WO2018120337A1 - Terpène synthase et son utilisation - Google Patents

Terpène synthase et son utilisation Download PDF

Info

Publication number
WO2018120337A1
WO2018120337A1 PCT/CN2017/071449 CN2017071449W WO2018120337A1 WO 2018120337 A1 WO2018120337 A1 WO 2018120337A1 CN 2017071449 W CN2017071449 W CN 2017071449W WO 2018120337 A1 WO2018120337 A1 WO 2018120337A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
gene
synthase
nucleic acid
acid molecule
Prior art date
Application number
PCT/CN2017/071449
Other languages
English (en)
Chinese (zh)
Inventor
刘天罡
卞光凯
韩以超
侯安伟
苑玉杰
刘然
Original Assignee
武汉臻智生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 武汉臻智生物科技有限公司 filed Critical 武汉臻智生物科技有限公司
Publication of WO2018120337A1 publication Critical patent/WO2018120337A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P15/00—Preparation of compounds containing at least three condensed carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/002—Preparation of hydrocarbons or halogenated hydrocarbons cyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic

Definitions

  • the invention relates to the field of biology.
  • the invention relates to an indole synthase and its use. More specifically, the invention relates to steroid synthase, nucleic acid molecules, constructs, recombinant cells, and uses thereof, and methods of synthesizing terpenoids.
  • Terpenoids are a general term for compounds containing isoprene units. To date, approximately 76,000 terpenoids have been found in animals, plants and microorganisms. It is widely used in the perfume production industry, health care products industry, agricultural production and medical industry.
  • the present invention aims to solve at least one of the technical problems existing in the prior art at least to some extent.
  • the invention proposes a terpenoid synthase.
  • the catalytic substrate of the quinone synthase is a compound having 10 to 25 carbon atoms.
  • the indole synthase of the present invention is capable of catalyzing a compound of a long carbon chain and has a broad spectrum and is capable of catalyzing various substrates in order to obtain different terpenoids.
  • the above-described steroid synthase may further have the following additional technical features:
  • the catalytic substrate is selected from the group consisting of: geranyl pyrophosphate; geranyl pyrophosphate; farnesyl pyrophosphate; geranylgeranyl pyrophosphate; and geranyl method Nicot pyrophosphate.
  • Each of the terpene synthases according to embodiments of the present invention is capable of catalyzing the synthesis of terpenoids from the above substrates, and different terpenoid synthases catalyze the different terpenoids for the same substrate.
  • the steroid synthase has the amino acid sequence of any one of SEQ ID NOS: 1 to 6.
  • the hydrazine synthase according to an embodiment of the present invention has a broad spectrum and is capable of catalyzing various substrates in order to obtain different terpenoids.
  • the invention provides a nucleic acid molecule.
  • the nucleic acid molecule encodes a steroid synthase as described above.
  • a nucleic acid molecule according to an embodiment of the invention is capable of efficiently encoding a terpene synthase to catalyze a variety of substrates in order to obtain different terpenoids.
  • the nucleic acid molecule described above may also have the following additional technical features:
  • the nucleic acid molecule has the nucleotide sequence shown in any one of SEQ ID NOS: 7 to 12.
  • a nucleic acid molecule according to an embodiment of the invention is capable of efficiently encoding a terpene synthase to catalyze a variety of substrates in order to obtain different terpenoids.
  • the invention proposes a construct.
  • the construct contains a nucleic acid molecule as described above.
  • a construct according to an embodiment of the invention can encode a plurality of substrates by expressing a nucleic acid molecule encoding a synthetic terpene synthase to obtain different terpenoids.
  • the invention provides a recombinant cell.
  • the recombinant cell comprises: a first nucleic acid molecule encoding a purine synthase.
  • a hydrazine synthase is obtained, thereby catalyzing various substrates to obtain different terpenoids.
  • the steroid synthase has the amino acid sequence of any one of SEQ ID NOS: 1 to 6.
  • a hydrazine synthase is obtained, thereby catalyzing various substrates to obtain different terpenoids.
  • the first nucleic acid molecule has the nucleotide sequence shown in any one of SEQ ID NOS: 7 to 12.
  • a hydrazine synthase is obtained, thereby catalyzing a plurality of substrates to obtain different hydrazines. Class of compounds.
  • the recombinant cell further comprises: a second nucleic acid molecule selected from at least one of the following: an atoB gene derived from Escherichia coli XL1-blue or an idi gene; The erg13 gene, tHMG1 gene, erg12 gene, erg8 gene or mvd1 gene of yeast INVSC1. High yield of terpenoids is achieved by over-expression of the above genes in order to synthesize a large amount of catalytic substrate.
  • a second nucleic acid molecule selected from at least one of the following: an atoB gene derived from Escherichia coli XL1-blue or an idi gene; The erg13 gene, tHMG1 gene, erg12 gene, erg8 gene or mvd1 gene of yeast INVSC1. High yield of terpenoids is achieved by over-expression of the above genes in order to synthesize a large amount of catalytic substrate.
  • the invention provides the use of a steroid synthase or nucleic acid molecule or construct or recombinant cell described above for the synthesis of a steroid. Thereby, in order to obtain different terpenoids.
  • the synthesis is carried out in a host cell, and the catalytic substrate of the steroid synthase is obtained by overexpressing at least one of the following genes in a host cell: from the large intestine The atoB gene or idi gene of Bacillus XL1-blue; the erg13 gene, tHMG1 gene, erg12 gene, erg8 gene or mvd1 gene derived from Saccharomyces cerevisiae INVSC1. High yield of terpenoids is achieved by over-expression of the above genes in order to synthesize a large amount of catalytic substrate.
  • the terpenoid has a structure of one of the following:
  • the invention provides a method of synthesizing the above described terpenoids.
  • the method comprises: cultivating the recombinant cells described above under conditions suitable for expression of the terpenoid to obtain a culture product; and isolating the terpenoid from the culture product .
  • FIG. 1 shows a GC-MS detection FgMS in vitro reaction chromatogram according to an embodiment of the present invention
