglial cells BV2 is 41.11%, which is obviously higher than 31.16% of that of a positive drug nitric oxide synthase (nitric oxide synthase) inhibitor L-NMMA; and their inhibition rates for inflammatory factors IL-1, IL-6 and TNF-alpha are 75.00%, 16.67% and 28.57%, respectively. From the above, the effect of the trichoderma harzianum acid a compound on inhibiting tumor cells was not good, indicating that the compound has low cytotoxicity. The trichoderma harzianum acid A compound has better inhibition effect on NO and inflammatory factors IL-1, IL-6 and TNF-alpha, which shows that the compound has potential anti-inflammatory activity.
The second aspect of the invention relates to a trichoderma harzianum mutant strain for producing the trichoderma harzianum acid A compound, which is trichoderma harzianum with the histone deacetylase gene HOS2 knocked out.
Trichoderma harzianum (Trichoderma harzianum) (wild type) used in the invention is purchased from China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC), the collection number is CGMCC3.9236, the invention is called Trichoderma harzianum (Trichoderma harzianum) WT strain (wild type) or wild Trichoderma harzianum WT strain, and the sequence of a DNA fragment containing HOS2 in the genome (HOS 2 in the genome of the wild Trichoderma harzianum WT strain and DNA fragments of 665bp upstream and 1179bp downstream) is shown as SEQ No. 1. The gene knocked out from the bacterium is HOS2 (protein number: M431 DRAGT_ 478849), the function of which is histone deacetylase, the gene is also called histone deacetylase gene HOS2 in the invention, and the sequence of the gene HOS2 is shown in SEQ No. 3.
It is easy to understand that the Trichoderma harzianum mutant strain producing the Trichoderma harzianum acid A compound according to the present invention is a transformant obtained by knocking out the histone deacetylase gene HOS2 (M431 DRAGT_ 478849) from the genome of Trichoderma harzianum (Trichoderma harzianum) 3.9236, and is also called transformant TMY3 according to the present invention, wherein the sequences of DNA fragments of the genome from which HOS2 was knocked out (DNA fragments of 611bp upstream and 580bp downstream of HOS2 in the genome of transformant TMY 3) are shown in SEQ No. 2.
Furthermore, the trichoderma harzianum acid A compound is produced by knocking out the apparent regulatory factor HOS2 in trichoderma harzianum (Trichoderma harzianum) 3.9236 by using molecular biological gene manipulation to activate a new natural product, and has low cytotoxicity and high anti-inflammatory activity.
Knockout of HOS2 in the present invention: and (3) carrying out HOS2 knockout by utilizing a PEG-mediated protoplast transformation method, a homologous fragment at the upstream and downstream of the transferred gene and a CRISPR/Cas9 plasmid. As shown in FIG. 1, the HOS2 knockout transformant TMY3 was obtained by PCR.
The third aspect of the invention relates to an application of the trichoderma harzianum mutant strain in the second aspect of the invention in the production of trichoderma harzianum A compounds with the molecular structure shown in the formula (I).
According to some embodiments of the invention, the application comprises inoculating the trichoderma harzianum mutant strain into a fermentation medium for fermentation culture, and then separating and purifying the obtained fermentation culture solution to obtain the trichoderma harzianum acid A compound with the molecular structure shown in the formula (I).
In the present invention, the fermentation medium includes one or more of PDA, PDB and rice medium, preferably rice medium.
In some embodiments of the invention, the fermentation temperature is 25-30 ℃; the fermentation time is 10-14 days.
In some embodiments of the invention, separating and purifying the obtained fermentation broth comprises performing UPLC detection by extracting the crude extract with ethyl acetate, separating the metabolite by ODS column, gradient eluting with 30% -100% methanol/water, concentrating the fraction, and separating by semi-preparative liquid chromatography.
