WO2024082757A1 - 黄酮类化合物及其生物合成相关基因以及其应用 - Google Patents

黄酮类化合物及其生物合成相关基因以及其应用 Download PDF

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WO2024082757A1
WO2024082757A1 PCT/CN2023/109563 CN2023109563W WO2024082757A1 WO 2024082757 A1 WO2024082757 A1 WO 2024082757A1 CN 2023109563 W CN2023109563 W CN 2023109563W WO 2024082757 A1 WO2024082757 A1 WO 2024082757A1
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compound
structural formula
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flavonoid
seq
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French (fr)
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吕雪峰
黄雪年
张伟
张璇
周宇
郭勍
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中国科学院青岛生物能源与过程研究所
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Definitions

  • the invention belongs to the field of microbial gene resources and genetic engineering, and in particular, relates to flavonoid compounds, genes related to their biosynthesis and applications thereof.
  • Flavonoids are a large class of natural products mainly found in plants. They have diverse structural types and a wide range of biological activities. They play an important physiological role in plant growth, regulation and defense. The biosynthetic pathway of flavonoids in plants has been studied very clearly. First, L-phenylalanine is converted into p-coumaroyl-CoA by the action of phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-hydroxycinnamoyl-CoA ligase (4CL). Then, it is condensed with three molecules of malonyl-CoA to form chalcone under the action of chalcone synthase (CHS, III PKS).
  • PAL phenylalanine ammonia lyase
  • C4H cinnamate 4-hydroxylase
  • 4CL 4-hydroxycinnamoyl-CoA ligase
  • CHI Chalcone isomerase
  • FNS flavonoid synthase
  • flavonoids have many applications in food, health care and pharmaceutical development, and play a very important role in people's health. It is reported that flavonoids have a market of about US$200 million per year. Their sources are mainly extracted from plants. Due to the low content in plants and the complex extraction process, flavonoids have not been able to meet the large market demand. Therefore, it is possible to produce flavonoids through microorganisms using synthetic biology technology. Microorganisms lack endogenous genes for synthesizing flavonoids.
  • flavonoids in microorganisms is based on the synthesis pathway of flavonoids in plants. Due to the low matching of the microbial chassis and the enzymatic elements in plants, the output of microbial flavonoid cell factories is very low and cannot meet the requirements of commercialization.
  • the applicant discovered a fungus that can produce flavonoids.
  • the analysis of the synthesis pathway found that the synthesis of fungal flavonoids is significantly different from that of plants, which is a new biosynthetic mechanism.
  • CfoA, CfoK and CfoJ are responsible for catalyzing the synthesis of the core skeleton of flavonoids.
  • the hosts can produce flavonoids.
  • resistance tags are one of the basic elements in the development of microbial genetic manipulation systems and are the most basic guarantee for screening positive transformants during genetic transformation.
  • resistance tags there are few types of resistance tags that can be used for genetic manipulation of filamentous fungi and their universality is poor, which seriously restricts the development of genetic manipulation systems in filamentous fungi.
  • chlorflavonin also known as chlorflavonin, CAS: 23363-64-6
  • dechlorochlorflavonin also known as dechlorochlorflavonin, CAS: 51724-52-8
  • the antibacterial activity evaluation of the compound showed that it has very strong inhibitory activity against some plant pathogenic fungi (Sclerotium uniformis) and human pathogenic fungi (Candida albicans, Aspergillus fumigatus), and has the potential to be developed into pesticide fungicides, antibiotics and food preservatives.
  • the gene cluster responsible for the biosynthesis of chlorflavin contains a self-resistance gene cfoL encoding acetolactate synthase, which can effectively confer resistance to chlorflavin. Therefore, the combination of chlorflavin and cfoL can be used for genetic manipulation of filamentous fungi.
  • the discovery of cfoL enriched the types of genetic manipulation resistance tags and laid the foundation for the development of genetic manipulation systems in fungi.
  • the present invention provides new uses of chlorflavonoids or its derivatives.
  • the present invention provides the use of chloranil or dechloranil in inhibiting the growth of microorganisms, or in preparing an agent for inhibiting the growth of microorganisms, or in preparing a drug for treating a disease caused by a microorganism.
  • the agent that inhibits microbial growth is an antibiotic.
  • the microorganism is Sclerotium rolfsii Sacc., Candida albicans or Aspergillus fumigatus.
  • the disease is a plant disease, for example, Sclerotium ruthenicum infects crops and causes white rot in peanut, rice, cucumber, etc.
  • the reagents and drugs of the present invention can be prepared into different dosage forms, including liquid preparations, solid preparations, semi-solid preparations or gaseous preparations.
  • the reagent and medicine of the present invention may contain other auxiliary agents in addition to chloranil or dechloranil as active ingredients.
  • the minimum inhibitory concentration of chloranthoxamic acid to inhibit Sclerotium uniformis can be as low as 2 ⁇ g/mL or less; the minimum inhibitory concentration of dechlorochloranthoxamic acid to inhibit Sclerotium uniformis can be as low as 4 ⁇ g/mL or less.
  • the present invention provides the use of chlorflavin in inhibiting plant growth, or in inhibiting plant seed germination and rhizome sprouting, or in preparing an agent for inhibiting plant growth, or in preparing a herbicide; in a preferred embodiment, the plant is Arabidopsis thaliana.
  • the formulation is preferably a liquid formulation.
  • the present invention provides a gene cluster related to flavonoid biosynthesis or a gene related to flavonoid synthesis and applications thereof, and provides novel flavonoid compounds.
  • the present invention provides a gene cluster related to flavonoid biosynthesis or a gene related to flavonoid synthesis.
  • nucleic acid sequence of the gene cluster is shown as SEQ ID No.1.
  • the gene related to flavonoid synthesis is selected from FAD-dependent oxidase CfoG, monooxygenase CfoF, SAM-dependent methyltransferase CfoD, cytochrome P450 enzyme CfoH, ester hydrolase CfoK, Scytalone dehydratase CfoI, SAM-dependent methyltransferase CfoC, NADPH-dependent FMN reductase CfoJ, SAM-dependent methyltransferase CfoB, FAD-dependent oxidase CfoE, hybrid non-ribosomal peptide synthase and polyketide synthase CfoA.
  • FAD-dependent oxidase CfoG FAD-dependent oxidase CfoG
  • monooxygenase CfoF SAM-dependent methyltransferase CfoD
  • cytochrome P450 enzyme CfoH
  • the amino acid sequence of CfoG has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 14; preferably, the CfoG is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoG has at least 99% sequence identity compared to SEQ ID No. 14, and the CfoG is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoG is as shown in SEQ ID No. 14, and its encoding gene sequence is as shown in SEQ ID No. 2.
  • the amino acid sequence of CfoF has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No.15; preferably, the CfoF is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoF has at least 99% sequence identity compared to SEQ ID No.15, and the CfoF is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoF is as shown in SEQ ID No.15, and its encoding gene sequence is as shown in SEQ ID No.3.
  • the amino acid sequence of CfoD has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No.16; preferably, the CfoD is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoD has at least 99% sequence identity compared to SEQ ID No.16, and the CfoD is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoD is as shown in SEQ ID No.16, and its encoding gene sequence is as shown in SEQ ID No.4.
  • the amino acid sequence of CfoH has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 17; preferably, the CfoH is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoH has at least 99% sequence identity compared to SEQ ID No. 17, and the CfoH is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoH is as shown in SEQ ID No. 17, and its encoding gene sequence is as shown in SEQ ID No. 5.
  • the amino acid sequence of CfoK has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No.18; preferably, the CfoK is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoK has at least 99% sequence identity compared to SEQ ID No.18, and the CfoK is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoK is as shown in SEQ ID No.18, and its encoding gene sequence is as shown in SEQ ID No.6.
  • the amino acid sequence of CfoI has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No.19; preferably, the CfoI is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoI has at least 99% sequence identity compared to SEQ ID No.19, and the CfoI is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoI is as shown in SEQ ID No.19, and its encoding gene sequence is as shown in SEQ ID No.7.
  • the amino acid sequence of CfoC has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 20; preferably, the CfoC is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoC has at least 99% sequence identity compared to SEQ ID No. 20, and the CfoC is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoC is as shown in SEQ ID No. 20, and its encoding gene sequence is as shown in SEQ ID No. 8.
  • the amino acid sequence of CfoJ has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 21; preferably, the CfoJ is derived from Aspergillus blanckenii, for example, Aspergillus blanckenii More preferably, the amino acid sequence of CfoJ has at least 99% sequence identity with SEQ ID No.21, and the CfoJ is derived from Aspergillus blancense; the Aspergillus blancense includes Aspergillus blancense MEFC1001. More preferably, the amino acid sequence of CfoJ is as shown in SEQ ID No.21, and the coding gene sequence thereof is as shown in SEQ ID No.9.
  • the amino acid sequence of CfoB has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 22; preferably, the CfoB is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoB has at least 99% sequence identity compared to SEQ ID No. 22, and the CfoB is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoB is as shown in SEQ ID No. 22, and its encoding gene sequence is as shown in SEQ ID No. 10.
  • the amino acid sequence of CfoE has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 23; preferably, the CfoE is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoE has at least 99% sequence identity compared to SEQ ID No. 23, and the CfoE is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoE is as shown in SEQ ID No. 23, and its encoding gene sequence is as shown in SEQ ID No. 11.
  • the amino acid sequence of CfoA has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 24; preferably, the CfoA is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoA has at least 99% sequence identity compared to SEQ ID No. 24, and the CfoA is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoA is as shown in SEQ ID No. 24, and its encoding gene sequence is as shown in SEQ ID No. 12.
  • the Aspergillus flavus MEFC1001 with a preservation number of CGMCC No. 3.15294, is commercially available and is a fungus preserved in the General Microbiology Center of the China Culture Collection Administration.
  • the present invention also provides a new flavonoid compound, wherein the flavonoid compound is selected from one or any several of Compound 2 to Compound 23.
  • the present invention also provides uses of the above-mentioned new flavonoid compounds, which uses include uses in inhibiting the growth of microorganisms, or uses in preparing agents for inhibiting the growth of microorganisms, or uses in preparing drugs for treating diseases caused by microorganisms; in one embodiment, the agent for inhibiting the growth of microorganisms is an antibiotic; in a preferred embodiment, the microorganism is selected from one or any several of Candida albicans, Aspergillus fumigatus, Sclerotium rolfsii Sacc., and Botrytis cinerea.
  • compounds 3, 4 and 13 can inhibit the growth of Candida albicans, Sclerotium solani and Botrytis cinerea.
  • Compound 6 could inhibit the growth of Candida albicans and Aspergillus fumigatus.
  • Compounds 7 and 8 could inhibit the growth of Candida albicans, Aspergillus fumigatus, Sclerotium sclerotiorum and Botrytis cinerea.
  • Compound 10 could inhibit the growth of Candida albicans and Sclerotium uniformis.
  • Compound 14 could inhibit the growth of Candida albicans, Aspergillus fumigatus and Sclerotium sclerotiorum.
  • Compound 19 could inhibit the growth of Candida albicans and Sclerotium uniformis.
  • the reagents and drugs of the present invention can be prepared into different dosage forms, including liquid preparations, solid preparations, semi-solid preparations or gaseous preparations.
  • the reagents and drugs of the present invention may contain other auxiliary agents in addition to the above-mentioned compounds as active ingredients.
  • the present invention also provides a genetically engineered strain, wherein the genetically engineered strain is obtained by subjecting the above-mentioned gene related to flavonoid synthesis in Aspergillus leucoderma to gene mutation.
  • the starting strain of the genetically engineered strain can also be selected from Aspergillus, Aspergillus nidulans, yeast, Escherichia coli, plants or animals; preferably, Aspergillus, for example, Aspergillus leucophylla and Aspergillus oryzae.
  • the gene related to flavonoid synthesis is selected from one or any several of the above-mentioned FAD-dependent oxidase CfoG, monooxygenase CfoF, SAM-dependent methyltransferase CfoD, cytochrome P450 enzyme CfoH, ester hydrolase CfoK, Scytalone dehydratase CfoI, SAM-dependent methyltransferase CfoC, NADPH-dependent FMN reductase CfoJ, SAM-dependent methyltransferase CfoB, FAD-dependent oxidase CfoE, and hybrid non-ribosomal peptide synthase and polyketide synthase CfoA.
  • the mutation described in the present invention includes the loss of gene function or activity caused by gene deletion, gene insertion or gene replacement.
  • the gene mutation can be achieved by conventional techniques in the art, for example, gene knock-in or gene knock-out by homologous recombination to cause loss of gene function or activity; or, gene editing, such as zinc finger endonucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or CRISPR technology mutates the above genes, resulting in loss of gene function or activity.
  • gene editing such as zinc finger endonucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or CRISPR technology mutates the above genes, resulting in loss of gene function or activity.
  • the present invention also provides the use of the above genetically engineered strain in the production of flavonoid compounds.
  • the present invention also provides a method for preparing flavonoid compounds, which comprises the step of fermenting the above genetically engineered strain.
  • the flavonoid compound is selected from one or any several of Compound 2 to Compound 23.
  • the mutated gene is CfoB
  • the flavonoid compound is one or any combination of Compound 5, Compound 6, and Compound 7;
  • the mutated gene is CfoC
  • the flavonoid compound is one or any combination of Compound 8, Compound 9, Compound 10, Compound 11, and Compound 12;
  • the mutated gene is CfoD
  • the flavonoid compound is one or any combination of Compound 3, Compound 4, Compound 12, Compound 13, and Compound 14;
  • the mutated gene is CfoE
  • the flavonoid compound is one or any combination of Compound 2 and Compound 4;
  • the mutated gene is CfoF
  • the flavonoid compound is one or any combination of Compound 3, Compound 4, Compound 12, and Compound 13;
  • the mutated gene is CfoG
  • the flavonoid compound is one or any combination of Compound 12 and Compound 15;
  • the mutated gene is CfoH
  • the flavonoid compound is one or any combination of Compound 4 and Compound 12;
  • the mutated gene is CfoI
  • the flavonoid compound is one or any combination of Compound 3, Compound 4, and Compound 12;
  • the mutated gene is CfoJ
  • the flavonoid compound is one or any combination of Compound 16, Compound 17, Compound 18, and Compound 19;
  • the mutated gene is CfoK
  • the flavonoid compound is one or any several of Compound 20, Compound 21, Compound 22, and Compound 23.
  • the present invention provides genes related to flavonoid synthesis and their application in the preparation of flavonoid compounds.
  • the gene related to flavonoid synthesis is selected from one or any combination of ester hydrolase CfoK, NADPH-dependent FMN reductase CfoJ, hybrid non-ribosomal peptide synthase and polyketide synthase CfoA.
  • the amino acid sequence of CfoK has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No.18; preferably, the CfoK is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoK has at least 99% sequence identity compared to SEQ ID No.18, and the CfoK is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoK is as shown in SEQ ID No.18, and its encoding gene sequence is as shown in SEQ ID No.6.
  • the amino acid sequence of CfoJ has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No.21; preferably, the CfoJ is derived from Aspergillus, for example, Aspergillus MEFC1001; more preferably, the amino acid sequence of CfoJ has at least 99% sequence identity compared to SEQ ID No.21, and the CfoJ is derived from Aspergillus; the Aspergillus includes Aspergillus MEFC1001. More preferably, the amino acid sequence of CfoJ is as shown in SEQ ID No.21, and its encoding gene sequence is as shown in SEQ ID No.9.
  • the amino acid sequence of CfoA has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No.24; preferably, the CfoA is derived from Aspergillus blanckenii, for example, Aspergillus blanckenii MEFC1001; more preferably, the amino acid sequence of CfoA has at least 99% sequence identity compared to SEQ ID No.24.
  • the CfoA is derived from Aspergillus blanckenii, and the Aspergillus blanckenii includes Aspergillus blanckenii MEFC1001. More preferably, the amino acid sequence of CfoA is shown in SEQ ID No.24, and the coding gene sequence thereof is shown in SEQ ID No.12.
  • the Aspergillus flavus MEFC1001 with a preservation number of CGMCC No. 3.15294, is commercially available and is a fungus preserved in the General Microbiology Center of the China Culture Collection Administration.
  • the present invention provides the use of the above-mentioned gene related to flavonoid synthesis in the preparation of flavonoid compounds.
  • the present invention also provides the use of the above-mentioned genes related to flavonoid synthesis in the preparation of genetically engineered strains capable of producing flavonoid compounds.
  • the present invention also provides a genetically engineered strain capable of producing flavonoid compounds, wherein the genetically engineered strain is a genetically engineered strain obtained by introducing the above-mentioned gene related to flavonoid synthesis into a starting strain.
  • the "introduction” includes the step of expressing the above-mentioned target gene in the starting strain, preferably, overexpressing it.
  • the target gene is constructed into an expression vector, and the expression vector is transferred into a host cell to express the target gene, preferably, overexpressing it.
  • the "introduction" includes inserting the target gene into the genome of the host cell; preferably, the insertion into the genome of the host cell can be carried out by a homologous recombination double exchange method; in one embodiment, the target gene and the homologous arms can be inserted into the vector, and then the vector can be transferred into the host cell, and the homologous arms are used to undergo homologous recombination double exchange with the host cell genome to insert the target gene into the appropriate genomic location; in other embodiments, gene editing can also be used, for example, using the CRISPR/Cas system to cut at the desired genomic site, and inserting the target gene as an exogenous donor into the cutting site.
  • the present invention also provides the use of the above genetically engineered strain in the production of flavonoid compounds.
  • the starting strain of the genetically engineered strain is selected from Aspergillus, Aspergillus nidulans, yeast, Escherichia coli, plants or animals; preferably, Aspergillus, such as Aspergillus leucovorus, Aspergillus oryzae, Aspergillus terreus.
  • the gene related to flavonoid synthesis is selected from one or any combination of CfoA, CfoK and CfoJ.
  • the present invention also provides a method for preparing flavonoid compounds, which comprises the step of fermenting the above genetically engineered strain.
  • the flavonoid compound is selected from one or any several of Compound 22, Compound 24, Compound 25 and Compound 26.
  • the introduction of CfoA alone can produce compound 22 and compound 25
  • the simultaneous introduction of CfoA and CfoK can produce compound 24
  • the simultaneous introduction of CfoA, CfoK and CfoJ can produce compound 24 and compound 26.
