WO2021027771A1 - 乙二醛酶spg的功能及应用 - Google Patents

乙二醛酶spg的功能及应用 Download PDF

Info

Publication number
WO2021027771A1
WO2021027771A1 PCT/CN2020/108157 CN2020108157W WO2021027771A1 WO 2021027771 A1 WO2021027771 A1 WO 2021027771A1 CN 2020108157 W CN2020108157 W CN 2020108157W WO 2021027771 A1 WO2021027771 A1 WO 2021027771A1
Authority
WO
WIPO (PCT)
Prior art keywords
spg
glyoxalase
catalyzed
carbonyl
hydroxy
Prior art date
Application number
PCT/CN2020/108157
Other languages
English (en)
French (fr)
Inventor
陈晓亚
黄金泉
方欣
田秀
郭晓祥
林嘉玲
陈志文
上官小霞
王凌健
Original Assignee
中国科学院分子植物科学卓越创新中心
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 中国科学院分子植物科学卓越创新中心 filed Critical 中国科学院分子植物科学卓越创新中心
Publication of WO2021027771A1 publication Critical patent/WO2021027771A1/zh

Links

Images

Classifications

    • 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
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
    • 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
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/02—Thioester hydrolases (3.1.2)
    • C12Y301/02006—Hydroxyacylglutathione hydrolase (3.1.2.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y404/00—Carbon-sulfur lyases (4.4)
    • C12Y404/01—Carbon-sulfur lyases (4.4.1)
    • C12Y404/01005—Lactoylglutathione lyase (4.4.1.5)

Definitions

  • the present invention belongs to the fields of botany and molecular biology. More specifically, the present invention relates to the function and application of glyoxalase SPG or its homologs.
  • the juniperene synthase CDN is a sesquiterpene cyclase that catalyzes the formation of juniperene from FPP.
  • Methyglyoxal (Methyglyoxal, MG), also known as pyruvaldehyde, contains two active carbonyl groups. It is a compound that is toxic to cells and an inherently toxic by-product produced during glycolysis. It is inevitable that sugar is a source of energy for living organisms [2]. In the long-term evolutionary process of organisms, the MG detoxification system was produced, called the glyoxalase system.
  • the glyoxalase system consists of two enzymes, Glyoxalase I (Glyoxalase I, GLO1, GLXI, EC 4.4.1.5) and Glyoxalase II (Glyoxalase II, GLO2, GLXII, EC 3.1.2.6).
  • Glyoxalase I Glyoxalase I, GLO1, GLXI, EC 4.4.1.5
  • Glyoxalase II Glyoxalase II, GLO2, GLXII, EC 3.1.2.6
  • MG and reduced glutathione GSH produce thiohemiacetal through non-enzymatic reaction, which is catalyzed by GLXI to produce SD-lactyl glutathione, and then GLXII catalyzes SD-lactyl glutathione to produce non-toxic D -Lactic acid, and release reducing glutathione for repeated use, so as to achieve the effect of detoxification [3
  • Mycotoxins are secondary metabolites of fungi formed by infected plants and crops such as Fusarium, Penicillium and Alternaria, polluting grain, oil, food, livestock feed, fruits and vegetables, and seriously affecting food safety and human and animal health.
  • Deoxynivalenol (DON) and its acylated derivatives 3ADON and 15ADON are the most important mycotoxins, which are very serious to grain pollution. It is stable in nature and highly toxic. It is listed as the third category carcinogen by the International Agency for Research on Cancer IARC, and has been identified as one of the most dangerous natural food contaminants by the United Nations Food and Agriculture Organization and the World Health Organization WHO.
  • the use of biological detoxification technology to degrade mycotoxins has the advantages of green and high efficiency and is highly valued internationally, but such research is currently progressing slowly.
  • the purpose of the present invention is to provide the function and application of glyoxalase SPG or its homologues.
  • the use of glyoxalase SPG or its homologues is provided to catalyze the aromatization, isomerization, dehydration and/or cyclic compounds with ortho-hydroxyketone structure Epoxidation reaction; or for the preparation of preparations for catalyzing the aromatization reaction, isomerization reaction, dehydration reaction and/or epoxidation reaction of cyclic compounds with ortho-hydroxy ketone structures.