In some specific embodiments, the transformant TMY3 is cultured using a nutrient-rich, inexpensive, readily available rice medium. The secondary metabolite analysis is shown in FIG. 2. The specific process comprises the following steps:
(1) Fermentation culture: fermenting and culturing Trichoderma harzianum mutant strain at 28 deg.C or 25 deg.C in rice culture medium for 10-14 days to obtain secondary metabolite;
(2) Extraction and separation of secondary metabolites: the crude extract is extracted by ethyl acetate for UPLC detection, the metabolite is separated by ODS column chromatography, 30% -100% methanol/water is selected for gradient elution. Concentrating the fraction, and further separating by semi-preparative liquid chromatography to obtain pure product of the new compound;
(3) And (3) structural identification:
the sample was prepared with deuterated chloroform (CDCl) 3 ) Dissolving, obtaining one-dimensional and two-dimensional nuclear magnetic data by analyzing with a Bruce Avance III HD 500MHz Nuclear Magnetic Resonance (NMR) spectrometer (Bruck Bruker) 1 H-NMR、 13 C-NMR、Two-dimensional spectrogram (500 MHz)]As shown in fig. 4-8;
high-resolution mass spectrometry test is carried out by adopting Agilent 6520 precise mass QTOF LC/MS system (Agilent), and the molecular formula of the obtained compound is C 13 H 18 O 3 Molecular weight 223.1331 (calculated molecular weight 222.1329) and ionic form [ M+H ]] + As shown in table 7; the structure of the new product obtained by analysis is shown in figure 3, and the new product is named as trichoderma harzianum acid A compound in the invention.
Optical rotation analysis was performed using a J-810-150s spectropolarimeter (JASCO, japan spectro Co., ltd.) and showed that the compound had an optical rotation value of [ alpha ]] 20 D ＝28(C＝0.01g/100mL，CH 3 OH)。
The reagents described in the present invention are used directly or formulated in commercial concentrations.
III. Examples
The present invention will be specifically described below by way of specific examples. The experimental methods described below, unless otherwise specified, are all laboratory routine methods. The experimental materials described below, unless otherwise specified, are commercially available.
Example 1:
1. schematic diagram of HOS2 knockout transformant construction, as shown in FIG. 9
2. Construction of homology arm fragments
Constructing homologous fragments of the target knockout gene by using a fusion PCR technology, amplifying 5F and 3F fragments on the upstream and downstream of the target knockout gene according to a PCR system shown in table 1, performing agarose gel electrophoresis analysis on the fragments, recovering the fragments by using a kit, taking the recovered fragments as templates of the fusion PCR, and amplifying the fragments by using a PCR system prepared as shown in table 2 to obtain the homologous fragments. When the forward primer is 5F-F and the reverse primer is 5F-R in Table 1, amplifying an upstream 5F fragment of the target knocked-out gene HOS 2; when the forward primer is 3F-F and the reverse primer is 3F-R in Table 1, the downstream 3F fragment of the target knockdown gene HOS2 is amplified. The template DNA was genomic DNA of Trichoderma harzianum 3.9236.
TABLE 1 first round PCR System
TABLE 2 second round fusion PCR System
Construction of CRISPR/Cas9 knockout plasmid
The forward and reverse primers for the sgRNA were designed based on the selected sgRNA using the website (https:// bioinfo gp. Cnb. Csic. Es/tools/breakingcas) design sgRNA (small guide RNA).
TABLE 3 primer annealing System
TABLE 4 phosphorylation System of annealed products
Table 5 enzyme digestion System
Primer annealing: the synthesized primers were prepared according to the system of Table 3, and after controlling the temperature at 95℃for 3 minutes using a PCR apparatus, transferred to a 95℃water bath, the temperature control system was turned off, and the temperature was allowed to cool slowly from 95℃to 25℃at a rate of-1℃per 30 seconds, and after annealing, a DNA double strand containing cohesive ends was formed. The preparation of the phosphorylation system of the annealed product was carried out according to Table 4 and placed in a water bath at 37℃for 30 minutes.
The digestion backbone vector pHS-BVC-LW209: and (3) enzyme digestion is carried out at 37 ℃ overnight, gel digestion is carried out after electrophoresis, and an enzyme digestion system is shown in Table 5.
And (3) connection: the PCR instrument was kept at a constant temperature of 16℃for 1 hour, and the connection system was shown in Table 6.
Table 6 connection system
Plasmid transformation into E.coli:
mu.L of the ligation product was transferred to 50. Mu.L of DH5a competent cells, and cultured overnight in a 37℃incubator. After monoclonal is grown, picking colony extracting plasmid and sequencing and verifying.
4. Protoplast preparation and transformation
(1) Trichoderma harzianum mycelium is selected in an EP tube (a microcentrifuge tube), 3mL of PDB culture medium is added for homogenate, 3mL of bacterial liquid is transferred into the PDB culture medium, and the culture is carried out for 24 hours at 28 ℃ and 200 rpm.