  • the gene related to flavonoid synthesis is selected from CfoA, and the flavonoid compound is selected from one or any several of compound 22 and compound 25; the gene related to flavonoid synthesis is selected from CfoA and CfoK, and the flavonoid compound is selected from compound 24; the gene related to flavonoid synthesis is selected from CfoA, CfoK and CfoJ, and the flavonoid compound is selected from one or any several of compound 24 and compound 26.
  • the present invention also provides the use of CfoK and CfoJ.
  • the present invention provides the use of CfoK in the catalytic synthesis of compound 27 and/or compound 24; in other embodiments, the present invention provides the use of CfoJ in the catalytic synthesis of compound 26 and/or compound 3.
  • CfoK can catalyze compound 20 to generate compound 27; CfoK can catalyze compound 22 to generate compound 24; CfoJ can catalyze compound 24 to generate compound 26; CfoJ can catalyze compound 18 to generate compound 3.
  • the present invention also provides a genetically engineered strain introduced with CfoK and/or CfoJ, wherein the starting strain of the genetically engineered strain may be yeast or Escherichia coli.
  • the present invention also provides the use of the above-mentioned genetically engineered strain introduced with CfoK and/or CfoJ in the preparation of Compound 27, Compound 24, Compound 26 or Compound 3.
  • the present application found that the amino acid H at position 33 and the amino acid Y at position 50 from the N-terminus of CfoK are the key sites for its activity.
  • the amino acid at position 33 is mutated (for example, to A or Q) or the amino acid at position 50 is mutated (for example, to A or F)
  • the mutated CfoK cannot catalyze compound 22 to synthesize compound 24.
  • the amino acid P at position 112 and the amino acid W at position 154 from the N-terminus of CfoJ are the key sites for its activity. If the amino acid at position 112 is mutated (for example, mutated to A) or the amino acid at position 154 is mutated (for example, mutated to A), the mutated CfoJ cannot catalyze compound 24 to synthesize compound 26.
  • the present invention provides a resistance gene cfoL and its application.
  • the amino acid sequence of the resistance gene cfoL has at least 80%, 85%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity compared to SEQ ID No. 25; preferably, the resistance gene cfoL is derived from Aspergillus blanc, for example, Aspergillus blanc MEFC1001; more preferably, the amino acid sequence of the resistance gene cfoL has at least 99% sequence identity compared to SEQ ID No.
  • the resistance gene cfoL is derived from Aspergillus blanc; the Aspergillus blanc includes Aspergillus blanc MEFC1001. More preferably, the amino acid sequence of the resistance gene cfoL is as shown in SEQ ID No. 25, and its encoding gene sequence is as shown in SEQ ID No. 13.
  • the Aspergillus fumigatus MEFC1001 with a preservation number of CGMCC 3.15294, is commercially available and is a fungus preserved in the General Microbiology Center of the China Culture Collection Administration.
  • the present invention also provides a vector or a recombinant host cell containing the above-mentioned resistance gene cfoL.
  • the present invention also provides the use of the above-mentioned resistance gene cfoL in resisting chlorflavin or in conferring resistance to chlorflavin to microorganisms sensitive to chlorflavin.
  • a microorganism sensitive to chlorflavin refers to a microorganism whose growth or activity is inhibited in the presence of chlorflavin. For example, compared with the absence of chlorflavin, the growth level of the strain of the microorganism in the presence of chlorflavin is reduced to 0%, 5%, 10%, 20% or 30% of the control level.
  • the dosage of chlorflavin can be 1 ⁇ g/mL-200 ⁇ g/mL, for example, 2 ⁇ g/mL, 5 ⁇ g/mL, 10 ⁇ g/mL, 15 ⁇ g/mL, 20 ⁇ g/mL, 25 ⁇ g/mL, 30 ⁇ g/mL, 35 ⁇ g/mL, 40 ⁇ g/mL, 45 ⁇ g/mL, 50 ⁇ g/mL, 60 ⁇ g/mL, 70 ⁇ g/mL, 80 ⁇ g/mL, 90 ⁇ g/mL, 100 ⁇ g/mL or 150 ⁇ g/mL.
  • the present invention does not impose a strict limit on the dosage level of chlorflavin.
  • the dosage level of chlorflavin that can make microorganisms sensitive or lethal can be obtained according to conventional techniques and operations in the art.
  • To confer resistance to chlorflavin to chlorflavin refers to introducing the resistance gene cfoL into the chlorflavin-sensitive microorganism, which can alleviate or release the inhibition of growth or activity of the microorganism caused by chlorflavin.
  • the present invention also provides the use of the resistance gene cfoL as an antibiotic resistance screening marker or screening tag; preferably, the antibiotic is chlorflavin.
  • the selection marker or selection tag can be placed in a vector/plasmid, and the vector/plasmid is introduced into a target microorganism, which can cause the microorganism to produce resistance to antibiotics.
  • the screening marker or screening label can be understood as that the target microorganism introduced with the resistance gene cfoL can maintain the growth state in the presence of chlorflavin, while the microorganism without the resistance gene cfoL cannot maintain the growth state in the presence of chlorflavin, so that the target microorganism introduced with the resistance gene cfoL can be screened; based on this, the resistance gene cfoL can be used as a resistance screening marker or screening label for chlorflavin.
  • the present invention also provides the use of the resistance gene cfoL in preparing a genetically engineered strain that resists/tolerates chlorflavin or produces resistance to chlorflavin.
  • the present invention also provides a method for preparing a genetically engineered strain that is resistant to/tolerant to or resistant to chlorflavin, the method comprising the step of introducing the resistance gene cfoL into the strain.
  • the "introduction” includes the step of expressing the above-mentioned target gene in the starting strain, preferably, overexpressing it.
  • the target gene is constructed into an expression vector, and the expression vector is transferred into a host cell to express the target gene, preferably, overexpressing it.
  • the "introduction" includes inserting the target gene into the genome of the host cell; preferably, the insertion into the genome of the host cell can be carried out by a homologous recombination double exchange method; in one embodiment, the target gene and the homologous arms can be inserted into the vector, and then the vector can be transferred into the host cell, and the homologous arms are used to undergo homologous recombination double exchange with the host cell genome to insert the target gene into the appropriate genomic location; in other embodiments, gene editing can also be used, for example, using the CRISPR/Cas system to cut at the desired genomic site, and inserting the target gene as an exogenous donor into the cutting site.
  • the starting strain of the genetically engineered strain is a strain sensitive to chlorflavin; introducing the above-mentioned resistance gene cfoL into the strain sensitive to chlorflavin can make the strain resistant to chlorflavin.
  • being sensitive to chlorflavin means that the growth or activity of the strain or microorganism is inhibited in the presence of chlorflavin.
  • the growth level of the strain or microorganism is reduced to 0%, 5%, 10%, 20% or 30% of the control level compared to the absence of chlorflavin.
  • the dosage of chlorflavin can be 1 ⁇ g/mL-200 ⁇ g/mL, for example, 2 ⁇ g/mL, 5 ⁇ g/mL, 10 ⁇ g/mL, 15 ⁇ g/mL, 20 ⁇ g/mL, 25 ⁇ g/mL, 30 ⁇ g/mL, 35 ⁇ g/mL, 40 ⁇ g/mL, 45 ⁇ g/mL, 50 ⁇ g/mL, 60 ⁇ g/mL, 70 ⁇ g/mL, 80 ⁇ g/mL, 90 ⁇ g/mL, 100 ⁇ g/mL or 150 ⁇ g/mL.
  • the present invention does not impose a strict limit on the dosage level of chlorflavin.
  • the dosage level of chlorflavin that can make microorganisms sensitive or lethal can be obtained according to conventional techniques and operations in the art.
  • resistance to chlorflavin, tolerance to chlorflavin or resistance to chlorflavin are similar in meaning, and all refer to the target microorganism or target strain being able to maintain growth in the presence of chlorflavin.
  • resistance to chlorflavin, tolerance to chlorflavin or resistance to chlorflavin means that the above-mentioned resistance gene cfoL is introduced into the above-mentioned chlorflavin-sensitive microorganism, which can alleviate or relieve the inhibition of growth or activity of the microorganism caused by chlorflavin.
  • a conventional method in the art can be used to obtain a strain sensitive to chlorflavin.
  • the starting strain of the genetically engineered strain comprises a fungus, such as Aspergillus, such as Aspergillus fumigatus.
  • the starting strain of the genetically engineered strain can be Candida albicans or Sclerotium rolfsii Sacc.
  • the present invention also provides a genetically engineered strain capable of resisting, tolerating or developing resistance to chlorflavin, wherein the genetically engineered strain contains the resistance gene cfoL.
  • Figure 1 The biosynthetic pathway of flavonoids in plants.
  • the substrate phenylalanine undergoes the phenylalanine aminolysis pathway to generate p-coumaroyl CoA, which is then converted into the flavonoid core skeleton by the sequential action of chalcone synthase CHS, chalcone isomerase CHI and flavonoid synthase FNS.
  • FIG. 1 Analysis of the Aspergillus flavonoid biosynthesis gene cluster cfo and its metabolites.
  • a The composition of each gene in the gene cluster cfo.
  • cfo contains 12 genes, of which the core gene is cfoA and the self-resistance gene is cfoL.
  • P450 enzyme genes cfoH
  • cfoJ NADPH-dependent FMN reductase genes
  • cfoI NTF2-like protein genes
  • cfoK CocE/NonD family hydrolase genes
  • cfoE monooxygenase genes
  • cfoF cfoF and cfoG
  • cfoB three monooxygenase genes
  • FIG. 3 Metabolite analysis of Aspergillus blanc and different mutants and their corresponding flavonoid compounds. The metabolites of all gene deletion mutants were analyzed, and 23 flavonoid compounds were found through separation and identification, all of which are intermediates in the biosynthesis of final product 1.
  • FIG. 4 Biosynthetic pathway of flavonoids in fungi.
  • the hybrid enzyme CfoA is the core enzyme, which uses benzoic acid or p-hydroxybenzoic acid as the starting unit to condense with four molecules of malonyl CoA to generate the key intermediate chalcone, which is then formed into the flavonoid core skeleton under the action of CfoK and CfoJ.
  • the core skeleton forms the final product 1 under the catalysis of different post-modification enzymes.
  • Figure 5 Isotope distribution in the structures of fungal flavonoids and plant flavonoids.
  • the bold chemical bonds in compound 1 represent those from [1,2- 13 C 2 ] sodium acetate, and the black dots in plant naringenin represent those from [1,3- 14 C 2 ] malonyl-CoA.
  • FIG. 7 In vitro enzyme activity analysis of CfoK and its mutants and their catalytic mechanism.
  • a. In vitro enzyme activity analysis of CfoK and its mutants;
  • b. The catalytic mechanism of CfoK, the nitrogen on H33 removes the hydrogen on the C2′-OH of the substrate, inducing an oxa-Michael addition reaction,
  • the 6-endo-trig ring-closing mode formed a C-ring intermediate containing an enolate anion, which was stabilized by forming a hydrogen bond with Y50.
  • the hydrogen ion on the imidazole ring of H33 was replenished and the enol tautomerization finally formed the flavanone product 24 containing a tetrahydropyrone form.
  • FIG. 8 Phylogenetic tree analysis of CfoJ and FNS currently found in nature.
  • CfoJ is located in the same branch as the NADPH-dependent FMN reductase family, while FNS I and FNS II are located on two other independent branches.
  • FIG. 9 In vitro enzyme activity analysis of CfoJ and mutants and their catalytic mechanism.
  • a. In vitro enzyme activity analysis of CfoJ and different cofactors and in vitro enzyme activity analysis of CfoJ mutants;
  • b. The catalytic mechanism of CfoJ: first, H 2 O removes the hydrogen atom at the C3 position of the activated substrate, and then the electron flows to between C2 and C3, causing the hydrogen atom at the C2 position to flow to the N5 position of FMN in the form of a hydrogen negative with a pair of electrons, and at the same time a double bond is formed between C2 and C3, and FMN is reduced to FMNH 2 .
  • Figure 10 In vitro enzymatic activity verification of CfoK and CfoJ. a. Isolation and purification of CfoK protein and in vitro enzymatic activity analysis; b. Isolation and purification of CfoJ protein and in vitro enzymatic activity analysis.
  • Figure 11 Analysis of metabolites of engineered strains and their corresponding flavonoids.
  • FIG. 12 Compounds chloranthoxaline (1) and dechloranthoxaline (2) inhibit the germination of Arabidopsis seeds; a. Germination of Arabidopsis seeds on MS plate medium, negative control dimethyl sulfoxide (DMSO), positive control glufosinate (GA), test compounds chloranthoxaline (1) and dechloranthoxaline (2). b. Growth inhibition curves of chloranthoxaline (1) and dechloranthoxaline (2) on seed germination at different concentrations.
  • DMSO dimethyl sulfoxide
  • GA positive control glufosinate
  • FIG. 14 Verification that the gene cfoL is a self-resistance gene to chlorflavin (1).
  • Figure 15 The gene cfoL confers resistance to chlorflavin (1) to Aspergillus fumigatus; a. Growth of wild-type Aspergillus fumigatus on PDA plates and PDA plates containing compound 1. b. Screening of transformants introduced with the cfoL gene into Aspergillus fumigatus on resistance plates containing compound 1, and PCR verification of the transformants. c. Growth of wild-type Aspergillus fumigatus and Aspergillus fumigatus overexpressing the cfoL gene on PDA plates.
  • Enzyme hydrolysate Weigh 0.4 g of cellulase (Sigma product, product catalog number: C1184), 0.4 g of lyase (Sigma product, product catalog number: L1412), and 0.2 g of snail enzyme (Sangon Biotechnology Co., Ltd., Shanghai, product catalog number: SB0870) and dissolve them in 50 ml of 0.6 M MgSO4 aqueous solution, and filter and sterilize through a 0.22 ⁇ m sterile filter.
  • plasmid extraction uses the Plasmid Mini Kit I kit (D6942-01) of OMEGA
  • DNA fragment recovery uses the Cycle-Pure Kit kit (D6492-01) of OMEGA
  • gel recovery uses the Gel Extraction Kit kit (D2500-01) of OMEGA.
  • PDBS plate 24 g/L potato culture medium PDB dry powder (BD product, product catalog number: 7114771), 1.2 M sorbitol, 4 g/L agarose, the balance is deionized water, autoclaved at 121°C for 20 minutes and then kept warm at 48°C.
  • PDB dry powder BD product, product catalog number: 7114771
  • 1.2 M sorbitol 4 g/L agarose
  • PDA plate 39 g/L potato culture medium PDA dry powder (BD product, product catalog number: 633840), the balance is deionized water, autoclave at 121°C for 20 minutes, and cool to about 60°C to prepare the plate.
  • PDA dry powder BD product, product catalog number: 633840
  • PDAS plate 39 g/L potato culture medium PDA dry powder (BD product, product catalog number: 633840), 1.2 M sorbitol, the balance is deionized water, autoclave at 121° C. for 20 minutes, and cool to about 60° C. to prepare the plate.
  • PDA dry powder BD product, product catalog number: 633840
  • 1.2 M sorbitol the balance is deionized water, autoclave at 121° C. for 20 minutes, and cool to about 60° C. to prepare the plate.
  • SGCY medium 2% sucrose, 1% glucose, 0.5% casein hydrolyzate, 0.5% yeast extract powder, 1% MgCl 2 ⁇ 6H 2 O, the balance is deionized water, autoclaved at 121°C for 20 minutes, cooled to about 30°C and inoculated for culture.
  • SYT medium 1% soluble starch, 0.4% yeast extract powder, 0.2% tryptone, 0.1% CaCO 3 , 0.004% FeSO 4 ⁇ 7H 2 O, 0.1% KBr, the balance is deionized water, sterilized by high pressure at 121°C for 20 minutes, and inoculated and cultured after cooling to about 30°C.
  • PPM medium 15% sucrose, 2.5% soybean meal, 0.5% peptone, 0.1% NaNO 3, the balance is deionized water, autoclave at 121°C for 20 minutes, and inoculate and culture after cooling to about 30°C.
  • YPED medium 2% sucrose, 1% casein hydrolysate, 1% polypeptone, 1% yeast extract, the balance is deionized water, autoclave at 121°C for 20 minutes, cool to about 30°C and then inoculate for culture.
  • MM medium 0.2% NH 4 Cl, 0.1% (NH 4 ) 2 SO 4 , 0.05% KCl, 0.05% NaCl, 0.1% KH 2 PO 4 , 0.05% Mg 2 SO 4 ⁇ 7H 2 O, 0.002% FeSO 4 ⁇ 7H 2 O and 2% sucrose, the balance is deionized water, autoclaved at 121° C. for 20 minutes.
  • CD medium 0.2% soluble starch, 0.1% KH 2 PO 4 , 0.1% yeast extract, 0.05% Mg 2 SO 4 ⁇ 7H 2 O, 0.01% FeSO 4 ⁇ 7H 2 O, 0.055% CaCl 2 , 2% maltose, 1% polypeptone, the balance is deionized water, autoclave at 121°C for 20 minutes, and inoculate and culture after cooling to about 30°C.
  • Example 1 Discovery of cfo gene cluster from co-localization of resistance genes
  • ALS acetolactate synthase
  • cfo gene cluster was located (the nucleic acid sequence is shown in SEQ ID No.1), which contains 12 genes ( Figure 2a, Table 1), of which 11 genes, including cfoA, cfoB, cfoC, cfoD, cfoE, cfoF, cfoG, cfoH, cfoI, cfoJ, and cfoK, are responsible for flavonoid biosynthesis, and cfoL is a self-resistance gene encoding ALS protein.
  • the sequence information is as follows:
  • a pair of external primers were designed at the upstream and downstream of each gene.
  • the left and right homologous exchange arms for homologous recombination were obtained by PCR amplification, each of which was about 1500 base pairs in length.
  • the homologous arms were fused with the hygromycin resistance tag hph by fusion PCR, and the targeting element obtained by purifying and concentrating the above PCR reaction products was knocked into the MEFC1001 strain to screen for gene deletion mutants.
  • the MEFC1001 strain was cultured in SGCY medium at 28°C and 220rpm for 2 days, the mycelia was collected by a 100-mesh sterile filter cloth, and the cell wall was digested with an enzymatic solution of 10 times the volume of the mycelia weight, and the enzymatic digestion conditions were 30°C and 130rpm for 2 hours.
  • the enzymatic solution was filtered by a 500-mesh sterile filter cloth, and the filtrate was centrifuged at 4000rpm to obtain protoplasts, which were washed once with a precooled 1.0M sorbitol solution, and then washed once with a precooled STC (1.0M sorbitol, 50mM Tris ⁇ HCl-pH 8.0, 50mM CaCl2 ), and finally the protoplasts were resuspended in precooled STC, and the concentration was adjusted to 5 ⁇ 107 /mL to obtain a protoplast suspension.
  • each gene targeting element (about 3 ⁇ g) to the suspension, then add 50 ⁇ L PSTC (40% PEG 4000, 1.2M sorbitol, 50mM Tris ⁇ HCl-pH 8.0, 50mM CaCl 2 ), gently mix, and place on ice for 30 minutes.
  • PSTC 50% PEG 4000, 1.2M sorbitol, 50mM Tris ⁇ HCl-pH 8.0, 50mM CaCl 2
  • 1mL PSTC mix, place at room temperature for 20 minutes, mix with 15mL PDBS, pour into 5 PDAS screening plates (containing 50mg/L hygromycin B), and culture at 30°C in the dark for 5 days.
  • Transformants with hygromycin resistance were selected from the screening plate and transferred to PDAH plates (PDA + 50 mg/L hygromycin B). After 5 days of culture, part of the hyphae on the single colony was picked to extract the genome, which was used as a template for PCR amplification using external primers on the homologous arms. If the random insertion PCR product size is about 3.5 kb, if homologous recombination occurs, the PCR product size is about 5.0 kb, indicating that the target gene has been knocked out.
  • the constructed gene deletion mutants and wild-type MEFC1001 strain were inoculated in PPM medium, and the number of spores in the medium was 10 7/50 mL.
  • the culture was carried out at 220 rpm and 30°C for 7 days to obtain the fermentation broth of each strain.
  • the fermentation broth was extracted with the same volume of ethyl acetate and concentrated to obtain a crude extract. After dissolution in methanol, the solution was filtered through a 0.22 ⁇ M organic membrane and analyzed using Waters ACQUITY UPLC.
  • a Eclipse Plus C18RRHD column 50 mm ⁇ 2.1 mm, 1.8 ⁇ m) was used.
  • the analysis method was as follows: mobile phase A (95% water + 5% acetonitrile + 0.05% formic acid), mobile phase B (100% acetonitrile + 0.05% formic acid), gradient elution: 0-0.58 min 100%-80% A, 0.58-4.05 min 80%-40% A, 4.05-5.79 min 40% A, 5.79-6.37 min 40%-0% A, 6.95-7.53 min 100% A, flow rate: 0.6 mL/min, and detection wavelength: 345 nm.
  • the fermentation scale of the mutant strain was expanded.
  • the fermentation conditions were the same as above.
  • the fermentation broth was extracted three times with the same volume of ethyl acetate and concentrated to obtain a crude extract.
  • the crude extract was subjected to column chromatography using a dry column packing method.
  • the filler was octadecylsilane bonded silica gel filler, and the methanol-water gradient elution (methanol volume 10% to 100%) was performed. Each gradient elution was 10 column volumes. A small amount of HPLC analysis was taken.
  • the analysis method was the same as above.
  • the optimal preparation conditions were determined according to the polarity and retention time of the flavonoid compounds in each component.
  • mutants with three monooxygenases and P450 enzymes knocked out were analyzed.
  • mutant ⁇ cfoE compound 1 disappeared and 2 was accumulated, so CfoE performed the function of a halogenase and was responsible for adding a chlorine atom to the C3' position of 2 to form the final product 1.
  • mutant ⁇ cfoF compounds 3, 4, 12 and 13 were accumulated, and none of them had a substituent group at the C3 position, indicating that CfoF was responsible for the hydroxylation at the C3 position.
  • Compounds 12 and 15 were produced in the mutant with cfoG knocked out.
  • the mutant strain with cfoK knockout accumulated compounds 20-23 in its metabolites, of which 20 and 22 were chalcone compounds, indicating that the key intermediates of flavonoid synthesis in fungi are similar to those in plants, both of which are chalcone. It also indicates that the non-ribosomal peptide-polyketide hybrid enzyme (NRPS-PKS) encoded by the core gene cfoA in the gene cluster cfo can synthesize chalcone and has the function of chalcone synthase (CHS), while the CHS in plants is a type III PKS.
  • NRPS-PKS non-ribosomal peptide-polyketide hybrid enzyme
  • CfoK has a function similar to that of chalcone isomerase (CHI) in plants, which can catalyze the intramolecular oxa-Michael addition reaction and convert chalcone into tricyclic flavanone.
  • CHI chalcone isomerase
  • the amino acid sequence similarity between CfoK and CHI found in nature is very low.
  • Phylogenetic tree analysis shows that CfoK is located on different branches from plant CHI and bacterial CHI ( Figure 6). To further verify the function of the cfoK gene, the CDS sequence of cfoK was first obtained by 5′-race and 3′-race technology.
  • CfoK is a new type of chalcone isomerase.
  • CfoJ has a function similar to that of flavonoid synthase (FNS) in plants, which can catalyze desaturation reactions to form double bonds at C2 and C3 positions, converting flavanones into flavonoids.
  • FNS flavonoid synthase
  • FNS II cytochrome P450
  • CfoJ is annotated as an NADPH-dependent FMN reductase. Phylogenetic tree analysis shows that CfoJ is located on different branches from FNS I and FNS II ( Figure 8).
  • CfoJ is a new type of flavonoid synthase.
  • the applicant used three key enzymes in the fungal flavonoid synthesis pathway, CfoA (CHS), CfoK (CHI) and cfoJ (FNS) as enzymatic elements, and constructed flavonoid-producing chassis cells in Aspergillus oryzae, Aspergillus terreus and Saccharomyces cerevisiae, respectively, laying the foundation for constructing plant-derived flavonoid compounds through synthetic biology.
  • CfoA CfoA
  • CHI CfoK
  • FNS cfoJ
  • the nucleotide sequences of genes cfoA, cfoK and cfoJ were amplified by PCR using specific primers and the genome of MEFC1001 as a template.
  • the pTAex3 vector used for heterologous expression was digested with restriction endonuclease KpnI, and the linearized pTAex3 vector was recovered.
  • the cfoA, cfoK and cfoJ genes were connected to the linearized pTAex3 vector using the Seamless Assembly Cloning (Clone Smarter) kit to obtain plasmids pTAex3-cfoA, pTAex3-cfoK and pTAex3-cfoJ.
  • the pAdeA vector used for heterologous expression was digested with restriction endonuclease XbaI, and the linearized pAdeA vector was recovered.
  • the cfoK gene containing the amyB promoter and terminator on the pTAex3-cfoK plasmid was amplified and connected to the linearized pAdeA vector using the Seamless Assembly Cloning kit to construct the pAdeA-cfoK plasmid.
  • the pBARI vector used for heterologous expression was digested with restriction endonuclease HindIII, and the linearized pBARI vector was recovered.
  • the cfoJ gene containing the amyB promoter and terminator on the pTAex3-cfoJ plasmid was amplified and connected to the linearized pBARI vector using the Seamless Assembly Cloning Kit to construct the pBARI-cfoJ plasmid.
  • Aspergillus oryzae was inoculated into YPED liquid medium at a spore inoculum of 1 ⁇ 10 7/50 mL and cultured at 28°C and 220 rpm for 12 hours. Collection of mycelium, enzymatic hydrolysis of protoplasts, and plasmid transformation experiments were performed by PEG-CaCl 2 -mediated protoplast transformation method. When the pTAex3-cfoA plasmid was transformed, the protoplast regeneration and screening culture were MMAS (MM+0.2% agar+1.2M sorbitol+0.15% methionine+0.01% adenine). The transformant was verified by PCR to obtain a mutant strain Ao-cfoA expressing the cfoA gene.
  • the mutant strain Ao-cfoA On the basis of the mutant strain Ao-cfoA, the pAdeA-cfoK plasmid was transformed, and the protoplast regeneration and screening culture were MMAS (MM+0.2% agar+1.2M sorbitol+0.15% methionine). The transformant was verified by PCR to obtain a mutant strain Ao-cfoA-cfoK expressing both cfoA and cfoK genes.
  • the mutant strain Ao-cfoA-cfoK On the basis of the mutant strain Ao-cfoA-cfoK, the pBARI-cfoJ plasmid was transformed, and the protoplast regeneration and screening culture was MMAS (MM+0.2% agar+1.2M sorbitol+0.15% methionine+500 ⁇ g/mL glufosinate). The transformant was verified by PCR to obtain the mutant strain Ao-cfoA-cfoK-cfoJ expressing cfoA, cfoK and cfoJ genes simultaneously.
  • MMAS MM+0.2% agar+1.2M sorbitol+0.15% methionine+500 ⁇ g/mL glufosinate
  • the mutant strains At-cfoA, At-cfoA-cfoK and At-cfoA-cfoK-cfoJ were transformed in Aspergillus terreus, and the mutant strains Sc-cfoA, Sc-cfoA-cfoK and Sc-cfoA-cfoK-cfoJ were transformed in Saccharomyces cerevisiae.
  • the fermentation broth was extracted three times with the same volume of ethyl acetate and concentrated to obtain a crude extract.
  • the crude extract was subjected to column chromatography using a dry column, with octadecylsilane bonded silica gel as the filler, and methanol-water gradient elution (methanol volume 10% to 100%), with each gradient elution lasting 10 column volumes.
  • the target compound 1 was in the 80% methanol/water component
  • compound 2 was in the 60% methanol/water component.
  • the 80% methanol/water component and the 60% methanol/water component were concentrated and dissolved in methanol, and then filtered with a 0.22 ⁇ m filter membrane for semi-preparative liquid phase purification.
  • Example 5 Evaluation of the inhibitory activity of chloranil and dechloranil against pathogenic fungi
  • Candida albicans and Aspergillus fumigatus Two human pathogenic fungi strains, Candida albicans and Aspergillus fumigatus, and four plant pathogenic fungi strains, Sclerotium rolfsii Sacc., Botrytis cinerea, Fusarium oxysporum f.sp.cucumerinum, FOC, and Colletotrichum gloeosporioides, were selected as test strains.
  • Candida albicans was inoculated in PDB medium and cultured at 28°C and 220rpm for 12 hours, then diluted to 5 ⁇ 10 5 /mL with sterile PDB medium to obtain a bacterial suspension for use.
  • test strains were inoculated on PDA plates and cultured at 28°C for 5-7 days until the hyphae or spores covered the plate. Wash and scrape off the mycelium with sterile 0.85% NaCl solution (containing 0.25% Tween20), add 50 mL of sterile PDB medium to obtain a bacterial suspension stock solution, and further dilute with sterile PDB medium to obtain a bacterial suspension with a concentration of 5 ⁇ 10 5 cells/mL for use.
  • the 96-well plate was sealed and cultured at 28°C for 72 hours. The absorbance of each well was measured at a wavelength of 600nm using an ELISA reader (or the solution in the well was observed to be turbid in a bright place).
  • the lowest sample concentration that can completely inhibit the growth of indicator bacteria in the well is the minimum inhibitory concentration (MIC) of the compound.
  • MIC minimum inhibitory concentration
  • Compound 1 showed very strong inhibitory activity against C.albicans, A.fumigatus and S.rolfsii Sacc.
  • compound 2 showed strong inhibitory activity against C.albicans and S.rolfsii Sacc.
  • flavonoids inhibit C.albicans and A.fumigatus
  • the inhibitory activity of compounds 1 and 2 against S.rolfsii Sacc. is firstly found in this patent. Therefore, compounds 1 and 2 have the potential to be developed into pesticides, antibiotics and food preservatives.
  • Example 6 Chloroflavone has the activity of inhibiting the germination of Arabidopsis seeds
  • Compound 1 (chloroflavin) and Compound 2 (dechlorochloroflavin) were obtained by the method of Example 1; Compound 1 (chloroflavin) and Compound 2 (dechlorochloroflavin) were dissolved in DMSO, respectively, and sterilized by membrane filtration.
  • compound 2 (dechlorochloroflavin) also has inhibitory activity, which can completely inhibit the germination of Arabidopsis seeds at a concentration of 20 ⁇ g/mL ( Figure 12). Therefore, chloroflavin and dechlorochloroflavin have herbicidal activity and have the potential to be developed into herbicides.
  • Example 7 Isolation and Identification of Flavonoids from Aspergillus leucophylla MEFC1001
  • Aspergillus mefc1001 strain (strain collection number: CGMCC No.3.15294) on a PDA plate for static culture, wash the spores with sterile water, inoculate them in SM medium, culture the seed liquid at 28°C and 220rpm, inoculate them in PPM medium, ferment them at 28°C and 220rpm for 14 days to obtain the fermentation liquid of Aspergillus mefc1001 strain.
  • the fermentation broth was extracted three times with the same volume of ethyl acetate and concentrated to obtain a crude extract.
  • the crude extract was subjected to column chromatography using a dry column, with octadecylsilane bonded silica gel as the filler, and methanol-water gradient elution (methanol volume 10% to 100%), with each gradient elution lasting 10 column volumes. After each component was concentrated, it was dissolved in methanol and purified by semi-preparative liquid phase after filtration with a 0.22 ⁇ m filter membrane.
  • Example 8 Construction of engineered strains producing different flavonoids in Aspergillus glaucosa and product analysis
  • a pair of external primers were designed at their upstream and downstream, respectively.
  • the left and right homologous exchange arms for homologous recombination were obtained by PCR amplification, each of which was about 1500 base pairs in length.
  • the homologous arms were fused with the hygromycin resistance tag hph by fusion PCR, the targeting element obtained by purifying and concentrating the above PCR reaction products was knocked into the MEFC1001 strain, and the gene deletion mutant was screened.
  • the MEFC1001 strain was cultured in SGCY medium at 28°C and 220rpm for 2 days, the mycelia was collected by a 100-mesh sterile filter cloth, and the cell wall was digested with an enzymatic solution of 10 times the volume of the mycelia weight, and the enzymatic digestion conditions were 30°C and 130rpm for 2 hours.
  • the enzymatic solution was filtered by a 500-mesh sterile filter cloth, and the filtrate was centrifuged at 4000rpm to obtain protoplasts, which were washed once with a precooled 1.0M sorbitol solution, and then washed once with a precooled STC (1.0M sorbitol, 50mM Tris ⁇ HCl-pH 8.0, 50mM CaCl2 ), and finally the protoplasts were resuspended in precooled STC, and the concentration was adjusted to 5 ⁇ 107 /mL to obtain a protoplast suspension.
  • each gene targeting element (about 3 ⁇ g) to the suspension, then add 50 ⁇ L PSTC (40% PEG 4000, 1.2M sorbitol, 50mM Tris ⁇ HCl-pH 8.0, 50mM CaCl 2 ), gently mix, and place on ice for 30 minutes.
  • PSTC 50% PEG 4000, 1.2M sorbitol, 50mM Tris ⁇ HCl-pH 8.0, 50mM CaCl 2
  • 1mL PSTC mix, place at room temperature for 20 minutes, mix with 15mL PDBS, pour into 5 PDAS screening plates (containing 50mg/L hygromycin B), and culture at 30°C in the dark for 5 days.
  • Transformants with hygromycin resistance were selected from the screening plate and transferred to PDAH plates (PDA + 50 mg/L hygromycin B) for 5 days of culture. Part of the hyphae on the single colony was picked to extract the genome, which was used as a template for PCR amplification using external primers on the homologous arms. If the random insertion PCR product size is about 3.5 kb, if homologous recombination occurs, the PCR product size is about 5.0 kb, indicating that the target gene has been knocked out.
  • the Aspergillus glaucosa engineered strain constructed in 2.2 and the wild-type MEFC1001 strain were inoculated in PPM medium, respectively, with the number of spores in the medium being 10 7/50 mL.
  • the culture was carried out at 220 rpm and 30°C for 7 days to obtain the fermentation broth of each strain.
  • the fermentation broth was extracted with the same volume of ethyl acetate and concentrated to obtain a crude extract.
  • the fermentation scale of the mutant strain with changes in metabolites was expanded, and the culture medium and fermentation conditions were the same as 8.2.
  • the fermentation broth was extracted three times with the same volume of ethyl acetate and concentrated to obtain a crude extract.
  • the crude extract was subjected to column chromatography using a dry column, and the filler was octadecylsilane bonded silica gel filler, and the methanol-water gradient elution (methanol volume 10% to 100%), each gradient elution 10 column volumes.
  • a small amount was taken for HPLC analysis, and the analysis method was the same as in Example 2.3.
  • the optimal preparation conditions were determined. Purification conditions: mobile phase A (100% water + 0.05% formic acid), mobile phase B (100% acetonitrile + 0.05% formic acid), the ratio of the mobile phase was adjusted according to the polarity of the target compound, elution at a flow rate of 2mL/min, detection wavelength 345nm, chromatographic column Waters X-bridge C18 (100mm ⁇ 10mm, 5 ⁇ m). The corresponding compound was collected at the corresponding retention time.
  • CfoK has a similar function to chalcone isomerase (CHI) in plants, which can catalyze the intramolecular oxa-Michael addition reaction and convert chalcone into tricyclic flavanone.
  • CHI chalcone isomerase
  • the amino acid sequence similarity between CfoK and CHI found in nature is very low.
  • Phylogenetic tree analysis shows that CfoK is located on a different branch from plant CHI and bacterial CHI. It is speculated that CfoK is a new type of chalcone isomerase.
  • CfoJ has a function similar to that of flavonoid synthase (FNS) in plants, which can catalyze the desaturation reaction to form double bonds at C2 and C3 positions, converting flavonoids into flavonoids.
  • FNS flavonoid synthase
  • FNS II cytochrome P450
  • CfoJ is annotated as an NADPH-dependent FMN reductase. Phylogenetic analysis shows that CfoJ is located on a different branch from FNS I and FNS II; it is speculated that CfoJ is a new type of flavonoid synthase.
  • Candida albicans and Aspergillus fumigatus were selected as test strains.
  • Sclerotium rolfsii Sacc. was selected as test strains.
  • Botrytis cinerea was selected as test strains.
  • Fusarium oxysporum f.sp.cucumerinum (FOC) was selected as test strains.
  • Candida albicans was inoculated in PDB medium and cultured at 28°C and 220rpm for 12 hours, then diluted to 5 ⁇ 10 5 /mL with sterile PDB medium to obtain a bacterial suspension for use.
  • test strains were inoculated on PDA plates and cultured at 28°C for 5-7 days until the hyphae or spores covered the plate. Wash and scrape off the mycelium with sterile 0.85% NaCl solution (containing 0.25% Tween20), add 50 mL of sterile PDB medium to obtain a bacterial suspension stock solution, and further dilute with sterile PDB medium to obtain a bacterial suspension with a concentration of 5 ⁇ 10 5 cells/mL for use.
  • the final concentrations of the compounds to be tested are 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, 0.24, 0.12, 0.06 and 0.03 ⁇ g/mL.
  • the 96-well plate is sealed and cultured at 28°C for 72 hours.
  • the absorbance of each well was measured at a wavelength of 600 nm using an ELISA reader (or the solution in the well was observed to see if it was turbid in a bright place).
  • the lowest sample concentration that could completely inhibit the growth of the indicator bacteria in the well was the minimum inhibitory concentration (MIC) of the compound.
  • MIC minimum inhibitory concentration
  • Chloroflavone was prepared using Aspergillus mefc1001 in the manner of Example 4. Specifically, Aspergillus mefc1001 was placed on a PDA plate for static culture, spores were washed with sterile water, inoculated into SM medium, seed liquid was cultured at 28°C and 220 rpm, inoculated into PPM medium, and fermented at 28°C and 220 rpm for 14 days to obtain fermentation liquid.
  • the fermentation broth was extracted three times with the same volume of ethyl acetate and concentrated to obtain a crude extract.
  • the crude extract was subjected to column chromatography using a dry column, with octadecylsilane bonded silica gel as the filler, and methanol-water gradient elution (methanol volume 10% to 100%), with each gradient elution lasting 10 column volumes.
  • the target compound 1 was in the 80% methanol/water component.
  • the 80% methanol/water component was concentrated and dissolved in methanol, and then filtered with a 0.22 ⁇ m filter membrane for semi-preparative liquid phase purification.
  • Example 12 cfoL gene is a self-resistance gene of Aspergillus leucophylla to chlorflavin
  • the gene cluster cfo responsible for the synthesis of chlorflavin contains a gene cfoL encoding acetolactate synthase (the nucleic acid sequence is shown in SEQ ID No.13, and the amino acid sequence is shown in SEQ ID No.25) (gene The cluster structure is shown in Figure 2a).
  • cfoL is the self-resistance gene of Aspergillus blancmange to chlorflavin
  • different gene deletion mutants were constructed, and it was finally found that cfoL can confer resistance to chlorflavin to Aspergillus blancmange. The details are as follows:
  • a pair of external primers were designed upstream and downstream.
  • the left and right homologous exchange arms for homologous recombination were obtained by PCR amplification, each with a length of about 1500 base pairs, and the hygromycin resistance marker hph was connected in the middle of the homologous arms.
  • the PCR product was purified and concentrated to obtain the targeting element, which was knocked into the MEFC1001 strain to screen for the mutant strain ⁇ cfoL with the cfoL gene deleted.
  • the MEFC1001 strain was cultured in SGCY medium at 28°C and 220rpm for 2 days, the mycelia was collected by a 100-mesh sterile filter cloth, the cell wall was digested with an enzymatic solution of 10 times the volume of the mycelia weight, and the enzymatic digestion conditions were 30°C and 130rpm for 2 hours.
  • the enzymatic solution was filtered by a 500-mesh sterile filter cloth, and the filtrate was centrifuged at 4000rpm to obtain protoplasts, which were washed once with a precooled 1.0M sorbitol solution, and then washed once with a precooled STC (1.0M sorbitol, 50mM Tris ⁇ HCl-pH 8.0, 50mM CaCl 2 ), and finally the protoplasts were re-spinned in a precooled STC, and the concentration was adjusted to 5 ⁇ 10 7 cells/mL to obtain a protoplast suspension.
  • Transformants with hygromycin resistance were selected from the screening plate and transferred to PDAH plates (PDA + 50 mg/L hygromycin B). After 5 days of culture, part of the hyphae on the single colony was picked to extract the genome, which was used as a template for PCR amplification using external primers on the homologous arms. If the random insertion PCR product size is about 3.5 kb, if homologous recombination occurs, the PCR product size is about 5.0 kb, indicating that the target gene has been knocked out.
  • cfoA is the core gene responsible for the synthesis of chlorflavin. After cfoA was knocked out, the strain would no longer produce flavonoids such as chlorflavin.
  • the cfoL gene was amplified from the Aspergillus genome, and the ⁇ cfoA- ⁇ cfoL mutant was transformed to obtain the cfoL-complemented mutant ⁇ cfoA- ⁇ cfoL::cfoL.
  • the mutant strain ⁇ cfoA- ⁇ cfoL::cfoL with complementation of cfoL could grow on both plates.
  • the above experiments show that the cfoL gene is a self-resistance gene in Aspergillus Brilliant, and its presence can resist the inhibition of chlorflavin on the strain itself.
  • Example 13 The self-resistance gene cfoL can confer resistance to chlorflavin in Aspergillus fumigatus
  • the applicant introduced the cfoL gene into Aspergillus fumigatus, which is sensitive to chlorflavin, and found that the mutant strain of Aspergillus fumigatus acquired resistance to chlorflavin. Therefore, the combination of the cfoL gene and chlorflavin can be used for genetic transformation of Aspergillus fumigatus.
  • the applicant introduced the cfoL gene into Aspergillus fumigatus, which is sensitive to chlorflavin. Specifically, Aspergillus fumigatus MEFC1001 was inoculated into PPM medium, cultured at 28°C and 220rpm for 2 days, and mRNA was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa) kit, and reverse transcribed into cDNA using the PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa) kit, and the CDS sequence of the cfoL gene was obtained by amplification with specific primers.
  • Aspergillus fumigatus MEFC1001 was inoculated into PPM medium, cultured at 28°C and 220rpm for 2 days, and mRNA was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa) kit, and reverse transcribed into cDNA using the PrimeScript TM RT reagent Kit with gDNA Er
  • the cfoL-CDS was connected to the constitutive promoter PgpdAt commonly used in Aspergillus by fusion PCR to construct an overexpression element.
  • the PEG-CaCl 2 -mediated protoplast transformation method in Aspergillus fumigatus is basically the same as the transformation method of Aspergillus fumigatus in Example 2.1.
  • the screening plate is PDAS (containing 10 ⁇ g/mL of chlorflavin), and it is cultured for 5 days under dark conditions at 30°C, and transformants can be observed to grow on the screening plate. Part of the mycelium was selected to extract the genome, and PCR amplification was performed using primers that could obtain the cfoL-CDS sequence.
  • Example 5 of the present application records that chlorflavin has a significant inhibitory effect on Candida albicans and Sclerotium rolfsii Sacc.

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Abstract

提供了氯黄菌素及其衍生物的应用，尤其是在抑制微生物生长以及在抑制植物生长中的用途；还提供了一种与黄酮合成相关的基因以及新的黄酮类的化合物，在目的菌株中对上述与黄酮合成相关的基因进行基因操作，可以得到新的黄酮类化合物，并且，这些黄酮类化合物具有良好的抑菌效果；另外，还提供了一种抗性基因cfoL，其能够赋予微生物对氯黄菌素产生抗性，从而能够作为筛选标签用于微生物的遗传转化操作。

Description

黄酮类化合物及其生物合成相关基因以及其应用 技术领域
本发明属于微生物基因资源和基因工程领域，具体地，涉及黄酮类化合物、其生物合成相关基因以及其应用。
背景技术
黄酮类化合物是主要存在于植物中的一大类天然产物，具有多样的结构类型和广泛的生物学活性，它们在植物的生长、调控和防御中具有重要生理作用。黄酮类化合物在植物中的生物合成途径已经研究的非常清楚。首先L-苯丙氨酸经过苯丙氨酸解氨酶(PAL)、肉桂酸4-羟化酶(C4H)和4-羟基肉桂酰-CoA连接酶(4CL)的作用，形成对香豆酰辅酶A，然后在查耳酮合酶(CHS，III PKS)的作用下与三分子丙二酰CoA缩合生成查耳酮，查耳酮异构酶(CHI)催化查耳酮形成构型专一的黄烷酮，最后在黄酮合酶(FNS)的作用下形成黄酮骨架。在此骨架的基础上经过羟化、甲基化和糖基化等修饰，形成了结构类型和生物活性多样的黄酮类化合物(如图1所示)。
目前市场上已经有很多黄酮类相关的功能性食品和药物，例如抗炎抗衰老作用的花青素和槲皮素，具有植物雌激素功能的大豆异黄酮，以及具有保肝作用的水飞蓟宾等。黄酮类化合物在食品、保健和医药开发中存在诸多的应用，对人们的健康发挥着非常重要的作用。据报道黄酮类化合物每年大约有2亿美元的市场。它们的来源主要是从植物中提取获得，由于植物中含量较低、提取工艺复杂等原因，黄酮类化合物一直无法满足市场的大量需求。因此，运用合成生物学技术通过微生物生产黄酮类化合物成为一种可能。微生物中缺少合成黄酮的内源基因，目前在微生物中黄酮的构建都是基于植物中黄酮的合成途径。由于微生物底盘和植物中的酶学元件匹配性较低，因此微生物黄酮细胞工厂的产量很低，无法达到商业化的要求。在本发明中，申请人发现一株能产生黄酮类化合物的真菌。合成途径解析发现真菌黄酮与植物的合成明显不同，是一种全新的生物合成机制。其中CfoA、CfoK和CfoJ负责催化合成黄酮的核心骨架。通过将这三种酶在米曲霉、土曲霉和酿酒酵母中异源重构，宿主可以产生黄酮类化合物。
另外，抗性标签是微生物遗传操作体系开发中基本的元件之一，是在遗传转化过程中筛选获得阳性转化子最基本的保障。目前可用于丝状真菌遗传操作的抗性标签种类少、普适性差，因此严重制约了丝状真菌中遗传操作体系的开发。在本专利中申请人在一株亮白曲霉MEFC1001(保藏于中国微生物菌种保藏管理委员会普通微生物中心的真菌，登记入册编号为：CGMCC 3.15294)中分离获得了氯黄菌素(又称为氯黄酮chlorflavonin，CAS：23363-64-6)和去氯氯黄菌素(又称为去氯氯黄酮dechlorochlorflavonin，CAS:51724-52-8)，通过该化合物的抗菌活性评价，显示其对部分植物病原真菌(齐整小核菌)和人类治病真菌(白色念珠菌、烟曲霉)具有非常强的抑制活性，具有开发成为农药杀菌剂、抗生素和食品防腐剂的潜力。另外，申请人发现，负责氯黄菌素生物合成的基因簇中含有一个编码乙酰乳酸合酶的自抗性基因cfoL，该基因可以很好的赋予真菌对氯黄菌素的抗性，因此氯黄菌素和cfoL的组合使用可用于丝状真菌的遗传操作，cfoL的发现丰富了遗传操作抗性标签的类型，为在真菌中遗传操作体系的开发奠定了基础。
此外，利用合成生物学技术改造微生物，使其生产黄酮类化合物成为一种可能，目前已经报道了很多在微生物中合成植物源黄酮的研究。本专利中申请人在一株亮白曲霉中发现了氯代黄酮类化合物氯黄菌素，又称为氯黄酮(chlorflavonin，CAS：23363-64-6)及其生物合成基因簇cfo，通过对cfo中的基因敲除，构建了生产不同黄酮的工程菌株。分离获 得了一系列黄酮类化合物，其中含有结构新颖的黄酮类化合物，活性测试显示它们具有抑菌活性，因此工程菌株和黄酮类化合物均具有潜在的应用价值。
发明内容
在第一组技术方案中，本发明提供了氯黄菌素或其衍生物的新用途。
一方面，本发明提供了氯黄菌素或去氯氯黄菌素在抑制微生物生长中的用途，或者，在制备抑制微生物生长的试剂中的用途，或者，在制备治疗由微生物所导致的疾病的药物中的用途。
在一个实施方式中，所述抑制微生物生长的试剂为抗生素。
在优选的实施方式中，所述微生物为齐整小核菌(Sclerotium rolfsii Sacc.)、白色念珠菌(Candida albicans)或烟曲霉(Aspergillus fumigatus)。
在一个实施方式中，所述疾病为植物疾病，例如，由齐整小核菌侵染作物导致花生、水稻、黄瓜等的白绢病。
本发明中的试剂和药物可以制备成不同的剂型，包括为液体制剂、固体制剂、半固体制剂或气体制剂。
本发明的试剂和药物，除了含有氯黄菌素或去氯氯黄菌素作为活性成分之外，还可以含有其他的辅助试剂。
本发明中，氯黄菌素抑制齐整小核菌的最小抑菌浓度可以低至2μg/mL以下；去氯氯黄菌素抑制齐整小核菌的最小抑菌浓度可以低至4μg/mL以下。
另一方面，本发明提供了氯黄菌素在抑制植物生长中的用途，或者，在抑制植物种子萌发和根茎发芽中的用途，或者，在制备抑制植物生长的试剂中的用途，或者，在制备除草剂中的用途；在优选的实施方式中，所述植物为拟南芥。
在制备为抑制植物生长的试剂时，优选剂型为液体剂型。
本发明中，所述氯黄菌素的结构式如下所示：
所述去氯氯黄菌素的结构式如下所示：
在第二组技术方案中，本发明提供了与黄酮生物合成相关的基因簇或者与黄酮合成相关的基因以及其应用，并提供了新型的黄酮类化合物。
一方面，本发明提供了与黄酮生物合成相关的基因簇或者与黄酮合成相关的基因。
在一个实施方式中，所述基因簇的核酸序列如SEQ ID No.1所示。
在其他的实施方式中，所述与黄酮合成相关的基因选自FAD依赖的氧化酶CfoG、单加氧酶CfoF、SAM依赖的甲基转移酶CfoD、细胞色素P450酶CfoH、酯水解酶CfoK、Scytalone脱水酶CfoI、SAM依赖的甲基转移酶CfoC、NADPH依赖的FMN还原酶CfoJ、SAM依赖的甲基转移酶CfoB、FAD依赖的氧化酶CfoE、杂合的非核糖体肽合酶与聚酮合酶CfoA中 的一种或任意几种组合。
在一个实施方式中，所述CfoG的氨基酸序列与SEQ ID No.14相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoG来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoG的氨基酸序列与SEQ ID No.14相比具有至少99％的序列同一性，并且所述CfoG来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoG的氨基酸序列如SEQ ID No.14所示，其编码基因序列如SEQ ID No.2所示。
在一个实施方式中，所述CfoF的氨基酸序列与SEQ ID No.15相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoF来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoF的氨基酸序列与SEQ ID No.15相比具有至少99％的序列同一性，并且所述CfoF来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoF的氨基酸序列如SEQ ID No.15所示，其编码基因序列如SEQ ID No.3所示。
在一个实施方式中，所述CfoD的氨基酸序列与SEQ ID No.16相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoD来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoD的氨基酸序列与SEQ ID No.16相比具有至少99％的序列同一性，并且所述CfoD来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoD的氨基酸序列如SEQ ID No.16所示，其编码基因序列如SEQ ID No.4所示。
在一个实施方式中，所述CfoH的氨基酸序列与SEQ ID No.17相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoH来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoH的氨基酸序列与SEQ ID No.17相比具有至少99％的序列同一性，并且所述CfoH来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoH的氨基酸序列如SEQ ID No.17所示，其编码基因序列如SEQ ID No.5所示。
在一个实施方式中，所述CfoK的氨基酸序列与SEQ ID No.18相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoK来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoK的氨基酸序列与SEQ ID No.18相比具有至少99％的序列同一性，并且所述CfoK来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoK的氨基酸序列如SEQ ID No.18所示，其编码基因序列如SEQ ID No.6所示。
在一个实施方式中，所述CfoI的氨基酸序列与SEQ ID No.19相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoI来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoI的氨基酸序列与SEQ ID No.19相比具有至少99％的序列同一性，并且所述CfoI来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoI的氨基酸序列如SEQ ID No.19所示，其编码基因序列如SEQ ID No.7所示。
在一个实施方式中，所述CfoC的氨基酸序列与SEQ ID No.20相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoC来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoC的氨基酸序列与SEQ ID No.20相比具有至少99％的序列同一性，并且所述CfoC来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoC的氨基酸序列如SEQ ID No.20所示，其编码基因序列如SEQ ID No.8所示。
在一个实施方式中，所述CfoJ的氨基酸序列与SEQ ID No.21相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoJ来源于亮白曲霉，例如，亮白曲霉 MEFC1001；更优选的，所述CfoJ的氨基酸序列与SEQ ID No.21相比具有至少99％的序列同一性，并且所述CfoJ来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoJ的氨基酸序列如SEQ ID No.21所示，其编码基因序列如SEQ ID No.9所示。
在一个实施方式中，所述CfoB的氨基酸序列与SEQ ID No.22相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoB来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoB的氨基酸序列与SEQ ID No.22相比具有至少99％的序列同一性，并且所述CfoB来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoB的氨基酸序列如SEQ ID No.22所示，其编码基因序列如SEQ ID No.10所示。
在一个实施方式中，所述CfoE的氨基酸序列与SEQ ID No.23相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoE来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoE的氨基酸序列与SEQ ID No.23相比具有至少99％的序列同一性，并且所述CfoE来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoE的氨基酸序列如SEQ ID No.23所示，其编码基因序列如SEQ ID No.11所示。
在一个实施方式中，所述CfoA的氨基酸序列与SEQ ID No.24相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoA来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoA的氨基酸序列与SEQ ID No.24相比具有至少99％的序列同一性，并且所述CfoA来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoA的氨基酸序列如SEQ ID No.24所示，其编码基因序列如SEQ ID No.12所示。
所述亮白曲霉MEFC1001，保藏编号为CGMCC No.3.15294，可市售获得，为保藏于中国微生物菌种保藏管理委员会普通微生物中心的真菌。
另一方面，本发明还提供了一种新的黄酮类化合物，所述黄酮类化合物选自化合物2-化合物23中的一种或任意几种。
所述化合物2结构式如下所示：
所述化合物3结构式如下所示：
所述化合物4结构式如下所示：
所述化合物5结构式如下所示：
所述化合物6结构式如下所示：
所述化合物7结构式如下所示：
所述化合物8结构式如下所示：
所述化合物9结构式如下所示：
所述化合物10结构式如下所示：
所述化合物11结构式如下所示：
所述化合物12结构式如下所示：
所述化合物13结构式如下所示：
所述化合物14结构式如下所示：
所述化合物15结构式如下所示：
所述化合物16结构式如下所示：
所述化合物17结构式如下所示：
所述化合物18结构式如下所示：
所述化合物19结构式如下所示：
所述化合物20结构式如下所示：
所述化合物21结构式如下所示：
所述化合物22结构式如下所示：
所述化合物23结构式如下所示：
另一方面，本发明还提供了上述新的黄酮类化合物的用途，所述用途包括在抑制微生物生长中的用途，或者，在制备抑制微生物生长的试剂中的用途，或者，在制备治疗由微生物所导致的疾病的药物中的用途；在一个实施方式中，所述抑制微生物生长的试剂为抗生素；在优选的实施方式中，所述微生物选自白色念珠菌(Candida albicans)、烟曲霉(Aspergillus fumigatus)、齐整小核菌(Sclerotium rolfsii Sacc.)、灰霉病菌(Botrytis cinerea)中的一种或任意几种。
例如，化合物3、化合物4和化合物13可以抑制白色念珠菌、齐整小核菌和灰霉病菌的生长。
化合物6可以抑制白色念珠菌和烟曲霉的生长。
化合物7和化合物8可以抑制白色念珠菌、烟曲霉、齐整小核菌和灰霉病菌的生长。
化合物10可以抑制白色念珠菌和齐整小核菌的生长。
化合物14可以抑制白色念珠菌、烟曲霉和齐整小核菌的生长。
化合物19可以抑制白色念珠菌和齐整小核菌的生长。
本发明中的试剂和药物可以制备成不同的剂型，包括为液体制剂、固体制剂、半固体制剂或气体制剂。
本发明的试剂和药物，除了含有上述化合物作为活性成分之外，还可以含有其他的辅助试剂。
另一方面，本发明还提供了一种基因工程菌株，所述基因工程菌株为将亮白曲霉中的上述与黄酮合成相关的基因进行基因突变所得到的基因工程菌株。
所述基因工程菌株的出发菌株还可以选自曲霉、构巢曲霉、酵母、大肠杆菌、植物或动物；优选，曲霉，例如，亮白曲霉、米曲霉。
在一个实施方式中，所述与黄酮合成相关的基因选自上述FAD依赖的氧化酶CfoG、单加氧酶CfoF、SAM依赖的甲基转移酶CfoD、细胞色素P450酶CfoH、酯水解酶CfoK、Scytalone脱水酶CfoI、SAM依赖的甲基转移酶CfoC、NADPH依赖的FMN还原酶CfoJ、SAM依赖的甲基转移酶CfoB、FAD依赖的氧化酶CfoE、杂合的非核糖体肽合酶与聚酮合酶CfoA中的一种或任意几种。
本发明所述的突变包括通过基因缺失、基因插入或基因取代的方式导致基因功能或活性丧失。
在一个实施方式中，所述基因突变可以采用本领域常规的技术操作来实现，例如，通过同源重组的方式进行基因敲入或基因敲除从而导致基因功能或活性丧失；或者，采用基因编辑的方式，如锌指核酸内切酶(ZFN)、类转录激活因子效应物核酸酶(TALEN)或 CRISPR技术对上述基因进行突变从而导致基因功能或活性丧失。
另一方面，本发明还提供了上述基因工程菌株在生产黄酮类化合物中的应用。
另一方面，本发明还提供了一种制备黄酮类化合物的方法，所述方法包括对上述基因工程菌株进行发酵的步骤。
在一个实施方式中，所述黄酮类化合物选自化合物2-化合物23中的一种或任意几种。
在一个实施方式中，所述突变的基因为CfoB，所述黄酮类化合物为化合物5、化合物6、化合物7中的一种或任意几种；
在一个实施方式中，所述突变的基因为CfoC，所述黄酮类化合物为化合物8、化合物9、化合物10、化合物11、化合物12中的一种或任意几种；
在一个实施方式中，所述突变的基因为CfoD，所述黄酮类化合物为化合物3、化合物4、化合物12、化合物13、化合物14中的一种或任意几种；
在一个实施方式中，所述突变的基因为CfoE，所述黄酮类化合物为化合物2、化合物4中的一种或任意几种；
在一个实施方式中，所述突变的基因为CfoF，所述黄酮类化合物为化合物3、化合物4、化合物12、化合物13中的一种或任意几种；
在一个实施方式中，所述突变的基因为CfoG，所述黄酮类化合物为化合物12、化合物15中的一种或任意几种；
在一个实施方式中，所述突变的基因为CfoH，所述黄酮类化合物为化合物4、化合物12中的一种或任意几种；
在一个实施方式中，所述突变的基因为CfoI，所述黄酮类化合物为化合物3、化合物4、化合物12中的一种或任意几种；
在一个实施方式中，所述突变的基因为CfoJ，所述黄酮类化合物为化合物16、化合物17、化合物18、化合物19中的一种或任意几种；
在一个实施方式中，所述突变的基因为CfoK，所述黄酮类化合物为化合物20、化合物21、化合物22、化合物23中的一种或任意几种。
在第三组技术方案中，本发明提供了与黄酮合成相关的基因及其在制备黄酮类化合物中的应用。
在一个实施方式中，所述与黄酮合成相关的基因选自酯水解酶CfoK、NADPH依赖的FMN还原酶CfoJ、杂合的非核糖体肽合酶与聚酮合酶CfoA中的一种或任意几种组合。
在一个实施方式中，所述CfoK的氨基酸序列与SEQ ID No.18相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoK来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoK的氨基酸序列与SEQ ID No.18相比具有至少99％的序列同一性，并且所述CfoK来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoK的氨基酸序列如SEQ ID No.18所示，其编码基因序列如SEQ ID No.6所示。
在一个实施方式中，所述CfoJ的氨基酸序列与SEQ ID No.21相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoJ来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoJ的氨基酸序列与SEQ ID No.21相比具有至少99％的序列同一性，并且所述CfoJ来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoJ的氨基酸序列如SEQ ID No.21所示，其编码基因序列如SEQ ID No.9所示。
在一个实施方式中，所述CfoA的氨基酸序列与SEQ ID No.24相比具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述CfoA来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述CfoA的氨基酸序列与SEQ ID No.24相比具有至少99％的序列 同一性，并且所述CfoA来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述CfoA的氨基酸序列如SEQ ID No.24所示，其编码基因序列如SEQ ID No.12所示。
所述亮白曲霉MEFC1001，保藏编号为CGMCC No.3.15294，可市售获得，为保藏于中国微生物菌种保藏管理委员会普通微生物中心的真菌。
另一方面，本发明提供了上述与黄酮合成相关的基因在制备黄酮类化合物中的应用。
另一方面，本发明还提供了上述与黄酮合成相关的基因在制备能够产黄酮类化合物的基因工程菌株中的应用。
另一方面，本发明还提供了一种能够产黄酮类化合物的基因工程菌株，所述基因工程菌株为在出发菌株中引入上述与黄酮合成相关的基因所得到的基因工程菌株。
所述的“引入”包括将上述目的基因在出发菌株中进行表达的步骤，优选，过表达。例如，将目的基因构建到表达载体上，将表达载体转入到宿主细胞中以表达目的基因，优选，过表达。在其他的实施方式中，所述的“引入”包括将目的基因插入到宿主细胞的基因组中；优选的，所述插入到宿主细胞的基因组中可以采用同源重组双交换的方法；在一个实施方式中，可以采用将目的基因以及同源臂插入到载体上，然后将载体转入到宿主细胞中，利用同源臂与宿主细胞基因组发生同源重组双交换从而将目的基因插入到合适的基因组的位置；在其他的实施方式中，还可以采用基因编辑的方式，例如，利用CRISPR/Cas系统在期望的基因组位点上进行切割，同时将目的基因作为外源供体插入到切割位点上。
另一方面，本发明还提供了上述基因工程菌株在生产黄酮类化合物中的应用。
所述基因工程菌株的出发菌株选自曲霉、构巢曲霉、酵母、大肠杆菌、植物或动物；优选，曲霉，例如，亮白曲霉、米曲霉、土曲霉。
优选的，所述与黄酮合成相关的基因选自CfoA、CfoK和CfoJ中的一种或任意几种组合。
另一方面，本发明还提供了一种制备黄酮类化合物的方法，所述方法包括对上述基因工程菌株进行发酵的步骤。
在一个实施方式中，所述黄酮类化合物选自化合物22、化合物24、化合物25和化合物26中的一种或任意几种。
具体而言，单独引入CfoA可以产生化合物22和化合物25，同时引入CfoA和CfoK可以产生化合物24，同时引入CfoA、CfoK和CfoJ可以产生化合物24和化合物26。
在优选的实施方式中，所述与黄酮合成相关的基因选自CfoA，所述黄酮类化合物选自化合物22和化合物25中的一种或任意几种；所述与黄酮合成相关的基因选自CfoA和CfoK，所述黄酮类化合物选自化合物24；所述与黄酮合成相关的基因选自CfoA、CfoK和CfoJ，所述黄酮类化合物选自化合物24和化合物26中的一种或任意几种。
所述化合物22结构式如下所示：
所述化合物24结构式如下所示：
所述化合物25结构式如下所示：
所述化合物26结构式如下所示：
另一方面，本发明还提供了CfoK和CfoJ的应用。
在一个实施方式中，本发明提供了CfoK在催化合成化合物27和/或化合物24中的应用；在其他的实施方式中，本发明提供了CfoJ在催化合成化合物26和/或化合物3中的应用。
具体的，CfoK可以催化化合物20生成化合物27；CfoK可以催化化合物22生成化合物24；CfoJ可以催化化合物24生成化合物26；CfoJ可以催化化合物18生成化合物3。
另一方面，本发明还提供了一种引入CfoK和/或CfoJ的基因工程菌株，所述基因工程菌株的出发菌株可以为酵母或大肠杆菌。
另一方面，本发明还提供了上述引入CfoK和/或CfoJ的基因工程菌株在制备化合物27、化合物24、化合物26或化合物3中的应用。
本申请中，化合物27结构式如下所示：
此外，本申请发现，CfoK自N端第33位的氨基酸H和第50位的氨基酸Y是其发挥活性的关键位点，将第33位氨基酸进行突变(例如，突变为A或Q)或将第50位氨基酸进行突变(例如，突变为A或F)，突变的CfoK无法将化合物22催化合成化合物24。
CfoJ自N端第112位的氨基酸P和第154位的氨基酸W是其发挥活性的关键位点，将第112位氨基酸进行突变(例如，突变为A)或将第154位氨基酸进行突变(例如，突变为A)，突变的CfoJ无法将化合物24催化合成化合物26。
在第四组技术方案中，本发明提供了一种抗性基因cfoL及其应用。
在一个实施方式中，抗性基因cfoL的氨基酸序列与SEQ ID No.25相比，具有至少80％、85％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述抗性基因cfoL来源于亮白曲霉，例如，亮白曲霉MEFC1001；更优选的，所述抗性基因cfoL的氨基酸序列与SEQ ID No.25相比具有至少99％的序列同一性，并且所述抗性基因cfoL来源于亮白曲霉；所述亮白曲霉包括亮白曲霉MEFC1001。更优选的，所述抗性基因cfoL的氨基酸序列如SEQ ID No.25所示，其编码基因序列如SEQ ID No.13所示。
所述亮白曲霉MEFC1001，保藏编号为CGMCC 3.15294，可市售获得，为保藏于中国微生物菌种保藏管理委员会普通微生物中心的真菌。
另一方面，本发明还提供了含有上述抗性基因cfoL载体或重组宿主细胞。
另一方面，本发明还提供了上述抗性基因cfoL在抵抗氯黄菌素或者在赋予氯黄菌素敏感的微生物对氯黄菌素产生抗性中的应用。
本发明中，对氯黄菌素敏感的微生物，是指在氯黄菌素存在的情况下，所述微生物的生长或者活性受到抑制。比如，与不存在氯黄菌素的情况相比，微生物在氯黄菌素存在的情况下，菌株的生长水平降低至对照水平的0％、5％、10％、20％或30％。
在一些实施方式中，氯黄菌素的用量可以为1μg/mL-200μg/mL，例如，2μg/mL、5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL、30μg/mL、35μg/mL、40μg/mL、45μg/mL、50μg/mL、60μg/mL、70μg/mL、80μg/mL、90μg/mL、100μg/mL或150μg/mL。
本发明不对氯黄菌素的用量水平做严格限定，本领域根据常规技术和常规操作即可获得可以使得微生物敏感或致死的氯黄菌素的用量水平。
赋予氯黄菌素敏感的微生物对氯黄菌素产生抗性，是指在上述氯黄菌素敏感的微生物中引入上述抗性基因cfoL，能够缓解或者解除氯黄菌素对所述微生物造成的生长或者活性的抑制。
另一方面，本发明还提供了上述抗性基因cfoL作为抗生素抗性筛选标记或筛选标签中的应用；优选的，所述抗生素为氯黄菌素。
在一个实施方式中，所述筛选标记或筛选标签可以置于载体/质粒中，所述载体/质粒引入目的微生物中，可以导致所述微生物产生对抗生素的抗性。
本发明中，所述筛选标记或筛选标签，可以理解为，引入抗性基因cfoL的目标微生物能够在氯黄菌素存在的情况下维持生长状态，而没有引入抗性基因cfoL的微生物无法在氯黄菌素存在的情况维持生长状态，从而可以筛选得到引入抗性基因cfoL的目标微生物；基于此，使得抗性基因cfoL可以作为氯黄菌素的抗性筛选标记或筛选标签。
另一方面，本发明还提供了上述抗性基因cfoL在制备抵抗/耐受氯黄菌素或对氯黄菌素产生抗性的基因工程菌株中的应用。
另一方面，本发明还提供了一种制备抵抗氯黄菌素/耐受氯黄菌素或对氯黄菌素产生抗性的基因工程菌株中的方法，所述方法包括在所述菌株中引入所述抗性基因cfoL的步骤。
所述的“引入”包括将上述目的基因在出发菌株中进行表达的步骤，优选，过表达。例如，将目的基因构建到表达载体上，将表达载体转入到宿主细胞中以表达目的基因，优选，过表达。在其他的实施方式中，所述的“引入”包括将目的基因插入到宿主细胞的基因组中；优选的，所述插入到宿主细胞的基因组中可以采用同源重组双交换的方法；在一个实施方式中，可以采用将目的基因以及同源臂插入到载体上，然后将载体转入到宿主细胞中，利用同源臂与宿主细胞基因组发生同源重组双交换从而将目的基因插入到合适的基因组的位置；在其他的实施方式中，还可以采用基因编辑的方式，例如，利用CRISPR/Cas系统在期望的基因组位点上进行切割，同时将目的基因作为外源供体插入到切割位点上。
在一个实施方式中，所述基因工程菌株的出发菌株为对氯黄菌素敏感的菌株；在对氯黄菌素敏感的菌株中引入上述抗性基因cfoL，可以导致所述菌株能够抵抗氯黄菌素。
本发明中，对氯黄菌素敏感，是指在氯黄菌素存在的情况下，所述菌株或微生物的生长或者活性受到抑制。比如，与不存在氯黄菌素的情况相比，菌株或微生物在氯黄菌素存在的情况下，菌株的生长水平降低至对照水平的0％、5％、10％、20％或30％。
在一些实施方式中，氯黄菌素的用量可以为1μg/mL-200μg/mL，例如，2μg/mL、5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL、30μg/mL、35μg/mL、40μg/mL、45μg/mL、50μg/mL、60μg/mL、70μg/mL、80μg/mL、90μg/mL、100μg/mL或150μg/mL。
本发明不对氯黄菌素的用量水平做严格限定，本领域根据常规技术和常规操作即可获得可以使得微生物敏感或致死的氯黄菌素的用量水平。
本发明中，抵抗氯黄菌素、耐受氯黄菌素或对氯黄菌素产生抗性，含义是相似的，均是指代，目标微生物或目标菌株能够在氯黄菌素存在的情况下，还能维持生长。尤其是，针对氯黄菌素敏感的微生物，当引入本发明的抗性基因后，其“抵抗氯黄菌素、耐受氯黄菌素或对氯黄菌素产生抗性”是指在上述氯黄菌素敏感的微生物中引入上述抗性基因cfoL，能够缓解或者解除氯黄菌素对所述微生物造成的生长或者活性的抑制。
本发明中，可以采用本领域常规的方法获得对氯黄菌素敏感的菌株。
在一个实施方式中，所述基因工程菌株的出发菌株包括真菌，例如，曲霉，例如，烟曲霉。
在其他的实施方式中，所述基因工程菌株的出发菌株可以为白色念珠菌(Candida albicans)或齐整小核菌(Sclerotium rolfsii Sacc.)。
另一方面，本发明还提供了一种能够抵抗氯黄菌素、耐受氯黄菌素或对氯黄菌素产生抗性的基因工程菌株，所述基因工程菌株中含有上述抗性基因cfoL。
附图说明
图1.植物中黄酮类化合物的生物合成途径。底物苯丙氨酸经过苯丙氨酸氨解途径生成对香豆酰CoA，然后在查尔酮合酶CHS、查尔酮异构酶CHI和黄酮合酶FNS的依次作用下生成黄酮核心骨架。
图2.亮白曲霉黄酮生物合成基因簇cfo及其代谢产物分析。a.基因簇cfo中各基因的组成，cfo中包含了12个基因，其中核心基因是cfoA、自抗性基因是cfoL，此外，还包括P450酶基因(cfoH)，NADPH-依赖的FMN还原酶基因(cfoJ)，NTF2-like蛋白基因(cfoI)，CocE/NonD家族水解酶基因(cfoK)，三个单加氧酶基因(cfoE，cfoF和cfoG)和三个甲基转移酶基因(cfoB，cfoC和cfoD)；b.亮白曲霉野生型和突变株ΔcfoA的代谢产物分析及其对应的黄酮类化合物。与野生型相比突变株ΔcfoA中的代谢产物1-4消失，经过分离鉴定发现它们均为黄酮类化合物，其中化合物1氯黄酮为终产物。
图3.亮白曲霉和不同突变株的代谢产物分析及其对应的黄酮类化合物。对所有基因缺失突变株的代谢产物进行分析，通过分离鉴定发现了23个黄酮类化合物，它们均是在终产物1的生物合成过程中的中间产物。
图4.真菌中黄酮类化合物的生物合成途径。杂合酶CfoA作为核心酶，以苯甲酸或对羟基苯甲酸作为起始单元与4分子的丙二酰CoA缩合，生成关键中间体查尔酮，然后在CfoK、CfoJ作用下形成黄酮核心骨架，在此基础上，核心骨架在不同后修饰酶的催化下形成终产物1。
图5.真菌黄酮与植物黄酮结构中的同位素分布。化合物1中化学键的加粗代表来自[1,2-13C2]乙酸钠，植物柚皮素中的黑点代表来自[1,3-14C2]丙二酰辅酶A。
图6.CfoK与目前在自然界已发现的CHI进化树分析。CfoK与CocE/NonD家族水解酶位于同一个分支，而植物CHI和细菌CHI分别位于另外两个独立的分支上。
图7.CfoK与突变体的体外酶活分析及其催化机制。a.CfoK及其突变体的体外酶活分析；b.CfoK的催化机制，H33上的N拔除底物C2′-OH上的氢，诱发oxa-Micheal加成反应，以 6-endo-trig的关环方式形成含有烯醇负离子的C环中间体，并通过与Y50形成氢键得到稳定，H33咪唑环上氢离子的回补和烯醇互变，最终形成了含有四氢吡喃酮形式的黄烷酮产物24。
图8.CfoJ与目前在自然界已发现的FNS进化树分析。CfoJ与NADPH依赖的FMN还原酶家族位于同一个分支，而FNS I和FNS II分别位于另外两个独立的分支上。
图9.CfoJ与突变体的体外酶活分析及其催化机制。a.CfoJ与不同辅因子的体外酶活分析以及CfoJ突变体的体外酶活分析；b.CfoJ的催化机制，首先H2O拔除活化的底物C3位上的氢原子，然后电子流向C2和C3之间，导致C2位上的氢原子以氢负的形式带着一对电子流向FMN的N5位，同时C2和C3之间形成双键，FMN被还原为FMNH2。
图10.CfoK和CfoJ的体外酶活验证。a.CfoK蛋白的分离纯化与体外酶活分析；b.CfoJ蛋白的分离纯化与体外酶活分析。
图11.工程菌株代谢产物分析及其相应的黄酮类化合物。(a)米曲霉工程菌株代谢产物分析；(b)土曲霉工程菌株代谢产物分析；(c)酿酒酵母工程菌株代谢产物分析；(d)工程菌株中分离获得的黄酮类化合物。
图12.化合物氯黄菌素(1)和去氯氯黄菌素(2)抑制拟南芥种子的萌发；a.拟南芥种子在MS平板培养基上的萌发，阴性对照二甲基亚砜(DMSO)，阳性对照草铵膦(GA)，供试化合物氯黄菌素(1)和去氯氯黄菌素(2)。b.不同浓度下氯黄菌素(1)和去氯氯黄菌素(2)对种子萌发的生长抑制曲线。
图13.不同亮白曲霉工程菌株的代谢产物HPLC分析。
图14.基因cfoL是氯黄菌素(1)的自抗性基因验证。
图15.基因cfoL赋予烟曲霉对氯黄菌素(1)的抗性；a.野生型烟曲霉在PDA平板和含化合物1的PDA平板上的生长。b.烟曲霉中导入cfoL基因在含化合物1的抗性平板上的转化子筛选，以及转化子PCR验证。c.野生型烟曲霉和过表达cfoL基因的烟曲霉在PDA平板上的生长。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
下列实施例中未注明具体条件的实验方法，通常按照常规条件或丝状真菌标准操作的条件或按照制造厂商所建议的条件。除非另外说明，百分比和份数按重量计。下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
酶解液：称取0.4g纤维素酶(Sigma产品，产品目录号：C1184)、0.4g裂解酶(Sigma产品，产品目录号：L1412)、0.2g蜗牛酶(生工生物工程股份有限公司产品，上海，产品目录号：SB0870)溶解于50ml的0.6M MgSO4水溶液中，经由0.22μm的无菌过滤器过滤除菌。
本发明中质粒提取采用OMEGA公司Plasmid Mini Kit I试剂盒(D6942-01)，DNA片段回收是采用OMEGA公司Cycle-Pure Kit试剂盒(D6492-01)，凝胶回收是采用OMEGA公司Gel Extraction Kit试剂盒(D2500-01)。
PDBS平板：24g/L马铃薯土豆培养基PDB干粉(BD公司产品，产品目录号：7114771)，1.2M山梨醇，4g/L琼脂糖，余量为去离子水，121℃高压灭菌20分钟后48℃保温。
PDA平板：39g/L马铃薯土豆培养基PDA干粉(BD公司产品，产品目录号：633840)，余量为去离子水，121℃高压灭菌20分钟，待冷却至约60℃制备平板。
PDAS平板：39g/L马铃薯土豆培养基PDA干粉(BD公司产品，产品目录号：633840)，1.2M山梨醇，余量为去离子水，121℃高压灭菌20分钟，待冷却至约60℃制备平板。
SGCY培养基：2％蔗糖，1％葡萄糖，0.5％酪蛋白水解物，0.5％酵母浸出粉，1％MgCl2·6H2O，余量为去离子水，121℃高压灭菌20分钟，待冷却至约30℃接种培养。
SYT培养基：1％可溶性淀粉，0.4％酵母浸出粉，0.2％胰蛋白胨，0.1％CaCO3，0.004％ FeSO4·7H2O，0.1％KBr，余量为去离子水，121℃高压灭菌20分钟，待冷却至约30℃接种培养。
PPM培养基：15％蔗糖，2.5％豆粕，0.5％蛋白胨，0.1％NaNO3，余量为去离子水，121℃高压灭菌20分钟，待冷却至约30℃接种培养。
YPED培养基：2％蔗糖、1％酪蛋白水解物、1％多聚蛋白胨，1％酵母提取物，余量为去离子水，121℃高压灭菌20分钟，待冷却至约30℃接种培养。
MM培养基：0.2％NH4Cl、0.1％(NH4)2SO4、0.05％KCl、0.05％NaCl、0.1％KH2PO4、0.05％Mg2SO4·7H2O、0.002％FeSO4·7H2O和2％蔗糖，余量为去离子水，121℃高压灭菌20分钟。
CD培养基：0.2％可溶性淀粉、0.1％KH2PO4、0.1％酵母浸出汁、0.05％Mg2SO4·7H2O、0.01％FeSO4·7H2O、0.055％CaCl2、2％麦芽糖，1％多聚蛋白胨，余量为去离子水，121℃高压灭菌20分钟，待冷却至约30℃接种培养。
实施例1、自抗性基因共定位发现cfo基因簇
1.1首先在459株已经测序的真菌基因组中寻找ALS(乙酰乳酸合酶)基因，发现49株菌的基因组中ALS基因是多拷贝的。通过antiSMASH预测发现49个基因组中存在2765个次级代谢产物生物合成基因簇。通过共定位分析最终锁定来源于亮白曲霉的cfo基因簇中含有ALS基因。
1.2通过对实验室保存的一株亮白曲霉MEFC1001(可市售获得，保藏于中国微生物菌种保藏管理委员会普通微生物中心的真菌，登记入册编号为：CGMCC No.3.15294)进行基因组测序及生物信息学分析，定位了cfo基因簇(核酸序列如SEQ ID No.1所示)，它包含了12个基因(图2a，表1)，其中cfoA、cfoB、cfoC、cfoD、cfoE、cfoF、cfoG、cfoH、cfoI、cfoJ、cfoK共11个基因是负责黄酮生物合成的基因，以及cfoL是自抗性基因，编码ALS蛋白。
表1.基因簇cfo中各基因编码蛋白的功能分析
序列信息如下：
SEQ ID No.1







SEQ ID No.2
SEQ ID No.3

SEQ ID No.4
SEQ ID No.5
SEQ ID No.6
SEQ ID No.7
SEQ ID No.8
SEQ ID No.9
SEQ ID No.10
SEQ ID No.11

SEQ ID No.12

SEQ ID No.13
SEQ ID No.14
SEQ ID No.15
SEQ ID No.16
SEQ ID No.17
SEQ ID No.18
SEQ ID No.19
SEQ ID No.20
SEQ ID No.21
SEQ ID No.22
SEQ ID No.23

SEQ ID No.24

SEQ ID No.25
实施例2、真菌黄酮生物合成途径解析
2.1基因簇cfo中各基因缺失突变株的构建
根据同源重组双交换技术，对亮白曲霉MEFC1001中cfo基因簇包含的12个基因进行了敲除，构建不同基因缺失的突变株。
具体的，根据cfo基因簇的核苷酸序列，在各基因的上下游分别设计一对外部引物。通过PCR扩增获得用于同源重组的左右同源交换臂，长度分别大约为1500个碱基对，通过融合PCR将同源臂与潮霉素抗性标签hph融合，纯化浓缩上述PCR反应产物获得的打靶元件，将打靶元件敲入MEFC1001菌株中，筛选获得基因缺失突变株。
具体的将MEFC1001菌株于SGCY培养基中28℃220rpm培养2天，100目的无菌滤布收集菌丝，采用菌丝重量10倍体积的酶解液消化细胞壁，酶解条件为30℃130rpm消化2小时。采用500目无菌滤布过滤酶解液，滤液4000rpm离心收集获得原生质体，用预冷1.0M山梨醇溶液洗涤一次，再用预冷的STC(1.0M山梨醇，50mM Tris·HCl-pH 8.0，50mM CaCl2)洗涤一次，最后把原生质体重悬于预冷的STC中，浓度调整为5×107个/mL，得到原生质体悬液。向悬液中加入10μL的各基因打靶元件(约3μg)，再加入50μL PSTC(40％PEG 4000，1.2M山梨醇，50mM Tris·HCl-pH 8.0，50mM CaCl2)，轻轻混匀，冰浴30min。加入1mL PSTC，混匀后室温放置20min，与15mL PDBS混合倒入5块PDAS筛选平板(含50mg/L潮霉素B)，30℃黑暗条件下培养5天。
从筛选平板挑选具有潮霉素抗性的转化子转接至PDAH平板(PDA+50mg/L潮霉素B)上，培养5天后，挑取单菌落上的部分菌丝，提取基因组，以此为模板采用同源臂上的外部引物PCR扩增。若是随机插入PCR产物大小大约为3.5kb，若发生同源重组PCR产物大小大约为5.0kb，则表明目的基因被敲除。
2.2基因敲除突变株的代谢产物分析
在PPM培养基中分别接种构建的各基因缺失突变株和野生型MEFC1001菌株，它们在培养基中的孢子数为107个/50mL。220rpm 30℃培养7天，获得各菌株的发酵液。采用同体积的乙酸乙酯对发酵液萃取，浓缩获得粗提物。甲醇溶解后0.22μM的有机膜过滤，Waters ACQUITY UPLC分析，Eclipse Plus C18RRHD色谱柱(50mm×2.1mm,1.8μm)，分析方法：流动相A(95％水+5％乙腈+0.05％甲酸)，流动相B(100％乙腈+0.05％甲酸)，梯度洗脱0-0.58min 100％-80％A,0.58-4.05min 80％-40％A,4.05-5.79min 40％A,5.79-6.37min 40％-0％A,6.95-7.53min 100％A，流速0.6mL/min，检测波长345nm。
根据分析结果，对突变株的发酵规模扩大，发酵条件同上，采用相同体积的乙酸乙酯对发酵液萃取三次，浓缩获得粗提物。采用干法装柱对粗提物进行柱层析，填料为十八烷基硅烷键合硅胶填料，甲醇水梯度洗脱(甲醇体积10％～100％)，每个梯度洗脱10个柱体积。取少量HPLC分析，分析方法同上，根据各组分中黄酮类化合物的极性大小和保留时间，确定最佳制备条件。纯化条件：流动相A(100％水+0.05％甲酸)，流动相B(100％乙腈+0.05％甲酸)，根据目标化合物的极性调整流动相的比例，2mL/min的流速洗脱，检测波长345nm，色谱柱Waters X-bridge C18(100mm×10mm,5μm)。在相应保留时间收集获得相应的黄酮类化合物。分离和鉴定突变株和野生型中的代谢产物(图3)。
首先对敲除三个甲基转移酶的突变株发酵产物进行分析。在ΔcfoB中积累了化合物5-7，它们的C7位与1相比由原来的甲氧基变为羟基，表明CfoB负责催化C7-OH的甲基化。在ΔcfoC中积累了化合物8-12，其中8、9和11的C6-OH以及10的C8-OH与终产物1相比均缺少了甲基，表明CfoC负责C6-OH或C8-OH的甲基化。在突变株ΔcfoD中积累了化合物14，在14中C3位由甲氧基变为羟基，因此CfoD负责C3-OH的甲基化。
对敲除三个单加氧酶和P450酶的突变株发酵产物进行分析。在突变株ΔcfoE中，化合物1消失，2得到了积累，因此CfoE行使卤化酶的功能，负责在2的C3'位加上氯原子形成终产物1。在突变株ΔcfoF中，积累了化合物3、4、12和13，它们的C3位均没有取代基团，表明CfoF负责C3位的羟基化。在敲除cfoG的突变株中产生了化合物12和15，与其他中间体相比，它们的C6和C8位均缺少了羟基，表明CfoG负责黄酮类化合物C6或C8位的羟基化。敲除P450基因cfoH的突变株中化合物4和12的C2'位未被氧化，表明CfoH负责C2'位的羟基化。至此基于基因敲除实验和中间产物的结构信息，确定了cfo基因簇中三个甲基转移酶、三个氧化酶和一个P450酶的功能(图4)。
2.3真菌黄酮关键中间体查尔酮骨架的装配机制分析
敲除cfoK的突变株代谢产物中积累了化合物20-23，其中20和22是查耳酮类化合物，表明在真菌中黄酮合成关键中间体与植物类似，都是查耳酮，同时也说明基因簇cfo中的核心基因cfoA编码的非核糖体肽-聚酮杂合酶(NRPS-PKS)可以合成查尔酮，具有查尔酮合酶(CHS)的功能，而植物中的CHS是III型的PKS。进一步的通过[1,2-13C2]乙酸钠标记，发现真菌中的查尔酮是由CfoA催化苯甲酸或对羟基苯甲酸与4分子丙二酰辅酶A缩合形成的，明显不同于植物中III型PKS催化对香豆酰辅酶A与3分子丙二酰辅酶A形成查尔酮的组装模式(图5)。
2.4真菌CHI和FNS体外酶活分析
在突变株ΔcfoK中积累了查尔酮20和22，推测CfoK具有与植物中查尔酮异构酶(CHI)相似的功能，可以催化分子内的oxa-Michael加成反应，将查尔酮转化为三环体系的黄烷酮。CfoK与目前自然界中已发现的CHI氨基酸序列相似性非常低，进化树分析CfoK与植物CHI和细菌CHI位于不同的分支上(图6)。为进一步验证cfoK基因的功能，首先采用5′-race和3′-race技术获得cfoK的CDS序列，密码子优化后在大肠杆菌中诱导表达获得可溶性蛋白，分离纯化获得CfoK，体外酶活分析和点突变实验表明CfoK确实具有CHI的活性，同时其催化机制是组氨酸残基介导的oxa-Michael加成反应，与植物CHI中H2O介导的反应机制完全不同(图7)。综述所述，CfoK是一种新型的查尔酮异构酶。
在突变株ΔcfoJ中积累了黄烷酮16-19，推测CfoJ具有与植物中黄酮合酶(FNS)相似的功能，可以催化脱饱和反应在C2和C3位形成双键，将黄烷酮转化为黄酮。目前自然界发现了两类FNS，分别是FNS I(α-KG依赖的双加氧酶)和FNS II(细胞色素P450)。而CfoJ注释为NADPH依赖的FMN还原酶，进化树分析CfoJ与FNS I和FNS II位于不同的分支上(图8)。为进一步验证cfoJ基因的功能，首先采用5′-race和3′-race技术获得cfoJ的CDS序列，密码子优化后在大肠杆菌中诱导表达获得可溶性蛋白，分离纯化获得CfoJ，体外酶活分析和点突变实验表明CfoJ确实具有FNS的活性，同时其催化机制是依赖于FMN介导的C2-C3位的脱氢反应，与自然界中的两类FNS自由基催化机制完全不同(图9)。 综上所述，CfoJ是一种新型的黄酮合酶。
为了进一步验证cfoK和cfoJ基因的功能，我们进行了各自编码蛋白的体外酶活实验，首先采用5′-race和3′-race技术获得cfoK和cfoJ的CDS序列，密码子优化后在大肠中诱导表达获得可溶性蛋白，分离纯化获得CfoK和CfoJ(图10)。以相应中间体为底物构建酶活反应体系。结果显示CfoK可以催化查耳酮20和22生成相应柚皮素(27)和乔松素(24)(图10a)，CfoJ可以催化乔松素(24)和化合物18生成相应白杨素(26)和3(图10b)。进一步验证了CfoK和CfoJ在黄酮生物合成中的功能。
至此，通过基因敲除、同位素标记，体外酶活及突变体活性分析等实验，解析了真菌中黄酮类化合物完整的生物合成途径，如图4所示，真菌黄酮完全不同于植物黄酮的生物合成。
实施例3、黄酮合成关键基因的异源表达
为验证利用真菌生物合成途径构建黄酮细胞工厂的可行性，申请人利用真菌黄酮合成途径中的三个关键酶CfoA(CHS)、CfoK(CHI)和cfoJ(FNS)作为酶学元件，分别在米曲霉(Aspergillus oryzae)、土曲霉(Aspergillus terreus)和酿酒酵母(Saccharomyces cerevisiae)中构建了产黄酮的底盘细胞，为通过合成生物学构建植物源的黄酮类化合物奠定了基础。具体如下：
3.1构建含关键基因的异源表达质粒
采用特异性引物以MEFC1001的基因组为模板PCR扩增基因cfoA,cfoK和cfoJ的核苷酸序列。用限制性内切酶KpnI酶切用于异源表达的pTAex3载体，回收线性化的pTAex3载体。通过Seamless Assembly Cloning(Clone Smarter)试剂盒分别将cfoA,cfoK和cfoJ基因与线性化的pTAex3载体连接，获得质粒pTAex3-cfoA，pTAex3-cfoK和pTAex3-cfoJ。限制性内切酶XbaI酶切用于异源表达的pAdeA载体，回收线性化的pAdeA载体。扩增pTAex3-cfoK质粒上含有amyB启动子和终止子的cfoK基因，通过Seamless Assembly Cloning试剂盒连接到线性化的pAdeA载体上，构建pAdeA-cfoK质粒。限制性内切酶HindIII酶切用于异源表达的pBARI载体，回收线性化的pBARI载体。扩增pTAex3-cfoJ质粒上含有amyB启动子和终止子的cfoJ基因，通过Seamless Assembly Cloning试剂盒连接到线性化的pBARI载体上，构建pBARI-cfoJ质粒。
3.2异源表达菌株的构建及其代谢产物分析
将米曲霉按孢子接种量1×107个/50mL接种至YPED液体培养基中，28℃220rpm培养12小时。菌丝体的收集、原生质体的酶解、以及通过PEG-CaCl2介导的原生质体转化方法进行质粒的转化实验。其中转化pTAex3-cfoA质粒时，原生质体再生和筛选培养为MMAS(MM+0.2％琼脂+1.2M山梨醇+0.15％甲硫氨酸+0.01％腺嘌呤)。转化子通过PCR验证获得表达cfoA基因的突变株Ao-cfoA。在突变株Ao-cfoA的基础上转化pAdeA-cfoK质粒，原生质体再生和筛选培养为MMAS(MM+0.2％琼脂+1.2M山梨醇+0.15％甲硫氨酸)。转化子通过PCR验证获得同时表达cfoA和cfoK基因的突变株Ao-cfoA-cfoK。在突变株Ao-cfoA-cfoK的基础上转化pBARI-cfoJ质粒，原生质体再生和筛选培养为MMAS(MM+0.2％琼脂+1.2M山梨醇+0.15％甲硫氨酸+500μg/mL草铵膦)。转化子通过PCR验证获得同时表达cfoA、cfoK和cfoJ基因的突变株Ao-cfoA-cfoK-cfoJ。采用与米曲霉工程菌株相似的构建方式，分别在土曲霉中转化获得突变株At-cfoA，At-cfoA-cfoK和At-cfoA-cfoK-cfoJ，在酿酒酵母中转化获得突变株Sc-cfoA，Sc-cfoA-cfoK和Sc-cfoA-cfoK-cfoJ。
分别将米曲霉、土曲霉、酿酒酵母的野生菌株和工程菌株接种至CD液体培养基中，28℃220rpm培养3天，培养结束后的发酵液处理方法、粗提物分析方法、化合物的分离纯化方法等均同于实施例2.2，结果显示异源表达的工程菌株中均能产生黄酮类化合物(图11)。具体而言，单独表达cfoA可以产生化合物22和化合物25，同时表达cfoA和cfoK可以产生化合物24，同时表达cfoA、cfoK和cfoJ可以产生化合物24和化合物26。
关键基因的异源表达实验进一步验证了真菌黄酮的生物合成途径，同时证明该途径可 以为黄酮细胞工厂的构建提供酶学元件，为通过合成生物学在微生物中生产植物源的黄酮类化合物奠定了基础。
实施例4、氯黄菌素和去氯氯黄菌素的分离与纯化
4.1将亮白曲霉MEFC1001菌株(可市售获得，保藏于中国微生物菌种保藏管理委员会普通微生物中心的真菌，登记入册编号为：CGMCC 3.15294)置于PDA平板上静止培养，用无菌水洗涤孢子，接种于SM培养基，28℃，220rpm培养种子液，接种于PPM培养基中，28℃，220rpm发酵14天，获得发酵液。
4.2采用相同体积的乙酸乙酯对发酵液萃取三次，浓缩获得粗提物。采用干法装柱对粗提物进行柱层析，填料为十八烷基硅烷键合硅胶填料，甲醇水梯度洗脱(甲醇体积10％～100％)，每个梯度洗脱10个柱体积。目标化合物1在80％甲醇/水组分中，化合物2在60％甲醇/水组分中。将80％甲醇/水组分和60％甲醇/水组分浓缩后甲醇溶解，0.22μm滤膜过滤后半制备液相进行纯化。纯化方法：流动相A(100％水+0.05％甲酸)，流动相B(100％乙腈+0.05％甲酸)，色谱柱Waters X-bridge C18(100mm×10mm,5μm)，流速2mL/min，检测波长345nm，根据化合物1和2的极性大小，调整流动相A和B的比例，在相应保留时间收集各化合物。根据HRESIMS和NMR鉴定，最终确定化合物1和2分别是氯黄菌素(chlorflavonin，CAS：23363-64-6)和去氯氯黄菌素(dechlorochlorflavonin，CAS:51724-52-8)。
化合物1的结构式如下所示：
所述化合物2的结构式如下所示：
实施例5、氯黄菌素和去氯氯黄菌素抑制病原真菌活性评价
5.1选取两株人类致病真菌白色念珠菌(Candida albicans)、烟曲霉(Aspergillus fumigatus)，以及4株植物病原真菌齐整小核菌(Sclerotium rolfsii Sacc.)、灰霉病菌(Botrytis cinerea)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cucumerinum,FOC)和苹果炭疽病菌(Colletotrichum gloeosporioides)作为供试菌株。将白色念珠菌接种于PDB培养基中，28℃220rpm培养12小时，然后用无菌PDB培养基稀释至5×105个/mL，获得菌悬液备用。其他供试菌株接种于PDA平板上，28℃培养5-7天，待菌丝或孢子长满平板。用无菌0.85％NaCl溶液(含0.25％Tween20)洗涤并刮下菌丝，加入50mL无菌PDB培养基，得到菌悬液母液，进一步以无菌PDB培养基稀释得到5×105个/mL浓度的菌悬液备用。
5.2分别取1mg待测样品(化合物1和2)和阳性对照(两性霉素B)，溶解于100μL DMSO中，配成浓度为10mg/mL的溶液。充分混匀后，吸取50μL样品溶液到另一只离心管中，接着加入50μL DMSO，得到浓度减半的样品溶液。按照此方法，得到15组浓度依次减半的样品溶液。无菌条件下，取95μL待测的菌悬液依次加入到96孔板中，取5μL稀释后待测样品依次加入含菌悬液的96孔板中，待测化合物的终浓度依次为500、250、125、 62.5、31.25、15.63、7.81、3.91、1.95、0.98、0.49、0.24、0.12、0.06和0.03μg/mL。轻轻震荡混匀后，将96孔板密封至于28℃培养72小时。使用酶标仪在600nm波长下测定每孔的吸光值(或者在明亮处肉眼观察小孔内溶液是否浑浊)，能够在小孔内完全抑制指示菌生长的最低样品浓度即为该化合物的最小抑菌浓度(MIC)。上述检测平行测定三次，检测结果如下表所示，化合物1对C.albicans、A.fumigatus和S.rolfsii Sacc.均表现出非常强的抑制活性，化合物2对C.albicans和S.rolfsii Sacc.表现出强的抑制活性。尽管黄酮类化合物抑制C.albicans和A.fumigatus已有文献报道(J.Antibiot.2001,54,1031-1035.)，但在本专利中化合物1和2对S.rolfsii Sacc.的抑制活性为首次发现。因此化合物1和2具有开发成为农药杀菌剂、抗生素和食品防腐剂的潜力。
表2.化合物1和化合物2对病原真菌的抑制活性