  • the cyclic compound compound is a monocyclic compound or a polycyclic compound (including a bicyclic compound, such as containing 2-10 rings).
  • the ortho-hydroxy ketone structure includes: Wherein X is a ring structure, such as a 4-7 membered ring, more preferably a 5-6 membered ring.
  • the aromatization reaction or isomerization reaction includes: forming a benzene ring, increasing the number of unsaturated bonds in the ring structure, changing the position of the unsaturated bonds in the ring structure, or changing the ring structure or The optical conformation or chirality of the group on it.
  • the cyclic compound with ortho-hydroxy ketone structure includes: 3-hydroxy-2-carbonyl-furan Karaman or its analogue, which is subjected to glyoxalase SPG or its homologue Catalyzes the aromatization reaction and/or isomerization reaction to produce deoxyhemigossypol (dHG) or its analogues; or, 8,11-dihydroxy-7-carbonyl-cadinene or its analogues, which Catalyzed by glyoxalase SPG or its homologue, isomerization reaction, dehydration reaction and/or epoxidation reaction to produce furocalamen or its analogues; or acetyl deoxynivalenol (3A-DON) or its analogue, which undergoes an isomerization reaction catalyzed by glyoxalase SPG to produce isomerized acetyl deoxynivalenol or its analogue.
  • the 3-hydroxy-2-carbonyl-furan Karaman and 8,11-dihydroxy-7-carbonyl-cadinene are intermediate substances involved in the biosynthesis of gossypol, so that the The glyoxalase SPG or its homologues are used in gossypol biosynthesis.
  • the glyoxalase SPG includes: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2; or (b) passing the amino acid sequence shown in SEQ ID NO: 2 through one or more (E.g.
  • the ID of the gene encoding glyoxalase SPG includes : Gh_A03G0247, Gh_A03G0248, Gh_D03G1320, Gh_D03G1321, Gh_D03G1322, Gorai.003G145000.1, Gorai.003G145100.1, Gorai.003G145200.1, Ga_01G2453, Ga_01G_03D03386, GB_AGB_160GB_03D03386, GB_A160GB_03G0384
  • the homolog of the glyoxalase SPG includes: glyoxalase I (GLXI), glyoxalase GLO1; preferably, the glyoxalase I includes GhGLXI, CrGLXI, SmGLXI, OSGLXI, AtGLXI, DzGLXI, DmGLXI, DrGLXI; preferably, the glyoxalase GLO1 includes HsGLO1.
  • the homolog of the glyoxalase SPG also includes a variant of the glyoxalase I or glyoxalase GLO1, such as having more than 80% of its amino acid sequence ( Preferably 85% or more; more preferably 90% or more; more preferably 95% or more; such as 98% or more or 99% or more) polypeptides that are identical and have their functions.
  • a variant of the glyoxalase I or glyoxalase GLO1 such as having more than 80% of its amino acid sequence ( Preferably 85% or more; more preferably 90% or more; more preferably 95% or more; such as 98% or more or 99% or more) polypeptides that are identical and have their functions.
  • glyoxalase SPG is provided to reduce or detoxify the toxicity of the acylated derivative of the toxin deoxynivalenol (DON).
  • a method for catalyzing the aromatization reaction, isomerization reaction, dehydration reaction and/or epoxidation reaction of a cyclic compound having an ortho-hydroxy ketone structure including: using glyoxal Enzyme SPG or its homologues process the cyclic compound with ortho-hydroxy ketone structure.
  • the cyclic compound compound is a monocyclic compound or a polycyclic compound (including a bicyclic compound, such as containing 2-10 rings); or, the ortho-hydroxy ketone structure includes: Wherein X is a ring structure (e.g. 4-7 membered ring, more preferably 5-6 membered ring).
  • the aromatization reaction or isomerization reaction includes: forming a benzene ring, increasing the number of unsaturated bonds in the ring structure, changing the position of the unsaturated bonds in the ring structure, or changing the ring structure or The optical conformation or chirality of the group on it.