(2) Filtering the bacterial liquid, putting the bacterial balls on the filter cloth into 3mL of TG solution, homogenizing, transferring 2.5mL of bacterial liquid into the TG solution, and culturing at 28 ℃ and 200rpm for 12 hours.
(3) Filtering bacterial liquid, putting the bacterial balls on the filter cloth into Trichoderma lysing enzyme N-M solution for enzymolysis, carrying out timing microscopic examination, observing cell morphology, generating enough protoplast, and filtering. Centrifuging, discarding supernatant, and resuspending STC solution. And after full resuspension, split charging into 15mL EP tubes, respectively adding the constructed CRISPR/Cas9 plasmid and homologous fragments, and adding no negative control. After mixing, the mixture was ice-bathed for 50 minutes.
(4) Adding a proper amount of 60% PEG solution, mixing, standing at room temperature for 20 min, adding a proper amount of STC solution, mixing, and inoculating on SPDA (hygromycin concentration is 100 μg/mL) plate. SPDA (hygromycin concentration 100. Mu.g/mL) was added to each plate. Culturing at 25 ℃ for 5 days.
(5) Selecting monoclonal transformant, screening hygromycin, preserving bacteria, inoculating mycelium into PDB culture medium, and culturing for genome extraction.
5. Genomic DNA extraction
Extracting genome by phenol chloroform extraction method. The method comprises the following specific steps:
(1) The PDB strain was cultured for 2-3 days.
(2) The thalli are picked up by a gun head or a flat toothpick, and the water is removed by absorbent paper and then put into a 1.5mL EP tube.
(3) Each tube was placed with 2-3 steel balls, 400. Mu.L LEST buffer was added and the tissue mill was broken.
(4) After the disruption, 300. Mu.L of LEST buffer was added and mixed well, and the mixture was left at room temperature for 5 minutes.
(5) Add 500 μl PCI solution (phenol: chloroform: isoamyl alcohol=25:24:1), mix upside down 10-15 times, and leave at room temperature for 5 minutes.
(6) Centrifuge at 13000rpm at 4℃for 10 min.
(7) The supernatant was transferred to a fresh EP tube, 1mL of pure ethanol was added, and the mixture was centrifuged at 13000rpm at 4℃for 10 minutes.
(8) The supernatant was discarded, 500. Mu.L of 70% ethanol was added, and after slight shaking, the mixture was centrifuged at 4℃at 13000rpm for 2 minutes.
(9) The supernatant was discarded and dried at room temperature.
(10) Add 20. Mu.L TE buffer, 0.5. Mu.L RNase A, water bath at 37℃for 30 min, preserve at-20 ℃.
6. Verification of transformants
According to the schematic diagram, two pairs of primers of 5F-F and 3F-R, RT-F and RT-R are used for verification, and the result is shown in FIG. 1, and as can be seen from FIG. 1, the RT-F and RT-R can amplify the band in the wild type, but the band cannot be amplified in the transformant TMY3. Meanwhile, 5F-F and 3F-R amplified more bands in the wild type than in the transformant TMY3. Indicating that the invention successfully obtained positive transformants (HOS 2 knocked out transformant TMY 3).
7. Fermentation culture
The secondary metabolite is obtained after fermenting and culturing the transformant TMY3 strain in rice culture medium at 28 ℃ or 25 ℃ for 10-14 days. The secondary metabolite analysis is shown in FIG. 2.
8. Extraction and separation of secondary metabolites
The crude extract is extracted by ethyl acetate for UPLC detection, the metabolite is separated by ODS column, 30% -100% methanol/water is selected for gradient elution. Concentrating the fraction, and further separating by semi-preparative liquid chromatography to obtain a novel compound (Trichoderma harzianum acid A compound) pure product.
9. Structural identification
The sample was prepared with deuterated chloroform (CDCl) 3 ) Dissolving, obtaining one-dimensional and two-dimensional nuclear magnetic data by analysis of Nuclear Magnetic Resonance (NMR) spectrometer 1 H-NMR、 13 C-NMR, two-dimensional spectrogram (500 MHz)]As shown in fig. 4-8; high resolution mass spectrometry to obtain molecular weight and molecular formula of the compound (see table 7); the structure of the new product Trichoderma harzianum acid A obtained by analysis is shown in figure 3, and the new product is named as Trichoderma harzianum acid A compound in the invention.