  • Figure 3 shows a schematic diagram of the structure of plasmid pMH1 according to one embodiment of the present invention
  • Figure 4 is a schematic view showing the structure of plasmid pFZ81 according to an embodiment of the present invention.
  • Figure 5 is a schematic view showing the structure of plasmid pGB309 according to an embodiment of the present invention.
  • Figure 6 shows a schematic diagram of the structure of plasmid pGB310 according to one embodiment of the present invention
  • Figure 7 is a schematic view showing the structure of plasmid pGB311 according to an embodiment of the present invention.
  • Figure 8 is a schematic view showing the structure of plasmid pGB312 according to an embodiment of the present invention.
  • Figure 9 is a schematic view showing the structure of plasmid pGB313 according to an embodiment of the present invention.
  • Figure 10 is a schematic view showing the structure of plasmid pGB314 according to an embodiment of the present invention.
  • Figure 11 is a schematic view showing the structure of plasmid pGB147 according to an embodiment of the present invention.
  • Figure 12 is a schematic view showing the construction of a plasmid and a mutant strain for synthesizing different types of products for FgMS and FgGS fermentation according to an embodiment of the present invention
  • Figure 13 is a schematic view showing a method for synthesizing a terpenoid compound according to an embodiment of the present invention, wherein (a) is a schematic diagram of a highly efficient synthesis of a terpenoid chassis of the MVA pathway; (b) is a purine synthase with different chain lengths a schematic diagram of a combination of olefin pyrophosphate synthase, a compound of sesquiterpene (C15), diterpene (C20), and a sesquiterpene (C25); (c) a fermentation product of six E. coli strains (T7-T12) GS-MS chromatogram
  • Figure 14 shows a mass spectrum of FgMS and FgGS fermentation products according to one embodiment of the present invention
  • Figure 15 shows the product of AaTS synthesis in accordance with one embodiment of the present invention
  • Figure 16 shows a spectrum of compound (1) according to one embodiment of the present invention, wherein a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400MHz); c is the carbon spectrum ( 13 C NMR, CDCl 3 , 101MHz); d is the HSQC spectrum (CDCl 3 ); e is the 1 H- 1 H COSY spectrum (CDCl 3 ); f is the HMBC spectrum (CDCl 3 );
  • Figure 17 shows a spectrum of compound (2) according to one embodiment of the present invention, wherein a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400MHz); c is the carbon spectrum ( 13 C NMR, CDCl 3 , 101MHz); d is the HSQC spectrum (CDCl 3 ); e is the 1 H- 1 H COSY spectrum (CDCl 3 ); f is the HMBC spectrum (CDCl 3 );
  • Figure 18 shows a spectrum of compound (3) according to one embodiment of the present invention, wherein a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400MHz); c is the carbon spectrum ( 13 C NMR, CDCl 3 , 101MHz); d is the HSQC spectrum (CDCl 3 ); e is the 1 H- 1 H COSY spectrum (CDCl 3 ); f is the HMBC spectrum (CDCl 3 );
  • Figure 19 shows a spectrum of a compound (4) according to one embodiment of the present invention, wherein a is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400 MHz); b is a carbon spectrum ( 13 C NMR, CDCl 3 , 101 MHz) );
  • Figure 20 shows a spectrum of compound (5) according to one embodiment of the present invention, wherein a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400MHz); c is the carbon spectrum ( 13 C NMR, CDCl 3 , 101MHz); d is the HSQC spectrum (CDCl 3 ); e is the 1 H- 1 H COSY spectrum (CDCl 3 ); f is the HMBC spectrum (CDCl 3 );
  • Figure 21 shows a spectrum of the compound (6) according to one embodiment of the present invention, wherein a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400MHz); c is the carbon spectrum ( 13 C NMR, CDCl 3 , 101MHz); d is the HSQC spectrum (CDCl 3 ); e is the 1 H- 1 H COSY spectrum (CDCl 3 ); f is the HMBC spectrum (CDCl 3);
  • Figure 22 shows a spectrum of the compound (7) according to one embodiment of the present invention, wherein a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400MHz); c is the carbon spectrum ( 13 C NMR, CDCl 3 , 101MHz); d is the HSQC spectrum (CDCl 3 ); e is the 1 H- 1 H COSY spectrum (CDCl 3 ); f is the HMBC spectrum (CDCl 3 );
  • Figure 23 shows a spectrum of compound (8) according to one embodiment of the present invention, wherein a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400MHz); c is the carbon spectrum ( 13 C NMR, CDCl 3 , 101MHz); d is the HSQC spectrum (CDCl 3 ); e is the 1 H- 1 H COSY spectrum (CDCl 3 ); f is the HMBC spectrum (CDCl 3 );
  • Figure 24 shows a spectrum of compound (9) according to one embodiment of the present invention, wherein a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400MHz); c is the carbon spectrum ( 13 C NMR, CDCl 3 , 101MHz); d is the HSQC spectrum (CDCl 3 ); e is the 1 H- 1 H COSY spectrum (CDCl 3 ); f is the HMBC spectrum (CDCl 3 );
  • Figure 25 shows a spectrum of compound (10) according to one embodiment of the present invention, wherein a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( 1 H NMR, CDCl 3, 400MHz); c is carbon spectra (13 C NMR, CDCl 3, 101MHz); d is a HSQC spectrum (CDCl 3); e is a spectrum of 1 H- 1 H COSY (CDCl 3); f is the HMBC spectrum Figure (CDCl 3 );
  • Figure 26 shows a spectrum of the compound (11) according to one embodiment of the present invention, wherein a is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400 MHz); b is a carbon spectrum ( 13 C NMR, CDCl 3 , 101 MHz) );
  • Figure 27 shows a spectrum of the compound (12) according to one embodiment of the present invention, wherein a is a hydrogen spectrum ( 1 H NMR, CDCl 3 , 400 MHz); b is a carbon spectrum ( 13 C NMR, CDCl 3 , 101 MHz) );
  • a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation
  • b is a hydrogen spectrum ( 1 H NMR, CDCl 3 ) according to an embodiment of the present invention. , 400MHz);
  • c is the carbon spectrum ( 13 C NMR, CDCl 3 , 101MHz);
  • d is the HSQC spectrum (CDCl 3 );
  • e is the 1 H- 1 H COSY spectrum (CDCl 3 );
  • f is the HMBC spectrum (CDCl 3);
  • a is a planar structure and 1 H- 1 H COSY and a critical HMBC correlation
  • b is a hydrogen spectrum ( 1 H NMR, CDCl 3 ) according to an embodiment of the present invention. , 400MHz);
  • c is the carbon spectrum ( 13 C NMR, CDCl 3 , 101MHz);
  • d is the DEPT 135° spectrum (CDCl 3 );
  • e is the HSQC spectrum (CDCl 3 );
  • f is 1 H- 1 H COSY Spectrum (CDCl 3 );
  • g is the HMBC spectrum (CDCl 3 ).
  • the invention provides a steroid synthase.
  • the catalytic substrate of the steroid synthase is a compound having 10 to 25 carbon atoms.