  • the cyclic compound with ortho-hydroxy ketone structure includes: 3-hydroxy-2-carbonyl-furan Karaman, which undergoes aromatization reaction and/or isomerization reaction through catalysis to produce Deoxyhemigossypol (deoxyhemigossypol, dHG); or, 8,11-dihydroxy-7-carbonyl-junibinene, which undergoes isomerization, dehydration and/or epoxidation through catalysis to form furan Karaman (furocalamen); or acetyl deoxynivalenol (3A-DON), which undergoes an isomerization reaction through catalysis to generate isomerized acetyl deoxynivalenol.
  • a method for reducing or deactivating the toxicity of deoxynivalenol (DON) or its acylated derivatives including treatment with glyoxalase SPG.
  • a method for improving the ability of glyoxalase SPG to reduce or detoxify methylglyoxal (MG) toxicity includes sequence modification of glyoxalase SPG so that it occurs M69F/F101N mutation, F101N/I121R mutation or M69F/F101N/I121R, and 4 amino acids GKMK (corresponding to SEQ ID NO: 2 sequence) is added to its Loop13, and GKMK is inserted between positions 153 to 154.
  • a glyoxalase SPG mutant which has the ability to reduce or detoxify the toxicity of methylglyoxal (MG), which corresponds to the sequence of SEQ ID NO: 2 and has M69F/F101N Mutation, F101N/I121R mutation or M69F/F101N/I121R, and 4 amino acids GKMK is added to its Loop13.
  • MG methylglyoxal
  • a method for synthesizing gossypol including:
  • FPP farnesyl diphosphate
  • CDN juniperene synthase
  • SPG is positively correlated with the accumulation of gossypol. SPG is almost not expressed in the leaves of glandless cotton and PGF suppression plants, and can be induced by the elicitor VdNEP.
  • CDN juniperene synthase
  • DH1 alcohol dehydrogenase-1
  • 2-ODD-1 2-oxoglutarate/Fe(ii)-dependent dioxygenase-1.
  • LC-MS detects the enzyme activity products of SPG and GLXI.
  • the glyoxalase I GhGLXI, SPG and human glyoxalase HsGLO1 activities of G. hirstum were measured.
  • Enzyme kinetic determination The denatured protein was subjected to an enzyme activity experiment as a control for the experiment.
  • SPG can use 3A-DON as a substrate for catalytic reactions.
  • SPG catalyzes the conversion of 3A-DON to transfer the double bond from C9-C10 to C8-C9, and the C8 carbonyl group to C7.
  • the isomerization of compounds 12 to 13 involves the transfer of the carbonyl group from C-2 to C-3, forcing the C1-C11 double bond to transfer to C1-C2, and then the enolization of the C-3 carbonyl group provides ring aromatization .
  • the green color represents the protein sequence of SPG
  • the blue color represents the protein sequence of glyoxalase I GhGLXI in upland cotton
  • the other black color represents the glyoxalase I in other species.
  • Each subgenome has one GLXI on chromosome 13, and two (A subgenome) and four (D subgenome) SPGs on chromosome 3. This indicates local amplification after the birth of the ancestor.
  • variable shear model of upland cotton GhGLXI the variable shear model of upland cotton GhGLXI.
  • the present invention discloses a glyoxalase I (SPG), which is an enzyme capable of catalyzing the isomerization, dehydration and epoxidation of compounds with ortho-hydroxy ketone structures.
  • SPG glyoxalase I
  • the research of the present invention proves that SPG can catalyze the aromatization reaction, isomerization reaction, dehydration reaction and/or epoxidation reaction of natural products.
  • homologs of glyoxalase I (SPG), including glyoxalase I (GLXI) family proteins, glyoxalase GLO1 can also catalyze reactions independently of GSH.
  • the SPG can also use 3A-DON as a substrate to perform a catalytic reaction to generate iso-3A-DON, a low-toxicity product with the same molecular weight. Therefore, SPG has broad application prospects in biotoxin degradation, disease-resistant crop engineering and fragrance design.
  • glycoxalase I SPG
  • Glyoxalase SPG Glyoxalase SPG
  • SPG SPG
  • Polypeptides (proteins) with relatively high origin such as 80% or more; preferably 85% or more; more preferably 90% or more; more preferably 95% or more; such as 98% or 99% or more).
  • the "glyoxalase SPG homologue” refers to the glyoxalase SPG from a different species, but has sequence homology (such as 40% or more; preferably 50% or more; more Preferably more than 55%) of the polypeptide (protein).