HRMS analysis of the compounds of table 7
The optical rotation detection result shows that the optical rotation value of the compound is [ alpha ]] 20 D ＝28(C＝0.01g/100mL，CH 3 OH)。
10. Activity analysis of the trichoderma harzianum acid a compound:
preparing the trichoderma harzianum acid A compound to a concentration of 10mM (the solvent is DMSO), detecting biological activity, and detecting the inhibition rate of the compound on tumor cells A549, HCT-8, hepG2 and MCF-7 by an MTT method to evaluate the anti-tumor activity; the anti-inflammatory activity of this compound was evaluated by detecting the inhibition rate of lipopolysaccharide-induced microglial BV 2-produced NO and inflammatory factors IL-1, IL-6 and TNF-alpha by Griess method and ELISA kit, and the results are shown in Table 8.
TABLE 8 Activity detection results of Trichoderma harzianum acid A Compounds
As can be seen from table 8, the trichoderma harzianum a compound has a poor inhibitory effect on tumor cells, indicating that the compound has low cytotoxicity. The trichoderma harzianum acid A compound has better inhibition effect on NO and inflammatory factors IL-1, IL-6 and TNF-alpha, which shows that the compound has better anti-inflammatory activity.
It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.
Sequence listing
<110> university of Beijing chemical industry, university of Beijing
<120> Trichoderma harzianum acid A compound and Trichoderma harzianum mutant strain producing the same
<130> RB2005421-FF
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3335
<212> DNA
<213> (HOS 2-containing DNA fragment in the genome of wild Trichoderma harzianum WT strain)
<400> 1
gctgtgagtg tgagcgaata ggtcagtatg aaggtttgaa ggaggtgaag atattggata 60
gacaatgtta aagagcagcc accaagagag atgccttgtg cttactcatt atatcaaacc 120
tgaggccagt caggacctgc agtttccctt acatggacat ggtacatact ccttccagtg 180
ggttcgataa accttatctc agcagtccat aagcccagcc agccagtagc ttttcaatct 240
cacgtgaccc cagccagatt ggcagccccg gagcttttcg atggcgttcg ctcactcact 300
cttctcggtc ccttcctcat accaccatct acaactttat ttttatttct tacgcatcca 360
aagtgctcaa acgaaaaggc caggctggct tcctcaaata ccattacaat ctcggccgct 420
cacattcaca ttgggatgca tttgatcaag cagcataatg cttgctgtag ctgaatcagc 480
agcctcaccc cttgaccctt gacccttgac ccttgacagt gatagctgtg tatcagtcat 540
tcatctcgtt catcttctca acgctatttc ctcagtcgtt gatctaataa caatagagcg 600
ccaatctgac gaggacattt tttatacaat tttaatcacc cagccgattc acctctctgc 660
ccaccatgga caactacagg tatcgtgtgc ctaagccaaa ctacctccct catagactgg 720
acatcgaccc cgatgccgac atagtagagg aatatgccca gcccctcggc attgccagcg 780
atgcggaaaa cgcaaagttc taccagcagt gcaaacgcct ggctgaatcg tcaggcatta 840
ctcgccccaa gggctacaat gtgtcttttc attgcaatcc cgagatggag aagcaccact 900
tcggcatgac acatcctatg aagccctggc gcctgacgct gtccaagagc ctgatttact 960
catacggcat gtcatttgcc atggacaact acctgacccg ggccgctacg tacgaggagc 1020
tggcgtcttt ccactccacc gactatctag actttctggg cactgttctc ccagaggcag 1080
ttcctcgaga tcttgagaac cagaaccctg acctgaaata caacttggga ggctccgatt 1140
gtcctttgtt cgacggcctc tataattact gctccatgtc tgccggcagt gcgcttgatg 1200
ccgcccggaa aatcgttagc aaacagtctg acattgccat tgcgtggggg ggcggcttgc 1260
accacgcaaa aaaggccgag gcctctggtt tttgctacat caacgacatt gtcatcgcca 1320
ttctggagct tctgcgcttc tacccacgtg ttttatacat tgacattgat gtacatcacg 1380
gcgatggtgt tgaggaggcc tttttctcaa ccgacagggt catgacagtc tcctttcata 1440
aatacgatcc caacaacttc tttcccggca ctggtgcgtt ggacgataat gggcctaaga 1500