  • the inventors have found that most of the existing terpenoid synthase can only catalyze the substrate of short carbon chain (for example, 5 to 10 carbon atoms), and the specificity is strong, and only a specific substrate can be catalyzed, and the corresponding axene can be obtained.
  • the hydrazine synthase of the present invention is not only capable of catalyzing compounds of short carbon chains, such as isopentenyl pyrophosphate and olefins.
  • Propyl pyrophosphate which is also capable of catalyzing long carbon chain compounds (e.g., 10 to 25 carbon atoms), has a broad spectrum and is capable of catalyzing a variety of substrates to obtain different terpenoids.
  • the catalytic substrate is selected from one of the following: geranyl pyrophosphate (GPP); farnesyl pyrophosphate (FPP); geranylgeranyl pyrophosphate (GGPP); and geranyl Farnesyl pyrophosphate (GFPP).
  • GPP geranyl pyrophosphate
  • FPP farnesyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • GFPP geranyl Farnesyl pyrophosphate
  • the steroid synthase has the amino acid sequence of any one of SEQ ID NOS: 1 to 6.
  • the inventors have found that the above-mentioned steroidal synthase is provided with farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), geranyl farnesyl pyrophosphate (GFPP), and isopentenyl pyrophosphate ( IPP), allyl pyrophosphate (DMAPP) and geranyl pyrophosphate (GPP) are converted to terpenoid functions. Further, the terpene synthase can efficiently obtain a plurality of different terpenoids by catalyzing a substrate.
  • FPP farnesyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • GFPP geranyl farnesyl pyrophosphate
  • IPP isopentenyl pyrophosphate
  • DMAPP allyl pyrophosphate
  • GPP geranyl pyrophosphate
  • FgMS Indole synthase 1
  • SEQ ID NO: 1 amino acid sequence shown in SEQ ID NO: 1, as follows:
  • Indole synthase 2 (abbreviated as D510A) has the amino acid sequence shown in SEQ ID NO: 2, as follows:
  • FgGS Indole synthase 3
  • SEQ ID NO: 3 amino acid sequence shown in SEQ ID NO: 3, as follows:
  • Indole synthase 4 (GGPPS-Aa for short) has the amino acid sequence shown in SEQ ID NO: 4, as follows:
  • AaTS Indole synthase 5
  • SEQ ID NO: 5 amino acid sequence shown in SEQ ID NO: 5, as follows:
  • FgAS Indole synthase 6
  • SEQ ID NO: 6 amino acid sequence shown in SEQ ID NO: 6, as follows:
  • FgMS, D510A, FgGS and FgAS are isolated from Fusarium graminearum of Taxus, and GGPPS-Aa and AaTS are isolated from Alternaria alternata of Taxus.
  • the serial number is found to be XP_018034954.1, AHY23929.1, XP_002846409.1, XP_003025181.
  • the invention provides a nucleic acid molecule.
  • the nucleic acid molecule encodes a preceding purine synthase.
  • a nucleic acid molecule according to an embodiment of the present invention is capable of efficiently encoding a terpene synthase, and the obtained purine synthase has a broad spectrum and is capable of catalyzing various substrates to obtain different terpenoids.
  • the nucleic acid molecule has the nucleotide sequence shown in any one of SEQ ID NOS: 7 to 12.
  • a nucleic acid molecule according to an embodiment of the invention is capable of efficiently encoding a terpene synthase to catalyze a variety of substrates in order to obtain different terpenoids.
  • Nucleic acid molecule 1 has the nucleotide sequence shown in SEQ ID NO: 7, and is capable of encoding FgMS, a specific nucleotide sequence. as follows:
  • Nucleic acid molecule 2 has the nucleotide sequence shown in SEQ ID NO: 8, encoding D510A, and the specific nucleotide sequence is as follows:
  • Nucleic acid molecule 3 has the nucleotide sequence shown in SEQ ID NO: 9, encoding FgGS, and the specific nucleotide sequence is as follows:
  • the nucleic acid molecule 4 has the nucleotide sequence shown in SEQ ID NO: 10 and encodes GGPPS-Aa, and the specific nucleotide sequence is as follows:
  • Nucleic acid molecule 5 has the nucleotide sequence shown in SEQ ID NO: 11, encoding AaTS, and the specific nucleotide sequence is as follows:
  • the nucleic acid molecule 6 has the nucleotide sequence shown in SEQ ID NO: 12 and encodes FgAS.
  • the specific nucleotide sequence is as follows:
  • the invention proposes a construct.
  • the construct comprises a nucleic acid molecule as described above.
  • a construct according to an embodiment of the invention can encode a plurality of substrates by expressing a nucleic acid molecule encoding a synthetic terpene synthase to obtain different terpenoids.
  • nucleic acid molecules are equally applicable to such constructs and will not be described herein.
  • the invention provides a recombinant cell.
  • the recombinant cell comprises: a first nucleic acid molecule, the first nucleic acid molecule encoding a purine synthase.
  • a hydrazine synthase is obtained, thereby catalyzing various substrates to obtain different terpenoids.
  • the steroid synthase has the amino acid sequence of any one of SEQ ID NOS: 1 to 6, and according to another embodiment of the present invention, the first nucleic acid molecule has SEQ ID NOs: 7 to 12 A nucleotide sequence as shown.
  • a second nucleic acid molecule selected from at least one of the following: an atoB gene derived from Escherichia coli XL1-blue (acetoacetyl-CoA thioesterase), an idi gene ( Isopentenyl pyrophosphate isomerase) gene; erg13 (HMG-CoA synthase) gene derived from Saccharomyces cerevisiae INVSC1, tHMG1 gene (HMG-CoA reductase, deleted transmembrane region of HMG1), erg12 gene (metholone) Acid kinase), erg8 gene (mevalonate-5-phosphate kinase), mvd1 gene (mevalonate-5-pyrophosphate kinase).
  • an atoB gene derived from Escherichia coli XL1-blue
  • idi gene Isopentenyl pyrophosphate isomerase
  • erg13
  • the inventors have found that the above genes are closely related to the catalytic substrate of the steroid synthase, and a large amount of the catalytic substrate can be obtained by overexpressing the above gene. Further, under the catalysis of a hydrazine synthase, a large amount of a quinone compound having a small amount of synthesis can be synthesized in a large amount.
  • the invention provides the use of a steroid synthase or nucleic acid molecule or construct or recombinant cell described above for the synthesis of a steroid.
  • a hydrazine synthase is obtained, thereby catalyzing various substrates to obtain different terpenoids.
  • the synthesis is carried out in a host cell, and the catalytic substrate of the steroid synthase is obtained by overexpressing at least one of the following genes in the host cell: derived from Escherichia coli XL1-blue atoB gene or idi gene; erg13 gene, tHMG1 gene, erg12 gene, erg8 gene or mvd1 gene derived from Saccharomyces cerevisiae INVSC1.
  • a large amount of substrate is obtained by overexpressing the above genes.