  • the “homologs” include homologous polypeptides of glyoxalase SPG in multiple species, such as but not limited to: glyoxalase I (GLXI), glyoxalase GLO1.
  • the glyoxalase SPG may be a polypeptide of the amino acid sequence shown in SEQ ID NO: 2.
  • they can also be homofunctional polypeptides from the same species, including but not limited to polypeptides encoded by the following gene IDs: Gh_A03G0247, Gh_A03G0248, Gh_D03G1320, Gh_D03G1321, Gh_D03G1322, Gorai.003G145000.1, Gorai.003G145100.1, Gorai .003G145200.1, Ga_01G2453, Ga_01G2453, GB_A03G0384, GB_A03G0385, GB_A03G0386, GB_D03G1602, GB_D03G1603, GB_D03G1604.
  • the glyoxalase SPG homologue may include glyoxalase I (GLXI), glyoxalase GLO1; more specifically, it may include: GhGLXI, CrGLXI, SmGLXI, OSGLXI, AtGLXI, DzGLXI , DmGLXI, DrGLXI; HsGLO1.
  • GLXI glyoxalase I
  • the present invention also includes variant forms having the same functions as the above-mentioned polypeptides.
  • These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • amino acids with similar or similar properties are substituted
  • the function of the protein is usually not changed.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the present invention also provides analogs of the polypeptides.
  • the difference between these analogs and the natural polypeptide may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained through various techniques, such as random mutagenesis through radiation or exposure to mutagens, site-directed mutagenesis or other known molecular biology techniques.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • the present invention also includes polynucleotides encoding the above-mentioned polypeptides.
  • the polynucleotide encoding the glyoxalase SPG or its homologue may be in the form of DNA or RNA.
  • the polynucleotide encoding the mature polypeptide of glyoxalase SPG or its homologue includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optionally Additional coding sequence) and non-coding sequence.
  • the present invention also includes expression vectors and host cells containing the above-mentioned polynucleotides, which can be used to obtain active polypeptides through recombinant expression.
  • the inventors screened the glyoxalase I (SPG) of the present invention through bioinformatics analysis and virus-induced transgene silencing technology (VIGS), which can participate in gossypol. Biosynthesis.
  • VIGS virus-induced transgene silencing technology
  • the inventors found that SPG inhibited the content of gossypol, hemigossypolone and noctidin in plants from a sharp decrease, which proved that SPG participated in the biosynthesis of terpenoids such as gossypol in cotton.
  • the present invention provides the use of glyoxalase SPG or its homologues to catalyze the aromatization, isomerization, and dehydration reactions of cyclic compounds with ortho-hydroxy ketone structures. And/or epoxidation reaction.
  • the glyoxalase SPG or its homologues can also be used to prepare and catalyze the aromatization, isomerization, dehydration and/or epoxidation reactions of cyclic compounds with ortho-hydroxyketone structures. Preparations.
  • the number of rings contained in the cyclic compound is not particularly limited, and may be a monocyclic compound or a polycyclic compound, such as containing 2-10 rings, more specifically such as 3, 4, 5, and 6. Ring.
  • the X ring may be a 4- to 7-membered ring, more preferably a 5- to 6-membered ring.
  • the aromatization reaction is also called "aromatization reaction”, which mainly refers to the reaction of converting alkanes or cycloalkanes into aromatic hydrocarbons or similar compounds.
  • the isomerization reaction refers to the change of the three-dimensional structure (configuration) of the compound after being catalyzed by the glyoxalase SPG or its homologue in the present invention.
  • the aromatization reaction or isomerization reaction includes: forming a benzene ring, increasing the number of unsaturated bonds in the ring structure, changing the position of the unsaturated bond in the ring structure, or changing the ring structure or its groups Optical conformation or chirality.
  • a cyclic compound with an ortho-hydroxy ketone structure has undergone the aromatization reaction, isomerization reaction, dehydration reaction and/or epoxidation reaction, including: Hydroxy-2-carbonyl-furan Karaman is the substrate, which undergoes aromatization and/or isomerization catalyzed by glyoxalase SPG to generate deoxylhemigossypol (dHG); with 8,11-two Hydroxy-7-carbonyl-cadinene is the substrate, which undergoes isomerization, dehydration and/or epoxidation reactions catalyzed by glyoxalase SPG to produce furan Karaman; or acetyl deoxynival sickle Bactenol is a substrate, which undergoes an isomerization reaction through catalysis to produce isomerized acetyl deoxynivalenol.
  • the 3-hydroxy-2-carbonyl-furan Karaman and 8,11-dihydroxy-7-carbonyl-cadinene are intermediate substances involved in the biosynthesis of gossypol. Therefore, the glyoxalase SPG plays a catalytic role in the biosynthesis of gossypol. Therefore, glyoxalase SPG or its homologues can be applied to the biosynthesis of gossypol, including in vitro synthesis or artificial synthesis.
  • Glyoxalase SPG or its homologues catalyze the aromatization reaction of natural products containing ortho-hydroxy ketone structures to form benzene rings, and has important application value for the artificial synthesis of aromatized chemicals. At the same time, it can catalyze the isomerization reaction, dehydration reaction and/or epoxidation reaction of the substrate, and also has significant application value.
  • the inventors also found that the toxin produced by Fusarium graminearum (DON) and its acylated derivatives have similar ortho hydroxyl groups by searching for substrate analogs.
  • DON Fusarium graminearum
  • the structure of ketones and in vitro enzyme activity experiments show that SPG can use 3A-DON as a substrate to catalyze the reaction to produce iso-3A-DON with the same molecular weight. It can also produce another compound with a molecular weight of 304 but unknown structure (predicted to be Unstable anti-compounds can be).
  • the toxicity experiment of animal cell culture shows that the toxicity of iso-3A-DON is much lower than that of 3A-DON.
  • GLXI in other species cannot catalyze the conversion of 3A-DON, so this activity may be unique to SPG.
  • the present invention also provides the use of glyoxalase SPG to reduce or deactivate the toxicity of the toxin deoxynivalol or its acylated derivatives.
  • SPG can be used as a detoxification agent for the biological detoxification of food mycotoxins, and it can also be used to cultivate crops that are resistant to Fusarium graminearum or inhibit the accumulation of mycotoxins.
  • SPG lost the binding site of GSH, and SPG also lost its detoxification activity to MG.
  • SPG loses the signal peptide for plastid localization.
  • Subcellular localization experiments show that SPG is localized in the cytoplasm, which is consistent with the biosynthesis of gossypol in the cytoplasm.
  • the inventors also modified SPG, predicted some of its sites that may be related to the detoxification function, and performed mutations and verifications one by one. The results found that SPG lost its detoxification ability to MG, but after replacing the GSH binding site , Some mutants partially restored the ability to detoxify MG.
  • the present invention also provides a method for improving the ability of glyoxalase SPG in reducing or detoxifying methylglyoxal (MG) toxicity, including sequence modification of glyoxalase SPG so that it generates M69F/ F101N mutation, F101N/I121R mutation or M69F/F101N/I121R, and its Loop13 adds 4 amino acids GKMK (between its 153-154).
  • the invention also provides the prepared mutant polypeptide. This discovery of the present invention provides a new way to change the biological activity of glyoxalase SPG for MG detoxification.
  • SPG gene ID As cotton has had multiple genome doubling events during its evolution, there are many SPG homologous genes. In Gossypium hirsutum, the SPG gene ID is: A subgenome Gh_A03G0247 and Gh_A03G0248, D subgenome Genomics of Gh_D03G1320, Gh_D03G1321 and Gh_D03G1322.
  • the IDs in Raymond's cotton are: Gorai.003G145000.1, Gorai.003G145100.1, Gorai.003G145200.1.
  • the gene IDs in Asian cotton are: Ga_01G2453 and Ga_01G2453.
  • the gene IDs in sea island cotton are: GB_A03G0384, GB_A03G0385, GB_A03G0386, GB_D03G1602, GB_D03G1603 and GB_D03G1604.
  • the proteins in Gossypium plants that are more than 90% homologous to the SPG sequence, preferably more than 95%, are considered to have the same function as SPG.
  • SPG take Gh_D03G1322 as an example
  • coding protein sequence SEQ ID NO: 2
  • the expression of the gossypol gene is positively correlated with the accumulation of gossypol.
  • SPG is the same as cotton
  • PGF is a bHLH transcription factor that positively regulates cotton gland development and gossypol biosynthesis [Ma, D.et al. Nat Commun 7, 10456 (2016)].