a两性霉素B；NI:无抑制活性
实施例6、氯黄菌素具有抑制拟南芥种子萌发的活性
采用实施例1的方法获得化合物1(氯黄菌素)和化合物2(去氯氯黄菌素)；分别将化合物1(氯黄菌素)和化合物2(去氯氯黄菌素)溶于DMSO中，滤膜过滤除菌。加入到MS(0.216％Murashige and Skoog基础培养基+0.8％蔗糖+0.8琼脂)培养基中，稀释成不同浓度梯度(0.001μg/mL、0.02μg/mL、0.1μg/mL、0.2μg/mL、0.4μg/mL、0.6μg/mL、0.8μg/mL、1μg/mL、2μg/mL、10μg/mL)的平板。将野生型拟南芥种子用75％乙醇-10％次氯酸钠溶液消毒清洗，放置在配制好的不同浓度梯度的培养基中，4℃放置48小时。然后转移到植物培养间，20℃培养条件下，光照16小时黑暗8小时，共培养120小时，统计种子萌发情况。抑制率＝未正常萌发种子数/萌发种子数×100％。草铵膦为阳性对照，DMSO为阴性对照。实验结果显示当化合物1(氯黄菌素)在浓度为1μg/mL时可以完全抑制拟南芥种子的萌发，明显低于阳性药草铵膦的浓度(图12)。此外化合物2(去氯氯黄菌素)也具有抑制活性，其在浓度为20μg/mL时能够完全抑制拟南芥种子的萌发(图12)。因此，氯黄菌素和去氯氯黄菌素具有除草活性，具有开发成为除草剂的潜力。
实施例7、亮白曲霉MEFC1001黄酮类化合物的分离鉴定
7.1将亮白曲霉MEFC1001菌株(菌种保藏号：CGMCC No.3.15294)置于PDA平板上静止培养，用无菌水洗涤孢子，接种于SM培养基，28℃，220rpm培养种子液，接种于PPM培养基中，28℃，220rpm发酵14天，获得亮白曲霉MEFC1001菌株发酵液。
7.2采用相同体积的乙酸乙酯对发酵液萃取三次，浓缩获得粗提物。采用干法装柱对粗提物进行柱层析，填料为十八烷基硅烷键合硅胶填料，甲醇水梯度洗脱(甲醇体积10％～100％)，每个梯度洗脱10个柱体积。各组分浓缩后甲醇溶解，0.22μm滤膜过滤后半制备液相进行纯化。纯化方法：流动相A(100％水+0.05％甲酸)，流动相B(100％乙腈+0.05％甲酸)，色谱柱Waters X-bridge C18(100mm×10mm,5μm)，流速2mL/min，检测波长345nm，根据化合物的极性大小，调整流动相A和B的比例，在相应保留时间收集各组分中的化合物。共分离纯化获得4个化合物，根据HRESIMS和NMR鉴定它们均为黄酮类化合物，其中氯黄菌素(化合物1)为终产物，化合物2-4为化合物1生物合成途径中的中间体(如图2所示)。
实施例8、亮白曲霉中生产不同黄酮工程菌株的构建与产物分析
8.1亮白曲霉中黄酮类化合物生物合成基因簇的确定。基于亮白曲霉在PPM培养基中 的转录组分析和生物信息学预测，发现亮白曲霉中cfo基因簇可能负责黄酮类化合物的生物合成。于是根据同源重组双交换技术，对cfo基因簇中的核心基因cfoA进行了敲除，代谢产物分析发现突变株ΔcfoA与野生型相比黄酮类化合物消失，证明该基因簇负责黄酮类化合物的生物合成，且合成的终产物为氯黄菌素(图2)。
8.2亮白曲霉工程菌株的构建。为了在亮白曲霉中构建生产不同黄酮类化合物的工程菌株，首先对cfo中的基因(表1中的CfoG、CfoF、CfoD、CfoH、CfoK、CfoI、CfoC、CfoJ、CfoB、CfoE)分别进行了敲除，获得不同的基因缺失突变株。
具体的，根据cfo基因簇中各基因的核苷酸序列，分别在它们的上下游设计一对外部引物。通过PCR扩增获得用于同源重组的左右同源交换臂，长度分别大约为1500个碱基对，通过融合PCR将同源臂与潮霉素抗性标签hph融合，纯化浓缩上述PCR反应产物获得的打靶元件，将打靶元件敲入MEFC1001菌株中，筛选获得基因缺失突变株。
具体的将MEFC1001菌株于SGCY培养基中28℃220rpm培养2天，100目的无菌滤布收集菌丝，采用菌丝重量10倍体积的酶解液消化细胞壁，酶解条件为30℃130rpm消化2小时。采用500目无菌滤布过滤酶解液，滤液4000rpm离心收集获得原生质体，用预冷1.0M山梨醇溶液洗涤一次，再用预冷的STC(1.0M山梨醇，50mM Tris·HCl-pH 8.0，50mM CaCl2)洗涤一次，最后把原生质体重悬于预冷的STC中，浓度调整为5×107个/mL，得到原生质体悬液。向悬液中加入10μL的各基因打靶元件(约3μg)，再加入50μL PSTC(40％PEG 4000，1.2M山梨醇，50mM Tris·HCl-pH 8.0，50mM CaCl2)，轻轻混匀，冰浴30min。加入1mL PSTC，混匀后室温放置20min，与15mL PDBS混合倒入5块PDAS筛选平板(含50mg/L潮霉素B)，30℃黑暗条件下培养5天。
从筛选平板挑选具有潮霉素抗性的转化子转接至PDAH平板(PDA+50mg/L潮霉素B)上培养5天后，挑取单菌落上的部分菌丝，提取基因组，以此为模板采用同源臂上的外部引物PCR扩增。若是随机插入PCR产物大小大约为3.5kb，若发生同源重组PCR产物大小大约为5.0kb，则表明目的基因被敲除。
8.3亮白曲霉工程菌株的代谢产物分析。在PPM培养基中分别接种在2.2中构建的亮白曲霉工程菌株和野生型MEFC1001菌株，它们在培养基中的孢子数为107个/50mL。220rpm30℃培养7天，获得各菌株的发酵液。采用同体积的乙酸乙酯对发酵液萃取，浓缩获得粗提物。甲醇溶解后0.22μM的有机膜过滤，Waters ACQUITY UPLC分析，Eclipse Plus C18RRHD色谱柱(50mm×2.1mm,1.8μm)，分析方法：流动相A(95％水+5％乙腈+0.05％甲酸)，流动相B(100％乙腈+0.05％甲酸)，梯度洗脱0-0.58min 100％-80％A,0.58-4.05min80％-40％A,4.05-5.79min 40％A,5.79-6.37min 40％-0％A,and 6.95-7.53min 100％A，流速0.6mL/min，检测波长345nm。分析结果显示工程菌株中产生了与野生型不同的黄酮类代谢产物(图13)。
实施例9、工程菌株中黄酮类化合物的分离纯化
根据8.3中各突变株的产物分析结果，对代谢产物存在变化的突变株扩大发酵规模，培养基和发酵条件同8.2。发酵结束后，采用相同体积的乙酸乙酯对发酵液萃取三次，浓缩获得粗提物。采用干法装柱对粗提物进行柱层析，填料为十八烷基硅烷键合硅胶填料，甲醇水梯度洗脱(甲醇体积10％～100％)，每个梯度洗脱10个柱体积。取少量HPLC分析，分析方法同实施例2.3，根据各组分中黄酮类化合物的极性大小和保留时间，确定最佳制备条件。纯化条件：流动相A(100％水+0.05％甲酸)，流动相B(100％乙腈+0.05％甲酸)，根据目标化合物的极性调整流动相的比例，2mL/min的流速洗脱，检测波长345nm，色谱柱Waters X-bridge C18(100mm×10mm,5μm)。在相应保留时间收集获得相应的化合物。经过HRESIMS和NMR分析，最终在工程菌株中确定了22个黄酮类化合物，分别是化合物2-23，其中工程菌株ΔcfoB能够产生化合物5-7，工程菌株ΔcfoC能够产生化合物8-12,工程菌株ΔcfoD能够产生化合物3-4和12-14，工程菌株ΔcfoE能够产生化合物2和4，工程菌株ΔcfoF能够产生化合物3-4和12-13，工程菌株ΔcfoG能够产生化合物12和15，工程 菌株ΔcfoH能够产生化合物4和12，工程菌株ΔcfoI能够产生化合物3-4和12，工程菌株ΔcfoJ能够产生化合物16-19，工程菌株ΔcfoK能够产生化合物20-23。不同菌株与对应的化合物结构如表3所示，其中化合物6-8，10和19为新化合物。
在突变株ΔcfoK中积累了查尔酮20和22，推测CfoK具有与植物中查尔酮异构酶(CHI)相似的功能，可以催化分子内的oxa-Michael加成反应，将查尔酮转化为三环体系的黄烷酮。CfoK与目前自然界中已发现的CHI氨基酸序列相似性非常低，进化树分析CfoK与植物CHI和细菌CHI位于不同的分支上；推测CfoK是一种新型的查尔酮异构酶。
在突变株ΔcfoJ中积累了黄烷酮16-19，推测CfoJ具有与植物中黄酮合酶(FNS)相似的功能，可以催化脱饱和反应在C2和C3位形成双键，将黄烷酮转化为黄酮。目前自然界发现了两类FNS，分别是FNS I(α-KG依赖的双加氧酶)和FNS II(细胞色素P450)。而CfoJ注释为NADPH依赖的FMN还原酶，进化树分析CfoJ与FNS I和FNS II位于不同的分支上；推测CfoJ是一种新型的黄酮合酶。
表3.不同突变体中对应的黄酮类化合物
实施例10.黄酮类化合物抑制病原真菌活性评价
10.1选取两株人类致病真菌白色念珠菌(Candida albicans)、烟曲霉(Aspergillus fumigatus)，以及4株植物病原真菌齐整小核菌(Sclerotium rolfsii Sacc.)、灰霉病菌(Botrytis cinerea)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cucumerinum，FOC)和苹果炭疽病菌(Colletotrichum gloeosporioides)作为供试菌株。将白色念珠菌接种于PDB培养基中，28℃220rpm培养12小时，然后用无菌PDB培养基稀释至5×105个/mL，获得菌悬液备用。其他供试菌株接种于PDA平板上，28℃培养5-7天，待菌丝或孢子长满平板。用无菌0.85％NaCl溶液(含0.25％Tween20)洗涤并刮下菌丝，加入50mL无菌PDB培养基，得到菌悬液母液，进一步以无菌PDB培养基稀释得到5×105个/mL浓度的菌悬液备用。
10.2分别取亮白曲霉工程菌株中获得的化合物(3-23)和阳性对照(两性霉素B)，溶解于100μL DMSO中，配成浓度为10mg/mL的溶液。充分混匀后，吸取50μL样品溶液 到另一只离心管中，接着加入50μL DMSO，得到浓度减半的样品溶液。按照此方法，得到15组浓度依次减半的样品溶液。无菌条件下，取95μL待测的菌悬液依次加入到96孔板中，取5μL稀释后待测样品依次加入含菌悬液的96孔板中，待测化合物的终浓度依次为500、250、125、62.5、31.25、15.63、7.81、3.91、1.95、0.98、0.49、0.24、0.12、0.06和0.03μg/mL。轻轻震荡混匀后，将96孔板密封至于28℃培养72小时。使用酶标仪在600nm波长下测定每孔的吸光值(或者在明亮处肉眼观察小孔内溶液是否浑浊)，能够在小孔内完全抑制指示菌生长的最低样品浓度即为该化合物的最小抑菌浓度(MIC)。上述检测平行测定三次，检测结果如表4所示。
表4.黄酮类化合物对病原真菌的抑制活性

a两性霉素B；NI：无抑制活性
实施例11、氯黄菌素的发酵制备
11.1按照实施例4的方式利用亮白曲霉MEFC1001菌株制备氯黄菌素，具体而言：将亮白曲霉MEFC1001菌株置于PDA平板上静止培养，用无菌水洗涤孢子，接种于SM培养基，28℃，220rpm培养种子液，接种于PPM培养基中，28℃，220rpm发酵14天，获得发酵液。
11.2采用相同体积的乙酸乙酯对发酵液萃取三次，浓缩获得粗提物。采用干法装柱对粗提物进行柱层析，填料为十八烷基硅烷键合硅胶填料，甲醇水梯度洗脱(甲醇体积10％～100％)，每个梯度洗脱10个柱体积。目标化合物1在80％甲醇/水组分中。将80％甲醇/水组分浓缩后甲醇溶解，0.22μm滤膜过滤后半制备液相进行纯化。纯化方法：流动相A(100％水+0.05％甲酸)，流动相B(100％乙腈+0.05％甲酸)，色谱柱Waters X-bridge C18(100mm×10mm,5μm)，流速2mL/min，检测波长345nm，根据化合物1极性大小，调整流动相A和B的比例，在相应保留时间收集各化合物。根据HRESIMS和NMR鉴定，最终确定化合物1为氯黄菌素(chlorflavonin，CAS：23363-64-6)。
化合物1的结构式如下所示：
实施例12、cfoL基因是亮白曲霉抵抗氯黄菌素的自抗性基因
在亮白曲霉MEFC1001中，负责合成氯黄菌素的基因簇cfo中含有一个编码乙酰乳酸合酶的基因cfoL(核酸序列如SEQ ID No.13所示，氨基酸序列如SEQ ID No.25所示)(基因 簇结构如图2a所示)。
为证明cfoL基因是亮白曲霉对氯黄菌素的自抗性基因，构建了不同的基因缺失突变株，最终发现cfoL能够赋予亮白曲霉对氯黄菌素的抗性。具体如下：
12.1基因簇cfo中目标基因敲除和回补突变株的构建
根据cfoL的核苷酸序列，在其上下游分别设计一对外部引物。通过PCR扩增获得用于同源重组的左右同源交换臂，长度分别大约为1500个碱基对，同源臂中间连接潮霉素抗性标hph。纯化浓缩PCR产物获得打靶元件，将打靶元件敲入MEFC1001菌株中，筛选获得cfoL基因缺失的突变株ΔcfoL。
具体的，将MEFC1001菌株于SGCY培养基中28℃220rpm培养2天，100目的无菌滤布收集菌丝，采用菌丝重量10倍体积的酶解液消化细胞壁，酶解条件为30℃130rpm消化2小时。采用500目无菌滤布过滤酶解液，滤液4000rpm离心收集获得原生质体，用预冷1.0M山梨醇溶液洗涤一次，再用预冷的STC(1.0M山梨醇，50mM Tris·HCl-pH 8.0，50mM CaCl2)洗涤一次，最后把原生质体重旋于预冷的STC中，浓度调整为5×107个/mL，得到原生质体悬液。向悬液中加入10μL的基因打靶元件(约3μg)，再加入50μL PSTC(40％PEG 4000，1.2M山梨醇，50mM Tris·HCl-pH 8.0，50mM CaCl2),轻轻混匀，冰浴30min。加入1mL PSTC，混匀后室温放置20min，与15mL PDBS混合倒入5块PDAS筛选平板(含50mg/L潮霉素B)，30℃黑暗条件下培养5天。
从筛选平板挑选具有潮霉素抗性的转化子转接至PDAH平板(PDA+50mg/L潮霉素B)上，培养5天后，挑取单菌落上的部分菌丝，提取基因组，以此为模板采用同源臂上的外部引物PCR扩增。若是随机插入PCR产物大小大约为3.5kb，若发生同源重组PCR产物大小大约为5.0kb，则表明目的基因被敲除。
采用上述同样的方法，构建获得同时敲除cfoA和cfoL的双敲菌株ΔcfoA-ΔcfoL。cfoA是负责合成氯黄菌素的核心基因，敲除cfoA后菌株将不再产生氯黄菌素等黄酮类化合物。另外在亮白曲霉基因组上扩增cfoL基因，转化ΔcfoA-ΔcfoL突变株，获得回补cfoL的突变株ΔcfoA-ΔcfoL::cfoL。
12.2突变株的平板抗性实验
将12.1中获得的所有亮白曲霉突变株ΔcfoL、ΔcfoA-ΔcfoL、ΔcfoA-ΔcfoL::cfoL和野生型(WT)分别接种于PDA平板和含有终浓度10μg/mL氯黄菌素(1)的PDA平板，观察野生型和各突变株的生长状况(图14)。突变株ΔcfoL在两种平板上均生长缓慢，而不产化合物1(氯黄菌素)的突变株ΔcfoA-ΔcfoL在PDA中可以生长，在含化合物1(氯黄菌素)的PDA平板上生长受到抑制。回补cfoL的突变株ΔcfoA-ΔcfoL::cfoL在两种平板上均可生长。上述实验表明cfoL基因是亮白曲霉中的自抗性基因，它的存在可以抵抗氯黄菌素对菌株自身的抑制。
实施例13、自抗性基因cfoL能够赋予烟曲霉对氯黄菌素的抗性
为验证cfoL基因能否赋予其他菌株对氯黄菌素的抗性，申请人将cfoL基因导入了对氯黄菌素敏感的烟曲霉中，发现烟曲霉突变株获得了对氯黄菌素的抗性。因此cfoL基因与氯黄菌素的组合可用于烟曲霉的遗传转化。
为验证cfoL基因是否能够赋予其他菌株对氯黄菌素的抗性，申请人将cfoL基因导入了对氯黄菌素敏感的烟曲霉中。具体的，将亮白曲霉MEFC1001接种至PPM培养基中，28℃220rpm培养2天，采用MiniBEST Plant RNA Extraction Kit(TaKaRa)试剂盒提取mRNA，采用PrimeScriptTM RT reagent Kit with gDNA Eraser(TaKaRa)试剂盒反转录成cDNA，通过特异性引物扩增获得cfoL基因的CDS序列，通过融合PCR将cfoL-CDS与曲霉中常用的组成型启动子PgpdAt连接，构建过表达元件。烟曲霉中PEG-CaCl2介导的原生质体转化方法与实施例2.1中亮白曲霉的转化方法基本相同，筛选平板为PDAS(含10μg/mL的氯黄菌素)，30℃黑暗条件下培养5天，可观察到筛选平板上长出转化子。挑取部分菌丝提取基因组，采用可以获得cfoL-CDS序列的引物PCR扩增，结果显示与野生型相比所有的转化 子中均含有cfoL-CDS序列(图15)。以上结果表明cfoL基因能够赋予烟曲霉菌株对氯黄菌素的抗性，因此cfoL基因与氯黄菌素组合可以用于真菌的遗传操作，其中cfoL基因可作为基因敲除或外源基因导入时的抗性标签，氯黄菌素可用于阳性转化子的筛选。
除了上述提到的对氯黄菌素敏感的烟曲霉，本领域技术人员也可以通过常规技术手段获得其他对氯黄菌素敏感的微生物，从而利用cfoL基因作为筛选标记进行遗传转化。例如，本申请实施例5中记载了氯黄菌素对白色念珠菌(Candida albicans)和齐整小核菌(Sclerotium rolfsii Sacc.)具有显著的抑制作用。
基于上述教导，本领域技术人员可以采用cfoL基因和氯黄菌素针对氯黄菌素敏感的微生物建立遗传转化体系。
虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明，在不脱离本发明精神和范围内，本领域技术人员都可以在此基础上做出各种改动与变型，因此，本发明的保护范围应该以权利要求书所界定的为准。










Claims (36)

  1. 氯黄菌素或去氯氯黄菌素的用途，所述用途选择以下i-iii任意一组：
    i、在抑制微生物生长中的用途；
    ii、在制备抑制微生物生长的试剂中的用途；
    iii、在制备治疗由微生物所导致的疾病的药物中的用途；
    其特征在于，所述微生物为齐整小核菌(Sclerotium rolfsii Sacc.)、白色念珠菌(Candida albicans)或烟曲霉(Aspergillus fumigatus)。
  2. 根据权利要求1所述的用途，其特征在于，所述抑制微生物生长的试剂为抗生素。
  3. 根据权利要求1所述的用途，其特征在于，所述疾病为植物疾病。
  4. 氯黄菌素或去氯氯黄菌素的用途，所述用途选自以下a-c任意一组：
    a、在抑制植物生长中的用途；
    b、在制备抑制植物生长的试剂中的用途；
    c、在制备除草剂中的用途。
  5. 根据权利要求1-4任一所述的用途，其特征在于，
    所述氯黄菌素的结构式如下所示：
  6. 根据权利要求1-4任一所述的用途，其特征在于，
    所述去氯氯黄菌素的结构式如下所示：
  7. 一种与黄酮合成相关的基因，其特征在于，
    所述与黄酮合成相关的基因选自FAD依赖的氧化酶CfoG、单加氧酶CfoF、SAM依赖的甲基转移酶CfoD、细胞色素P450酶CfoH、酯水解酶CfoK、Scytalone脱水酶CfoI、SAM依赖的甲基转移酶CfoC、NADPH依赖的FMN还原酶CfoJ、SAM依赖的甲基转移酶CfoB、FAD依赖的氧化酶CfoE、杂合的非核糖体肽合酶与聚酮合酶CfoA中的一种或任意几种组合；
    所述CfoG的氨基酸序列与SEQ ID No.14相比具有至少80％的序列同一性，所述CfoF的氨基酸序列与SEQ ID No.15相比具有至少80％的序列同一性，所述CfoD的氨基酸序列与SEQ ID No.16相比具有至少80％的序列同一性，所述CfoH的氨基酸序列与SEQ ID No.17相比具有至少80％的序列同一性，所述CfoK的氨基酸序列与SEQ ID No.18相比具有至少80％的序列同一性，所述CfoI的氨基酸序列与SEQ ID No.19相比具有至少80％的序列同一性，所述CfoC的氨基酸序列与SEQ ID No.20相比具有至少80％的序列同一性，所述CfoJ的氨基酸序列与SEQ ID No.21相比具有至少80％的序列同一性，所述CfoB的氨基酸序列与SEQ ID No.22相比具有至少80％的序列同一性，所述CfoE的氨基酸序列与SEQ ID No.23相比具有至少80％的序列同一性，所述CfoA的氨基酸序列与SEQ ID No.24相比具有至少80％的序列同一性；
    优选的，所述与黄酮合成相关的基因来源于亮白曲霉。
  8. 权利要求7所述的与黄酮合成相关的基因在制备黄酮类化合物中的应用。
  9. 权利要求7所述的与黄酮合成相关的基因在制备能够产黄酮类化合物的基因工程菌株中的应用。
  10. 根据权利要求8或9所述的应用，其特征在于，所述黄酮类化合物选自化合物2-化合物23中的一种或任意几种。
  11. 一种新的黄酮类化合物，所述化合物选自化合物6、化合物7、化合物8、化合物10、化合物19、化合物2-化合物5、化合物9、化合物11-化合物18、化合物20-化合物23中的一种或任意几种；优选的，所述黄酮类化合物选自化合物6、化合物7、化合物8、化合物10或化合物19中的一种或任意几种。
  12. 权利要求11所述的黄酮类化合物的用途，所述用途包括在抑制微生物生长中的用途，或者，在制备抑制微生物生长的试剂中的用途，或者，在制备治疗由微生物所导致的疾病的药物中的用途；优选的，所述微生物选自白色念珠菌(Candida albicans)、烟曲霉(Aspergillus fumigatus)、齐整小核菌(Sclerotium rolfsii Sacc.)、灰霉病菌(Botrytis cinerea)中的一种或任意几种
  13. 一种基因工程菌株，所述基因工程菌株为将亮白曲霉中的与黄酮合成相关的基因进行突变所得到的基因工程菌株；所述与黄酮合成相关的基因选自权利要求7中的FAD依赖的氧化酶CfoG、单加氧酶CfoF、SAM依赖的甲基转移酶CfoD、细胞色素P450酶CfoH、酯水解酶CfoK、Scytalone脱水酶CfoI、SAM依赖的甲基转移酶CfoC、NADPH依赖的FMN还原酶CfoJ、SAM依赖的甲基转移酶CfoB、FAD依赖的氧化酶CfoE、杂合的非核糖体肽合酶与聚酮合酶CfoA中的一种或任意几种。
  14. 权利要求13所述的基因工程菌株在生产黄酮类化合物中的应用；优选的，所述黄酮类化合物选自化合物2-化合物23中的一种或任意几种。
  15. 一种制备黄酮类化合物的方法，所述方法包括对权利要求13所述的基因工程菌株进行发酵的步骤；优选的，所述黄酮类化合物选自化合物2-化合物23中的一种或任意几种。
  16. 根据权利要求11所述的黄酮类化合物，或者，权利要求14所述的应用，或者权利要求15所述的方法，其特征在于，
    所述化合物2结构式如下所示：
    所述化合物3结构式如下所示：
    所述化合物4结构式如下所示：
    所述化合物5结构式如下所示：
    所述化合物6结构式如下所示：
    所述化合物7结构式如下所示：
    所述化合物8结构式如下所示：
    所述化合物9结构式如下所示：
    所述化合物10结构式如下所示：
    所述化合物11结构式如下所示：
    所述化合物12结构式如下所示：
    所述化合物13结构式如下所示：
    所述化合物14结构式如下所示：
    所述化合物15结构式如下所示：
    所述化合物16结构式如下所示：
    所述化合物17结构式如下所示：
    所述化合物18结构式如下所示：
    所述化合物19结构式如下所示：
    所述化合物20结构式如下所示：
    所述化合物21结构式如下所示：
    所述化合物22结构式如下所示：
    所述化合物23结构式如下所示：
  17. 一种与黄酮合成相关的基因，其特征在于，
    所述与黄酮合成相关的基因选自酯水解酶CfoK、NADPH依赖的FMN还原酶CfoJ、杂合的非核糖体肽合酶与聚酮合酶CfoA中的一种或任意几种组合；
    所述CfoK的氨基酸序列与SEQ ID No.18相比具有至少80％的序列同一性；
    所述CfoJ的氨基酸序列与SEQ ID No.21相比具有至少80％的序列同一性；
    所述CfoA的氨基酸序列与SEQ ID No.24相比具有至少80％的序列同一性；
    优选的，所述与黄酮合成相关的基因来源于亮白曲霉。
  18. 权利要求17所述的与黄酮合成相关的基因在制备黄酮类化合物中的应用。
  19. 权利要求17所述的与黄酮合成相关的基因在制备能够产黄酮类化合物的基因工程菌株中的应用。
  20. 一种能够产黄酮类化合物的基因工程菌株，所述基因工程菌株为在出发菌株中引入权利要求17所述的与黄酮合成相关的基因所得到的基因工程菌株。
  21. 根据权利要求20所述的基因工程菌株，其特征在于，所述出发菌株选自曲霉、酵母、大肠杆菌中的一种或任意几种。
  22. 权利要求20或21所述的基因工程菌株在生产黄酮类化合物中的应用。
  23. 根据权利要求18-19或22任一所述的应用，其特征在于，所述黄酮类化合物选自化合物22、化合物24、化合物25和化合物26中的一种或任意几种；
    所述化合物22结构式如下所示：
    所述化合物24结构式如下所示：
    所述化合物25结构式如下所示：
    所述化合物26结构式如下所示：
  24. 一种制备黄酮类化合物的方法，所述方法包括对权利要求20或21所述的基因工程菌株进行发酵的步骤。
  25. 权利要求17中的CfoK在催化合成化合物27和/或化合物24中的应用；
    所述化合物24结构式如下所示：
    所述化合物27结构式如下所示：
  26. 权利要求17中的CfoJ在催化合成化合物26和/或化合物3中的应用；
    所述化合物26结构式如下所示：
    所述化合物3结构式如下所示：
  27. 一种抗性基因，所述抗性基因的氨基酸序列与SEQ ID No.25相比，具有至少80％的序列同一性。
  28. 根据权利要求27所述的抗性基因，其特征在于，所述抗性基因来源于亮白曲霉。
  29. 权利要求27或28所述的抗性基因在抵抗氯黄菌素或者在赋予氯黄菌素敏感的微生物对氯黄菌素产生抗性中的应用。
  30. 权利要求27或28所述的抗性基因在作为抗生素抗性筛选标记或筛选标签中的应用；优选的，所述抗生素为氯黄菌素。
  31. 根据权利要求30所述的应用，其特征在于，所述筛选标记或筛选标签置于载体/质粒中。
  32. 权利要求27或28所述的抗性基因在制备抵抗氯黄菌素或对氯黄菌素产生抗性或能够耐受氯黄菌素的基因工程菌株中的应用。
  33. 一种制备抵抗氯黄菌素或对氯黄菌素产生抗性或耐受氯黄菌素的基因工程菌株的方法，所述方法包括在所述菌株的出发菌株中引入权利要求27或28所述的抗性基因的步骤。
  34. 根据权利要求33所述的方法，其特征在于，所述基因工程菌株的出发菌株为对氯黄菌素敏感的菌株。
  35. 根据权利要求33所述的方法，其特征在于，所述引入包括将所述抗性基因在出发菌株中进行表达的步骤。
  36. 一种能够抵抗氯黄菌素或对氯黄菌素产生抗性或能够耐受氯黄菌素的基因工程菌株，所述基因工程菌株由权利要求33-35任一方法制备得到。
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