  • a large amount of a quinone compound having a small amount of synthesis can be synthesized in a large amount.
  • the quinone compound has a structure of one of the following.
  • the inventors have found that new terpenoids, such as compounds (5), (6), (7), (8), (9), (10), have been obtained under the catalytic action of the hydrazine synthase described above. (11), (12) and (54).
  • the invention provides a method of synthesizing the above described terpenoids.
  • the method comprises: culturing the preceding recombinant cells under conditions suitable for expression of the terpenoid to obtain a culture product; and isolating the terpenoid from the culture product.
  • the expression vector containing the gene of interest (the nucleotide sequence shown in any one of SEQ ID NOS: 7 to 12) is transformed into the expression host E. coli BL21 (DE3), and after transformation, the monoclonal antibody is picked up to the LB culture containing the corresponding antibiotic.
  • Base 37 ° C, 220 rpm overnight culture.
  • the cells were collected by centrifugation at 8 000 rpm for 5 min, and then the cells were completely resuspended with 30-40 mL of Protein Purification Buffer A (Buffer A: 50 mM Tris-HCl, 300 mM NaCl, 4 mM ⁇ -mercaptoethanol, pH 7.6), sonicated (pulse 5 s, pause for 8 s). , ultrasonic disruption 5min).
  • Buffer A 50 mM Tris-HCl, 300 mM NaCl, 4 mM ⁇ -mercaptoethanol, pH 7.6
  • the histidine-tagged protein was purified using Bio-Rad's Biologic DuoFlow Chromatography System.
  • the protein separation column was loaded onto the FPLC for control.
  • the flow rate of the FPLC was always 1.5 mL/min, and the flow rate of the sample was automatically loaded at 2 mL/min.
  • the obtained supernatant sample was purified by Biorad using a 5 mL Hitrap HP Ni-NTA column, which was first passed through 30 mL (6 column volumes) of buffer A (Buffer A: 50 mM Tris-HCl, 300 mM).
  • the volume of protein from the PD-10 column was diluted to 3.5 mL, and the sample was loaded onto an ion exchange column Hitrap 16/10Q/FF and purified by FPLC.
  • the ion exchange column was flushed with buffer C (20 mL (1 column volume) after completion of sample loading, and then started to elute with buffer D (20 mM Tris-HCl, 1 M NaCl, pH 7.6) at a flow rate of 20 mL.
  • buffer D increased from 0% to 30%; after the flow rate of 40mL (2 column volume), the buffer D increased from 30% to 50%; then the flow rate of 20mL (1 column volume), buffer D by 50% increase to 100%; finally use 100% 20mL buffer D to clean the column.
  • the eluted protein of interest was collected by UV absorption and detected by SDS-PAGE.
  • the resulting protein was further concentrated by centrifugation to 2 mL and loaded onto a gel filtration column (gel filtration Superdex 200 column) using buffer E (50 mM phosphate buffer containing 10% glycerol) on FPLC before loading. , pH 7.6) equilibrated 240 mL (2 column volumes) and the eluted proteins of interest (FgMS, D510A, FgGS, GGPPS-Aa and AaTS) were recovered according to UV absorption.
  • the sample was concentrated to 2 mL with a protein centrifugation concentrating column, and the liquid nitrogen was quickly frozen after storage and stored in a -80 ° C refrigerator.
  • the GC-MS used for the detection of terpenoids was a Thermo TRACE GC ULTRA gas chromatograph equipped with TSQ QUANTUM XLS MS and the gas chromatographic column was TRACE TR-5MS (30 m x 0.25 mm x 0.25 um). 1 ⁇ L of each injection was injected, and high-purity helium was used as a carrier gas, and the flow rate was set to 1 mL/min.
  • the GC conditions were maintained at 80 ° C for 1 min, then ramped to 220 ° C at a rate of 10 ° C/min and maintained at 220 ° C for 15 min.
  • the injector and transfer line temperatures were set to 230 ° C and 240 ° C, respectively.
  • FgMS and FgGS enzymes have a very broad substrate specificity. They are capable of synthesizing the corresponding monoterpenes, sesquiterpenes, diterpenes, and diquinones using four substrates of GPP, FPP, GGPP, and GFPP (Fig. 1, Fig. 2). This is the most widely used terpenoid synthase found in the substrate to date.
  • Escherichia coli XL1-blue genomic DNA and S. cerevisiae INVSC1 genomic DNA were obtained by purification using Qiagen's Blood and Cell Culture DNA Mini Kit.
  • Plasmid pMH1 contains the first three genes of the mevalonate pathway: the atoB gene derived from E. coli XL1-blue (acetoacetyl-CoA thioesterase, https://www.ncbi.nlm.nih.gov/nucleotide/313848522?
  • Plasmid pGB309 contains three genes for the synthesis of a sesquiterpene compound, which is a FgMS gene derived from Fusarium graminearum (SEQ ID NO. 7), the amino acid sequence of which is SEQ ID NO.
  • Plasmid pGB310 contains three genes for the synthesis of diterpenoids, respectively FgMS derived from Fusarium graminearum and subjected to site-directed mutagenesis (amino acid D mutation at position 510 to A), abbreviated as D510A (SEQ. ID NO.8), whose amino acid sequence is SEQ ID NO.
  • Plasmid pGB311 contains three genes for the synthesis of sesquiterpene compounds, respectively D510A derived from Fusarium graminearum and subjected to site-directed mutagenesis of the deletion strand extension domain, abbreviated as D510A; idi gene derived from Escherichia coli XL1-blue; FPPS derived from E. coli XL1-blue can synthesize farnesyl pyrophosphate using IPP and DMAPP as substrates for the synthesis of sesquiterpene.
  • D510A derived from Fusarium graminearum and subjected to site-directed mutagenesis of the deletion strand extension domain
  • D510A idi gene derived from Escherichia coli XL1-blue
  • FPPS derived from E. coli XL1-blue can synthesize farnesyl pyrophosphate using IPP and DMAPP as substrates for the synthesis of sesquit
  • the plasmid pGB312 contains three genes for synthesizing a sesquiterpene compound, which is a FgGS gene derived from Fusarium graminearum (SEQ ID NO. 9), and its amino acid sequence is SEQ ID NO.
  • Plasmid pGB313 contains three genes for the synthesis of diterpenoids, which are the FgGS gene derived from Fusarium graminearum; the idi gene derived from Escherichia coli XL1-blue; and the yak based on Taxus canadensis.
  • the geranyl pyrophosphate synthase GGPPS is capable of synthesizing geranylgeranyl pyrophosphate using IPP and DMAPP as a substrate for the synthesis of diterpene products.
  • Plasmid pGB314 contains three genes for the synthesis of sesquiterpene compounds, namely FgGS derived from Fusarium graminearum; idi gene derived from Escherichia coli XL1-blue; FPPS derived from Escherichia coli XL1-blue, capable of IPP and DMAPP Synthesis of farnesyl pyrophosphate as a substrate for the synthesis of sesquiterpene.
  • Plasmid pBBR1MCS was used as template for amplification with primer P1/P2, and p15A replicon was amplified with primer P3/P4. After purification by PCR product, DNA concentration was determined by Nanodrop, then 20 ng pCR amplified p15A fragment and equimolar pBBR1MCS fragment were determined.
  • the amplification conditions were: 98 ° C, 2 min pre-denaturation, then 30 PCR cycles 98 ° C, 20 s; 60 ° C, 20 s; 72 ° C, 6 min, and finally 72 ° C fully extended for 10 min. Subsequently, E. coli XL1-blue was transformed to obtain plasmid pBBR1MCS/p15A.
  • the pMH1 plasmid backbone was amplified with the primer P5/P6 using pBBR1MCS/p15A as a template, and the corresponding genes were amplified using P7/P8, P9/P10 and P11/P12 as primers.
  • 50 ng of pBBR1MCS/p15A amplification product and equimolar amplification products of each gene were mixed, and the volume was adjusted to 5 ⁇ L with deionized water, then added to 15 ⁇ L of Gibson buffer and mixed, and reacted at 50 ° C for 1 h.
  • clones were picked and positive clones were sequenced to obtain plasmid pMH1 (Fig. 3).
  • the pFZ81 plasmid backbone was amplified with primers P13/P14 using pBBR1MCS-2 as a template, and the corresponding genes were amplified using P15/P16, P17/P18, P19/P20 and P21/P22 as primers.
  • 50 ng of pBBR1MCS-2 amplification product and equimolar amplification products of each gene were mixed, and the volume was adjusted to 5 ⁇ L with deionized water, then added to 15 ⁇ L of Gibson buffer and mixed, and reacted at 50 ° C for 1 h.
  • Gibson buffer After transformation of E. coli XL1-blue, clones were picked and positive clones were sequenced to obtain plasmid pFZ81 (Fig. 4).
  • FgMS F. graminearum mangicdiene synthase
  • GFPPS SEQ ID NO. 13
  • pGB307 scaffold with primers P33/P39, P40/P41 and P37/P38, respectively.
  • the above three fragments were assembled to obtain plasmid pGB309 (Fig. 5).
  • F510 fragments of D510A site-directed mutagenesis were amplified by P23/P26 and P24/P25, respectively, and the two fragments were ligated by SOE-PCR to obtain the mutated D510A, which was subsequently cloned into Plasmid pGB302 was obtained on the pET21a(+) plasmid.
  • the idi gene was cloned into pETduet-1 to obtain plasmid pGB307.
  • the D510A, GGPPS and plasmid pGB307 backbones were amplified with primers P33/P34, P35/P36 and P37/P38, and the above three fragments were assembled to obtain plasmid pGB310 (Fig. 6).
  • the fpps gene and the idi gene were amplified from the E. coli BL21 (DE3) genome by primers P29/P30 and P31/P32, respectively, and then the fpps gene was cloned into pET21a to obtain plasmid pGB305; idi was cloned into pET21a (+) to obtain plasmid pGB306. .
  • Plasmids pGB305 and pGB306 were digested with XbaI/XhoI, SpeI/XhoI, respectively, and then the idi fragment digested with pGB306 was ligated to plasmid pGB305 by means of homologous enzyme to obtain plasmid pGB308. Subsequently, the fpps-idi fragment was digested with pba308 by XbaI/XhoI and ligated to plasmid pGB302 by homologous enzyme, respectively, to obtain plasmid pGB311 (Fig. 7).
  • FgGS F. graminearum GJ1012 synthase
  • GFPPS GFPPS
  • pGB307 plasmid backbone were amplified with primers P42/46, P41/P47 and P37/P45, and the above three fragments were assembled to obtain plasmid pGB312 (Fig. 8).
  • the FgGS, GGPPS and pGB307 plasmid backbones were amplified with primers P42/43, P44/P36 and P37/P45, and the above three fragments were assembled to obtain plasmid pGB313 (Fig. 9).
  • the FgGS gene was digested with SacI/HindIII from plasmid pUC57-FgGS (pGB303) containing codon-optimized FgGS and ligated into pET21a(+) to obtain plasmid pGB304.
  • the fpps-idi fragment was subsequently digested from pGB308 by XbaI/XhoI and ligated to plasmid pGB304 by homologous enzymes, respectively, to obtain plasmid pGB314 (Fig. 10).
  • BL21(DE3)/pMH1/pFZ81 was obtained in Bacillus sp. BL21 (DE3) and designated as PS, and then pGB309 was transformed into strain PS to obtain strain T7 (Fig. 12).
  • the two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transferred into E. coli BL21 (DE3) to obtain BL21(DE3)/pMH1/pFZ81, designated PS, and then pGB310 was transformed into the strain PS.
  • BL21(DE3)/pMH1/pFZ81 designated PS
  • pGB310 was transformed into the strain PS.
  • T8 was obtained (Fig. 12). Subsequently, fermentation and product extraction were carried out in the same manner as in Example 3.
  • the main product 4 is identical to the compound synthesized by Streptomyces-derived DtcycB reported in the literature, and is a 14-member macrocyclic compound cembrene A (Meguro A, Tomita T, Nishiyama M, et al. Identification and characterization of bacterial diterpene cyclases). That synthesize the cembrane skeleton [J]. ChemBioChem, 2013, 14(3): 316-321.).
  • the NIST search results of the compound (16-19) showed that the compound (16) was Trachylobane, the compound (18) was Cyclotetradecatetraene, and the compound (17) was E, E-7, 11 , 15-Trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene, compound (19) is (3E,7E,11E)-1-Isopropyl-4,8,12-trimethylcyclotetradeca-3,7, 11-trienol.
  • the remaining structure is unknown for compounds 32-36.
  • GPP was synthesized based on FPPS with IPP and DMAPP as substrates, and then 1 molecule of IPP was used to generate FPP, and the two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transferred into the large intestine.
  • BL21(DE3)/pMH1/pFZ81 was obtained in Bacillus sp. BL21 (DE3) and designated as PS, and then pGB311 was transformed into strain PS to obtain strain T9 (Fig. 12). Subsequently, fermentation and product extraction were carried out in the same manner as in Example 3.
  • the two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transferred into E. coli BL21 (DE3) to obtain BL21(DE3)/pMH1/pFZ81, designated PS, and then pGB312 was transformed into In strain PS, strain T10 was obtained (Fig. 12). Subsequently, fermentation and product extraction were carried out in the same manner as in Example 3.
  • the two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transformed into E. coli BL21 (DE3) to obtain BL21(DE3)/pMH1/pFZ81, designated PS, and then pGB313 was transformed into the strain PS.
  • strain strain T11 was obtained (Fig. 12). Subsequently, fermentation and product extraction were carried out in the same manner as in Example 3.
  • the mutant strain E. coli T11 containing FgGS was able to synthesize 14 diterpenoids using GGPP as a substrate, and we purified and identified compound 5-10 (Fig. 13c, Fig. 14).
  • the results show that the compound (5-10) is a novel class of skeleton compounds.
  • the compound (5) is a quaternary diterpene compound of 5-5-5-5 ring;
  • the compounds (6) and (8) are ternary diterpene compounds having 5-5-9 rings having different double arrow positions;
  • Compound (7) is a quaternary diterpene compound of 5-5-7-4 ring;
  • compounds (9) and (10) are 5-5-7-4 and 5-5-6-5 ring-free double bonds a quaternary sterol compound.
  • the first two 5-5 membered rings of these six compounds are their common structure. It shows that they have some common cyclization steps at the beginning of the synthesis, which is named GJ1012A–F.
  • GPP was synthesized based on FPPS with IPP and DMAPP as substrates, and then 1 molecule of IPP was used to generate FPP, and the two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transferred.
  • BL21(DE3)/pMH1/pFZ81 was obtained in E. coli BL21 (DE3) and designated as PS, and then pGB314 was transformed into strain PS to obtain strain T12 (Fig. 12). Subsequently, fermentation and product extraction were carried out in the same manner as in Example 3.
  • the structure of two diterpenoids and the compound of FgMS product (11) is consistent with (23); the compounds (23), (43), and (44) are divalent quinone compounds of unknown structure.
  • the three compounds synthesized are compounds (13) and (14) and linalool, respectively.
  • AaTS is a newly discovered terpenoid synthase of the present invention derived from Alternaria alternata.
  • AaTS was codon-optimized and ligated to vector pET28a by restriction enzyme site NdeI/EcoRI to obtain plasmid pGB136.
  • the AaTS protein was subsequently purified as described in Example 1 and subjected to an in vitro reaction. The results of the in vitro reaction showed that AaTS was able to synthesize diterpene compounds using GGPP as a substrate (Fig. 15).
  • Indole synthase 6 (FgAS, F. graminearum AJ1012 synthase) is an enzyme highly similar to indole synthase 1 (FgMS). It differs from FgMS in amino acid 65, the amino acid sequence of this site in FgMS is F, and the amino acid sequence of this site in FgAS is L. Using the same strategy, a second sesquiterpene novel skeleton compound (54) was detected in the diclofenium-producing strain containing FgAS by the method of Example 3.
  • Figure 16 shows the spectrum of the compound (1), and the 1 H NMR data indicates that the compound (1) has four single-peak methyl signals (Me-22, Me-23, Me-24, Me-25), 2 Bimodal methyl signals (Me-20 and Me-21), and 2 olefinic hydrogens (H-1 and H-18) (Table 2).
  • 13 C NMR and heteronuclear single quantum correlation spectroscopy (HSQC) confirmed the presence of 25 carbon atoms, of which 3 sp 3 hybridized quaternary carbon atoms (C-6, C-11 and C-15), 2 sp 2 hetero The quaternary carbon atoms (C-2 and C-19), 4 aliphatic methine groups, 2 enemethine groups, 8 methylene groups and 6 methyl groups.
  • the coupling relationship of 1 H- 1 H COSY is: H-20/H-3/H-4/H-5, H-7/H-21, H-10/H-14 and H-17/H- 18.
  • the HMBC map shows that the related signals of methyl hydrogen are: Me-20 and C-2, C-3, C-4; Me-21 and C-3, C-6, C-7, C-8; Me -22 and C-1, C-11, C-12, C-14; Me-23 and C-13, C-14, C-15, C-16; Me-24 and C-18, C-19 , C-25; Me-25 and C-18, C-19, C-24.
  • the HMBC map suggests a coupling relationship between H-5 and C-4, C-5, C-7; H-1 and C-3, C-6, C-11; H-18 and C-16. Therefore, the planar structure of the compound (1) is a tetracyclic sesquiterpene.
  • Figure 17 shows the spectrum of the compound (2), and the 1 H NMR data indicates that the compound (2) has 6 methyl signals (Me-20, Me-21, Me-22, Me-23, Me-24, Me- 25) and 4 olefinic hydrogens (H-2, H-6, H-9 and H-18) (Table 3).
  • 13 C NMR and HSQC confirmed the presence of 25 carbon atoms, of which 2 sp 3 hybrids and 4 sp 2 hybridized quaternary carbon atoms, 4 enemethine groups, 9 methylene groups and 6 methyl groups.
  • the coupling relationship of 1 H- 1 H COSY is: H-1/H-2, H-5/H-6, H-8/H-9, H-16/H-17/H-18.
  • the HMBC map shows that the related signals of methyl hydrogen are: Me-20 and C-2, C-3, C-4; Me-21 and C-6, C-7, C-8; Me-22 and C. -1, C-11, C-12; M-23 and C-13, C-14, C-15, C-16; Me-24 and C-18, C-19, C-25; Me-25 With C-18, C-19, C-24.
  • the chemical shifts of C-20 and C-21 were 14.96 and 18, suggesting that the double bonds between C-2 and C-3, C-6 and C-7 are in the E configuration. Therefore, the planar structure of the compound (2) is a 11-6 membered bicyclic sesquiterpene.
  • 1 H NMR data indicated the presence of 6 methyl signals (Me-20, Me-21, Me-22, Me-23, Me-24, Me-25) and 5 olefins (Table 4).
  • 13 C NMR and HSQC confirmed the presence of 25 carbon atoms, of which 5 sp 2 hybridized quaternary carbon atoms (C-3, C-7, C-11, C-15, C-19), 1 aliphatic And 4 enemethyl groups, 8 methylene groups and 6 methyl groups.
  • the coupling relationship of 1 H- 1 H COSY is: H-1/H-2, H-5/H-6, H-9/H-10, H-12/H-13/H-14, H- 16/H-17/H-18.
  • the HMBC map shows that the related signals of methyl hydrogen are: Me-20 and C-2, C-3, C-4; Me-21 and C-6, C-7, C-8; Me-22 and C. -10, C-11, C-12; M-23 and C-14, C-15, C-16; Me-24 and C-18, C-19, C-25; Me-25 and C-18 , C-19, C-24.
  • the HMBC map suggests that H-2 is coupled with C-14, C-4; H-6 and C-4, C-8; H-10 and C-8. Therefore, the compound (3) is a 14-membered monocyclic sesquiterpene.
  • Figure 20 shows the spectrum of the compound (5), and the 1 H NMR data indicates that the compound (5) has four single-peak methyl signals (Me-17, Me-18, Me-19, Me-20), 1 Bimodal methyl signal (Me-16) and 1 olefinic hydrogen (H-6) (Table 5).
  • 13 C NMR and HSQC confirmed the presence of 20 carbon atoms, of which 3 sp 3 hybridized quaternary carbon atoms (C-3, C-12, C-15), 1 sp 2 hybridized quaternary carbon atom (C -7), 4 aliphatic methine groups, 1 enemethine group, 6 methylene groups and 5 methyl groups.
  • the coupling relationship of 1 H- 1 H COSY is: H-1/H-2/H-10/H-9/H-8, H-4/H-16.
  • the HMBC map shows that the signals related to methyl hydrogen are Me-16 and C-3, C-4, C-5; Me-17 and C-3, C-6, C-7; Me-18 and C- 11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13, C-18; Me-20 and C-1, C-11, C-14, C-15.
  • the HMBC map suggests that H-4 has a coupling relationship with C-2, C-6, and C-7. Therefore, the planar structure of the compound (5) is 5-5-5-5-membered tetracyclic diterpenes.
  • Figure 21 shows the spectrum of the compound (6), and the 1 H NMR data indicates that the compound (6) has five methyl signals (Me-16, Me-17, Me-18, Me-19 and Me-20), and 1 olefinic hydrogen (H-6) (Table 6).
  • 13 C NMR and HSQC confirmed the presence of 20 carbon atoms, of which 2 sp 3 hybridized quaternary carbon atoms (C-12, C-15), 3 sp 2 hybridized quaternary carbon atoms (C-2, C -3, C-7), 2 aliphatic methine groups, 1 enemethine group, 7 methylene groups and 5 methyl groups.
  • the coupling relationship of 1 H- 1 H COSY is: H-5/H-6, H-8/H-9.