gcgaacataa tcccggagct caccacgccg tcaatgttcc gctcaacgat ggtattacag 1560
acgaacaata cgacatgctc tttaacaaca tcatcagcaa gattgtcgag aaattccgac 1620
caagcgccat cgcactccaa tgcggtgccg attctctcgc gggagatcgc ctgggccgat 1680
tcaaccttca agtgcagggg catggtgcat gtgttgagtt ctgcaagaga cttggcattc 1740
ccatgatcct ctttggcggt ggtggttaca cccctcggaa tgttgcacga gcctggacat 1800
atgagacaag tatagccatc aactgccagg acaaaatcag ccctatatta ccagagcacg 1860
ctccgtggag agagcagttc cgccaggaca ccctcttccc gaccctcgaa cagattttgg 1920
gcgagccgcg acagaatcgt aacccccaga agcgtttgca ggaaatcatt caacacgtca 1980
cagagcaact acgtttcgtc gagagtgcgc caagcgtaca gatgcaggtc atccctcctg 2040
acttgggcag cttcagagat gatgtggaag aaaggttaaa agaggaccag gaggagagga 2100
acgagcagct acgaaaggag agggagtcgg gagtaggaac agccatggaa ttctaattta 2160
tcaaaacgac tcatcatacc ttcactccag aaccaactgc tgcgtgagca gactctttaa 2220
tttggaaata acccgtagac aacaagacaa aaaaaaaaga aaaaaagcat taaatgagat 2280
cccaagtact gtacaccacc agggcaaggc gtatgtctac tagtatatca cacacgacag 2340
catggcggcc atgcagccag gaatagcaga agagaggcgc aagtgcatta gtggaaggcc 2400
gcattatctt cgtccccaat ggccttcctg gtatcgcgag tttcctccct cgacccatcc 2460
cccagtcctc tttctctatg actcgggggg tccagccatt tttcaccccc cttccaccaa 2520
tgtgctgcga ggaaacgaat atgcacacga aaatatccaa ttgtatggta ctcgaaaaat 2580
aaaccaaaag taggaaagtt attccgaagg taccgtttta aaagtatcgg acaacggctg 2640
agccaccagc gggcatggca gtctattcac ataataaatt agcttttgac ctcacttctc 2700
ttccttttct ttaccctggg aaaggggatg cttaccgtgc aaccgttgtt gccgccagtc 2760
cacacaccgt tgatgtacga gcatttgctg ttaacatcac cagtaatctc aatgttgaag 2820
tccagcagag cagtgctagt gggaaggttc tggaagatgg agatgaaagt caagccacca 2880
gcggtcttac caagaccaac gatgacggga gaccagttac cagctccctt ggggtcgaga 2940
gggctgttcc agacgcaagc atctgcaact ccgtagccct tcttgttgac gtagtactgg 3000
gcggaggtac tcttgccatc ccacacatag tagtcagact cgtcggggtt gcagagagga 3060
atggtctgac cggcagtggc acaggcggga ataaccatgc tctcgatacc ggggtagtcg 3120
gttcggcacg tgcaaacgga atcgtcgagg tcgttctgga tggtaacacc gccagcacca 3180
gcctcgcaaa gcttggggtg gtcagggcga gtcagctcga ggaagccgtc cgagttgcag 3240
taaagaccgc caatggattg cagagtagat ccttggatcg ggggccactg ggtcttctgg 3300
tagccgagag ggcaagcata agagcacatg cctcc 3335
<210> 2
<211> 1191
<212> DNA
<213> (HOS 2-knocked-out DNA fragment in transformant TMY3 genome)
<400> 2
gctgtgagtg tgagcgaata ggtcagtatg aaggtttgaa ggaggtgaag atattggata 60
gacaatgtta aagagcagcc accaagagag atgccttgtg cttactcatt atatcaaacc 120
tgaggccagt caggacctgc agtttccctt acatggacat ggtacatact ccttccagtg 180
ggttcgataa accttatctc agcagtccat aagcccagcc agccagtagc ttttcaatct 240
cacgtgaccc cagccagatt ggcagccccg gagcttttcg atggcgttcg ctcactcact 300
cttctcggtc ccttcctcat accaccatct acaactttat ttttatttct tacgcatcca 360
aagtgctcaa acgaaaaggc caggctggct tcctcaaata ccattacaat ctcggccgct 420
cacattcaca ttgggatgca tttgatcaag cagcataatg cttgctgtag ctgaatcagc 480
agcctcaccc cttgaccctt gacccttgac ccttgacagt gatagctgtg tatcagtcat 540
tcatctcgtt catcttctca acgctatttc ctcagtcgtt gatctaataa caatagagcg 600
ccaatctgac gcagtccaca caccgttgat gtacgagcat ttgctgttaa catcaccagt 660
aatctcaatg ttgaagtcca gcagagcagt gctagtggga aggttctgga agatggagat 720
gaaagtcaag ccaccagcgg tcttaccaag accaacgatg acgggagacc agttaccagc 780
tcccttgggg tcgagagggc tgttccagac gcaagcatct gcaactccgt agcccttctt 840
gttgacgtag tactgggcgg aggtactctt gccatcccac acatagtagt cagactcgtc 900
ggggttgcag agaggaatgg tctgaccggc agtggcacag gcgggaataa ccatgctctc 960
gataccgggg tagtcggttc ggcacgtgca aacggaatcg tcgaggtcgt tctggatggt 1020
