Tom tat tiếng anh: Nghiên cứu đa dạng và sinh tổng hợp cyclooligomer depsipeptide của nấm ký sinh côn trùng tại Khu bảo tồn thiên nhiên Copia và Vườn quốc gia Xuân Sơn.

Nghiên cứu đa dạng và sinh tổng hợp cyclooligomer depsipeptide của nấm ký sinh côn trùng tại Khu bảo tồn thiên nhiên Copia và Vườn quốc gia Xuân Sơn.Nghiên cứu đa dạng và sinh tổng hợp cyclooligomer depsipeptide của nấm ký sinh côn trùng tại Khu bảo tồn thiên nhiên Copia và Vườn quốc gia Xuân Sơn.Nghiên cứu đa dạng và sinh tổng hợp cyclooligomer depsipeptide của nấm ký sinh côn trùng tại Khu bảo tồn thiên nhiên Copia và Vườn quốc gia Xuân Sơn.Nghiên cứu đa dạng và sinh tổng hợp cyclooligomer depsipeptide của nấm ký sinh côn trùng tại Khu bảo tồn thiên nhiên Copia và Vườn quốc gia Xuân Sơn.Nghiên cứu đa dạng và sinh tổng hợp cyclooligomer depsipeptide của nấm ký sinh côn trùng tại Khu bảo tồn thiên nhiên Copia và Vườn quốc gia Xuân Sơn.Nghiên cứu đa dạng và sinh tổng hợp cyclooligomer depsipeptide của nấm ký sinh côn trùng tại Khu bảo tồn thiên nhiên Copia và Vườn quốc gia Xuân Sơn.Nghiên cứu đa dạng và sinh tổng hợp cyclooligomer depsipeptide của nấm ký sinh côn trùng tại Khu bảo tồn thiên nhiên Copia và Vườn quốc gia Xuân Sơn.

MINISTRY OF EDUCATION AND TRAINING HANOI NATIONAL UNIVERSITY OF EDUCATION NGUYEN THI THUY VAN RESEARCH ON DIVERSITY AND BIOSYNTHESIS OF CYCLOOLIGOMER DEPSIPEPTIDE OF ENTOMOPATHOGENIC FUNGI IN COPIA NATURE RESERVE AND XUAN SON NATIONAL PARK Major: Microbiology Code: 9.42.01.07 SUMMARY OF PhD THESIS IN BIOLOGY HANOI - 2022 The thesis was completed at: HANOI NATIONAL UNIVERSITY OF EDUCATION Supervisors: Assoc Prof PhD Duong Minh Lam Prof PhD Ngo Sy Hien Reviewer 1: Assoc Prof PhD Le Thanh Binh Vietnam Academy of Science and Technology Reviewer 2: Assoc Prof PhD Vu Nguyen Thanh Food Industries Research Institute Reviewer 3: Assoc Prof PhD Tran Van Tuan Vietnam National University, Hanoi The thesis is defended at the doctoral thesis evaluation committee at At :00 on , 2022 The thesis could be found at: National Library of Vietnam, Hanoi Library of Hanoi National University of Education INTRODUCTION Entomopathogenic fungi (EPF) or insect fungi are a group of fungi that cause disease in insects This group of fungi plays a crucial role in the ecosystem Up to now, nearly 2000 species of insect parasitic fungi have been recorded Entomopathogenic fungi have the ability to biosynthesize a variety of secondary metabolites such as cyclic depsipeptide, peptides, amino acid derivatives, polyketides, hybrid peptides, terpenoids, and others Secondary metabolites from insect parasitic fungi with a strong spectrum of activity have been attracting the attention of researchers The most notable is the group of active ingredients called cyclooligomer depsipeptide Cyclooligomer depsipeptide (COD) are a special group of non-ribosomal peptides consisting of 2, 3, or monomers Each monomer is composed of at least one 2-hydroxycarboxylic acid and one 2-amino acid COD are biosynthesized by the non-ribosomal peptide synthetases (NRPSs) enzyme system COD are naturally occurring substances found in bacteria, fungi, plants, algae, sponges, and a number of other marine organisms Among the groups of COD, COD from fungi, especially parasitic fungi, are a very important group and accounting for the largest proportion Cyclooligomer depsipeptide from entomopathogenic fungi have a broad spectrum of biological activities, including phytotoxic, cytotoxic, antiviral, insecticidal, antimalarial, anti-tumor, and inhibitory activities of several enzymes as well as inhibiting amyloid formation in Alzheimer’s disease In particular, recent studies show that COD from entomopathogenic fungi have great potential in cancer