  • the HMBC map shows that the signals related to methyl hydrogen are Me-16 and C-2, C-3, and C-4; Me-17 and C-6, C-7, C-8; Me-18 and C. -11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13, C-18; Me-20 and C-1, C-11, C-14 , C-15.
  • the HMBC map suggests that H-6 has a coupling relationship with C-5, H-9 and C-7, and H-10 with C-1, C-2, C-3, C-9 and C-11. Therefore, the planar structure of the compound (6) is 5-5-9-membered tricyclic dioxime.
  • Figure 22 shows the spectrum of the compound (7), and the 1 H NMR data indicates that the compound (7) has five methyl signals Me-16, Me-17, Me-18, Me-19, Me-20 (Table 7). . 13 C NMR and HSQC confirmed the presence of 20 carbon atoms, of which 3 sp 3 hybridized quaternary carbon atoms (C-3, C-12, C-15), 2 sp 2 hybridized quaternary carbon atoms (C -6, C-7), 3 aliphatic methine groups, 7 methylene groups and 5 methyl groups. These data suggest that the compound (7) has a tetracyclic structure.
  • the coupling relationship of 1 H- 1 H COSY is: H-1/H-2/H-10/H-11, H-4/H-5 and H-8/H-9.
  • the HMBC map shows that the signals related to methyl hydrogen are Me-16 and C-2, C-3, C-4, C-6; Me-17 and C-6, C-7, C-8; Me- 18 with C-11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13, C-18; Me-20 and C-1, C-11, C-14, C-15. Therefore, the planar structure of the compound (7) is 5-5-7-4 membered tetracyclic diterpene.
  • Figure 23 shows the spectrum of the compound (8).
  • the 1 H NMR results indicate that the compound (8) has four methyl groups (Me-17, Me-18, Me-19 and Me-20), and one terminal olefin. Hydrogen (H-16) (Table 8).
  • 13 C NMR and HSQC confirm the presence of 20 carbon atoms, including two sp 3 hybridized quaternary carbon atom (C-12, C-15 ) and 2 an sp 2 hybrid quaternary carbon atom (C-3, C -7), 1 methine group, 3 aliphatic methine groups, 1 olefinic group, 7 aliphatic methylene groups and 4 methyl groups.
  • C-12, C-15 two sp 3 hybridized quaternary carbon atom
  • C-3, C -7 an sp 2 hybrid quaternary carbon atom
  • 1 methine group 3 aliphatic methine groups
  • 1 olefinic group 7 aliphatic methylene groups and 4 methyl groups.
  • the coupling relationship of 1 H- 1 H COSY is: H-1/H-2, H-5/H-6, H-8/H-9/H-10.
  • the HMBC map can be seen that the related signals of methyl hydrogen are Me-17 and C-6, C-7, C-8; Me-18 and C-11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13, C-18; Me-20 and C-1, C-11, C-14, C-15.
  • the HMBC map suggests a coupling relationship between H-5 and C-4; H-10 and C-2 and C-11; H-16 and C-2, C-4. Therefore, the planar structure of the compound (8) is 5-5-9-membered tricyclic diterpenes.
  • Figure 24 shows the spectrum of the compound (9).
  • the 1 H NMR results indicated that the compound (9) had five methyl signals Me-16, Me-17, Me-18, Me-19 and Me-20 (Table 9).
  • 13 C NMR and HSQC confirmed the presence of 20 carbon atoms, including 4 sp 3 hybrid quaternary carbon atoms (C-3, C-7, C-12 and C-15), 4 aliphatic methine groups, 7 methylene groups and 5 methyl groups.
  • the HMBC map can see that the related signals of methyl hydrogen are Me-16 and C-2, C-3, C-4, C-6; Me-17 and C-6, C-7, C-8; Me-18 and C-11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13 and C-18; Me-20 and C-1, C- 11, C-14, C-15.
  • the HMBC map suggests a coupling relationship between H-8 and C-2, C-8, C-9, C-10, and C-11. Therefore, the planar structure of the compound (9) is 5-5-7-4 membered tetracyclic diterpenes.
  • Figure 25 shows the spectrum of the compound (10), and the 1 H NMR results indicate that the compound (10) has five methyl groups (Me-16, Me-17, Me-18, Me-19 and Me-20), 1 A secondary alcohol H-6 (Table 10). 13 C NMR and HSQC confirmed the presence of 20 carbon atoms, including 4 sp 3 hybrid quaternary carbon atoms (C-3, C-7, C-12 and C-15), 4 aliphatic methine groups, 7 methylene groups and 5 methyl groups. These data suggest that compound (10) is a tetracyclic structure.
  • the coupling relationship indicated by 1 H- 1 H COSY is: H-4/H-5/H-6, H-8/H-9/H-10, H-1/H-2.
  • the HMBC map can be seen that the signals related to methyl hydrogen are Me-16 and C-2, C-3, C-4, C-7; Me-17 and C-3, C-6, C-7, C-8; Me-18 and C-11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13, C-18; Me-20 and C- 1, C-11, C-14, C-15.
  • the HMBC map suggests a coupling relationship between H-2 and C-10, H-10 and C-11. Therefore, the planar structure of the compound (10) is 5-5-6-5-membered tetracyclic diterpenes.
  • Figure 26 shows the spectrum of the compound (11), which is a color oil trans-nerolidol.
  • 1 H NMR 400MHz, deuterochloroform
  • Figure 27 shows the spectrum of the compound (12) which is the compound 2E, 6E-farnesol as a color oil.
  • 1 H NMR 400 MHz, deuterated chloroform
  • 2.12 - 2.08 m, 2H
  • 1.68 (s, 6H) 1.60 (s, 6H).
  • Figure 28 shows the spectrum of Compound (53), 1 H NMR data suggest that compound (53) there are three singlet methyl signals (Me-17, Me-18 , Me-20), 1 methyl doublets Base signal (Me-19), and 2 olefinic hydrogens (H-9 and H-16) (Table 11).
  • 13 C NMR and heteronuclear single quantum correlation spectroscopy (HSQC) confirmed the presence of 20 carbon atoms with one sp 3 hybrid quaternary carbon (C-11) and two sp 2 hybrid quaternary carbon atoms ( C-7 and C-14), 5 aliphatic methine groups, 1 enemethine group, 6 aliphatic methylene groups, 1 exo-methylene group and 4 methyl groups.
  • compound (53) is a tetracyclic structure.
  • the coupling relationship of 1 H- 1 H COSY is: H-1/H-2, H-3/H-19 and H-8/H-9.
  • the HMBC map shows that the related signals of methyl hydrogen are: Me-17 and C-14, C-15 and C-16; Me-18 and C-6, C-7 and C-8; M-19 and C -2, C-3 and C-4; Me-20 and C-1, C-10, C-11 and C-12.
  • the HMBC map suggests H-1 and C-2, C-6, C-10; H-3 and C-5, C-6; H-5 and C-6, C-7; H-8 and C- 10; H-10 and C-15; there is a coupling relationship between H-13 and C-15. Therefore, the planar structure of the compound (53) is a tetracyclic sesquiterpene.
  • Figure 29 shows the spectrum of the compound (54), and the 1 H NMR data indicates that the compound (54) has 6 methyl signals (Me-20, Me-21, Me-22, Me-23, Me-24, Me- 25).
  • 13 C NMR, heteronuclear single quantum correlation spectroscopy (HSQC) and DEPT 135° confirmed the presence of 25 carbon atoms with 3 sp 3 hybrid quaternary carbon atoms (C-11, C-14, C-17), 3 sp 2 hybrid quaternary carbon atoms (C-2, C-3, C-7), 3 aliphatic methine groups, 1 ene methine group, 9 aliphatic methylene groups, and 6 Methyl.
  • HSQC heteronuclear single quantum correlation spectroscopy
  • the coupling relationship of 1 H- 1 H COSY is: H-4/H-5/H-6, H-8/H-9 and H-15/H-16.
  • the HMBC map shows that the related signals of methyl hydrogen are: Me-20 and C-2, C-3 and C-4; Me-21 and C-6, C-7 and C-8; M-22 and C -1, C-10,
  • the planar structure of the compound (54) is a 5-8-6-6 membered tetracyclic sesquiterpene.