aacaccgcca gcaccagcct cgcaaagctt ggggtggtca gggcgagtca gctcgaggaa 1080
gccgtccgag ttgcagtaaa gaccgccaat ggattgcaga gtagatcctt ggatcggggg 1140
ccactgggtc ttctggtagc cgagagggca agcataagag cacatgcctc c 1191
<210> 3
<211> 1176
<212> DNA
<213> (Gene HOS 2)
<400> 3
atggacaact acctgacccg ggccgctacg tacgaggagc tggcgtcttt ccactccacc 60
gactatctag actttctggg cactgttctc ccagaggcag ttcctcgaga tcttgagaac 120
cagaaccctg acctgaaata caacttggga ggctccgatt gtcctttgtt cgacggcctc 180
tataattact gctccatgtc tgccggcagt gcgcttgatg ccgcccggaa aatcgttagc 240
aaacagtctg acattgccat tgcgtggggg ggcggcttgc accacgcaaa aaaggccgag 300
gcctctggtt tttgctacat caacgacatt gtcatcgcca ttctggagct tctgcgcttc 360
tacccacgtg ttttatacat tgacattgat gtacatcacg gcgatggtgt tgaggaggcc 420
tttttctcaa ccgacagggt catgacagtc tcctttcata aatacgatcc caacaacttc 480
tttcccggca ctggtgcgtt ggacgataat gggcctaaga gcgaacataa tcccggagct 540
caccacgccg tcaatgttcc gctcaacgat ggtattacag acgaacaata cgacatgctc 600
tttaacaaca tcatcagcaa gattgtcgag aaattccgac caagcgccat cgcactccaa 660
tgcggtgccg attctctcgc gggagatcgc ctgggccgat tcaaccttca agtgcagggg 720
catggtgcat gtgttgagtt ctgcaagaga cttggcattc ccatgatcct ctttggcggt 780
ggtggttaca cccctcggaa tgttgcacga gcctggacat atgagacaag tatagccatc 840
aactgccagg acaaaatcag ccctatatta ccagagcacg ctccgtggag agagcagttc 900
cgccaggaca ccctcttccc gaccctcgaa cagattttgg gcgagccgcg acagaatcgt 960
aacccccaga agcgtttgca ggaaatcatt caacacgtca cagagcaact acgtttcgtc 1020
gagagtgcgc caagcgtaca gatgcaggtc atccctcctg acttgggcag cttcagagat 1080
gatgtggaag aaaggttaaa agaggaccag gaggagagga acgagcagct acgaaaggag 1140
agggagtcgg gagtaggaac agccatggaa ttctaa 1176
<210> 4
<211> 21
<212> DNA
<213> (primer 5F-F)
<400> 4
gctgtgagtg tgagcgaata g 21
<210> 5
<211> 41
<212> DNA
<213> (primer 5F-R)
<400> 5
catcaacggt gtgtggactg cgtcagattg gcgctctatt g 41
<210> 6
<211> 41
<212> DNA
<213> (primer 3F-F)
<400> 6
caatagagcg ccaatctgac gcagtccaca caccgttgat g 41
<210> 7
<211> 20
<212> DNA
<213> (primer 3F-R)
<400> 7
<210> 8
<211> 22
<212> DNA
<213> (primer RT-F)
<400> 8
gcatgacaca tcctatgaag cc 22
<210> 9
<211> 20
<212> DNA
<213> (primer RT-R)
<400> 9
gacctgcatc tgtacgcttg 20
<210> 10
<211> 68
<212> DNA
<213> (primer sgRNA-F)
<400> 10
accgtagttt cctgatgagt ccgtgaggac gaaacgagta agctcgtcaa actacctccc 60
tcatagac 68
<210> 11
<211> 68
<212> DNA
<213> (primer sgRNA-R)
<400> 11
aaacgtctat gagggaggta gtttgacgag cttactcgtt tcgtcctcac ggactcatca 60
ggaaacta 68
Claims (3)
1. A trichoderma harzianum acid A compound has a molecular structure shown in a formula (I):
2. a trichoderma harzianum mutant strain producing trichoderma harzianum acid a compound, which is trichoderma harzianum (Trichoderma harzianum) from which histone deacetylase gene HOS2 has been knocked out;
the sequence of the trichoderma harzianum of the histone deacetylase gene HOS2 is shown as SEQ No. 3; the preservation number of the trichoderma harzianum is CGMCC 3.9236.
3. Use of the trichoderma harzianum mutant strain of claim 2 for the production of the trichoderma harzianum a compound of claim 1, said trichoderma harzianum a compound having the molecular structure of formula (i):
the application comprises the steps of inoculating the trichoderma harzianum mutant strain into a fermentation medium for fermentation culture, and then separating and purifying the obtained fermentation culture solution, wherein the molecular structure of the trichoderma harzianum compound A is shown as a formula (I);