  • PGF inhibits the absence of glands in the leaves of plants, nor does it contain them.
  • Gossypol the inventors found that SPG is not expressed in the leaves of PGF-inhibited plants.
  • gossypol can be induced by the fungal elicitor VdNEP.
  • VGS virus-induced transgene silencing
  • forward primer GAGTAAGGTTACCGAATTCTCTAGATGCCAATAACCCAGGCCTTC (SEQ ID NO: 7)
  • reverse primer CGCGTGAGCTCGGTACCGGATCCAACTCGGGATCGTTTTCGGT (SEQ ID NO: 8)
  • the forward primer introduces BamHI restriction site
  • SPG gene is connected to pTRV2 vector (Biovector) by homologous recombination method
  • the sequenced vector is transferred to Agrobacterium GV3101, and cultured upside down at 28°C for 2 to 3 days.
  • RNA of the second true leaf of the SPG inhibition plant was analyzed by qRT-PCR (Table 1), and it was found that the expression of SPG in the VIGS plant leaves was indeed down-regulated, as shown in Figure 1c.
  • leaf extract After the cotton leaves are ground with liquid nitrogen, 1 ml of leaf extract is added to every 100 mg of material.
  • HPLC analysis adopts Agilent 1200 system, Agilent ZORBAX Eclipse XDB-C18 analytical column (150mm ⁇ 4.6mm, 5 ⁇ m) reverse C18 analytical column.
  • Mass detection adopts Agilent 6120 quadrupole detector, API-ES ion source, positive ion mode, fragmentation voltage 70V.
  • the mobile phase flow rate is 1mL/min
  • the injection volume is 10 ⁇ L
  • the column temperature is 40°C
  • the detection time is 50min.
  • RNA extraction reagent preheated at 65°C, vortex vigorously immediately to mix, and place at 65°C for 30 minutes. Centrifuge at 13,500g for 2min. Transfer the supernatant to a new 2mL tube, add 0.6mL chloroform, 13,500g, centrifuge for 5min, carefully aspirate the supernatant to a new RNase-Free centrifuge tube. Repeat step 3. Transfer the supernatant to a new 2mL tube, add LiCl to a final concentration of 2M, and place at -20°C for 3h. Centrifuge at 13,500g for 10 minutes, discard the waste solution, wash the precipitate twice with 70% ethanol, dry at room temperature and add RNase-Free ddH 2 O.
  • RNA extraction reagents 0.2M Tris, 50mM EDTA, 1M NaCl, 1% CTAB, 1% ⁇ -mercaptoethanol, pH 8.0.
  • High-fidelity enzyme HS DNA Polymerase amplifies the full-length cDNA fragment (549bp) of SPG.
  • PCR reaction conditions were: 98°C, denaturation for 1 minute; 98°C, denaturation for 10 seconds; 57°C, renaturation for 10 seconds; 72°C, extension for 40 seconds; 72°C, 5 minutes; 4°C incubation.
  • the SPG was connected to the pET-32a prokaryotic expression vector by homologous recombination.
  • the sequenced SPG prokaryotic expression vector plasmid was transformed into BL21 strain, spread on Amp-resistant LB medium, at 37°C, inverted and cultured overnight.
  • Amp-resistant LB was added to a 96-well plate, and a single clone was picked for colony PCR.
  • the clones identified by PCR as positive were added to 2 mL of resistant liquid LB (100 ⁇ g/mL Ampr) and incubated overnight at 37°C. Transfer the large shake according to the ratio of 1:100. The volume of the large shake depends on the situation. It can be set to 100mL. Incubate at 37°C until OD600 ⁇ 0.6 (about 3h).
  • SPG enzyme activity system 25mM HEPES, 100 ⁇ M ZnCl 2 , 5 ⁇ g protein, 100 ⁇ M gossypol pathway intermediate substrate, including 3-hydro-furocalamen-2-one (3-hydro-furocalamen-2-one) and 8, 11-dihydroxy-7-carbonyl-cadinene (8,11-dihydroxy-7-keto-( ⁇ )-cadinene). Reacted at 30°C for two hours, extracted with ethyl acetate, drained and dissolved in acetonitrile, filtered, and detected by HPLC-MS.