prevention, inhibiting the growth of several different human cancer lines In other countries, the research on COD as well as the applications of COD started very early Typical examples are the study on extracting enniatin A from Fusarium orthocera var enniatinum by Gaumann E (1947), research on the biosynthesis of cyclodepsipeptide D- D-, L-L-, and D-L- Cyclodi- (β-seryloxypropionyl) by Hassal C.H et al (1967) or study on the structure of beauvericin from fungi by Hamill R.L et al (1969) After more than 70 years, COD in general and COD from entomopathogenic fungi are still attracting the attention of many scientists around the world In Vietnam, a number of studies on entomopathogenic fungi and their applications have been published The studies focus mainly on the diversity of entomopathogenic fungi or the use of fungal biomass to produce insecticides, such as the research of Nguyen Thi Loc (2006), Pham Thi Thuy (2010), Pham Van Nha (2013) Research on COD from entomopathogenic fungi is a novel issue; currently, there is no research on this topic Copia Nature Reserve (Son La) and Xuan Son National Park (Phu Tho) are two areas with outstanding biodiversity in Northern Vietnam Favored by nature in terms of natural conditions, the generations of flora and fauna in these two areas are very rich, valuable not only for scientific research or conservation of genetic resources but also for economic development and exploitation of natural resources (especially biological resources) With tropical and subtropical forests, there are many rare and precious species of flora and fauna that exist and are typical for the Northern mountainous region However, the fungal flora in Copia Nature Reserve and Xuan Son National Park has not been studied much The knowledge about species diversity, biological characteristics, and use-value is still very limited In particular, there are hardly any studies on entomopathogenic fungi in these two areas In order to supplement knowledge about entomopathogenic fungi and COD from entomopathogenic fungi as well as contribute to building a list of genetic resources and conserving the biodiversity of entomopathogenic fungi in the Nature Reserve Copia and Xuan Son National Park, we chose to study the topic of "Research on diversity and biosynthesis of cyclooligomer depsipeptide of entomopathogenic fungi in Copia Nature Reserve and Xuan Son National Park" Target Evaluation of species composition and biosynthetic ability of cyclooligomer depsipeptide of some entomopathogenic fungi isolated in Copia Nature Reserve (Son La) and Xuan Son National Park (Phu Tho) sObjectives, scopes, and delimitations - Research subject: Fungi were isolated from insect samples collected in Copia Nature Reserve (Son La Province) and Xuan Son National Park (Phu Tho Province) - Scope of research: Isolation, selection, and study of morphological and molecular characteristics of fungi strains capable of COD biosynthesis Research on environmental effects and culture conditions to collect COD, extract, purify and investigate biological activities of COD from selected fungal strains Research contents - Research on diversity of entomopathogenic fungi isolated from samples collected in Copia Nature Reserve and Xuan Son National Park - Selection of entomopathogenic fungal strains capable of biosynthesis of cyclooligomer depsipeptide - Study on the identification of selected fungal strains by morphological and molecular methods - Research on the selection of culture medium and conditions, biosynthesis of cyclooligomer depsipeptide for selected fungal strains at laboratory scale - Research on recovery, purification and determine the chemical structure of cyclooligomer depsipeptide - Research to