the fermentation medium is one or more of PDA, PDB and rice medium; the fermentation temperature is 25-30 ℃; and/or, the fermentation time is 10-14 days;
the obtained fermentation culture solution is separated and purified: extracting the crude extract with ethyl acetate, performing UPLC detection, separating metabolite by ODS column, gradient eluting with 30% -100% methanol/water, concentrating the fraction, and separating by semi-preparative liquid chromatography.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011082163.0A CN114315778B (en) | 2020-10-12 | 2020-10-12 | Trichoderma harzianum acid A compound and trichoderma harzianum mutant strain for producing same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011082163.0A CN114315778B (en) | 2020-10-12 | 2020-10-12 | Trichoderma harzianum acid A compound and trichoderma harzianum mutant strain for producing same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114315778A CN114315778A (en) | 2022-04-12 |
| CN114315778B true CN114315778B (en) | 2023-06-20 |
Family
ID=81032460
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011082163.0A Active CN114315778B (en) | 2020-10-12 | 2020-10-12 | Trichoderma harzianum acid A compound and trichoderma harzianum mutant strain for producing same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114315778B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4915944A (en) * | 1983-03-15 | 1990-04-10 | Yissum Research Development Corp. Of The Hebrew University Of Jerusalem | Novel isolate of trichoderma, fungicidal compositions containing said isolate and use thereof |
| CN1176248A (en) * | 1994-12-19 | 1998-03-18 | 美国氰胺公司 | Restricted 9-CIS-retinoids |
| CN103922967A (en) * | 2014-04-15 | 2014-07-16 | 北京化工大学 | Hydroxamic acid compounds and application thereof in preparation of medicines for inhibiting cancer cell proliferation and/or treating cancers |
| CN104116728A (en) * | 2014-06-28 | 2014-10-29 | 北京化工大学 | Pharmaceutical composition composed of hydroxamic acid compounds and vitamin A compounds and application thereof |
| CN107011238A (en) * | 2017-03-14 | 2017-08-04 | 北京化工大学 | Class I histone deacetylase inhibitor and its production and use |
| CN110616156A (en) * | 2019-10-22 | 2019-12-27 | 慕恩（广州）生物科技有限公司 | Trichoderma harzianum solid state fermentation culture medium, trichoderma harzianum conidium, biological agent comprising conidium and application of biological agent |
-
2020
- 2020-10-12 CN CN202011082163.0A patent/CN114315778B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4915944A (en) * | 1983-03-15 | 1990-04-10 | Yissum Research Development Corp. Of The Hebrew University Of Jerusalem | Novel isolate of trichoderma, fungicidal compositions containing said isolate and use thereof |
| CN1176248A (en) * | 1994-12-19 | 1998-03-18 | 美国氰胺公司 | Restricted 9-CIS-retinoids |
| CN103922967A (en) * | 2014-04-15 | 2014-07-16 | 北京化工大学 | Hydroxamic acid compounds and application thereof in preparation of medicines for inhibiting cancer cell proliferation and/or treating cancers |
| CN104116728A (en) * | 2014-06-28 | 2014-10-29 | 北京化工大学 | Pharmaceutical composition composed of hydroxamic acid compounds and vitamin A compounds and application thereof |
| CN107011238A (en) * | 2017-03-14 | 2017-08-04 | 北京化工大学 | Class I histone deacetylase inhibitor and its production and use |