Abstract

L'invention concerne une terpène synthase, une molécule d'acide nucléique qui code la terpène synthase, une construction qui contient la molécule d'acide nucléique, une cellule recombinante, et son utilisation, ainsi qu'un procédé de synthèse d'un composé terpénoïde, un substrat catalytique de la terpène synthase étant un composé ayant de 10 à 25 atomes de carbone.
PCT/CN2017/071449 2016-12-27 2017-01-17 Terpène synthase et son utilisation WO2018120337A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201611223498.3 2016-12-27
CN201611223498 2016-12-27

Publications (1)

Publication Number Publication Date
WO2018120337A1 true WO2018120337A1 (fr) 2018-07-05

Family

ID=62701923

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/071449 WO2018120337A1 (fr) 2016-12-27 2017-01-17 Terpène synthase et son utilisation

Country Status (2)

Country Link
CN (1) CN108239631B (fr)
WO (1) WO2018120337A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113430218A (zh) * 2021-04-30 2021-09-24 中国医学科学院药用植物研究所 倍半萜类化合物的生物酶催化合成方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106906201B (zh) * 2017-04-10 2020-03-10 武汉大学 一种生产橙花叔醇的萜类合酶及其应用
CN113480660B (zh) * 2021-06-03 2022-11-18 武汉大学 一种嵌合萜类合酶及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013000660A1 (fr) * 2011-06-27 2013-01-03 Firmenich Sa Micro-organismes modifiés et leur utilisation pour produire des terpènes
CN105154420A (zh) * 2015-08-20 2015-12-16 中国医学科学院药用植物研究所 赤芝萜类合酶GL22395编码基因cDNA序列及其应用

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2004277608A1 (en) * 2003-09-29 2005-04-14 The Regents Of The University Of California Methods for identifying a biosynthetic pathway gene product
JP5590769B2 (ja) * 2004-07-27 2014-09-17 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア イソプレノイド化合物を生産するための遺伝的に修飾された宿主細胞および同宿主細胞の使用方法
US20090053797A1 (en) * 2005-08-19 2009-02-26 Yoichiro Shiba Genetically modified host cells and use of same for producing isoprenoid compounds
CN103243065B (zh) * 2013-05-30 2014-12-03 武汉大学 一种生产法尼烯的菌株及其应用
CN105647879A (zh) * 2016-03-16 2016-06-08 武汉大学 一种二萜化合物variediene合成基因Au13192及其应用
CN106011090A (zh) * 2016-06-13 2016-10-12 西安电子科技大学 一种还原酶及其编码基因与应用
CN106906201B (zh) * 2017-04-10 2020-03-10 武汉大学 一种生产橙花叔醇的萜类合酶及其应用
CN107083378A (zh) * 2017-04-10 2017-08-22 武汉大学 一种生产Longiborneol的萜类合酶及其应用
CN106987578B (zh) * 2017-04-10 2020-01-07 武汉大学 一种生产koraiol的萜类合酶及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013000660A1 (fr) * 2011-06-27 2013-01-03 Firmenich Sa Micro-organismes modifiés et leur utilisation pour produire des terpènes
CN105154420A (zh) * 2015-08-20 2015-12-16 中国医学科学院药用植物研究所 赤芝萜类合酶GL22395编码基因cDNA序列及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SCHIFRIN A. ET AL.: "A single terpene synthase is responsible for a wide variety of sesquiterpenes in Sorangium cellulosum Soce56", ORGANIC & BIOMOLECULAR CHEMISTRY, vol. 14, no. 13, 25 February 2016 (2016-02-25), pages 3385 - 3393, XP055605518, ISSN: 1477-0520, DOI: 10.1039/c6ob00130k *
YANG TAO ET AL.: "Plant Terpenoid Synthases", MOLECULAR BIOLOGY AND GENETIC ENGINEERING, ACTA BOTANICA YUNNANICA, vol. 27, no. 1, 25 February 2005 (2005-02-25), pages 1 - 10, ISSN: 0253-2700 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113430218A (zh) * 2021-04-30 2021-09-24 中国医学科学院药用植物研究所 倍半萜类化合物的生物酶催化合成方法

Also Published As

Publication number Publication date
CN108239631B (zh) 2021-07-16
CN108239631A (zh) 2018-07-03

Similar Documents

Publication Publication Date Title
Kim et al. Cloning and heterologous expression of the cyclooctatin biosynthetic gene cluster afford a diterpene cyclase and two p450 hydroxylases
Sadre et al. Metabolite diversity in alkaloid biosynthesis: a multilane (diastereomer) highway for camptothecin synthesis in Camptotheca acuminata
Agger et al. Identification of sesquiterpene synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120
CN106906201B (zh) 一种生产橙花叔醇的萜类合酶及其应用
Kai et al. Biosynthesis and biotechnological production of anti-cancer drug Camptothecin
Picaud et al. Amorpha-4, 11-diene synthase: mechanism and stereochemistry of the enzymatic cyclization of farnesyl diphosphate
Toyomasu et al. Biosynthetic gene-based secondary metabolite screening: a new diterpene, methyl phomopsenonate, from the fungus Phomopsis amygdali
WO2018120337A1 (fr) Terpène synthase et son utilisation
CN112142585B (zh) Mangicols二倍半萜化合物、合成方法、基因簇、核酸分子、构建体及其应用
Petříčková et al. Biosynthesis of Colabomycin E, a New Manumycin‐Family Metabolite, Involves an Unusual Chain‐Length Factor
Maury et al. Reconstruction of a bacterial isoprenoid biosynthetic pathway in Saccharomyces cerevisiae
Luo et al. Characterization of a sesquiterpene cyclase from the glandular trichomes of Leucosceptrum canum for sole production of cedrol in Escherichia coli and Nicotiana benthamiana
CN106987578B (zh) 一种生产koraiol的萜类合酶及其应用
Jiang et al. Schultriene and nigtetraene: Two sesterterpenes characterized from pathogenetic fungi via genome mining approach
Nakano et al. Substrate specificity of the CYC2 enzyme from Kitasatospora griseola: production of sclarene, biformene, and novel bicyclic diterpenes by the enzymatic reactions of labdane-and halimane-type diterpene diphosphates
Li et al. Characterization of two chimeric sesterterpene synthases from a fungal symbiont isolated from a sesterterpenoid-producing Lamiaceae plant Leucosceptrum canum
Xing et al. Functional characterization and structural bases of two class I diterpene synthases in pimarane-type diterpene biosynthesis
WO2019148609A1 (fr) Terpène synthase et son application
JP6399315B2 (ja) テルペン合成酵素遺伝子、アセト酢酸エステル加水分解酵素遺伝子、及びテルペンの製造方法
CN110317765B (zh) 一种高产香叶醇葡萄糖苷的大肠杆菌表达菌株及其应用
CN109234216B (zh) 一种生产鲨烯的基因工程菌及其方法
CN107083378A (zh) 一种生产Longiborneol的萜类合酶及其应用
CN113265391B (zh) 一种芳樟醇合酶CcLS及其编码基因与应用
JP7294636B2 (ja) 新規カロテノイド及び該カロテロイドの製造方法
CN113402357A (zh) 一类5-12-5三环二倍半萜骨架化合物及其制备

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17886539

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17886539

Country of ref document: EP

Kind code of ref document: A1