investigate some biological activities of the fractions in the extraction process of cyclooligomer depsipeptide New contributions - This is the first thesis published on the biosynthesis of COD of fungal strains isolated from insect samples collected in Copia Nature Reserve (Son La Province) and Xuan Son National Park (Phu Tho Province) - For the first time, the presence of the fungus Cordyceps cateniannulata was recorded in Vietnam - It is the first publication in the world about Cordyceps cateniannulata having the ability to biosynthesize COD - This is the first thesis to comprehensively study an entomopathogenic fungus strain of the genus Cordyceps capable of COD biosynthesis and has potential applications (from isolation, selection, identification, and study characteristics of fermentation, collection, extraction, purification, and investigation of biological activity of COD) Structure of the thesis The thesis consists of 158 pages, including the following sections: Introduction: pages Chapter Overviews of research issues: 37 pages Chapter Materials and research methods: 15 pages Chapter Results and discussion: 71 pages Conclusions and Recommendations: pages References: The thesis cited 12 Vietnamese references, 209 foreign language references and 02 web pages CHAPTER 1: OVERVIEWS OF RESEARCH ISSUES In this chapter, six issues related to the research in the thesis have been reviewed, namely (1.1) Fungi and entomopathogenic fungi; (1.2) Entomopathogenic fungi that biosynthesize cyclooligomer depsipeptide; (1.3) Nutritional requirements and culture conditions of entomopathogenic fungi that biosynthesize cyclooligomer depsipeptide; (1.4) Extraction, purification, and structural study of cyclooligomer depsipeptide from entomopathogenic fungi; (1.5) Research situation in the world and Vietnam In section (1.1) Fungi and entomopathogenic fungi, an introduction to fungi (section 1.1.1), a brief overview of entomopathogenic fungi and secondary metabolites from parasitic fungi (section 1.1 2) have been specified In section (1.2) Entomopathogenic fungi that biosynthesize cyclooligomer depsipeptide, the contents of chemical structure, forms of COD from fungi and COD activity have been reviewed (section 1.2.1) Diversity of entomopathogenic fungi that biosynthesize cyclooligomer depsipeptide (Some common genera of entomopathogenic fungi biosynthesize COD, Cordyceps spp COD biosynthesis - section 1.2.2) and COD biosynthetic pathway in fungi (section 1.2.3) is presented in detail from page 18 to page 25 Section (1.3) Nutritional requirements and culture conditions of cyclooligomer depsipeptide biosynthetic entomopathogenic fungi, we reviewed the selection of culture medium, pH, carbon source, nitrogen source In section (1.4) Extraction, purification and structure study of cyclooligomer depsipeptide from entomopathogenic fungi, steps of extraction, purification, methods in studying COD structure were reviewed and described in detail In section (1.5) Research situation in the world and Vietnam, research directions on entomopathogenic fungi producing COD in the world and Vietnam have been reviewed CHAPTER II: MATERIALS AND METHODS In this chapter, the Materials and research subjects were presented in section (2.1) including (2.1.1) Research subjects; (2.1.2) Chemicals and equipment; (2.1.3) Medium Research Methods (Section 2.2) are divided into main groups as follows: 2.2.1 The microbiological method includes the following methods: Isolation method, Breeding method, Activation method of selected fungal strains, Method of selection of fungal strains producing cyclooligomer depsipeptide, Freeze-drying method, Method of selecting nutrient medium and culture conditions that are suitable for growth and accumulation of COD of selected strains, Evaluation of cytotoxic activity by MTT method, Method of assessment of tested antimicrobial activity, Assessment of antioxidant activity rate 2.2.2 Molecular biology methods include the following methods: Extraction of total DNA, DNA multiplication, and Sequencing methods 2.2.3 Methods for extraction and purification of cyclooligomer depsipeptide include the following methods: Method of extraction of compounds from fungal biomass, Method of determining the chemical structure of clean compounds, Experimental extraction and purification of COD from fungal biomass C cateniannulata CPA14V 2.2.4 Analytical methods include the following: Method of determining cell dry weight (CDW), Method of quantifying COD present in fungal biomass 2.2.5 Mathematical methods CHAPTER III: RESULTS AND DISCUSSION 3.1 Diversity of entomopathogenic fungi isolated from samples collected in Copia Nature Reserve and Xuan Son National Park 3.1.1 Results of isolation and characterization of entomopathogenic fungi in Copia Nature Reserve and Xuan Son National Park Results from 24 host samples belonging to the insect orders Blattodea, Hemiptera, Coleoptera, Hymenoptera, and Lepidoptera, 24 fungal strains were isolated, purified, and preserved for further studies The symbols of the strains are: CPA1, CPA3, CPA5, CPA13V, CPA14V, CPA15, CPA16, CPA31, CPA40, CPA44, XS01, XS07, XS12, XS36, XS37, XS38, XS57, XS65, XS66, XS67, XS69, XS71, XS77, XS83 Classify 24 isolates of entomopathogenic fungi based on morphological characteristics of asexual and sexual samples in the wild; Culture characteristics and ITS sequences of some fungal strains The results identified isolated strains belonging to the genera Aschersonia (01 samples), Purpureocillium (01 samples), Beauveria (08 samples), Cordyceps (06 samples), Isaria (05 samples), and Ophiocordyceps (03 samples) Specific results are shown in Table 3.1 and Figure 3.25 below Table 3.1 Isolated entomopathogenic fungi components Fungi Family Genus Number Strain Host of Sample Clavicipitaceae Aschersonia 01 XS01 Hemiptera Cordycipitaceae Beauveria 08 CPA5 Coleoptera CPA15 Coleoptera CPA16 Lepidoptera CPA44 Coleoptera XS36 Coleoptera XS37 Coleoptera XS38 Hymenoptera XS83 Lepidoptera Cordyceps 06 CPA3 Coleoptera CPA13V Lepidoptera CPA14V Blattodea CPA31 Lepidoptera XS57 Hymenoptera XS67 Hymenoptera Isaria 05 CPA40 Blattodea XS07 Lepidoptera XS66 Hymenoptera XS69 Lepidoptera XS71 Lepidoptera Ophiocodycipitaceae Ophiocordyceps 03 CPA1 Hymenoptera XS12 Hymenoptera XS65 Hymenoptera Purpureocillium 01 XS77 Coleoptera Figure 3.25 Phylogenetic relationships of entomopathogenic fungal strains isolated in the research area 3.1.2 Comments on the diversity of entomopathogenic fungi in Copia Nature Reserve and Xuan Son National Park The results suggested 33.3% of the isolates belonged to the genus Beauveria, followed by Cordyceps (25%) and Isaria (20.8%) The appearance of species of the genus Aschersonia parasitic on Hemiptera is first recorded in Vietnam Of the five host sets, Hymenoptera had 07/24 samples collected and was the host of the most fungal genera in the study, including Ophiocordyceps, Cordyceps, Beauveria, and Isaria Similarly, the host set Coleoptera has 07/24 samples collected and is the host of 3/6 fungal genera in the study (Beauveria, Cordyceps, and Purpureocillium) In this study, Cordyceps spp was found on host sets but not on Hemiptera The Isaria strain found in the order Blattodae in the study is a new record There are slight differences between the two study areas In Copia Nature Reserve, 10 samples of mycorrhizal fungi collected in sampling periods were classified into 4/6 studied genera Meanwhile, in Xuan Son National Park, 14 mushroom samples collected in one sampling period were classified into 6/6 studied genera 3.2 Biosynthetic ability of cyclooligomer depsipeptide of isolated entomopathogenic fungal strains The results showed that 19/24 (79%) of the isolates had the ability to synthesize COD on average from 0.11 to 4.98 mg/g All strains of the genus Beauveria were capable of producing COD, while 83.3% of Cordyceps strains and 60% of Isaria strains were capable of producing COD This finding demonstrates that Cordycipitaceae fungi have great potential for COD production and are worth investigating Four strains of Isaria sp XS69, Isaria sp CPA40, Cordyceps sp CPA14V and Beauveria sp CPA16 had the highest capacity to generate COD, reaching 3.48, 3.88, 4.81, and 4.98 mg/g, while the cumulative CDW biomass was 9.38, 10.42, respectively 84, and 5.81 g/l Of these strains, Cordyceps sp CPA14V was found in the host of the insect order Blattodea capable of producing 47.29 mg/l after days of culture Mushroom strain Cordyceps sp CPA14V has great potential, and further studies are needed on the culture technology and production of COD from this strain 11 Neighbor Joining method with bootstrap 1000 using Mega X software, as shown in figure 3.28, 3.29, and 3.30 Figure 3.28 The results of building a phylogenetic tree based on the ITS gene region 12 Figure 3.29 The results of building a phylogenetic tree based on the LSU gene region 13 Figure 3.30 Results of building phylogenetic tree based on gene regions of ITS, LSU and Rpb1 Through analyzing the phylogenetic tree when using each target gene region ITS, LSU, Rpb1 alone and combining all three gene regions above, it was confirmed that the studied fungus CPA14V belongs to the species Cordyceps cateniannulata, genus Cordyceps, family Cordycipitaceae Thus, through the analysis of phylogenetic tree and morphological characteristics, it can be concluded that strain CPA14V is Cordyceps cateniannulata CPA14V For the first time, the presence of this fungus was recorded in Vietnam 14 3.4 Selection of nutrient medium and culture conditions for cyclooligomer depsipeptide biosynthesis of C cateniannulata CPA14V We selected the nutrient medium and culture conditions for the biosynthesis of cyclooligomer depsipeptide of C cateniannulata CPA14V The selected nutrient media for research include 05 types, including PGB medium; Sabouraud medium (SBR); MM medium; Czapek-Dox medium (CzD); FDM medium The factors in the nutrient medium and culture conditions affecting the growth and biosynthesis of COD of the studied strains in the study include pH, carbon source, and nitrogen source The research results are presented on pages 99 to 108 of the full text of this thesis a) Growth ability of C cateniannulata CPA14V strain in 05 types of culture media 15 b) The ability to biosynthesize COD of C cateniannulata CPA14V strain in 05 types of culture media Figure 3.31 Growth ability of C cateniannulata CPA14V strain in 05 types of culture media Figure 3.32 Effect of pH on the growth and COD biosynthesis of C cateniannulata CPA14V 16 Figure 3.33 Effect of Carbon Source on Growth and COD Biosynthesis of C cateniannulata CPA14V Figure 3.34 Effect of Nitrogen Source on the Growth and COD Biosynthesis of C cateniannulata CPA14V The results showed that Czapek-Dox (CzD) medium with pH 8, carbon source is glucose, nitrogen source is yeast extract, very suitable for growth and COD biosynthesis of C cateniannulata CPA14V When using this medium in combination with shaking at 150 rpm for days at 25 oC, we obtained 65,789 ± 2,186g/l COD Specific results are shown in Figures 3.31, 3.32, 3.33, 3.34 17 3.5 Isolation, purification and determine the chemical structure cyclooligomer