| CN110616156A (en) * | 2019-10-22 | 2019-12-27 | 慕恩（广州）生物科技有限公司 | Trichoderma harzianum solid state fermentation culture medium, trichoderma harzianum conidium, biological agent comprising conidium and application of biological agent |
Non-Patent Citations (2)
| Title |
|---|
| 1株白木香内生真菌中抗肿瘤活性成分的分离纯化和结构鉴定;李冬利等;《微生物学杂志》;第30卷(第06期);第1-5页 * |
| Trichoderma harzianum中转录调控因子HOS2和PacC代谢调控功能解析与机制探究;吴梦月;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》(第3期);第B016-2085页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114315778A (en) | 2022-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110343709B (en) | Nocardiopsis arctica lasso peptide gene cluster and cloning and expression method thereof | |
| CN114315778B (en) | Trichoderma harzianum acid A compound and trichoderma harzianum mutant strain for producing same | |
| CN110484481B (en) | Method for improving lincomycin yield by modifying streptomyces lincolnensis SLCG _3128 gene | |
| CN110684795A (en) | Construction method of monascus purpureus comp50904_ c4 gene overexpression strain | |
| CN110791466B (en) | Recombinant bacterium for synthesizing butanetriol oleate as well as construction method and application thereof | |
| CN113604470B (en) | Recombinant yarrowia lipolytica T30pED for high yield of campesterol, construction method and application thereof | |
| CN113106109B (en) | Mutant enzyme CYP153A M228L and application thereof in synthesis of 10-hydroxy-2-decenoic acid | |
| CN112961816B (en) | Arthrobacter simplex engineering bacteria with steroid C1,2 dehydrogenation reaction capability | |
| CN115247168A (en) | Oat oxidation squalene cyclase AsHS2 and coding gene and application thereof | |
| CN113881619A (en) | Recombinant escherichia coli capable of synthesizing Bordetella pertussis oligosaccharide antigen | |
| CN109370969B (en) | Application of recombinant Klebsiella in preparation of 1, 3-propylene glycol | |
| CN107903227B (en) | Succinic anhydride compound, gene and protein related to succinic anhydride compound and preparation method of succinic anhydride compound | |
| CN113493758A (en) | Tyrosol-producing recombinant escherichia coli capable of shortening fermentation period and application thereof | |
| CN106434785B (en) | The preparation method of 2-n- heptyl -4- oxyquinoline | |
| CN112322564B (en) | Escherichia coli engineering bacterium and construction method and application thereof | |
| CN115181715B (en) | Recombinant escherichia coli capable of efficiently producing monophosphoryl lipid A vaccine adjuvant | |
| CN113817692B (en) | Diosgenin synthesis related protein, coding gene and application thereof | |
| CN101781648A (en) | Method for effectively inhibiting taxus media cell biosynthesis C-14 oxygenated taxane | |
| CN113583993B (en) | Polyketide synthase PreuA and application thereof in preparation of lichen ruber acid | |
| CN114807211B (en) | Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and construction method | |
| CN114540403B (en) | Method for producing esterified astaxanthin and use of esterified gene | |
| CN113652444A (en) | Method for constructing phaffia rhodozyma by using gene editing | |
| CN116732077A (en) | Cyclodiguanylate content detection model and construction method and application thereof | |
| CN116144637A (en) | Rumex olivaceus histone deacetylase and encoding gene and application thereof | |
| CN116875617A (en) | Coding gene, expression vector containing coding gene, protein coded by coding gene and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |