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Study on diversity and biosynthesis of Cyclooligomer depsipeptide of entomopathogenic fungi at Copia Nature Reserve and Xuan Son National Park - 2


LIST OF TABLES

Table 1.1. Some common cyclooligomers depsipeptide (COD) in fungi 11

Table 1.2. The ability to inhibit the growth of cancer cells of some CODs. 15 Table 1.3. The antibacterial ability of beauvericin 17

Table 1.4. Composition of some species of the genera Beauveria, Fusarium and Isaria

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COD 19 biosynthesis

Table 2.1. Fermentation medium for COD 43

Study on diversity and biosynthesis of Cyclooligomer depsipeptide of entomopathogenic fungi at Copia Nature Reserve and Xuan Son National Park - 2

Table 2.2. Sequences used to construct phylogenetic trees on three gene regions ITS, LSU, Rpb1 51

Table 2.3. Experimental methods of survey and post-survey experiments for extraction and purification of COD 53

Table 3.1. Composition of isolated entomopathogenic fungi 84

Table 3.2. COD biosynthesis ability of entomopathogenic fungi isolated in the study area 88

Table 3.3. 1H, 13C-NMR spectral data of CC1 and references 120

Table 3.4. In vitro cytotoxic activity of extracts and beauvericin from C. cateniannulata strain CPA14V 124

Table 3.5. In vitro antioxidant activity of extracts and beauvericin from C. cateniannulata strain CPA14V 125

Table 3.6. In vitro antimicrobial activity of extracts and beauvericin from C. cateniannulata CPA14V 126 strain


LIST OF IMAGES


Figure 1.1. Structure of some types of cyclooligomer depsipeptide 9

Figure 1.2. General structure of ester bonds between the carboxyl group of C-terminus α-hydroxy acid, ß-hydroxy acid and 10-chain hydroxy-acid

Figure 1.3. Structure of Pseudoxylallemmycin 12

Figure 1.4. Structures of some diketomorpholines 13

Figure 1.5. COD biosynthesis 25

Figure 1.6. Beauvericin 30 biosynthetic pathway

Figure 2.1. Standard curve graph for determining cumulative COD according to 56

Figure 3.1. Morphology of CPA1 sample 58

Figure 3.2. Morphology of CPA3 sample 59

Figure 3.3. Morphology of CPA5 60 sample

Figure 3.4. Morphology of CPA13V 61 sample

Figure 3.5. Morphology of CPA14V 62 sample

Figure 3.6. Morphology of CPA15 63 sample

Figure 3.7. Morphology of CPA16 64 sample

Figure 3.8. Morphology of CPA31 64 sample

Figure 3.9. Morphology of CPA40 65 sample

Figure 3.10. Morphology of CPA44 66 sample

Figure 3.11. Morphology of sample XS01 67

Figure 3.12. Morphology of sample XS07 68

Figure 3.13. Morphology of sample XS12 69

Figure 3.14. Morphology of sample XS36 70

Figure 3.15. Morphology of sample XS37 71

Figure 3.16. Morphology of sample XS38 72

Figure 3.17. Morphology of sample XS57 73

Figure 3.18. Morphology of sample XS65 74

Figure 3.19. Morphology of sample XS66 75

Figure 3.20. Morphology of sample XS67 76

Figure 3.21. Morphology of sample XS69 77


Figure 3.22. Morphology of sample XS71 78

Figure 3.23. Morphology of sample XS77 79

Figure 3.24. Morphology of sample XS83 80

Figure 3.25. Phylogenetic relationship of entomopathogenic fungi isolated in the study area 82

Figure 3.26. HPLC UV 203 nm chromatogram of standard substance (A) 90

Figure 3.27. Morphological characteristics of Cordyceps sp. CPA14V 92 strain

Figure 3.28. Results of constructing a phylogenetic tree based on gene region 94

Figure 3.29. Results of constructing a phylogenetic tree based on the LSU 97 gene region

Figure 3.30. Results of phylogenetic tree construction based on 3 gene regions ITS1-5.8S rDNA-ITS2, LSU and Rpb1 98

Figure 3.31. Growth and COD biosynthesis capacity of C. cateniannulata CPA14V strain in 05 types of research media 100

Figure 3.32. Effect of pH on growth and COD biosynthesis of the strain

C. cateniannulata CPA14V 103

Figure 3.33. Effect of carbon source on growth and COD biosynthesis of C. cateniannulata strain CPA14V 105

Figure 3.34. Effect of nitrogen source on growth and COD biosynthesis of C. cateniannulata strain CPA14V 107

Figure 3.35. Laboratory scale COD collection process (CC1) 111

Figure 3.36. Effect of extraction solvent on CC1 112 content

Figure 3.37. Effect of temperature on CC1 113 content

Figure 3.38. Effect of ultrasonic time on CC1 113 content

Figure 3.39. LC/MS Q-TOF spectrum of CC1 114

Figure 3.40. 1H-NMR spectrum of CC1 115

Figure 3.41. 13C-NMR spectrum, DEPT spectrum, HSQC spectrum of CC1 117

Figure 3.42. COSY spectrum, NOESY spectrum and HMBC spectrum of CC1 119

Figure 3.43. Chemical structure, 1H-1H COSY and main HMBC (HC) interactions of CC1 121

INTRODUCTION

1. Problem statement

Entomopathogenic fungi ( EPF ) or insect fungi ( Insect fungi ) are a group of fungi that cause diseases in insects [96]. This group of fungi plays a very important role in the ecosystem. Up to now, nearly 2000 species of insect parasitic fungi have been recorded [19, 222]. Entomopathogenic fungi have the ability to synthesize a variety of secondary metabolites such as: Cyclic depsipeptide, peptide, amino acid derivatives, polyketides, peptide hybrids, terpenoids... [48], [196]. Secondary metabolites from insect parasitic fungi with a strong spectrum of activity have attracted the attention of researchers. Notably, the group of active substances cyclooligomer depsipeptide.

Cyclooligomer depsipeptide (COD) is a special group of nonribosomal peptides consisting of 2, 3 or 4 monomers. Each monomer is composed of at least one 2-hydroxycarboxylic acid and one 2-amino acid. COD is biosynthesized by the enzyme system non-ribosomal peptide synthetases (NRPSs) [53]. COD is a natural active substance found in bacteria [55], fungi [168], plants [207], algae [46], sponges [17], and some other marine organisms [92, 141]. Among the COD groups, COD from entomopathogenic fungi is a very important group, accounting for the largest proportion [196].

Cyclooligomer depsipeptide from entomopathogenic fungi has a wide spectrum of biological activities, including phytotoxicity [116], cytotoxicity [176], antiviral [93], insecticidal [211], antimalarial [75], antitumor [106], inhibition of certain enzyme activities [128, 156] as well as inhibition of amyloid formation in Alzheimer's disease [73]. In particular, recent studies have shown that COD from entomopathogenic fungi has great potential in cancer prevention, inhibiting the growth of several different human cancer lines [72, 107, 197]. Currently, COD active ingredients from entomopathogenic fungi


Insect parasites are considered a new source of natural materials with potential applications in medicine.

In the world, research on entomopathogenic fungi as well as COD from entomopathogenic fungi started quite early. Typical examples include the study on the extraction of enniatin A from Fusarium orthocera var. enniatinum by Gaumann [57], the study on the biosynthesis of cyclodepsipeptide D- D-, LL-, and DL- Cyclodi- ( β -seryloxy- propionyl) by Hassal et al. [66] or the study on the structure of beauvericin from fungi by Hamill et al. [64],... After more than 70 years, entomopathogenic fungi and COD from entomopathogenic fungi still attract the attention of many scientists in the world. In Vietnam, there have been a number of research works on entomopathogenic fungi and their applications that have been published. Studies have focused mainly on the diversity of entomopathogenic fungi or the use of fungal biomass to produce preparations for use as pesticides, typically the studies of authors Nguyen Thi Loc [3], Pham Thi Thuy [10], Pham Van Nha [6], etc. Research on COD from entomopathogenic fungi is a very new issue, and there is currently no research work in Vietnam addressing this issue.

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 flora and fauna in these two areas are very rich, not only valuable for scientific research and gene conservation but also significant in economic development and exploitation of natural resources (especially biological resources). With tropical and subtropical forests, there are many rare and precious flora and fauna species present and typical of the northern mountainous region [2, 8, 223]. However, the fungal flora in Copia Nature Reserve and Xuan Son National Park has not been studied much. Knowledge about species diversity, biological characteristics and usage values ​​is still very limited. In particular, there is almost no research on entomopathogenic fungi in these two areas.