depsipeptide from C cateniannulata CPA14V 3.5.1 Isolation and purification cyclooligomer depsipeptide from C cateniannulata CPA14V The results of isolation and purification cyclooligomer depsipeptide from C cateniannulata CPA14V, including preparation of fungal biomass, extraction, isolation of substances from dichloromethane extract (CCD) by chromatography and some factors affecting COD collection were presented in pages 108 to 114 of the full text of this thesis The study established a procedure for extracting and isolating COD (purity 98.1%) from Cordyceps cateniannulata CPA14V by using combination of different chromatographic methods such as thin layer chromatography, normal-phase chromatogaraphy with silicagel stationary phase, reversed-phase chromatography with YMC RP 18 stationary phase and size exclusion chromatography with Sephadex LH-20 stationary phase at laboratory scale Extraction conditions were at 40-50oC, sonication for hours, using dichloromethane solvent (Figures 3.35, 3.36, 3.37, 3.38) 18 Figure 3.35 Isolation of COD from C cateniannulata CPA14V 19 Figure 3.36 Effect of extraction solvent on CC1 content Figure 3.37 Effect of temperature on CC1 content 20 Figure 3.38 Effect of ultrasound time on CC1 content 3.5.2 Determine the chemical structure cyclooligomer depsipeptide from C cateniannulata CPA14V The structure of cyclooligomer depsipeptide was elucidated based on 1D, 2D NMR spectroscopic data, HRESIMS, GC-MS, and comparison with reported data as well as some physical properties such as melting point (Mp), polarity ([α]D) Results were shown on pages 114 to 122 of the full text of this thesis The combination of spectral data (MS, 1H-NMR, 13C-NMR, DEPT, COSY, NOESY, HSQC and HMBC) together with the corresponding data of Beauver-icin in the references confirmed that CC1 was identified as a cyclic hexadepsipeptide, a mycotoxin named (3S,6R,9S,12R,15S,18R)-3,9,15tribenzyl-4,10,16-trimethyl -6,12,18-tri(propan-2-yl)-1,7,13-trioxa-4,10,16triazacyclooctadecane-2,5,8,11,14,17-hexone or beauvericin Table 3.3 Spectrum data 1H- and 13C-NMR of CC1 and reference compound Compound CC1 Beauvericin (Bogner et al., 2017 Positi on δH (mult., J Hz) #δH (mult., J Hz) δC #δC Phe (3 units): CO 170,9 - 169.5 5,83 (1H, dd, J= 12,5; 5.82 (1H, dd, J= 12.7, Α 57,8 56.5 4,5) 4.5) 3.42 (1H, dd, J= 14.8, 3,40 (1H, dd, J= 14,5, 4.5) Β 35,5 4,5) 34.7 3.06 (1H, dd, J= 14.8, 3,05 (1H, m) 12.7) 21 γ1 138,1 γ2 129,8 γ3 129,7 γ4 127,9 γ5 129,7 γ6 129,8 N32,2 CH3 Hiv (3 units): CO 173,0 Α 77,2 Β 31,2 7.25-7.28 (1H, m) 7.25-7.28 (1H, m) 7.19 (1H, m) 7.25-7.28 (1H, m) 7.25-7.28 (1H, m) 136.9 128.3 128.3 126.2 128.3 128.3 3,16 (1H, s) 31.2 7.26-7.30 (1H, m) 7.26-7.30 (1H, m) 7.20 (1H, tt, J= 6.0, 3.1) 7.26-7.30 (1H, m) 7.26-7.30 (1H, m) 3.17 (1H, s) - 172.3 4,88 (1H, d, J = 9,0) 75.5 1,84 (1H, m) 29.8 4.86 (1H, s) 1.83 (1H, dq, J= 8.8, 6.7) 0.87 (3H, d, J = γ1 17,2 0,88 (3H, d, J = 6,5 Hz) 17.8 6.6) 0.27 (3H, d, J = γ2 18,3 0,29 (3H, d, J = 7,0 Hz) 16.1 6.9) 13 #δH #δC of beauvericin ( H: 300 MHz, C: 75 MHz, methanol-d4) Figure 3.43 Chemical structure, important 1H-1H COSY and HMBC (HC) interactions of CC1 3.6 Studies on biological activities of extracts and cyclooligomer depsipeptide from C cateniannulata CPA14V In order to supplement the understanding of the biological activities of the active ingredients from C cateniannulata, we evaluated the cytotoxic activity, antioxidant activity, and antimicrobial activity of the extract of ethanol (CCM), n-hexane (CCH), dichloromethane (CCD), ethyl acetate (CCE), water (CCW) 22 and beauvericin (CC1) from C Cateniannulata CPA14V Specific results are presented in tables 3.4, 3.5, 3.6 Table 3.4 In vitro cytotoxic activity of the extracts from C cateniannulata CPA14V No Sample HepG2 cell PC3 cell Vero cell Cell Cell Cell IC50 IC50 IC50 inhibition inhibition inhibition (µg/mL) (µg/mL) (µg/mL) rate (%) rate (%) rate (%) CCM 80,55 ± 3,1 60,12 88,61 ± 0,6 80,97 22,86 ± 0,1 >100 CCH 53,17 ± 2,0 95,64 78,33 ± 1,8 69,37 31,31 ± 1,5 >100 CCD 96,56 ± 0,9 22,90 90,33 ± 1,5 45,29 26,25 ± 1,3 >100 35,43 ± 2,0 >100 CCE 56,49 ± 1,9 93,48 46,64 ± 2,1 >100 44,36 ± 2,3 >100 40,75 ± 2,2 >100 CCN 42,25 ± 2,4 >100 CC1 97,02 ± 1,8 19,17 95,16 ± 0,8 23,52 46,53 ± 1,8 >100 Paclitaxel 47,2 40,74 >100 54,2 ± 1,8 64,09 ± 2,4 39,16 ± 2,6 50nM nM nM Nm Table 3.4 In vitro antioxidant activity of the extracts from C cateniannulata CPA14V No Sample Ability to SC50 neutralize free (μg/mL) radicals (SC, %) Control (+) [axit ascorbic] 86,53 ± 0,3 12,6 μg/mL Control (-) [DPPH/EtOH+ 0,0 ± 0,0 DMSO] CCM 72,68 ± 2,8 102,95 CCH 1,54 ± 0,1 >200 CCD 79,07 ± 2,6 86,87 CCE 2,88 ± 1,5 >200 CCN 19,26 ± 1,8 >200 CC1 1,23 ± 0,6 >200 23 Table 3.5 In vitro antimicrobial activities of extracts from C Cateniannulata CPA14V Minimum inhibitor concentration (MIC: g/ml), Gram (-) Gram (+) Strains Yeast Sample bacteria bacteria s P B S A F S C E aeruginos subtilli Aureu Nige oxysporu cerevisia albican Coli a s s r m e s >200 >200 >200 100 >200 >200 200 CCM 200 >20 >200 >200 >200 200 >200 >200 >200 CCH 200 >200 200 100 100 >200 200 CCD 100 >20 >200 >200 >200 >200 >200 >200 >200 CCE >20 >200 >200 >200 >200 >200 >200 >200 CCN 100 200 100 100 100 200 200 CC1 100 The compound beauvericin (CC1) showed the highest inhibition activities on HepG2 and PC3 with IC50 values ranging from 19.17 to 45.29 µg/mL, which was a lower concentration of positive control Paclitaxel used in the study The results also showed that beauvericin (CC1) exhibited the best antimicrobial activity against all bacterial and fungal strains tested with MIC values of 100 g/ml (for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus niger, and Fusarium oxysporum) and MIC value of 200 g/ml (for Bacillus subtilis, Saccharomyces cerevisiae, and Candida albicans) Results were shown on pages 122 to 127 of the full text of this thesis CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS 1.1 Using 24 host samples belonging to the insect orders Blattodea, Hemiptera, Coleoptera, Hymenoptera, Lepidoptera collected in Copia Nature Reserve and Xuan Son National Park, we isolated 24 fungal strains The isolated fungal strains are diversely classified into genera, including Aschersonia, Purpureocillium, Beauveria, Cordyceps, Isaria, and Ophiocordyceps 1.2 The screening of entomopathogenic fungal strains in the study area identified 19/24 strains capable of COD biosynthesis Among them, the CPA14V strain isolated from the parasitic fungus on the insect order Blattodea has the best ability to biosynthesize COD for further research 24 1.3 Studying some morphological characteristics and identifying fungal strains selected by molecular biology method, the fungus strain CPA14V was identified as Cordyceps cateniannulata, genus Cordyceps, family Cordycipitaceae This is a new species recorded for Vietnam 1.4 Czapek-Dox (CzD) medium with pH 8, carbon source is glucose, nitrogen source is yeast, very suitable for growth and COD biosynthesis of C cateniannulata CPA14V When using this medium in combination with shaking at 150 rpm for days at 25oC, we obtained 65,789 ± 2,186g/l COD Equivalent to the studies of the fungal strains that have been published in the world 1.5 The conditions for extraction and recovery of COD from the biomass of C cateniannulata CPA14V have been determined Using dichloromethane extraction solvent, under the condition of temperature 40-50oC, ultrasonic for 2h and extraction process proposed to obtain 