Intermedeol synthetase and use thereof

Abstract

The present disclosure relates to the technical field of biology, and in particular to an intermedeol synthetase and a use thereof. A coding nucleic acid molecule is designed on the basis of an amino acid sequence of an intermedeol synthetase derived from a plant, and then a recombinant plasmid vector and an expression system which are used for expressing a novel enzyme are prepared. The intermedeol synthetase can be applied to synthesis of different citrus reticulata flavors, overcomes the time and labor waste of extracting essential oil from plants, and can be used for preparing various antibacterial, insecticidal or herbicidal products.

Description

Artemisinin synthase and its application Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to artemisinin synthase and applications thereof. Background Art

[0002] Artemisia is a traditional Chinese people's livelihood plant. Its extracts and combustion products are often used to repel mosquitoes. Existing studies have shown that Artemisia is rich in volatile terpenoid compounds, which have also attracted much attention due to their significant pharmacological activities. However, existing studies have focused less on the systematic characterization of Artemisia's terpenoid genes.

[0003] Artemisinin is a sesquiterpenoid compound, which is reported in various plant essential oils (e.g., grape seed essential oil, citrus essential oil, etc.), has a special smell, and can be used in food or daily chemicals. However, the whole process of plant essential oils from planting to refining is very complicated, and time-consuming and labor-intensive, requiring a large amount of plant peels, so its extraction cost is quite high, and its value is also quite expensive. Moreover, artemisinin content is very low in natural plant extracts, and the separation method using natural products is difficult to separate the plant essential oils containing high concentrations of artemisinin. Therefore, finding a more economical way to obtain the tool special smell essential oil substance of high artemisinin concentration is an inevitable trend to achieve economic efficiency and sustainable development.

[0004] Summary of the Invention

[0005] The purpose of the present disclosure is to provide an artemisinin synthase and the use of the enzyme in an expression system to prepare a composition with a special aroma and containing artemisinin as the main component, thereby achieving the purpose of efficient and continuous production.

[0006] In order to achieve the above objectives, the present disclosure provides the following technical solutions:

[0007] In a first aspect, the present disclosure provides an artemisinin synthase, comprising (a) a complete protein or functional protein fragment derived from a plant or fungus; or (b) a derived artemisinin synthase having at least 80% amino acid sequence homology with (a).

[0008] In an alternative embodiment, the plant is selected from the family Asteraceae.

[0009] In an alternative embodiment, the plant is selected from the genus Artemisia.

[0010] In an alternative embodiment, said plant comprises Artemisia argyi.

[0011] In an alternative embodiment, the artemisinin synthase catalyzes the synthesis of artemisinin in an expression system.

[0012] In an alternative embodiment, the expression system comprises a fungal expression system.

[0013] In an alternative embodiment, the fungal expression system comprises a yeast expression system.

[0014] In an alternative embodiment, the yeast expression system comprises a Saccharomyces cerevisiae expression system.

[0015] In an alternative embodiment, the Saccharomyces cerevisiae is a recombinant Saccharomyces cerevisiae containing a farnesene pyrophosphate synthase gene.

[0016] In an optional embodiment, the copy number of the farnesene pyrophosphate synthase gene in the recombinant Saccharomyces cerevisiae genome is 1 to 3, or further 2.

[0017] In an optional embodiment, the number of at least one mevalonate pathway-related gene in the recombinant Saccharomyces cerevisiae genome is greater than 1.

[0018] In an alternative embodiment, the copy number of at least one of the ERG10 gene, ERG13 gene, tHMG1 gene, ERG12 gene, ERG8 gene, MVD1 gene or IDI1 gene is 1 to 3, preferably 2 or 3.

[0019] In an optional embodiment, the repressor GAL80 gene and / or the activation cis-element or a fragment thereof within the range of -175 to -220 upstream of the ERG9 promoter is knocked out from the Saccharomyces cerevisiae genome.

[0020] In an optional embodiment, the amino acid sequence of the artemisinin synthase includes (a) or (b):

[0021] (a) SEQ ID NO. 1;

[0022] (b) a derivative artemisinin synthase having at least 80%, 81%, 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97% or 99% amino acid sequence identity to (a).

[0023] In a second aspect, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding the artemisinin synthase according to any one of the aforementioned embodiments or a nucleotide sequence complementary to the base thereof.

[0024] In an optional embodiment, the nucleic acid molecule comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO.3.

[0025] In a third aspect, the present disclosure provides a recombinant expression vector containing the nucleic acid molecule described in any one of the aforementioned embodiments, wherein the original vector of the recombinant expression vector includes pZY900, pYR017, pYR018 or pKlURA100.

[0026] In a fourth aspect, the present disclosure provides a transformant containing the nucleic acid molecule described in any one of the preceding embodiments, or a transformant containing the recombinant expression vector described in the preceding embodiments, wherein the host cell of the transformant is selected from a fungal expression system, preferably a yeast expression system, and more preferably a Saccharomyces cerevisiae expression system.

[0027] In a fifth aspect, the present disclosure provides a recombinant Saccharomyces cerevisiae, wherein the recombinant Saccharomyces cerevisiae contains the nucleic acid molecule described in any one of the aforementioned embodiments, or contains the recombinant expression vector described in the aforementioned embodiments, and the recombinant Saccharomyces cerevisiae further contains a farnesene pyrophosphate synthase gene.

[0028] In an optional embodiment, the copy number of the farnesene pyrophosphate synthase gene in the recombinant Saccharomyces cerevisiae genome is 1 to 3, or further 2.

[0029] In an optional embodiment, the number of at least one mevalonate pathway-related gene in the recombinant Saccharomyces cerevisiae genome is greater than 1.

[0030] In an alternative embodiment, the copy number of at least one of the ERG10 gene, ERG13 gene, tHMG1 gene, ERG12 gene, ERG8 gene, MVD1 gene or IDI1 gene is 1 to 3, preferably 2 or 3.

[0031] In an optional embodiment, the repressor GAL80 gene and / or the activation cis-element or a fragment thereof within the range of -175 to -220 upstream of the ERG9 promoter is knocked out from the Saccharomyces cerevisiae genome.

[0032] In a sixth aspect, the present disclosure provides a method for preparing the recombinant Saccharomyces cerevisiae according to any one of the aforementioned embodiments, the preparation method comprising the steps of:

[0033] (a) transforming the nucleic acid molecule described in any one of the preceding embodiments or the recombinant expression vector described in any one of the preceding embodiments into a Saccharomyces cerevisiae cell;

[0034] (b) increasing the copy number of farnesene pyrophosphate synthase gene and / or mevalonate pathway-related genes in the Saccharomyces cerevisiae genome by gene editing;

[0035] The order of implementing the steps (a) and (b) is not particular.

[0036] In an optional embodiment, the copy number of the farnesene pyrophosphate synthase gene is 1 to 3, further 2.

[0037] In an alternative embodiment, the copy number of at least one of the ERG10 gene, ERG13 gene, tHMG1 gene, ERG12 gene, ERG8 gene, MVD1 gene or IDI1 gene is 1 to 3, preferably 2 or 3.

[0038] In an optional embodiment, the preparation method comprises transforming a repressor GAL80 gene knockout expression cassette and / or an ERG9 promoter upstream activation cis-element knockout or partial knockout expression cassette into Saccharomyces cerevisiae cells.

[0039] In a seventh aspect, the present disclosure provides a method for preparing artemisinin, which comprises fermenting and culturing the transformant described in the aforementioned embodiment, or fermenting and culturing the recombinant brewer's yeast described in any of the aforementioned embodiments, or fermenting and culturing the recombinant brewer's yeast prepared by the preparation method described in any of the aforementioned embodiments, and recovering artemisinin from the fermentation product.

[0040] In an alternative embodiment, the method of recovering comprises extracting the fermentation product using an oil phase.

[0041] In an alternative embodiment, the oil phase comprises n-hexane, n-dodecane and / or isopropyl myristate.

[0042] In an optional embodiment, the recovery method is to add an oil phase during the fermentation process, collect the oil phase after the fermentation is completed, and separate the artemisinin enriched in the oil phase.

[0043] In an alternative embodiment, the method of collecting the oil phase comprises centrifugation.

[0044] In an alternative embodiment, the separation method comprises distillation.

[0045] In an eighth aspect, the present disclosure provides the use of artemisinin obtained by the preparation method described in any one of the aforementioned embodiments in the preparation of antibacterial products, insecticide products or herbicidal products.

[0046] In an alternative embodiment, the types of microorganisms that the antimicrobial product can inhibit or kill include fungi.

[0047] In an optional embodiment, the fungi include one or more of the genera Rhizoctonia, Plasmodium, Pythium, Phytophthora, Rhizopus, Black Coriolus, Venturia, Sclerotinia, Powdery Mildew, Pear-Shaped Spores, Powdery Molds, Verticillium, Botrytis, Alternaria, Fusarium, Cyclosporium, Gibberellum, Colletotrichum, and Puccinia.

[0048] In an alternative embodiment, the Rhizoctonia fungus includes Rhizoctonia solani.

[0049] In an alternative embodiment, the insect species that can be repelled or killed by the insecticidal product include insects of the Culicidae, Miridae or Delphacidae families.

[0050] In an optional embodiment, the Culicidae insects include one or more of the genera Aedes, Anopheles, Armigeres, Coquillettidia, Culex, Culiseta, Ficalbia, Heizmznnia, Hemagogus, Hodgesia, Malaya, Mansonia, Minmomyia, Orthopodomyia, Sabethes, Topomyia, Toxorhynchites, Tripteroides, Udaya, and Uranotaenia.

[0051] In an alternative embodiment, the insect of the genus Aedes includes Aedes aegypti.

[0052] In an optional embodiment, the Miridae insects include Genus Acomocera, Genus Adelphococris, Genus Agnocoris, Genus Alcecoris, Genus Alloeotomus, Genus Allorhinocoris, Genus Angerianus, Genus Apilophorus, Genus Apolygopsis, Genus Apolygus, Genus Arbolygus, Genus Argenis, Genus Astroscopometopus, Genus Bertsa), Blepharidopterus, Bothriomiris, Bothynotus, Brachycoleus, Bryocoris, Campylotropis, Capsodes, Capsus, Castanopsides, Charagochilus, Cheilocapsidea, Cheilocapsus, Chilocrates, Chrysorrhanis, and Cimicicapsus), Genus Cimidaeorus, Genus Closterotomus, Genus Coridromius, Genus Creontiades, Genus Cylapofulvuitius, Genus Cylapomorpha, Genus Cyllecoris, Genus Cyphodemidea, Genus Cyrtopeltis, Genus Cyrtorhinus, GenusDeraeocoris), Genus Dichrooscytus, Genus Dicyphus, Genus Dimia, Genus Dioclerus, Genus Dolichomiris, Genus Dortus, Genus Dryophilocoris, Genus Ectmetopterus, Genus Elthemidea, Genus Eolygus, Genus Erimiris, Genus Ernestinus, Genus Eupachypeltis, Genus Eurvopicoris, Genus Eurystylomorpha), Genus Eurystylopsis, Genus Eurystylus, Genus Excentricus, Genus Felisacus, Genus Finqulus, Genus Fulvius, Genus Globiceps, Genus Guisardinus, Genus Guisardus, Genus Halticus, Genus Harpedona, Genus Hekista, Genus Helopeltis, Genus Heterolyqus, Genus Heteropantilius), Genus Hyalopeplinus, Genus Hyalopeplus, Genus Hyoidea, Genus Isabel, Genus Isometopidea, Genus Isometopus, Genus Jessopocoris, Genus Labopidea, Genus Labops, Genus Lasiomiris, Genus Latizanchius, GenusLeptopterna), Genus Liistonotus, Genus Loristes, Genus Lygidea, Genus Lygocorides, Genus Lygocoris, Genus Lygus, Genus Macrolonius, Genus Macrolophus, Genus Macrolygus, Genus Malacocorisella, Genus Mansoniella, Genus Mecistoscelis, Genus Mecomma, Genus Megacoelum, Genus Mermitelocerus), Metasequoiamiris, Michailocoris, Monalocoris, Myiomma, Myrmecophyes, Myrmecoris, Mystilus, Neolygus, Nesidiocoris, Nicostratus, Notostir, Onomaus, Orientomiris, Orhocephalus, and Orthops), Genus Orthotylus, Genus Pachypeltis, Genus Paloniella, Genus Pantilius, Genus Paracyphodema, Genus Paramiridius, Genus Paranx, Genus Parapachypeltis, Genus Peltidolygus, Genus Peritropis, GenusPhytocoridea), Genus Phytocoris, Genus Pinalitus, Genus Polymerus, Genus Poppiocapsidea, Genus Proboscidocoris, Genus Prodromus, Genus Prolygus, Genus Psallops, Genus Pseudodoniella, Genus Pseudoloxops, Genus Raawelellus, Genus Reuterista, Genus Rhabdomiris, Genus Rhinocylapidius, Genus Rhinomiris), Genus Rhopaliceschatus, Genus Sabactiopus, Genus Salignus, Genus Scirtetellus, Genus Sinevi, Genus Singhalesia, Genus Sophianus, Genus Stenodema, Genus Stenotus, Genus Stethoconus, Genus Strongylocoris, Genus Sulawesifaulvius, Genus Tailorilygus, Genus Teratocoris, Genus The present invention relates to one or more of the genera Termatophylum, Genus Tinginotopsis, Genus Tinginotum, Genus Tolongia, Genus Totta, Genus Trigonotylus, Genus Tupiocoris, Genus Ulmica, Genus Ulmocyllus, Genus Zanchius and Genus Zonodoropsis.