In order to supplement the knowledge about entomopathogenic fungi and COD from entomopathogenic fungi as well as contribute to building a list of genetic resources and conserving biodiversity of entomopathogenic fungi in Copia Nature Reserve and Xuan Son National Park, we chose to research the topic: " Research on diversity and biosynthesis of cyclooligomer depsipeptide of entomopathogenic fungi in Copia Nature Reserve and Xuan Son National Park " .

2. Topic objectives

Evaluation of species composition and cyclooligomer depsipeptide biosynthesis ability of some entomopathogenic fungi isolated from Copia Nature Reserve (Son La) and Xuan Son National Park (Phu Tho).

3. Research object and scope

- Research subjects: Fungi were isolated from insect samples collected in Copia Nature Reserve (Son La) and Xuan Son National Park (Phu Tho).

- Scope of research: Isolation, selection, study of morphological characteristics and molecular biology of fungal strains capable of synthesizing COD. Study of environmental influences and culture conditions for COD collection, extraction, purification and investigation of biological activity of COD from selected fungal strains.

4. Research content

- Isolation and study of diversity of entomopathogenic fungi from samples collected in Copia Nature Reserve and Xuan Son National Park.

- Selection of entomopathogenic fungi strains capable of biosynthesizing cyclooligomer depsipeptide.

- Study on identification of selected fungal strains by morphological and molecular biological methods.


- Research on selection of culture medium and conditions, biosynthesis of cyclooligomer depsipeptide for selected fungal strains at laboratory scale.

- Study on recovery, purification and determination of chemical structure of cyclooligomer depsipeptide.

- Study on some biological activities of fractions in the extraction process of cyclooligomer depsipeptide.

5. New contributions of the thesis

- This is the first thesis to comprehensively research (from isolation, selection, classification, research on fermentation characteristics, extraction, purification, and investigation of biological activity of COD) on 01 entomopathogenic fungus strain of the genus Cordyceps that has the ability to synthesize COD and has potential for practical application.

- This is the first published thesis on the diversity of insect parasitic fungi and the COD biosynthesis ability of fungal strains isolated from insect samples collected in Copia Nature Reserve (Son La) and Xuan Son National Park (Phu Tho).

- First time recording the presence of Cordyceps cateniannulata mushroom in Vietnam.

- The first publication in the world about Cordyceps cateniannulata 's ability to biosynthesize COD.


CHAPTER 1. OVERVIEW OF RESEARCH MATERIALS


1.1. Fungi and entomopathogenic fungi

1.1.1. Introduction to mushrooms

Fungi are a group of eukaryotic microorganisms, unicellular or multicellular, heterotrophic, absorbing nutrients, with a thallus-shaped body, cell walls containing chitin and ß-glucan. Fungi are usually aerobic microorganisms, reproducing by spores (sexually and asexually) [43]. Fungi are very diverse, with an estimated 1.5 to 15 million species, and currently nearly 150 thousand species have been discovered and described [23, 67]...

Although humans know about mushrooms and use them as food, wine, etc., it was not until 1969 that mushrooms were classified as a separate kingdom in the five-kingdom system [200]. Currently, mushrooms are divided into five phyla including Chytridiomycota, Zygomycota, Glomeromycota, Ascomycota, and Basidiomycota [182]. Of these, nearly 84 thousand species belong to the Chytridiomycota phylum and 50 thousand species to the Basidiomycota phylum [222].

As a diverse biological sector, fungi have had many impacts on human life from the ecological environment, agriculture, food, pharmaceuticals and medicine. Fungi play an important role in the nutritional cycle, in ecological relationships, have the ability to decompose lignin, have the ability to live symbiotically to help protect plants and animals, and a few have the ability to cause diseases in plants and animals. The first antibiotic discovered, purified, and produced into a pharmaceutical product was penicillin produced by Penicillium . Since then, thousands of new active ingredients have been discovered from fungi. Fungi are a biological source with the potential to provide a variety of active ingredients that can be applied in human life [182].

1.1.2. Entomopathogenic fungi

1.1.2.1. Overview of entomopathogenic fungi

Entomopathogenic fungi ( EPF ) or insect fungi ( Insect fungi ) are a group of fungi that cause diseases in insects [97]. With the characteristic of causing diseases specific to different insect species, insect parasitic fungi have been

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