98.1% pure COD with recovery efficiency of 0.37% of dry biomass The chemical structure of COD obtained from C cateniannulata strain CPA14V has been determined COD have three groups of N-MePhe and three groups of Hiv, called beauvericin 1.6 The COD obtained from C cateniannulata strain CPA14V exhibited good biological activity Capable of causing hepatocellular toxicity Hep-G2 (IC50 = 19.17 µg/mL), prostate cancer (PC-3) (IC50 = 23.52 g/mL) cell lines, exhibited the best antimicrobial activity against all tested bacterial and fungal strains including E coli, P aeruginosa, B subtillis, S Cerevisiae, S aureus, A Niger, F Oxysporum, and C albicans with MIC= 100-200 µg/ml RECOMMENDATIONS The results obtained during the study show the abundance and potential of COD biosynthetic entomopathogenic fungi in Copia Nature Reserve and Xuan Son National Park In particular, the research results obtained on the biosynthesis, extraction, purification, and activity of COD compounds from C cateniannulata CPA14V showed potential in research and application of this compound in Vietnam However, to be able to develop this COD compound for application in medicine as well as other fields, there are still problems that need to be solved as follows: 2.1 Further testing and improvement of COD production, extraction, and purification processes is needed on an industrial scale 2.2 It is necessary to continue researching, testing, applying, and evaluating this COD compound in different fields, especially the medical field LIST OF PUBLICATION Nguyen Thi Thuy Van, Nguyen Dinh Viet, Duong Minh Lam (2021), “Structure, biosynthesis and bioactivity of fungal cyclooligomer depsipeptide compounds”, Scientific Journal of Hanoi National University of Education, 66(1), tr 124-134 Nguyen Dinh Viet, Nguyen Thi Thuy Van, Truong Xuan Lam, Duong Minh Lam (2021), “Morphological and molecular characteristics of Isaria fungal strains in Xuan Son National Park and Copia Nature Reserve”, Scientific Journal of Hanoi National University of Education, 66(1), tr 134-135 Nguyen Thi Thuy Van, Nguyen Dinh Viet, Duong Minh Lam (2021), “Morphological characteristics, growth and biosynthesis of cycooligomer depsipeptide of Cordyceps sp CPA14V”, Scientific Journal of Vinh University, 1A/2021, tr 80-87 Nguyen Thi Thuy Van, Duong Minh Lam, Dao Viet Hung, Vu Thi Thu Le, Pham Thi Hong Minh, Do Tien Lam (2021), Study on extraction and isolation of beauvericin from Cordyceps cateniannulata CPA14V, Journal of Science and Technology of Thai Nguyen University, 226 (14), tr 79-86 https://doi.org/10.34238/tnu-jst.4921 Van, N T T., Lam, D T, Minh, P T H., Le, V T T, Hoan, B V., Lam, D M (2021) Studies on biological activities of extracts and beauvericin from Cordyceps cateniannulata CPA14V International Journal of Agricultural Technology Vol 17(6): 2449-2460 Nguyen Thi Thuy Van, Nguyen Dinh Viet, Duong Minh Lam (2022), “Effects of Culture Conditions on Growth and Cyclooligomer Depsipeptide Biosynthesis of Cordyceps sp CPA14V”, VNU Journal of Science: Natural Sciences and Technology, Vol 38, No xx-xx https://doi.org/10.25073/25881140/vnunst.5273 ... of entomopathogenic fungi in the Nature Reserve Copia and Xuan Son National Park, we chose to study the topic of "Research on diversity and biosynthesis of cyclooligomer depsipeptide of entomopathogenic... in Copia Nature Reserve and Xuan Son National Park" Target Evaluation of species composition and biosynthetic ability of cyclooligomer depsipeptide of some entomopathogenic fungi isolated in Copia. .. (1.1) Fungi and entomopathogenic fungi; (1.2) Entomopathogenic fungi that biosynthesize cyclooligomer depsipeptide; (1.3) Nutritional requirements and culture conditions of entomopathogenic fungi