[0053] In an alternative embodiment, the insect of the genus Adelphococris includes the black stink bug.

[0054] In an optional embodiment, the planthopper family includes one or more of the genera Nilaparvata, Unkanodes, Neunkanodes, Laodelphax, Ribautodelphax, Megadelphax, Muirodelphax, Fangdelphax, Neoterthrona, Ramidelphax, Neuterthron, Miranus, Sogata, Parasogata, Neometopina, Changeondelphax, Chloriona, Paradelphacodes, and Muellerianella.

[0055] In an alternative embodiment, the Delphacidae insects include brown planthopper (Delphacidae).

[0056] In an optional embodiment, the plant species that the herbicidal product can inhibit or kill include one or more of the following: Cruciferae, Asteraceae, Solanaceae, Convolvulaceae, Rubiaceae, Crassulaceae, Cannabaceae, Polygonaceae, Caryophyllaceae, Amaranthaceae, Portulacaceae, Ranunculaceae, Rosaceae, Plantaginaceae, Vitaceae, Euphorbiaceae, Apiaceae, Lamiaceae, Malvaceae, Poaceae, Cyperaceae, Juncaceae, Liliaceae, Commelinaceae, Amaryllidaceae, and Alismataceae.

[0057] In an optional embodiment, the cruciferous plants include Arabidopsis, Alliaria, Alyssum, Aphragmus, Arabidopsis, Arabis, Armoracia, Atelanthera, Barbarea, Berteroa, Berteroella, Brassica, Braya, Bunias, Camelina, Capsicum, sella), Cardamine, Cardaria, Cheiranthus, Chorispora, Christolea, Clausia, Cochlearia, Coelonema, Conringia, Coronopus, Crambe, Cryptospora, Descurainia, Dilophia, Dimorphostemon ), Diplotaxis, Dipoma, Diptychocarpus, Dontostemon, Draba, Eruca, Erysimum, Euclidium, Eutrema, Goldbachia, Hedinia, Hemilophia, Hesperis, Hymenolobus, Iberis, Isatis, L achnoloma), Lepidium, Leptaleum, Lignariella, Litwinowia, Lobularia, Loxostemon, Macropodium, Malcolmia, Matthiola, Megacarpaea, Megadenia, Microsisymbrium, Microstigma, Nasturtium,Neomartinella, Neslia, Orychophragmus, Pachypterygium, Parrya, Parryodes, Pegaeophyton, Phaeonychium, Platycraspedum, Ptilotricum, Pugionium, Pycnoplinthus, Raphanus, Rorippa, Sinapis, Sisymbrium, Smelowskia, Sophora flavescens One or more of the genera lms-Laubachia, Sophiopsis, Spirorhynchus, Staintoniella, Sterigmostemum, Stevenia, Stroganowia, Synstemon, Syrenia, Taphrospermum, Tauscheria, Tetracme, Thellungiella, Thlaspi, Torularia, Turritis, and Yinshania.

[0058] In an optional embodiment, the cruciferous plant includes one or more of the genera Arabidopsis, Capsella, Cardamine, Lepidium, Aralia, Lepidium, Halorhizoma, Saccharina, Descurainia, and Herba Epipremnum.

[0059] In an alternative embodiment, the Arabidopsis plant includes Arabidopsis thaliana.

[0060] In a ninth aspect, the present disclosure provides an antibacterial agent comprising artemisinin obtained by the preparation method described in any one of the aforementioned embodiments.

[0061] In an optional embodiment, the effective dose of artemisinin is 10 to 100 ppm.

[0062] In an optional embodiment, the dosage form of the antibacterial agent includes tablets, injections, capsules, powders, premixes, granules, soluble powders, oral solutions, gels or ointments.

[0063] In an alternative embodiment, the types of microorganisms that the antimicrobial agent can inhibit or kill include fungi.

[0064] In an optional embodiment, the fungi include one or more of the genera Rhizoctonia, Plasmodium, Pythium, Phytophthora, Rhizopus, Black Coriolus, Venturia, Sclerotinia, Powdery Mildew, Pear-Shaped Spores, Powdery Molds, Verticillium, Botrytis, Alternaria, Fusarium, Cyclosporium, Gibberellum, Colletotrichum, and Puccinia.

[0065] In an alternative embodiment, the Rhizoctonia fungus includes Rhizoctonia solani.

[0066] In a tenth aspect, the present disclosure provides an insecticide comprising artemisinin obtained by the preparation method described in any one of the aforementioned embodiments.

[0067] In an optional embodiment, the effective dose of artemisinin is 500 to 5000 ppm.

[0068] In an optional embodiment, the formulation of the insecticide includes an aqueous emulsion, a suspension, a dry powder, a wettable powder, an emulsifiable concentrate, a thermal fog, an aerosol, a soluble granule, a fumigant, a bait, a granule or a slow-release agent.

[0069] In an alternative embodiment, the insects that the insecticide can repel or kill include insects from the Culicidae, Miridae, or Delphacidae families.

[0070] In an optional embodiment, the Culicidae insects include one or more of the genera Aedes, Anopheles, Armigeres, Coquillettidia, Culex, Culiseta, Ficalbia, Heizmznnia, Hemagogus, Hodgesia, Malaya, Mansonia, Minmomyia, Orthopodomyia, Sabethes, Topomyia, Toxorhynchites, Tripteroides, Udaya, and Uranotaenia.

[0071] In an alternative embodiment, the insect of the genus Aedes includes Aedes aegypti.

[0072] In an optional embodiment, the Miridae insects include Genus Acomocera, Genus Adelphococris, Genus Agnocoris, Genus Alcecoris, Genus Alloeotomus, Genus Allorhinocoris, Genus Angerianus, Genus Apilophorus, Genus Apolygopsis, Genus Apolygus, Genus Arbolygus, Genus Argenis, Genus Astroscopometopus, Genus Bertsa), Blepharidopterus, Bothriomiris, Bothynotus, Brachycoleus, Bryocoris, Campylotropis, Capsodes, Capsus, Castanopsides, Charagochilus, Cheilocapsidea, Cheilocapsus, Chilocrates, Chrysorrhanis, and Cimicicapsus), Genus Cimidaeorus, Genus Closterotomus, Genus Coridromius, Genus Creontiades, Genus Cylapofulvuitius, Genus Cylapomorpha, Genus Cyllecoris, Genus Cyphodemidea, Genus Cyrtopeltis, Genus Cyrtorhinus, GenusDeraeocoris), Genus Dichrooscytus, Genus Dicyphus, Genus Dimia, Genus Dioclerus, Genus Dolichomiris, Genus Dortus, Genus Dryophilocoris, Genus Ectmetopterus, Genus Elthemidea, Genus Eolygus, Genus Erimiris, Genus Ernestinus, Genus Eupachypeltis, Genus Eurvopicoris, Genus Eurystylomorpha), Genus Eurystylopsis, Genus Eurystylus, Genus Excentricus, Genus Felisacus, Genus Finqulus, Genus Fulvius, Genus Globiceps, Genus Guisardinus, Genus Guisardus, Genus Halticus, Genus Harpedona, Genus Hekista, Genus Helopeltis, Genus Heterolyqus, Genus Heteropantilius), Genus Hyalopeplinus, Genus Hyalopeplus, Genus Hyoidea, Genus Isabel, Genus Isometopidea, Genus Isometopus, Genus Jessopocoris, Genus Labopidea, Genus Labops, Genus Lasiomiris, Genus Latizanchius, GenusLeptopterna), Genus Liistonotus, Genus Loristes, Genus Lygidea, Genus Lygocorides, Genus Lygocoris, Genus Lygus, Genus Macrolonius, Genus Macrolophus, Genus Macrolygus, Genus Malacocorisella, Genus Mansoniella, Genus Mecistoscelis, Genus Mecomma, Genus Megacoelum, Genus Mermitelocerus), Metasequoiamiris, Michailocoris, Monalocoris, Myiomma, Myrmecophyes, Myrmecoris, Mystilus, Neolygus, Nesidiocoris, Nicostratus, Notostir, Onomaus, Orientomiris, Orhocephalus, and Orthops), Genus Orthotylus, Genus Pachypeltis, Genus Paloniella, Genus Pantilius, Genus Paracyphodema, Genus Paramiridius, Genus Paranx, Genus Parapachypeltis, Genus Peltidolygus, Genus Peritropis, GenusPhytocoridea), Genus Phytocoris, Genus Pinalitus, Genus Polymerus, Genus Poppiocapsidea, Genus Proboscidocoris, Genus Prodromus, Genus Prolygus, Genus Psallops, Genus Pseudodoniella, Genus Pseudoloxops, Genus Raawelellus, Genus Reuterista, Genus Rhabdomiris, Genus Rhinocylapidius, Genus Rhinomiris), Genus Rhopaliceschatus, Genus Sabactiopus, Genus Salignus, Genus Scirtetellus, Genus Sinevi, Genus Singhalesia, Genus Sophianus, Genus Stenodema, Genus Stenotus, Genus Stethoconus, Genus Strongylocoris, Genus Sulawesifaulvius, Genus Tailorilygus, Genus Teratocoris, Genus The present invention relates to one or more of the genera Termatophylum, Genus Tinginotopsis, Genus Tinginotum, Genus Tolongia, Genus Totta, Genus Trigonotylus, Genus Tupiocoris, Genus Ulmica, Genus Ulmocyllus, Genus Zanchius and Genus Zonodoropsis.

[0073] In an alternative embodiment, the insect of the genus Adelphococris includes the black stink bug.

[0074] In an optional embodiment, the planthopper family includes one or more of the genera Nilaparvata, Unkanodes, Neunkanodes, Laodelphax, Ribautodelphax, Megadelphax, Muirodelphax, Fangdelphax, Neoterthrona, Ramidelphax, Neuterthron, Miranus, Sogata, Parasogata, Neometopina, Changeondelphax, Chloriona, Paradelphacodes, and Muellerianella.

[0075] In alternative embodiments, the Delphacidae insect includes the brown planthopper (Delphacidae).

[0076] In an eleventh aspect, the present disclosure provides a herbicide comprising artemisinin obtained by the preparation method described in any one of the aforementioned embodiments.

[0077] In an optional embodiment, the effective dose of artemisinin is 25 to 200 ppm.

[0078] In an optional embodiment, the formulation of the herbicide includes an aqueous solution, an emulsifiable concentrate, a suspension, an aqueous emulsion, a microemulsion, an oil suspension, a microcapsule suspension, a soluble liquid, a film-spreading oil, a wettable powder, a soluble powder, a soluble granule, a dry suspension or a water-dispersible granule.

[0079] In an optional embodiment, the herbicide can inhibit or kill plant species including one or more of the following: Cruciferae, Asteraceae, Solanaceae, Convolvulaceae, Rubiaceae, Crassulaceae, Cannabaceae, Polygonaceae, Caryophyllaceae, Amaranthaceae, Portulacaceae, Ranunculaceae, Rosaceae, Plantaginaceae, Vitaceae, Euphorbiaceae, Apiaceae, Lamiaceae, Malvaceae, Poaceae, Cyperaceae, Juncaceae, Liliaceae, Commelinaceae, Amaryllidaceae, Alismataceae, etc.

[0080] In an optional embodiment, the cruciferous plants include Arabidopsis, Alliaria, Alyssum, Aphragmus, Arabidopsis, Arabis, Armoracia, Atelanthera, Barbarea, Berteroa, Berteroella, Brassica, Braya, Bunias, Camelina, Capsella, Cardamine, Cardaria, Cheiranthus, Chorispora, Chris tolea, Clausia, Cochlearia, Coelonema, Conringia, Coronopus, Crambe, Cryptospora, Descurainia, Dilophia, Dimorphostemon, Diplotaxis, Dipoma, Diptychocarpus, Dontostemon, Draba, Eruca, Erysimum, Euclidium, Eutrema, Goldbachia, Hedinia,Hemilophia, Hesperis, Hymenolobus, Iberis, Isatis, Lachnoloma, Lepidium, Leptaleum, Lignariella, Litwinowia, Lobularia, Loxostemon, Macropodium, Malcolmia, Ma tthiola, Megacarpaea, Megadenia, Microsisymbrium, Microstigma, Nasturtium, Neomartinella, Neslia, Orychophragmus, Pachypterygium, Parrya, Parryodes, Pegaeophyton, Ph aeonychium, Platycraspedum, Ptilotricum, Pugionium, Pycnoplinthus, Raphanus, Rorippa, Sinapis, Sisymbrium, Smelowskia, Solms-Laubachia, Sophiopsis, Spirohynchus, Staint oniella, Sterigmostemum, Stevenia, Stroganowia, Synstemon, Syrenia, Taphrospermum, Tauscheria, Tetracme, Thellungiella, Thlaspi, Torularia, Turritis, Yinshania, etc. or one or more thereof.

[0081] In an optional embodiment, the cruciferous plant includes one or more of the genera Arabidopsis, Capsella, Cardamine, Lepidium, Aralia, Lepidium, Halorhizoma, Saccharina, Descurainia, and Herba Lysimachiae.

[0082] In an alternative embodiment, the Arabidopsis plant includes Arabidopsis thaliana.

[0083] The present invention discloses a synthase with artemisinin synthesis activity screened from Artemisia argyi and fungi, respectively. A coding nucleic acid molecule is designed based on the amino acid sequence of the screened artemisinin synthase, and then a recombinant plasmid vector and expression system for expressing the artemisinin synthase are prepared. The synthase has the potential to be applied to the high-yield synthesis of artemisinin, making up for the time-consuming and labor-intensive plant extraction and promoting the industrial production of antibacterial, insect repellent and herbicide products. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0085] FIG1 is a schematic diagram of the construction of plasmids p900A19, p905A19, and p908A19;

[0086] FIG2 is a schematic diagram of the construction of plasmids p900-V5-15 and p900-VvPNSelnt;

[0087] FIG3 is the GC-MS detection results of the fermentation product of strain J900A19;

[0088] Figure 4 shows the A19 product 1 H NMR spectrum (600 MHz, CDCl3);

[0089] Figure 5 shows the A19 product 13 C NMR spectrum (150 MHz, CDCl3);

[0090] FIG6 is a topological diagram of the COSY and ROESY of the A19 product;

[0091] FIG7 is a COSY spectrum of the A19 product;

[0092] FIG8 is a ROESY spectrum of the A19 product;

[0093] FIG9 is an X-RAY crystal structure diagram of the A19 product;

[0094] Figure 10 shows the repellency rate of (+)-intermedeol against Aedes aegypti. The horizontal axis represents the (+)-intermedeol concentration, and the vertical axis represents the repellency rate. Different letters indicate significant differences (P<0.05).

[0095] Figure 11 shows the repellent effect of (+)-intermedeol on brown planthoppers. The horizontal axis represents the concentration of (+)-intermedeol, and the vertical axis represents the proportion of brown planthoppers choosing different odor sources. Different letters indicate significant differences (P<0.05).

[0096] Figure 12 shows the concentration of (+)-intermedeol on the horizontal axis and the proportion of black stink bugs choosing different odor sources on the vertical axis. Different letters indicate significant differences (P<0.05).

[0097] Figure 13 shows the antibacterial effect of (+)-intermedeol on Rhizoctonia solani, with the abscissa representing the (+)-intermedeol concentration and the ordinate representing the diameter of the inhibition zone and the inhibition rate;

[0098] FIG14 shows an experiment on the growth inhibition of Arabidopsis seedlings by (+)-intermedeol, wherein the abscissa represents the concentration of (+)-intermedeol and the ordinate represents the length of the seedling roots. DETAILED DESCRIPTION

[0099] In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in combination with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments.

[0100] (I) Definitions or terms

[0101] The following abbreviations shall apply to the relevant definitions or terms involved in this disclosure. Unless otherwise defined, all scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. The following terms are provided below.

[0102] 1. Artemisinin synthase

[0103] The artemisinin synthase described in the present disclosure refers to an artemisinin synthase that had not been successfully isolated and purified before the present disclosure. This is because artemisinin is a terpenoid compound, and traditional terpenoid synthases are generally natural enzymes. Enzymes capable of terpenoid synthesis expressed in plant cells under natural conditions are generally adapted to plant cell expression systems and terpenoid synthesis systems. The artemisinin synthase provided in the present disclosure is a novel enzyme that can be successfully expressed in an artificially constructed expression system and is capable of synthesizing artemisinin.

[0104] 2. Homology

[0105] The "homology" mentioned in the present disclosure has a meaning generally recognized in the art, and the percentage of sequence identity between two nucleic acids or polypeptides or regions can be calculated using disclosed techniques. For example, sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. Currently, there are many methods for measuring the identity between two polynucleotides or polypeptides that can be routinely selected by those skilled in the art according to actual needs.

[0106] 3. Expression

[0107] Expression as used herein refers to the process of producing a polypeptide through transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for measuring the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0108] 4. Host cells

[0109] The host cells described herein are cells that are used to receive, maintain, replicate, and amplify vectors. Host cells can also be used to express polypeptides encoded by the vectors. When the host cells divide, the nucleic acids contained in the vectors replicate, thereby amplifying the nucleic acids. The host cells can be eukaryotic or prokaryotic.

[0110] 5. Carrier

[0111] The vectors disclosed herein are replicable nucleic acids that, when transformed into appropriate host cells, can express one or more heterologous proteins from the vector. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction enzyme digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of polypeptides encoded by nucleic acids. Vectors typically remain episomal, but can be designed to integrate genes or portions thereof into chromosomes of the genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vectors are well known to those skilled in the art.

[0112] (II) Detailed technical plan

[0113] In a specific embodiment, in the first aspect, the present disclosure provides an artemisinin synthase, which includes (a) a complete protein or functional protein fragment derived from a plant or fungus; or (b) a derivative artemisinin synthase having at least 80% amino acid sequence homology with (a).

[0114] Because plants have long growth cycles and difficult to control yields, fungi are more difficult to cultivate and grow more slowly than bacteria. Therefore, developing alternative microbial production methods for plant or fungal proteins has greater economic value and social significance than methods for extracting corresponding proteins through cultivation of plants and fungi.

[0115] In some preferred embodiments, the above-mentioned derivatization process can be to replace the amino acid site with another amino acid of the same type (having similar chemical properties or functions). As an example, amino acids can be divided into the following categories according to their side chain properties: (1) non-polar amino acids: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar amino acids: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic amino acids: Asp (D), Glu (E); (4) basic amino acids: Lys (K), Arg (R), His (H). Alternatively, amino acids can be classified based on common side chain properties: (1) hydrophobic amino acids: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic amino acids: Cys, Ser, Thr, Asn, Gln; (3) acidic amino acids: Asp, Glu; (4) basic amino acids: His, Lys, Arg; (5) amino acids that affect chain orientation: Gly, Pro; (6) aromatic amino acids: Trp, Tyr, Phe. Conservative substitutions between amino acids in the same property category have little or no effect on the function, activity, or other biological properties of the polypeptide.

[0116] In an alternative embodiment, the plant is selected from the family Asteraceae.

[0117] In an alternative embodiment, the plant is selected from the genus Artemisia.

[0118] In an alternative embodiment, said plant comprises Artemisia argyi.

[0119] In an alternative embodiment, the artemisinin synthase catalyzes the synthesis of artemisinin in an expression system.

[0120] In an alternative embodiment, the expression system comprises a fungal expression system.

[0121] In an alternative embodiment, the fungal expression system comprises a yeast expression system.

[0122] In an alternative embodiment, the yeast expression system comprises a Saccharomyces cerevisiae expression system.

[0123] In an alternative embodiment, the Saccharomyces cerevisiae is a recombinant Saccharomyces cerevisiae containing a farnesene pyrophosphate synthase gene.

[0124] In an optional embodiment, the copy number of the farnesene pyrophosphate synthase gene in the recombinant Saccharomyces cerevisiae genome is 1 to 3, or further 2.

[0125] In an optional embodiment, the number of at least one mevalonate pathway-related gene in the recombinant Saccharomyces cerevisiae genome is greater than 1.

[0126] In an alternative embodiment, the copy number of at least one of the ERG10 gene, ERG13 gene, tHMG1 gene, ERG12 gene, ERG8 gene, MVD1 gene or IDI1 gene is 1 to 3, preferably 2 or 3.

[0127] The present invention introduces an artemisinin synthesis gene into Saccharomyces cerevisiae through gene editing, adjusts the metabolic pathway related to artemisinin expression in the Saccharomyces cerevisiae genome, and increases the copy number of genes involved in the mevalonate pathway and the farnesene pyrophosphate synthase gene in the recombinant Saccharomyces cerevisiae, thereby significantly improving the production of artemisinin.

[0128] In an optional embodiment, the repressor GAL80 gene and / or the activation cis-element or a fragment thereof within the range of -175 to -220 upstream of the ERG9 promoter is knocked out from the Saccharomyces cerevisiae genome.

[0129] In an optional embodiment, the amino acid sequence of the artemisinin synthase includes (a) or (b):

[0130] (a) SEQ ID NO. 1;

[0131] (b) a derivative artemisinin synthase having at least 80%, 81%, 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97% or 99% amino acid sequence identity to (a).

[0132] In a second aspect, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding the artemisinin synthase according to any one of the aforementioned embodiments or a nucleotide sequence complementary to the base thereof.

[0133] In an optional embodiment, the nucleic acid molecule comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO.3.

[0134] In a third aspect, the present disclosure provides a recombinant expression vector containing the nucleic acid molecule described in any one of the aforementioned embodiments, wherein the original vector of the recombinant expression vector includes pZY900, pYR017, pYR018 or pKlURA100.

[0135] In a fourth aspect, the present disclosure provides a transformant, which contains the nucleic acid molecule described in any one of the preceding embodiments, or a transformant containing the recombinant expression vector described in the preceding embodiments, wherein the host cell of the transformant is selected from a fungal expression system, preferably a yeast expression system, and more preferably a cerevisiae expression system.

[0136] In a fifth aspect, the present disclosure provides a recombinant Saccharomyces cerevisiae, wherein the recombinant Saccharomyces cerevisiae contains the nucleic acid molecule described in any one of the aforementioned embodiments, or contains the recombinant expression vector described in the aforementioned embodiments, and the recombinant Saccharomyces cerevisiae further contains a farnesene pyrophosphate synthase gene.

[0137] In an optional embodiment, the copy number of the farnesene pyrophosphate synthase gene in the recombinant Saccharomyces cerevisiae genome is 1 to 3, or further 2.

[0138] In an optional embodiment, the number of at least one mevalonate pathway-related gene in the recombinant Saccharomyces cerevisiae genome is greater than 1.

[0139] In an alternative embodiment, the copy number of at least one of the ERG10 gene, ERG13 gene, tHMG1 gene, ERG12 gene, ERG8 gene, MVD1 gene or IDI1 gene is 1 to 3, preferably 2 or 3.

[0140] In an optional embodiment, the repressor GAL80 gene and / or the activation cis-element or a fragment thereof within the range of -175 to -220 upstream of the ERG9 promoter is knocked out from the Saccharomyces cerevisiae genome.

[0141] In a sixth aspect, the present disclosure provides a method for preparing the recombinant Saccharomyces cerevisiae according to any one of the aforementioned embodiments, the preparation method comprising the steps of:

[0142] (a) transforming the nucleic acid molecule described in any one of the preceding embodiments or the recombinant expression vector described in any one of the preceding embodiments into a Saccharomyces cerevisiae cell;

[0143] (b) increasing the copy number of farnesene pyrophosphate synthase gene and / or mevalonate pathway-related genes in the Saccharomyces cerevisiae genome by gene editing;

[0144] The order of implementing the steps (a) and (b) is not particular.

[0145] In an optional embodiment, the copy number of the farnesene pyrophosphate synthase gene is 1 to 3, further 2.

[0146] In an alternative embodiment, the copy number of at least one of the ERG10 gene, ERG13 gene, tHMG1 gene, ERG12 gene, ERG8 gene, MVD1 gene or IDI1 gene is 1 to 3, preferably 2 or 3.

[0147] In an optional embodiment, the preparation method comprises transforming a repressor GAL80 gene knockout expression cassette and / or an ERG9 promoter upstream activation cis-element knockout or partial knockout expression cassette into Saccharomyces cerevisiae cells.

[0148] In a seventh aspect, the present disclosure provides a method for preparing artemisinin, which comprises fermenting and culturing the transformant described in the aforementioned embodiment, or fermenting and culturing the recombinant brewer's yeast described in any of the aforementioned embodiments, or fermenting and culturing the recombinant brewer's yeast prepared by the preparation method described in any of the aforementioned embodiments, and recovering artemisinin from the fermentation product.

[0149] In an alternative embodiment, the method of recovering comprises extracting the fermentation product using an oil phase.

[0150] In an alternative embodiment, the oil phase comprises n-hexane, n-dodecane and / or isopropyl myristate.

[0151] In an optional embodiment, the recovery method is to add an oil phase during the fermentation process, collect the oil phase after the fermentation is completed, and separate the artemisinin enriched in the oil phase.

[0152] In an alternative embodiment, the method of collecting the oil phase comprises centrifugation.

[0153] In an alternative embodiment, the separation method comprises distillation.

[0154] In an eighth aspect, the present disclosure provides the use of artemisinin obtained by the preparation method described in any one of the aforementioned embodiments in the preparation of antibacterial products, insecticide products or herbicidal products.

[0155] In an alternative embodiment, the types of microorganisms that the antimicrobial product can inhibit or kill include fungi.

[0156] In an optional embodiment, the fungi include one or more of the genera Rhizoctonia, Plasmodium, Pythium, Phytophthora, Rhizopus, Black Coriolus, Venturia, Sclerotinia, Powdery Mildew, Pear-Shaped Spores, Powdery Molds, Verticillium, Botrytis, Alternaria, Fusarium, Cyclosporium, Gibberellum, Colletotrichum, and Puccinia.

[0157] In an alternative embodiment, the Rhizoctonia fungus includes Rhizoctonia solani.

[0158] In an alternative embodiment, the insect species that can be repelled or killed by the insecticidal product include insects of the Culicidae, Miridae or Delphacidae families.

[0159] In an optional embodiment, the Culicidae insects include one or more of the genera Aedes, Anopheles, Armigeres, Coquillettidia, Culex, Culiseta, Ficalbia, Heizmznnia, Hemagogus, Hodgesia, Malaya, Mansonia, Minmomyia, Orthopodomyia, Sabethes, Topomyia, Toxorhynchites, Tripteroides, Udaya, and Uranotaenia.

[0160] In an alternative embodiment, the insect of the genus Aedes includes Aedes aegypti.

[0161] In an optional embodiment, the Miridae insects include Genus Acomocera, Genus Adelphococris, Genus Agnocoris, Genus Alcecoris, Genus Alloeotomus, Genus Allorhinocoris, Genus Angerianus, Genus Apilophorus, Genus Apolygopsis, Genus Apolygus, Genus Arbolygus, Genus Argenis, Genus Astroscopometopus, Genus Bertsa), Blepharidopterus, Bothriomiris, Bothynotus, Brachycoleus, Bryocoris, Campylotropis, Capsodes, Capsus, Castanopsides, Charagochilus, Cheilocapsidea, Cheilocapsus, Chilocrates, Chrysorrhanis, and Cimicicapsus), Genus Cimidaeorus, Genus Closterotomus, Genus Coridromius, Genus Creontiades, Genus Cylapofulvuitius, Genus Cylapomorpha, Genus Cyllecoris, Genus Cyphodemidea, Genus Cyrtopeltis, Genus Cyrtorhinus, GenusDeraeocoris), Genus Dichrooscytus, Genus Dicyphus, Genus Dimia, Genus Dioclerus, Genus Dolichomiris, Genus Dortus, Genus Dryophilocoris, Genus Ectmetopterus, Genus Elthemidea, Genus Eolygus, Genus Erimiris, Genus Ernestinus, Genus Eupachypeltis, Genus Eurvopicoris, Genus Eurystylomorpha), Genus Eurystylopsis, Genus Eurystylus, Genus Excentricus, Genus Felisacus, Genus Finqulus, Genus Fulvius, Genus Globiceps, Genus Guisardinus, Genus Guisardus, Genus Halticus, Genus Harpedona, Genus Hekista, Genus Helopeltis, Genus Heterolyqus, Genus Heteropantilius), Genus Hyalopeplinus, Genus Hyalopeplus, Genus Hyoidea, Genus Isabel, Genus Isometopidea, Genus Isometopus, Genus Jessopocoris, Genus Labopidea, Genus Labops, Genus Lasiomiris, Genus Latizanchius, GenusLeptopterna), Genus Liistonotus, Genus Loristes, Genus Lygidea, Genus Lygocorides, Genus Lygocoris, Genus Lygus, Genus Macrolonius, Genus Macrolophus, Genus Macrolygus, Genus Malacocorisella, Genus Mansoniella, Genus Mecistoscelis, Genus Mecomma, Genus Megacoelum, Genus Mermitelocerus), Metasequoiamiris, Michailocoris, Monalocoris, Myiomma, Myrmecophyes, Myrmecoris, Mystilus, Neolygus, Nesidiocoris, Nicostratus, Notostir, Onomaus, Orientomiris, Orhocephalus, and Orthops), Genus Orthotylus, Genus Pachypeltis, Genus Paloniella, Genus Pantilius, Genus Paracyphodema, Genus Paramiridius, Genus Paranx, Genus Parapachypeltis, Genus Peltidolygus, Genus Peritropis, GenusPhytocoridea), Genus Phytocoris, Genus Pinalitus, Genus Polymerus, Genus Poppiocapsidea, Genus Proboscidocoris, Genus Prodromus, Genus Prolygus, Genus Psallops, Genus Pseudodoniella, Genus Pseudoloxops, Genus Raawelellus, Genus Reuterista, Genus Rhabdomiris, Genus Rhinocylapidius, Genus Rhinomiris), Genus Rhopaliceschatus, Genus Sabactiopus, Genus Salignus, Genus Scirtetellus, Genus Sinevi, Genus Singhalesia, Genus Sophianus, Genus Stenodema, Genus Stenotus, Genus Stethoconus, Genus Strongylocoris, Genus Sulawesifaulvius, Genus Tailorilygus, Genus Teratocoris, Genus The present invention relates to one or more of the genera Termatophylum, Genus Tinginotopsis, Genus Tinginotum, Genus Tolongia, Genus Totta, Genus Trigonotylus, Genus Tupiocoris, Genus Ulmica, Genus Ulmocyllus, Genus Zanchius and Genus Zonodoropsis.

[0162] In an alternative embodiment, the insect of the genus Adelphococris includes the black stink bug.

[0163] In an optional embodiment, the planthopper family includes one or more of the genera Nilaparvata, Unkanodes, Neunkanodes, Laodelphax, Ribautodelphax, Megadelphax, Muirodelphax, Fangdelphax, Neoterthrona, Ramidelphax, Neuterthron, Miranus, Sogata, Parasogata, Neometopina, Changeondelphax, Chloriona, Paradelphacodes, and Muellerianella.

[0164] In an alternative embodiment, the Delphacidae insects include brown planthopper (Delphacidae).

[0165] In an optional embodiment, the plant species that the herbicidal product can inhibit or kill include one or more of the following: Cruciferae, Asteraceae, Solanaceae, Convolvulaceae, Rubiaceae, Crassulaceae, Cannabaceae, Polygonaceae, Caryophyllaceae, Amaranthaceae, Portulacaceae, Ranunculaceae, Rosaceae, Plantaginaceae, Vitaceae, Euphorbiaceae, Apiaceae, Lamiaceae, Malvaceae, Poaceae, Cyperaceae, Juncaceae, Liliaceae, Commelinaceae, Amaryllidaceae, and Alismataceae.

[0166] In an optional embodiment, the cruciferous plant includes Arabidopsis, Alliaria, Alyssum, Aphragmus, Arabidopsis, Arabis, Armoracia, Atelanthera, Barbarea, Berteroa, Berteroella, Brassica, Braya, Bunias, Camelina, Capsella, Cardamine, Cardaria, Cheiranthus, Chorispora, Christolea, Clausia, Cochlearia, Coelon ema), Conringia, Coronopus, Crambe, Cryptospora, Descurainia, Dilophia, Dimorphostemon, Diplotaxis, Dipoma, Diptychocarpus, Dontostemon , Draba, Eruca, Erysimum, Euclidium, Eutrema, Goldbachia, Hedinia, Hemilophia, Hesperis, Hymenolobus, Iberis, Isatis, Lachnoloma,Lepidium, Leptaleum, Lignariella, Litwinowia, Lobularia, Loxostemon, Macropodium, Malcolmia, Matthiola, Megacarpaea, Megadenia, Microsisymbrium, and Microstigma, Nasturtium, Neomartinella, Neslia, Orychophragmus, Pachypterygium, Parrya, Parryodes, Pegaeophyton, Phaeonychium, Platycraspedum, Ptilotri cum), Pugionium, Pycnoplinthus, Raphanus, Rorippa, Sinapis, Sisymbrium, Smelowskia, Solms-Laubachia, Sophiopsis, Spirorhynchus, Staintoniella, Sterigmost The invention further comprises one or more of the genera genus C. emum, Stevenia, Stroganowia, Synstemon, Syrenia, Taphrospermum, Tauscheria, Tetracme, Thellungiella, Thlaspi, Torularia, Turritis and Yinshania.

[0167] In an optional embodiment, the cruciferous plant includes one or more of the genera Arabidopsis, Capsella, Cardamine, Lepidium, Aralia, Lepidium, Halorhizoma, Saccharina, Descurainia, and Herba Epipremnum.

[0168] In an alternative embodiment, the Arabidopsis plant includes Arabidopsis thaliana.

[0169] In a ninth aspect, the present disclosure provides an antibacterial agent comprising artemisinin obtained by the preparation method described in any one of the aforementioned embodiments.

[0170] In an optional embodiment, the effective dose of artemisinin is 10 to 100 ppm.

[0171] In an optional embodiment, the dosage form of the antibacterial agent includes tablets, injections, capsules, powders, premixes, granules, soluble powders, oral solutions, gels or ointments.

[0172] In an alternative embodiment, the types of microorganisms that the antimicrobial agent can inhibit or kill include fungi.

[0173] In an optional embodiment, the fungi include one or more of the genera Rhizoctonia, Plasmodium, Pythium, Phytophthora, Rhizopus, Black Coriolus, Venturia, Sclerotinia, Powdery Mildew, Pear-Shaped Spores, Powdery Molds, Verticillium, Botrytis, Alternaria, Fusarium, Cyclosporium, Gibberellum, Colletotrichum, and Puccinia.

[0174] In an alternative embodiment, the Rhizoctonia fungus includes Rhizoctonia solani.

[0175] In a tenth aspect, the present disclosure provides an insecticide comprising artemisinin obtained by the preparation method described in any one of the aforementioned embodiments.

[0176] In an optional embodiment, the effective dose of artemisinin is 500 to 5000 ppm.

[0177] In an optional embodiment, the formulation of the insecticide includes an aqueous emulsion, a suspension, a dry powder, a wettable powder, an emulsifiable concentrate, a thermal fog, an aerosol, a soluble granule, a fumigant, a bait, a granule or a slow-release agent.

[0178] In an alternative embodiment, the insects that the insecticide can repel or kill include insects from the Culicidae, Miridae, or Delphacidae families.

[0179] In an optional embodiment, the Culicidae insects include one or more of the genera Aedes, Anopheles, Armigeres, Coquillettidia, Culex, Culiseta, Ficalbia, Heizmznnia, Hemagogus, Hodgesia, Malaya, Mansonia, Minmomyia, Orthopodomyia, Sabethes, Topomyia, Toxorhynchites, Tripteroides, Udaya, and Uranotaenia.

[0180] In an alternative embodiment, the insect of the genus Aedes includes Aedes aegypti.

[0181] In an optional embodiment, the Miridae insects include Genus Acomocera, Genus Adelphococris, Genus Agnocoris, Genus Alcecoris, Genus Alloeotomus, Genus Allorhinocoris, Genus Angerianus, Genus Apilophorus, Genus Apolygopsis, Genus Apolygus, Genus Arbolygus, Genus Argenis, Genus Astroscopometopus, Genus Bertsa), Blepharidopterus, Bothriomiris, Bothynotus, Brachycoleus, Bryocoris, Campylotropis, Capsodes, Capsus, Castanopsides, Charagochilus, Cheilocapsidea, Cheilocapsus, Chilocrates, Chrysorrhanis, and Cimicicapsus), Genus Cimidaeorus, Genus Closterotomus, Genus Coridromius, Genus Creontiades, Genus Cylapofulvuitius, Genus Cylapomorpha, Genus Cyllecoris, Genus Cyphodemidea, Genus Cyrtopeltis, Genus Cyrtorhinus, GenusDeraeocoris), Genus Dichrooscytus, Genus Dicyphus, Genus Dimia, Genus Dioclerus, Genus Dolichomiris, Genus Dortus, Genus Dryophilocoris, Genus Ectmetopterus, Genus Elthemidea, Genus Eolygus, Genus Erimiris, Genus Ernestinus, Genus Eupachypeltis, Genus Eurvopicoris, Genus Eurystylomorpha), Genus Eurystylopsis, Genus Eurystylus, Genus Excentricus, Genus Felisacus, Genus Finqulus, Genus Fulvius, Genus Globiceps, Genus Guisardinus, Genus Guisardus, Genus Halticus, Genus Harpedona, Genus Hekista, Genus Helopeltis, Genus Heterolyqus, Genus Heteropantilius), Genus Hyalopeplinus, Genus Hyalopeplus, Genus Hyoidea, Genus Isabel, Genus Isometopidea, Genus Isometopus, Genus Jessopocoris, Genus Labopidea, Genus Labops, Genus Lasiomiris, Genus Latizanchius, GenusLeptopterna), Genus Liistonotus, Genus Loristes, Genus Lygidea, Genus Lygocorides, Genus Lygocoris, Genus Lygus, Genus Macrolonius, Genus Macrolophus, Genus Macrolygus, Genus Malacocorisella, Genus Mansoniella, Genus Mecistoscelis, Genus Mecomma, Genus Megacoelum, Genus Mermitelocerus), Metasequoiamiris, Michailocoris, Monalocoris, Myiomma, Myrmecophyes, Myrmecoris, Mystilus, Neolygus, Nesidiocoris, Nicostratus, Notostir, Onomaus, Orientomiris, Orhocephalus, and Orthops), Genus Orthotylus, Genus Pachypeltis, Genus Paloniella, Genus Pantilius, Genus Paracyphodema, Genus Paramiridius, Genus Paranx, Genus Parapachypeltis, Genus Peltidolygus, Genus Peritropis, GenusPhytocoridea), Genus Phytocoris, Genus Pinalitus, Genus Polymerus, Genus Poppiocapsidea, Genus Proboscidocoris, Genus Prodromus, Genus Prolygus, Genus Psallops, Genus Pseudodoniella, Genus Pseudoloxops, Genus Raawelellus, Genus Reuterista, Genus Rhabdomiris, Genus Rhinocylapidius, Genus Rhinomiris), Genus Rhopaliceschatus, Genus Sabactiopus, Genus Salignus, Genus Scirtetellus, Genus Sinevi, Genus Singhalesia, Genus Sophianus, Genus Stenodema, Genus Stenotus, Genus Stethoconus, Genus Strongylocoris, Genus Sulawesifaulvius, Genus Tailorilygus, Genus Teratocoris, Genus The present invention relates to one or more of the genera Termatophylum, Genus Tinginotopsis, Genus Tinginotum, Genus Tolongia, Genus Totta, Genus Trigonotylus, Genus Tupiocoris, Genus Ulmica, Genus Ulmocyllus, Genus Zanchius and Genus Zonodoropsis.

[0182] In an alternative embodiment, the insect of the genus Adelphococris includes the black stink bug.

[0183] In an optional embodiment, the planthopper family includes one or more of the genera Nilaparvata, Unkanodes, Neunkanodes, Laodelphax, Ribautodelphax, Megadelphax, Muirodelphax, Fangdelphax, Neoterthrona, Ramidelphax, Neuterthron, Miranus, Sogata, Parasogata, Neometopina, Changeondelphax, Chloriona, Paradelphacodes, and Muellerianella.

[0184] In alternative embodiments, the Delphacidae insect includes the brown planthopper (Delphacidae).

[0185] In an eleventh aspect, the present disclosure provides a herbicide comprising artemisinin obtained by the preparation method described in any one of the aforementioned embodiments.

[0186] In an optional embodiment, the effective dose of artemisinin is 25 to 200 ppm.

[0187] In an optional embodiment, the formulation of the herbicide includes an aqueous solution, an emulsifiable concentrate, a suspension concentrate, an aqueous emulsion, a microemulsion, an oil suspension, a microcapsule suspension, a soluble liquid, a film-spreading oil, a wettable powder, a soluble powder, a soluble granule, a dry suspension, or a water-dispersible granule. In an optional embodiment, the plant species that the herbicide can inhibit or kill include one or more cruciferous plants of the family Cruciferae, Asteraceae, Solanaceae, Convolvulaceae, Rubiaceae, Crassulaceae, Cannabaceae, Polygonaceae, Caryophyllaceae, Amaranthaceae, Portulacaceae, Ranunculaceae, Rosaceae, Plantaginaceae, Vitaceae, Euphorbiaceae, Apiaceae, Lamiaceae, Malvaceae, Poaceae, Cyperaceae, Juncaceae, Liliaceae, Commelinaceae, Amaryllidaceae, and Alismataceae. In an optional embodiment, the cruciferous plants include Arabidopsis, Alliaria, Alyssum, Aphragmus, Arabidopsis, Arabis, Armoracia, Atelanthera, Barbarea, Berteroa, Berteroella, Brassica, Braya, Spoon Bunias, Camelina, Capsella, Cardamine, Cardaria, Cheiranthus, Chorispora, Christolea, Clausia, Cochlearia, Coelonema, Conringia, Coronopus, Crambe, Cryptopus tospora, Descurainia, Dilophia, Dimorphostemon, Diplotaxis, Dipoma, Diptychocarpus, Dontostemon, Draba, Eruca, Erysimum, Euclidium, Eutrema, Goldbachia, Tibetan shepherd's purse Hedinia, Hemilophia, Hesperis, Hymenolobus, Iberis, Isatis, Lachnoloma, Lepidium, Leptaleum, Lignariella, Litwinowia, Lobularia, Loxostemon, Macropodium,Malcolmia, Matthiola, Megacarpaea, Megadenia, Microsisymbrium, Microstigma, Nasturtium, Neomartinella, Neslia, Orychophragmus, Pachypterygium, Parrya, Parryodes, Pegaeophyton, Phaeonychium, Platycraspedum, Ptilotricum, Pugionium, Pycnoplinthus, Raphanus, R orippa, Sinapis, Sisymbrium, Smelowskia, Solms-Laubachia, Sophiopsis, Spirorhynchus, Staintoniella, Sterigmostemum, Stevenia, Stroganowia, Synstemon, Syrenia, Taphrospermum, Tauscheria, Tetracme, Thellungiella, Thlaspi, Torularia, Turritis, Yinshania, etc., or one or more thereof.

[0188] In an optional embodiment, the cruciferous plant includes one or more of the genera Arabidopsis, Capsella, Cardamine, Lepidium, Aralia, Lepidium, Halorhizoma, Saccharina, Descurainia, and Herba Lysimachiae.

[0189] In an alternative embodiment, the Arabidopsis plant includes Arabidopsis thaliana.

[0190] The following describes some embodiments of the present disclosure in detail with reference to the accompanying drawings. In the absence of conflict, the following embodiments and features therein may be combined with each other.

[0191] Example 1 Construction of an expression vector for artemisinin synthase derived from Artemisia argyi

[0192] The transcriptome data of Artemisia argyi (Artemisia argyi) from the NCBI database bioproject: PRJNA722539 were analyzed to obtain a synthase (AaTPS19) with potential artemisinin biosynthesis function, the amino acid sequence of which is shown in SEQ ID NO. 1:

[0193] The gene encoding the synthetase was synthesized after optimization according to the codons of Saccharomyces cerevisiae to obtain the nucleotide sequence shown in SEQ ID NO.3:

[0194] Using SEQ ID NO. 3 as a template, a specific gene primer pair P1 / P2 was designed. The codon-optimized synthetase gene fragment was amplified by PCR using VAZYME's Phanta high-fidelity enzyme. After gel recovery using a Tiangen Gum Recovery Kit, the fragment was ligated into the yeast expression vector pZY900 (using the same method as patent CN202210473488.4) after BsaI cleavage using a homologous recombination kit from Yisheng Company. After sequencing confirmation, a yeast expression vector containing the gene was obtained and named p900A19. A schematic diagram of the construction of plasmid p900A19 is shown in Figure 1.

[0195] Using primers P3 / P4 and P5 / P6, pYR017 (see Patent 202210473488.4) and p900A19 as templates were used to amplify the long fragment (containing the Ura3 left homology arm, histidine selection marker, CYC1 terminator, tHMG1 gene, GAL1-GAL10 promoter, PGK1 terminator, Ura3 right homology arm, and plasmid backbone) and the gene encoding AaTPS19. Subsequently, using yeast assembly methods, these fragments were recombined in Saccharomyces cerevisiae to construct p905A19. A schematic diagram of the construction of plasmid p905A19 is shown in Figure 1.

[0196] Primers P7 / P8 and P9 / P10 were used, respectively, with pYR018 (see Patent 202210473488.4) and p900A19 as templates to amplify a long fragment (containing the yprcdelta15 left homology arm, a tryptophan selection marker, a GPM1 terminator, a GAL1-GAL10 promoter, a PGK1 terminator, a yprcdelta15 right homology arm, and a plasmid backbone) and the AaTPS19 encoding gene. Subsequently, using yeast assembly methods, the above fragments were recombined in Saccharomyces cerevisiae to construct p908A19. A schematic diagram of the construction of plasmid p908A19 is shown in Figure 1.

[0197] Example 2 Construction of gene editing plasmid and knockout cassette

[0198] The fragment was amplified using primers P11 / P12 and plasmid pKlURA100 (see Zhang, Yueping et al. "A gRNA-tRNA array for CRISPR-Cas9based rapid multiplexed genome editing in Saccharomyces cerevisiae." Nature communications vol.10, 1 1053.5 Mar.2019, doi:10.1038 / s41467-019-09005-3) as a template. Subsequently, the above fragments and pCas were assembled using the Goldengate method (Zhang, Yueping et al. "A gRNA-tRNA array for CRISPR-Cas9based rapid multiplexed genome editing in Saccharomyces cerevisiae." Nature communications vol. 10, 1 1053.5 Mar. 2019, doi: 10.1038 / s41467-019-09005-3) to construct the plasmid pYH395.

[0199] Primers P13 / P14 and P15 / P16 were used to amplify the whole genome of Saccharomyces cerevisiae CEN.PK2-1D as a template to obtain two fragments. Subsequently, primers P13 and P16 were used to perform overlap extension PCR with the two obtained fragments as templates to obtain the ERG9 promoter upstream activation cis element (-220 to -175) knockout element.

[0200] Primers P33 / P34 were used to amplify the left homology arm of fragment X-4 using the yeast CEN.PK2-1D genome as a template, primers P35 / P36 were used to amplify the terminator TADH1 using the yeast CEN.PK2-1D genome as a template, primers P37 / P38 were used to amplify the pGAL1-pGAL10 and AaTPS19 gene fragment using p900A19 as a template, primers P39 / P40 were used to amplify the right homology arm containing X-4 using the yeast CEN.PK2-1D genome as a template, and primers P41 / P42 were used to amplify the terminator TCPS1 fragment using the yeast CEN.PK2-1D genome as a template. Overlap extension PCR was used with primers P33 / P40 and all the above fragments as templates to obtain the AaTPS19-C4 knock-in cassette.

[0201] Primers P43 / P44 were used to amplify the left homology arm of fragment XI-1 using the yeast CEN.PK2-1D genome as a template. Primers P45 / P46 were used to amplify the TADH1, pGAL1-pGAL10, AaTPS19 gene fragments and TCPS1 region using the AaTPS19-C4 knock-in cassette as a template. Primers P47 / P48 were used to amplify the right homology arm containing XI-1 using the yeast CEN.PK2-1D genome as a template. Overlap extension PCR was performed using primers P43 / P48 and all the above fragments as templates to obtain the AaTPS19-C5 knock-in cassette.

[0202] Primers P49 / P50 were used to amplify the left homology arm of fragment XII-5 using the yeast CEN.PK2-1D genome as a template. Primers P51 / P52 were used to amplify the TADH1, pGAL1-pGAL10, AaTPS19 gene fragments and TCPS1 region using the AaTPS19-C4 knock-in cassette as a template. Primers P53 / P54 were used to amplify the right homology arm containing XII-5 using the yeast CEN.PK2-1D genome as a template. Overlap extension PCR was performed using primers P49 / P54 and all the above fragments as templates to obtain the AaTPS19-C6 knock-in cassette.

[0203] Using primers 5211-F / R, 5212-F / R, and 5213-F / R, the whole genome of Saccharomyces cerevisiae CEN.PK2-1D, pRS426 plasmid, and the whole genome of Saccharomyces cerevisiae CEN.PK2-1D were amplified as templates to obtain three fragments. Subsequently, primers 5211-F and 5213-R were used as templates to perform overlap extension PCR to obtain the pZY521 knockout cassette.

[0204] pHM012 is a gRNA plasmid targeting X-4, XI-1, and XII-5 sites, and is a tool plasmid used for target recognition and cutting of Saccharomyces cerevisiae genomic DNA in Crispr-Cas9 gene editing technology. (1) Using the pstgRNA plasmid as a template, the plasmid was derived from the reference: A gRNA-tRNA array for CRISPR-Cas9based rapid multiplexed genome editing in Saccharomyces cerevisiae (DOI: 10.1038 / s41467-019-09005-3), primers X-4_scaffold F and tGly_XI-1R were used to amplify the plasmid fragment containing X-4 site gRNA, gRNA scaffold, tGly and part of XI-1 site gRNA by PCR; (2) Using the pstgRNA plasmid as a template, primers XI-1_scaffold F and tGly_XII-5R were used to amplify the plasmid fragment containing part of XI-1 site gRNA, gRNA scaffold, tGly and part of XII-5 site gRNA by PCR; (3) Using the pKlURA3 100 plasmid (the source of the pKlURA3 100: A gRNA-tRNA array for CRISPR-Cas9based rapid multiplexed genome editing in Saccharomyces cerevisiae) was used as a template, and primers XII-5_scaffold F and pKIURA3-R-common were used to amplify by PCR to obtain a fragment containing XII-5 site gRNA, gRNA scaffold, SUF17 terminator and URA screening tag); (4) The above three fragments were assembled with the pCas9 vector using the Golden Gate assembly method, and Escherichia coli was transformed. After enzyme digestion verification and sequencing, plasmid pHM012 was obtained.

[0205] 4) Strain Construction (Artemisia argyi-derived Genes)

[0206] The p900A19 plasmid was transformed into the competent yeast strain JCR27 by the PEG / LiAC method, plated on SD-URA screening plates, and cultured for 3 days. Colony PCR verification was performed using Novozyme PCR reagent, and the positive bacteria were named J900A19. (For the construction of yeast strain JCR27, see the literature Siemon, T., et al. (2020). "Semisynthesis of plant-derived englerin A enabled by microbe engineering of guaia-6,10(14)-diene as building block." Journal of the American Chemical Society 142(6):2760-2765.).

[0207] Plasmid p900A19 was linearized using the restriction endonuclease MssI, and a fragment containing the farnesyl pyrophosphate synthase gene and the AaTPS19 gene was recovered. The fragment was introduced into the competent yeast strain JCR27 by the PEG / LiAC method. After yeast colony PCR verification, the positive bacteria were named JA191.

[0208] Compared with the CEN.PK2-1D strain, the strain JA191 strengthened the MVA pathway and overexpressed the entire MVA pathway. On this basis, it also overexpressed the farnesyl pyrophosphate synthase gene and the AaTPS19 gene. The copy number of each gene in the strain JA191 is ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, ERG20, AaTPS19 = 2, 2, 2, 2, 2, 2, 2, 2, 1.

[0209] p908A19 was further digested with NotI endonuclease to recover the fragment containing the AaTPS19 gene. After similar transformation into yeast strain JA191 competent cells, yeast colony PCR verification was performed to obtain the positive bacteria JA192. The copy numbers of each gene in strain JA192 were ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, ERG20, and AaTPS19 = 2, 2, 2, 2, 2, 2, 2, 2, 2.

[0210] Similarly, p905A19 was digested with MssI endonuclease to recover a fragment containing the AaTPS19 gene. The fragment was introduced into the competent yeast strain JA192 by the PEG / LiAC method. After yeast colony PCR verification, the positive bacteria were named JA193. The copy numbers of each gene in strain JA193 were ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, ERG20, and AaTPS19 = 2, 2, 3, 2, 2, 2, 2, 2, 3.

[0211] The plasmid pYH395 and the knockout element of the upstream activation cis-element of the ERG9 promoter (-220 to -175) were co-transfected into the strain JA193 by the lithium acetate method. The colonies were verified by PCR to confirm the correctness of the knockout. Subsequently, the strain was cultured in YPD liquid medium (20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose) with a shaker at 220 rpm. After culturing at 30 degrees for 8 hours, the strain was washed with water and streaked onto 5-FOA plates (Li Xiaowei. Engineering acetyl-CoA pathway to construct an efficient synthesis platform for Saccharomyces cerevisiae [D]. Wuhan University, 2015.), cultured in a 30°C incubator for 3 days, and the colonies were picked from the plates to continue colony PCR to confirm the correctness of the knockout. The correct strain was named JA194.

[0212] This step is used to knock out the upstream activating cis-element of the ERG9 promoter (-220 to -175). The squalene synthase encoded by ERG9 uses FPP as a substrate to synthesize squalene, which is a competing pathway for the sesquiterpene synthesis pathway. Knocking out the upstream activating cis-element of the ERG9 promoter can downregulate the squalene synthesis pathway, reduce the competition of the squalene synthesis pathway for the substrate required for the synthesis of sesquiterpene compounds, and help further increase the yield of sesquiterpene compounds.

[0213] Plasmid pHM012 and the AaTPS19-C4, AaTPS19-C5, and AaTPS19-C6 knock-in cassettes were further co-transformed into strain JA194 using the lithium acetate method. The ERG9 promoter upstream activation cis-element (-220 to -175) was knocked out in the same manner as above, and strain JA196 was obtained by colony PCR verification. A positive strain JA196 was obtained, and the copy numbers of the genes in JA195 were ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, ERG20, and AaTPS19 = 2, 2, 3, 2, 2, 2, 2, 2, 6.

[0214] Finally, the pZY521 knockout cassette was transferred into strain JA196 using the lithium acetate method to generate strain JA197. This step knocks out the transcriptional repressor GAL80, allowing the target gene in the transferred strain to express autonomously without the need for an inducer, which helps reduce fermentation product yields and further increases the yield of sesquiterpenoid compounds.

[0215] Example 3 Shake flask fermentation

[0216] Strains J900A19 and J900V5-15 were inoculated into 50 mL of YPD medium supplemented with 1% (w / v) galactose and cultured at 30°C. After 3 days, the cells were harvested by centrifugation at 8000 rpm for 10 minutes. The cells were extracted with 10 mL of n-hexane, and samples were prepared for GC-MS analysis. A specific sesquiterpene product was detected in the J900A19 strain, as shown in Figure 3. This compound was tentatively designated A19. The same specific sesquiterpene product was detected in the J900V5-15 strain, as shown in Figure 4.

[0217] Strains JA191 to JA197 were fermented using a similar method, with 10% (w / w) isopropyl myristate (IPM) added as a cover, for 72 hours. The oil phase was centrifuged and analyzed by GC-MS. The yields of the sesquiterpene A19 product (finally identified as (+)-intermedeol) for strains JA191, JA192, JA193, JA194, JA196, and JA197 were 18.97 mg / L, 37.7 mg / L, 60.8 mg / L, 70.65 mg / L, 88.6 mg / L, 167 mg / L, and 186 mg / L, respectively.

[0218] Example 4 Fermentation in a fermenter

[0219] Reference literature (van Hoek, P.; de Hulster, E.; van Dijken, JP; Pronk, JT Fermentative capacity in high-cell-density fed-batch cultures of baker's yeast. Biotechnol. Bioeng. 2000, 68, 517-523.) was used to describe the fermentation medium. Constructed strain JA197 was subjected to batch fed fermentation. During the fermentation, a covering agent was added to achieve in-situ extraction. The covering agent was either n-dodecane or isopropyl myristate. The fermentation process was controlled to have dissolved oxygen above 20%, a pH of 5, a glucose concentration of 1-2 g / L, and an ethanol concentration of below 5 g / L. Finally, in a fermentor (15 L steel tank), the yield of the sesquiterpene product (finally identified as (+)-intermedeol) reached 12 g / L (the covering agent was n-dodecane) and 9.5 g / L (the covering agent was isopropyl myristate), respectively.

[0220] Example 5 Product Identification

[0221] In order to determine the structure of the sesquiterpene product, the strain JA197 was fermented under fermentation conditions without covering the organic solvent IPM. The fermentation control was the same as that when covering, except that no covering agent was added. After the fermentation was completed, the bacterial solution was extracted with an equal volume of methanol, followed by extraction with n-hexane, and static separation was performed. The upper organic phase was concentrated under reduced pressure and further purified using semi-preparative HPLC after reduced pressure concentration. The purified product was then subjected to 1 H and 13C The results of NMR, COSY, ROESY and X-RAY identification are shown in Figures 4 to 9, respectively, and the chemical structure of the sesquiterpene was determined to be It is named (+)-intermedeol.

[0222] Comparative Example Expression of Artemisinin Synthase from Termitomyces fungi and Vitis vinifera grapes

[0223] There are two reported intermedol synthases, one from Termitomyces and the other from Vitis vinifera.

[0224] The amino acid sequence of V5-15 from the fungus Termitomyces is shown in SEQ ID NO. 2:

[0225] The gene encoding this enzyme was synthesized after optimization of the Saccharomyces cerevisiae codons, and its nucleotide sequence is shown in SEQ ID NO.4:

[0226] Using SEQ ID NO.4 as a template, a specific gene primer pair P55 / P56 was designed. The V5-15 gene fragment was obtained by PCR amplification using VAZYME's Phanta high-fidelity enzyme. After gel recovery using a Tiangen Gum Recovery Kit, the fragment was ligated into the yeast expression vector pZY900 after BsaI incision using a homologous recombination kit from Yishen Company. After sequencing confirmation, the yeast expression vector containing the gene was obtained and named p900-V5-15. The schematic diagram of the construction of plasmid p900-V5-15 is shown in Figure 2.

[0227] The amino acid sequence of VvPNSeInt from grape Vitis vinifera is shown below:

[0228] The gene encoding this enzyme was synthesized after optimization of the Saccharomyces cerevisiae codon, and its nucleotide sequence is shown below:

[0229] Using the above sequence as a template, a specific gene primer pair P71 / P72 was designed. The gene fragment was obtained by PCR amplification using VAZYME's Phanta high-fidelity enzyme. After gel recovery using a Tiangen Gum Recovery Kit, the fragment was ligated into the yeast expression vector pZY900 after BsaI incision using a homologous recombination kit from Yishen Company. After sequencing confirmation, the yeast expression vector containing the gene was obtained and named p900-VvPNSeInt. The schematic diagram of the construction of plasmid p900-VvPNSeInt is shown in Figure 2.

[0230] Plasmid p900-V5-15 was linearized using the restriction endonuclease MssI, and a fragment containing the farnesyl pyrophosphate synthase gene and the V5-15 gene was recovered. The fragment was introduced into the competent yeast strain JCR27 by the PEG / LiAC method. After yeast colony PCR verification, the positive bacteria were named JV151.

[0231] Compared with the CEN.PK2-1D strain, the strain JV151 strengthened the MVA pathway and overexpressed the entire MVA pathway. On this basis, it also overexpressed the farnesyl pyrophosphate synthase gene and the V5-15 gene. The copy number of each gene in the strain JV151 is ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, ERG20, V5-15 = 2, 2, 2, 2, 2, 2, 2, 2, 1.

[0232] The plasmid p900-VvPNSeInt was linearized using the restriction endonuclease MssI, and the fragment containing the farnesyl pyrophosphate synthase gene and the VvPNSeInt gene was recovered. The fragment was introduced into the competent yeast strain JCR27 by the PEG / LiAC method. After yeast colony PCR verification, the positive bacteria were named JVVvPNSeInt.

[0233] Compared with strain CEN.PK2-1D, strain JVVvPNSeInt strengthened the MVA pathway and overexpressed the entire MVA pathway. On this basis, it also overexpressed the farnesyl pyrophosphate synthase gene and VvPNSeInt gene. The copy number of each gene in strain JVVvPNSeInt is ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, ERG20, VvPNSeInt = 2, 2, 2, 2, 2, 2, 2, 2, 1.

[0234] Strains JV151 and JVVvPNSeInt were fermented in shake flasks under the same conditions as strain JA191. The intermedeol production was detected at the end of fermentation and was 0.35 mg / L and 0.04 mg / L, respectively, which were much lower than that of strain JV191. This shows that AaTPS19 is superior to the currently reported intermedeol synthase.

[0235] Example 6 Monitoring of the insecticidal effect of A19

[0236] (1) Evaluation of the effectiveness of A19 ((+)-intermedeol) against Aedes aegypti using a hand-in-cage test

[0237] The hand-in-cage experimental setup consists of a 30 cm × 30 cm × 30 cm mosquito cage with a modified glove placed in it. A digital camera for video recording is mounted on the top of the cage. To create a test window, a square is cut from the back of a nitrile rubber glove and a magnetic frame slightly larger than the window size is glued to it, which serves as a base for stacking additional magnetic window frames. A piece of gauze mesh treated with the test compound is placed on the fixed magnetic frame, approximately 3.0 mm from the glove. A second piece of untreated gauze mesh is placed approximately 8.0 mm above the treated mesh using another magnetic frame. The stacked magnetic frames are secured with adhesive clips, ensuring sufficient space between the treated and untreated meshes to prevent mosquitoes from contacting the treated mesh or piercing the skin of the hand in the glove.

[0238] The experiment was conducted in a room with a relative humidity of approximately 50% and a temperature range of 28°C to 30°C. Mosquitoes that had not taken a blood meal and were fed 2% glucose were transferred to mosquito cages, approximately 10 to 20 days old (approximately 50 to 70 mosquitoes). The glucose was removed 8 hours before the test, and each test was conducted between 16:00 and 21:00. Before the experiment, a researcher took 800 μl of A19 dissolved in acetone from a 2 mL EP tube to treat a gauze mesh, with acetone as a control. After allowing the acetone to evaporate for approximately 7 minutes, the researcher assembled and put on the modified gloves. At the same time, a laboratory assistant transferred the prepared cages from the incubator to a bench in the laboratory.

[0239] At the beginning of the experiment, researchers inserted their modified glove-wearing hand into the cage. A digital camera recorded mosquito landings on the test window over a 10-minute period, and the number of landings was subsequently counted and recorded. For each cage, a solvent (acetone) control was tested twice, followed by the test compound treatment. At least 0.5 hours elapsed between trials to allow the mosquitoes to fully recover and ventilate the chamber of any residual vapors from the previous experiment. Data from the control group were also included in the statistics to establish baseline mosquito activity in response to the solvent, and any data from cages with low landing counts in the control group were excluded. The repellency rate for each cage was determined using the following formula: repellency rate = [1 - (number of mosquitoes landing on the dosing window / number of mosquitoes landing on the solvent window)] × 100. Each cage was tested by at least two different testers, including both male and female testers. Furthermore, each cage was tested only once per day. After the experiment, the cage was immediately returned to the incubator. A new modified glove and its magnetic frame were used for each experiment. This experiment tested two concentration gradients of (+)-intermedeol: 500 ppm and 5000 ppm. The test results, shown in Figure 10, show that A19 has a strong mosquito repellent effect at both concentrations, suggesting its potential for use as a mosquito repellent.

[0240] (2) Using the H-tube experiment to evaluate the repellent effect of A19 on brown planthoppers

[0241] The attractiveness of compound A19 to brown planthoppers (N. lugens) was tested using an H-tube olfactometer, modified from the method described by Lou and Cheng (Lou, Y.-G. and J.-A. Cheng (2003). "Role of rice volatiles in the foraging behavior of the predator Cyrtorhinus lividipennis for the rice brown planthopper Nilaparvata lugens." Biocontrol 48:73-86.). The H-tube setup was as follows: two cylindrical glass arms (3 cm diameter, 15 cm height), each open at the top and with a hole (3 cm diameter) 4.5 cm from the bottom. A second cylindrical "connector" glass tube (3 cm diameter, 17 cm length) connected the two arms via a hole 4.5 cm from the bottom. In the center of the connector, there was a 1 cm diameter hole for releasing the test insects. The experiment was conducted between 09:00 and 17:00, when brown planthoppers are most active. The final acetone-based concentration of the A19 compound was 500 ppm. Three-leaf rice seedlings were thoroughly immersed in the A19 solution for 20 seconds, followed by evaporation for 7 minutes. Acetone was used as a control. A double layer of gauze was placed on top of the glass arm and sealed with a rubber band. Brown planthoppers, starved for 0.5 hours, were introduced into the H tube through the release hole, which was then plugged with cotton. The apparatus was placed in an incubator maintained at 26 ± 2°C and 70 ± 5% relative humidity. After 1 hour, the number of brown planthoppers in the control and treatment groups was recorded. If the distance from the release hole to the insect resting area did not exceed 3 cm, it was considered "no choice." Otherwise, it was considered "choice." 17-20 individuals were tested in each replicate, with each treatment repeated at least seven times. The test results are shown in Figure 11. It can be seen that A19 has a strong repellent effect on the rice pest brown planthopper and has potential for use as a pesticide.

[0242] (3) Using the H-tube experiment to evaluate the repellent effect of A19 on the black stink bug

[0243] For the black stink bug, the H-tube setup was as described above. Experiments were conducted between 3:00 PM and 6:00 PM, as black stink bugs are most active during this time. 5-cm-long beans were placed in each arm of the H-tube to attract black stink bugs that had been starved for 1 hour. Simultaneously, 150 μl of A19 (500 ppm) and acetone were added to two 4 cm × 2 cm filter papers, respectively. The filter papers were allowed to evaporate for 2 minutes before being placed in the arms of the H-tube along with the beans. The apparatus was placed in an incubator maintained at 26 ± 2°C and 70 ± 5% relative humidity. Over the next 1.5 to 2.5 hours, black stink bugs selected between the treated and control groups. Fifteen individuals were tested in each replicate, with at least five replicates per treatment. The test results, shown in Figure 12, demonstrate that A19 has a strong repellent effect against cotton pests, suggesting potential for use as a pesticide.

[0244] Example 7 Antibacterial Effect Detection of A19

[0245] Evaluation of the inhibitory effect of A19 on Rhizoctonia solani using the inhibition zone test

[0246] To determine the inhibitory effect of A19 against Rhizoctonia solani (a plant pathogenic fungus that causes devastating diseases of various important economic crops worldwide), 7 mm mycelial plugs were cut from the edge of 3-day-old colonies and transferred to PDA medium (20% potato, 2% glucose and 2% agar) containing specific concentrations of the test compound. A19 was dissolved in DMSO at concentrations of 10 ppm, 25 ppm, 50 ppm and 100 ppm, respectively, and DMSO was used as a control. After incubation at 28°C for 24 h, the colony diameter was measured using the hybridization method described in a previous study (Liu, K., et al. (2022). "Biological and molecular characterizations of fluxapyroxad-resistant isolates of Botrytis cinerea." Phytopathology Research 4(1):2. The original mycelial plug diameter (7 mm) was subtracted from the measured data. At least three replicates were tested for each experiment. The test results are shown in Figure 13. It can be seen that A19 has a good inhibitory effect on Rhizoctonia solani and has the potential to be used as a pesticide.

[0247] Example 8A19 Herbicidal Effect Detection

[0248] Evaluation of the potential of A19 for herbicide development using an Arabidopsis seedling inhibition assay

[0249] Arabidopsis thaliana ecotype Columbia (Col-0) was selected for root system assay. The seeds were surface sterilized and incubated at 4°C for 3 days for vernalization. Then, they were sown in square Petri dishes (130 mm × 130 mm) containing half-strength MS medium (pH = 5.7). The Petri dishes were placed vertically in a growth chamber with a photoperiod of 16 h light (150 μmol m -2 S -1 ) / 8 hours light / dark, with a day / night temperature of 23°C / 21°C. Three to five days after germination, seedlings were transferred to 35 mm diameter Petri dishes containing 1 / 2 MS medium supplemented with A19 dissolved in DMSO at concentrations of 25 ppm, 50 ppm, 100 ppm, and 200 ppm. A DMSO control was used. After four to five days, root and leaf lengths were measured using ImageJ software (NIH, USA, http: / / rsb.info.nih.gov / ij) and a calibrated scale. At least ten seedlings were tested in each experiment. The results are shown in Figure 14. As shown, A19 exhibits a strong inhibitory effect on Arabidopsis seedlings and has the potential for use as a pesticide.

[0250] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. Artemisinin synthase, characterized in that The artemisinin synthase includes (a) a complete protein or a functional protein fragment derived from a plant; or (b) a derived artemisinin synthase having at least 80% amino acid sequence homology with (a).

2. The artemisinin synthase according to claim 1, characterized in that The plant is selected from the family Asteraceae.

3. The artemisinin synthase according to claim 1 or 2, characterized in that The plant is selected from the genus Artemisia.

4. The artemisinin synthase according to claim 3, characterized in that The plants described include Artemisia argyi.

5. The artemisinin synthase according to any one of claims 1 to 4, characterized in that The artemisinin synthase catalyzes the synthesis of artemisinin in the expression system.

6. The artemisinin synthase according to claim 5, characterized in that The expression system includes a fungal expression system.

7. The artemisinin synthase according to claim 6, characterized in that The fungal expression system includes a yeast expression system.

8. The artemisinin synthase according to claim 7, characterized in that The yeast expression system includes a Saccharomyces cerevisiae expression system.

9. The artemisinin synthase according to claim 8, characterized in that The saccharomyces cerevisiae is a recombinant saccharomyces cerevisiae, and the recombinant saccharomyces cerevisiae contains a farnesene pyrophosphate synthase gene.

10. The artemisinin synthase according to claim 9, characterized in that The copy number of the farnesene pyrophosphate synthase gene in the recombinant Saccharomyces cerevisiae genome is 1 to 3, further 2.

11. The artemisinin synthase according to claim 9 or 10, characterized in that The number of at least one mevalonate pathway-related gene in the recombinant Saccharomyces cerevisiae genome is greater than 1.

12. The artemisinin synthase according to claim 11, characterized in that The copy number of at least one of the ERG10 gene, ERG13 gene, tHMG1 gene, ERG12 gene, ERG8 gene, MVD1 gene, or IDI1 gene is 1 to 3, preferably 2 or 3.

13. The artemisinin synthase according to any one of claims 8 to 12, characterized in that The suppressor GAL80 gene and / or the activation cis-element or its fragment within the range of -175 to -220 upstream of the ERG9 promoter are knocked out in the Saccharomyces cerevisiae genome.

14. The artemisinin synthase according to any one of claims 1 to 13, wherein the amino acid sequence of the artemisinin synthase comprises (a) or (b): (a) SEQ ID NO.1 (b) a derivative artemisinin synthase having at least 80%, 81%, 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97% or 99% amino acid sequence identity with (a).

15. A nucleic acid molecule comprising a nucleotide sequence encoding the artemisinin synthase according to any one of claims 1 to 14 or a nucleotide sequence complementary to the base thereof.

16. The nucleic acid molecule according to claim 15, characterized in that The nucleic acid molecule comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO.

3.

17. A recombinant expression vector containing the nucleic acid molecule according to claim 15 or 16, characterized in that: The original vector of the recombinant expression vector includes pZY900, pYR017, pYR018 or pK1URA100.

18. A transformant containing the nucleic acid molecule according to claim 15 or 16, or containing the recombinant expression vector according to claim 17, wherein the host cell of the transformant is selected from a fungal expression system, preferably a yeast expression system, and more preferably a Saccharomyces cerevisiae expression system.

19. Recombinant Saccharomyces cerevisiae, characterized in that The recombinant Saccharomyces cerevisiae contains the nucleic acid molecule according to claim 15 or 16, or contains the recombinant expression vector according to claim 17, and the recombinant Saccharomyces cerevisiae also contains a farnesene pyrophosphate synthase gene.

20. The recombinant Saccharomyces cerevisiae according to claim 19, characterized in that The copy number of the farnesene pyrophosphate synthase gene in the recombinant Saccharomyces cerevisiae genome is 1 to 3, further 2.

21. The recombinant Saccharomyces cerevisiae according to claim 20, characterized in that The number of at least one mevalonate pathway-related gene in the recombinant Saccharomyces cerevisiae genome is greater than 1.

22. The recombinant Saccharomyces cerevisiae according to claim 21, characterized in that The copy number of at least one of the ERG10 gene, ERG13 gene, tHMG1 gene, ERG12 gene, ERG8 gene, MVD1 gene, or IDI1 gene is 1 to 3, preferably 2 or 3.

23. The recombinant Saccharomyces cerevisiae according to any one of claims 19 to 22, characterized in that: The suppressor GAL80 gene and / or the activation cis-element or its fragment within the range of -175 to -220 upstream of the ERG9 promoter are knocked out in the Saccharomyces cerevisiae genome.

24. The method for preparing the recombinant Saccharomyces cerevisiae according to any one of claims 19 to 23, characterized in that: The preparation method comprises the steps of: (a) transforming the nucleic acid molecule according to claim 15 or 16 or the recombinant expression vector according to claim 17 into a Saccharomyces cerevisiae cell; (b) increasing the copy number of farnesene pyrophosphate synthase gene and / or mevalonate pathway-related genes in the genome of Saccharomyces cerevisiae by gene editing; The order of implementing the steps (a) and (b) is not critical.

25. The preparation method according to claim 23, characterized in that: The copy number of the farnesene pyrophosphate synthase gene is 1 to 3, and further is 2.

26. The preparation method according to claim 24 or 25, characterized in that: The copy number of at least one of the ERG10 gene, ERG13 gene, tHMG1 gene, ERG12 gene, ERG8 gene, MVD1 gene, or IDI1 gene is 1 to 3, preferably 2 or 3.

27. The preparation method according to any one of claims 24 to 26, characterized in that: The preparation method comprises: transforming a suppressor GAL80 gene knockout expression cassette and / or an ERG9 promoter upstream activation cis-element knockout or partial knockout expression cassette into a cerevisiae yeast cell.

28. A method for preparing artemisinin, characterized in that: The preparation method comprises fermenting and culturing the transformant according to claim 18, or fermenting and culturing the recombinant brewer's yeast according to any one of claims 19 to 23, or fermenting and culturing the recombinant brewer's yeast prepared by the preparation method according to any one of claims 24 to 27, and recovering artemisinin from the fermentation product.

29. The preparation method according to claim 28, characterized in that: The recovery method includes extracting the fermentation product using an oil phase.

30. The preparation method according to claim 29, characterized in that: The oil phase includes n-hexane, n-dodecane and / or isopropyl myristate.

31. The preparation method according to claim 29, characterized in that: The recovery method is as follows: during the fermentation process, an oil phase is added, and after the fermentation is completed, the oil phase is collected to separate and obtain artemisinin enriched in the oil phase.

32. The preparation method according to claim 31, characterized in that: The method of collecting the oil phase includes centrifugation.

33. The preparation method according to claim 31, characterized in that: The separation method includes distillation.

34. Use of artemisinin obtained by the preparation method according to any one of claims 28 to 33 in the preparation of antibacterial products, insecticide products or herbicidal products.

35. The use according to claim 34, characterized in that The types of microorganisms that the antimicrobial product can inhibit or kill include fungi.

36. The use according to claim 35, characterized in that The fungi include one or more of the genera Rhizoctonia, Plasmodium, Pythium, Phytophthora, Rhizopus, Black Rot Shell, Black Star, Chain Sclerotinia, Powdery Mildew, Pear-shaped spores, Powdery Spores, Verticillium, Botrytis, Alternaria, Fusarium, Scabrospora, Gibberellum, Colletotrichum, and Puccinia.

37. The use according to claim 36, characterized in that The Rhizoctonia fungi include Rhizoctonia solani.

38. The use according to claim 34, characterized in that The insecticidal product can repel or kill insects of the family Culicidae, family Miridae or family Planthoppers.

39. The use according to claim 38, characterized in that The mosquitoes include one or more of the genera Aedes, Anopheles, Armigeres, Coquillettidia, Culex, culiseta, Ficalbia, Heizmznnia, Hemagogus, Hodgesia, Malaya, Mansonia, Minmomyia, Orthopodomyia, Sabethes, Topomyia, Toxorhynchites, Tripteroides, Udaya, and Uranotaenia.

40. The use according to claim 39, characterized in that The insects of the genus Aedes include Aedes aegypti.

41. The use according to claim 38, characterized in that The Miridae insects include Genus Acomocera, Genus Adelphococris, Genus Agnocoris, Genus Alcecoris, Genus Alloeotomus, Genus Allorhinocoris, Genus Angerianus, Genus Apilophorus, Genus Apolygopsis, Genus Apolygus, Genus Arbolygus, Genus Argenis, Genus Astroscopometopus ... Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Bertsa), Blepharidopterus, Bothriomiris, Bothynotus, Brachycoleus, Bryocoris, Campylotropis, Capsodes, Capsus, Castanopsides, Charagochilus, Cheilocapsidea, Cheilocapsus, Chilocrates, Chrysorrhanis, Cimicicapsus), Genus Cimidaeorus, Genus Closterotomus, Genus Coridromius, Genus Creontiades, Genus Cylapofulvuitius, Genus Cylapomorpha, Genus Cyllecoris, Genus Cyphodemidea, Genus Cyrtopeltis, Genus Cyrtorhinus ...Deraeocoris), Dichrooscytus, Dicyphus, Dimia, Dioclerus, Dolichomiris, Dortus, Dryophilocoris, Ectmetopterus, Elthemidea, Eolygus, Erimiris, Ernestinus, Eupachypeltis, Eurvopicoris, Eurystylomorpha), Genus Eurystylopsis, Genus Eurystylus, Genus Excentricus, Genus Felisacus, Genus Finqulus, Genus Fulvius, Genus Globiceps, Genus Guisardinus, Genus Guisardus, Genus Halticus, Genus Harpedona, Genus Hekista, Genus Helopeltis, Genus Heterolyqus, Genus Heteropantilius), Genus Hyalopeplinus, Genus Hyalopeplus, Genus Hyoidea, Genus Isabel, Genus Isometopidea, Genus Isometopus, Genus Jessopocoris, Genus Labopidea, Genus Labops, Genus Lasiomiris, Genus Latizanchius, GenusLeptopterna), Genus Liistonotus, Genus Loristes, Genus Lygidea, Genus Lygocorides, Genus Lygocoris, Genus Lygus, Genus Macrolonius, Genus Macrolophus, Genus Macrolygus, Genus Malacocorisella, Genus Mansoniella, Genus Mecistoscelis, Genus Mecomma, Genus Megacoelum, Genus Mermitelocerus), Metasequoiamiris, Michailocoris, Monalocoris, Myiomma, Myrmecophyes, Myrmecoris, Mystilus, Neolygus, Nesidiocoris, Nicostratus, Notostir, Onomaus, Orientomiris, Orhocephalus, Orthops), Genus Orthotylus, Genus Pachypeltis, Genus Paloniella, Genus Pantilius, Genus Paracyphodema, Genus Paramiridius, Genus Paranx, Genus Parapachypeltis, Genus Peltidolygus, Genus Peritropis, GenusPhytocoridea), Genus Phytocoris, Genus Pinalitus, Genus Polymerus, Genus Poppiocapsidea, Genus Proboscidocoris, Genus Prodromus, Genus Prolygus, Genus Psallops, Genus Pseudodoniella, Genus Pseudoloxops, Genus Raawelellus, Genus Reuterista, Genus Rhabdomiris, Genus Rhinocylapidius, Genus Rhinomiris), Genus Rhopaliceschatus, Genus Sabactiopus, Genus Salignus, Genus Scirtetellus, Genus Sinevi, Genus Singhalesia, Genus Sophianus, Genus Stenodema, Genus Stenotus, Genus Stethoconus, Genus Strongylocoris, Genus Sulawesifaulvius, Genus Tailorilygus, Genus Teratocoris, Genus The invention relates to one or more of the genera Termatophylum, Genus Tinginotopsis, Genus Tinginotum, Genus Tolongia, Genus Totta, Genus Trigonotylus, Genus Tupiocoris, Genus Ulmica, Genus Ulmocyllus, Genus Zanchius and Genus Zonodoropsis.

42. The use according to claim 41, characterized in that The insects of the genus Adelphococris include the black stink bug.

43. The use according to claim 38, characterized in that The planthopper family includes one or more of the genera Nilaparvata, Unkanodes, Neunkanodes, Laodelphax, Ribautodelphax, Megadelphax, Muirodelphax, Fangdelphax, Neoterthrona, Ramidelphax, Neuterthron, Miranus, Sogata, Parasogata, Neometopina, Changeondelphax, Chloriona, Paradelphacodes, and Muellerianella.

44. The use according to claim 38, characterized in that The Delphacidae insects include brown planthopper (Delphacidae).

45. The use according to claim 34, characterized in that The plant species that can be inhibited or killed by herbicidal products include one or more of the families Cruciferae, Asteraceae, Solanaceae, Convolvulaceae, Rubiaceae, Crassulaceae, Cannabaceae, Polygonaceae, Caryophyllaceae, Amaranthaceae, Portulacaceae, Ranunculaceae, Rosaceae, Plantaginaceae, Vitaceae, Euphorbiaceae, Apiaceae, Lamiaceae, Malvaceae, Poaceae, Cyperaceae, Juncaceae, Liliaceae, Commelinaceae, Amaryllis, and Alismataceae.

46. ​​The use according to claim 45, characterized in that The cruciferous plants include Arabidopsis, Alliaria, Alyssum, Aphragmus, Arabidopsis, Arabis, Armoracia, Atelanthera, Barbarea, Berteroa, Berteroella, Brassica, Braya, Bunias, Camelina, Capsella, Cardamine, Cardaria, Cheiranthus, Chorispora, Christolea, Clausia, Cochlearia, Coelonema, Conringia, Coronopus, Crambe, Cryptospora, Descurainia, Dilophia, Dimorphostemon, Diplotaxis, Dipoma, Diptychocarpus, Dontostemon, Draba, Eruca, Erysimum, Euclidium, Eutrema, Goldbachia, Hedinia, Hemilophia, Hesperis, Hymenolobus, Iberis, Isatis, Lachnos nucifera, noloma), Lepidium, Leptaleum, Lignariella, Litwinowia, Lobularia, Loxostemon, Macropodium, Malcolmia, Matthiola, Megacarpaea, Megadenia, Microsisymbrium, Microstigma, Nasturtium,Neomartinella, Neslia, Orychophragmus, Pachypterygium, Parrya, Parryodes, Pegaeophyton, Phaeonychium, Platycraspedum, Ptilotricum, Pugionium, Pycnoplinthus, Raphanus, Rorippa, Sinapis, Sisymbrium, Smelowskia, Sophora japonica, lms-Laubachia), Sophiopsis, Spirohynchus, Staintoniella, Sterigmostemum, Stevenia, Stroganowia, Synstemon, Syrenia, Taphrospermum, Tauscheria, Tetracme, Thellungiella, Thlaspi, Torularia, Turritis, Yinshania.

47. The use according to claim 46, characterized in that The cruciferous plants include one or more of Arabidopsis, Capsella, Cardamine, Lepidium, Oryza, Lepidium, Halona, ​​Saccharina, Descurainia, and Herba Epipremnum.

48. The use according to claim 47, characterized in that The Arabidopsis plant includes Arabidopsis thaliana.

49. An antibacterial agent, characterized in that The antibacterial agent comprises artemisinin obtained by the preparation method according to any one of claims 28 to 33.

50. The antibacterial agent according to claim 49, characterized in that The effective dosage of artemisinin is 10 to 100 ppm.

51. The antibacterial agent according to claim 49, characterized in that The dosage form of the antibacterial agent includes tablets, injections, capsules, powders, premixes, granules, soluble powders, oral solutions, gels or ointments.

52. The antibacterial agent according to any one of claims 49 to 51, characterized in that The types of microorganisms that the antimicrobial agent can inhibit or kill include fungi.

53. The antibacterial agent according to claim 52, characterized in that The fungi include one or more of the genera Rhizoctonia, Plasmodium, Pythium, Phytophthora, Rhizopus, Black Rot Shell, Black Star, Chain Sclerotinia, Powdery Mildew, Pear-shaped spores, Powdery Spores, Verticillium, Botrytis, Alternaria, Fusarium, Scabrospora, Gibberellum, Colletotrichum, and Puccinia.

54. The antibacterial agent according to claim 53, characterized in that The Rhizoctonia fungi include Rhizoctonia solani.

55. An insecticide, characterized in that The insecticide comprises artemisinin obtained by the preparation method according to any one of claims 28 to 33.

56. The insecticide according to claim 55, characterized in that The effective dosage of artemisinin is 500 to 5000 ppm.

57. The insecticide according to claim 55, characterized in that The dosage form of the insecticide includes water emulsion, suspension, dry powder, wettable powder, emulsifiable concentrate, thermal fog, aerosol, soluble granule, fumigant, bait, granule or slow-release agent.

58. The insecticide according to any one of claims 55 to 57, characterized in that The insecticide can repel or kill insects of the family Culicidae, family Miridae or family Delphacidae.

59. The insecticide according to claim 58, characterized in that The mosquitoes include one or more of the genera Aedes, Anopheles, Armigeres, Coquillettidia, Culex, culiseta, Ficalbia, Heizmznnia, Hemagogus, Hodgesia, Malaya, Mansonia, Minmomyia, Orthopodomyia, Sabethes, Topomyia, Toxorhynchites, Tripteroides, Udaya, and Uranotaenia.

60. The insecticide according to claim 59, characterized in that The insects of the genus Aedes include Aedes aegypti.

61. The insecticide according to claim 58, characterized in that The Miridae insects include Genus Acomocera, Genus Adelphococris, Genus Agnocoris, Genus Alcecoris, Genus Alloeotomus, Genus Allorhinocoris, Genus Angerianus, Genus Apilophorus, Genus Apolygopsis, Genus Apolygus, Genus Arbolygus, Genus Argenis, Genus Astroscopometopus ... Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Apolygus, Genus Bertsa), Blepharidopterus, Bothriomiris, Bothynotus, Brachycoleus, Bryocoris, Campylotropis, Capsodes, Capsus, Castanopsides, Charagochilus, Cheilocapsidea, Cheilocapsus, Chilocrates, Chrysorrhanis, Cimicicapsus), Genus Cimidaeorus, Genus Closterotomus, Genus Coridromius, Genus Creontiades, Genus Cylapofulvuitius, Genus Cylapomorpha, Genus Cyllecoris, Genus Cyphodemidea, Genus Cyrtopeltis, Genus Cyrtorhinus ...Deraeocoris), Dichrooscytus, Dicyphus, Dimia, Dioclerus, Dolichomiris, Dortus, Dryophilocoris, Ectmetopterus, Elthemidea, Eolygus, Erimiris, Ernestinus, Eupachypeltis, Eurvopicoris, Eurystylomorpha), Genus Eurystylopsis, Genus Eurystylus, Genus Excentricus, Genus Felisacus, Genus Finqulus, Genus Fulvius, Genus Globiceps, Genus Guisardinus, Genus Guisardus, Genus Halticus, Genus Harpedona, Genus Hekista, Genus Helopeltis, Genus Heterolyqus, Genus Heteropantilius), Genus Hyalopeplinus, Genus Hyalopeplus, Genus Hyoidea, Genus Isabel, Genus Isometopidea, Genus Isometopus, Genus Jessopocoris, Genus Labopidea, Genus Labops, Genus Lasiomiris, Genus Latizanchius, GenusLeptopterna), Genus Liistonotus, Genus Loristes, Genus Lygidea, Genus Lygocorides, Genus Lygocoris, Genus Lygus, Genus Macrolonius, Genus Macrolophus, Genus Macrolygus, Genus Malacocorisella, Genus Mansoniella, Genus Mecistoscelis, Genus Mecomma, Genus Megacoelum, Genus Mermitelocerus), Metasequoiamiris, Michailocoris, Monalocoris, Myiomma, Myrmecophyes, Myrmecoris, Mystilus, Neolygus, Nesidiocoris, Nicostratus, Notostir, Onomaus, Orientomiris, Orhocephalus, Orthops), Genus Orthotylus, Genus Pachypeltis, Genus Paloniella, Genus Pantilius, Genus Paracyphodema, Genus Paramiridius, Genus Paranx, Genus Parapachypeltis, Genus Peltidolygus, Genus Peritropis, GenusPhytocoridea), Genus Phytocoris, Genus Pinalitus, Genus Polymerus, Genus Poppiocapsidea, Genus Proboscidocoris, Genus Prodromus, Genus Prolygus, Genus Psallops, Genus Pseudodoniella, Genus Pseudoloxops, Genus Raawelellus, Genus Reuterista, Genus Rhabdomiris, Genus Rhinocylapidius, Genus Rhinomiris), Genus Rhopaliceschatus, Genus Sabactiopus, Genus Salignus, Genus Scirtetellus, Genus Sinevi, Genus Singhalesia, Genus Sophianus, Genus Stenodema, Genus Stenotus, Genus Stethoconus, Genus Strongylocoris, Genus Sulawesifaulvius, Genus Tailorilygus, Genus Teratocoris, Genus The invention relates to one or more of the genera Termatophylum, Genus Tinginotopsis, Genus Tinginotum, Genus Tolongia, Genus Totta, Genus Trigonotylus, Genus Tupiocoris, Genus Ulmica, Genus Ulmocyllus, Genus Zanchius and Genus Zonodoropsis.

62. The insecticide according to claim 61, characterized in that The insects of the genus Adelphococris include the black stink bug.

63. The insecticide according to claim 58, characterized in that The planthopper family includes one or more of the genera Nilaparvata, Unkanodes, Neunkanodes, Laodelphax, Ribautodelphax, Megadelphax, Muirodelphax, Fangdelphax, Neoterthrona, Ramidelphax, Neuterthron, Miranus, Sogata, Parasogata, Neometopina, Changeondelphax, Chloriona, Paradelphacodes, and Muellerianella.

64. The use according to claim 58, characterized in that The family Delphacidae includes the brown planthopper (Delphacidae).

65. A herbicide, characterized in that The herbicide comprises artemisinin obtained by the preparation method according to any one of claims 28 to 33.

66. The herbicide according to claim 65, characterized in that The effective dosage of artemisinin is 25 to 200 ppm.

67. The herbicide according to claim 65, characterized in that The dosage form of the herbicide includes aqueous solution, emulsifiable concentrate, suspension, aqueous emulsion, microemulsion, oil suspension, microcapsule suspension, soluble liquid, film-spreading oil, wettable powder, soluble powder, soluble granule, dry suspension or water-dispersible granule.

68. The herbicide according to any one of claims 65 to 67, characterized in that The herbicide can inhibit or kill plant species including one or more of the families Cruciferae, Asteraceae, Solanaceae, Convolvulaceae, Rubiaceae, Crassulaceae, Cannabaceae, Polygonaceae, Caryophyllaceae, Amaranthaceae, Portulacaceae, Ranunculaceae, Rosaceae, Plantaginaceae, Vitaceae, Euphorbiaceae, Apiaceae, Lamiaceae, Malvaceae, Poaceae, Cyperaceae, Juncaceae, Liliaceae, Commelinaceae, Amaryllisaceae, and Alismataceae.

69. The herbicide according to claim 68, characterized in that The cruciferous plants include Arabidopsis, Alliaria, Alyssum, Aphragmus, Arabidopsis, Arabis, Armoracia, Atelanthera, Barbarea, Berteroa, Berteroella, Brassica, Braya, Bunias, Camelina, Capsella, Cardamine, Cardaria, Cheiranthus, Chorispora, Christolea, Clausia, Cochlearia, Coelonema, Conringia, Coronopus, Crambe, Cryptospora, Descurainia, Dilophia, Dimorphostemon, Diplotaxis, Dipoma, Diptychocarpus, Dontostemon, Draba, Eruca, Erysimum, Euclidium, Eutrema, Goldbachia, Hedinia, Hemilophia, Hesperis, Hymenolobus, Iberis, Isatis, Lachnos nucifera, noloma), Lepidium, Leptaleum, Lignariella, Litwinowia, Lobularia, Loxostemon, Macropodium, Malcolmia, Matthiola, Megacarpaea, Megadenia, Microsisymbrium, Microstigma, Nasturtium,Neomartinella, Neslia, Orychophragmus, Pachypterygium, Parrya, Parryodes, Pegaeophyton, Phaeonychium, Platycraspedum, Ptilotricum, Pugionium, Pycnoplinthus, Raphanus, Rorippa, Sinapis, Sisymbrium, Smelowskia, Sophora japonica, lms-Laubachia), Sophiopsis, Spirohynchus, Staintoniella, Sterigmostemum, Stevenia, Stroganowia, Synstemon, Syrenia, Taphrospermum, Tauscheria, Tetracme, Thellungiella, Thlaspi, Torularia, Turritis, Yinshania.

70. The herbicide according to claim 69, characterized in that The cruciferous plants include one or more of Arabidopsis, Capsella, Cardamine, Lepidium, Oryza, Lepidium, Halona, ​​Saccharina, Descurainia, and Herba Epipremnum.

71. The herbicide according to claim 70, characterized in that The Arabidopsis plant includes Arabidopsis thaliana.

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