  • HPLC analysis adopts Agilent 1100 system, Agilent Eclipse XDB-C18 semi-preparative column (250mm ⁇ 9.4mm, 5 ⁇ m) reverse C18 analytical column.
  • the mobile phase is acetonitrile (B) and formic acid solution (A), the formic acid concentration is 0.1%, the mobile phase flow rate is 1 mL/min, and the injection volume is 10 ⁇ l.
  • Gradient elution conditions 0-3min, 20-70%B; 3-5min, 70-80%B; 5-7min, 80-84%B; 7-8min, 84-100%B; 8-10min, 100 -20%B.
  • HPLC analysis adopts Agilent 1100 system, Agilent Eclipse XDB-C18 semi-preparative column (250mm ⁇ 9.4mm, 5 ⁇ m) reverse C18 Analysis column.
  • the mobile phase is acetonitrile (B) and formic acid solution (A), the formic acid concentration is 0.1%, the mobile phase flow rate is 1 mL/min, and the injection volume is 10 ⁇ l.
  • the substrate is 3-hydroxy-2-carbonyl-furan Karaman
  • the substrate is 8,11-dihydroxy-7-carbonyl-cadinene.
  • Example 4 SPG can use 3A-DON as a substrate for catalytic reaction to reduce its toxicity
  • the inventors learned that the protein similarity between SPG and human glyoxalase I (GLO1) is 59%, indicating that the sequence of SPG is very conservative.
  • the inventors compared the crystal structures of SPG with human glyoxalase I and found that 4 sites related to GSH binding have changed.
  • the amino acid at position 69 of SPG protein becomes M, and the amino acid at this position of the corresponding GhGLXI and GLXI of other species is F; the amino acid at position 101 of SPG is F, and GhGLXI and GLXI of other species It is N at this position; the amino acid at position 121 of SPG is I, and the GLXI of GhGLXI and other species is R at this position.
  • SPG has deleted 4 amino acids in Loop13 (GKMK is deleted between positions 153 and 154), GhGLXI Here is GKMK. These sites may be closely related to GSH binding, as shown in Figure 6b.
  • the inventors performed combined mutations on SPG and GhGLXI, mutating the points corresponding to SPG to points corresponding to GhGLXI, and on the contrary, mutating the points corresponding to GhGLXI to points corresponding to SPG.
  • F119M represents changing the F at position 119 of GhGLXI to M
  • the SPG mutant M69F/F101N/I121R represents replacing the amino acids at the three positions of SPG with the amino acids at the corresponding positions of GhGLXI.
  • 1 unit (units/L) of glyoxalase I refers to the amount of protein that can produce 1 ⁇ M product per minute in a solution at 25°C and pH 7.0. After determination, the inventors found that Glyoxalase I GhGLXI of Gossypium hirsutum has MG detoxification activity, and its mutants lose their detoxification ability to MG to varying degrees, as shown in Figure 6c.
  • SPG presents tandem repeats in chromosomes, Gh_A03G0247 and Gh_A03G0248 in the A subgenome, and Gh_D03G1320, Gh_D03G1321, Gh_D03G1322, Gh_D03G1323 in the D subgenome.
  • Gh_D03G1323 is almost not expressed in various tissues, which is guessed to be a pseudogene.
  • Upland cotton glyoxalase I GhGLXI is located on chromosome 13, which is Gh_A13G2029 of A13 chromosome and Gh_D13G2432 of D13 chromosome, as shown in Figure 6e.
  • Arabidopsis thaliana has only one zinc ion-dependent glyoxalase I with variable cleavage, while GhGLXI also has variable cleavage, as shown in Figure 7a and b.
  • SPG lacks the signal peptide for plastid localization at the N-terminus, as shown in Figure 6f, Figure 7c, and d, which coincides with the biosynthesis of sesquiterpenoids located in the cytoplasm.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Nutrition Science (AREA)
  • Polymers & Plastics (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

提供了乙二醛酶SPG或其同源物用于催化具有邻位羟基酮结构的环状化合物发生芳香化反应、异构化反应、脱水反应和/或环氧化反应的用途，以及合成棉酚的方法。还提供了提高乙二醛酶SPG在降低或解除甲基乙二醛毒性方面的能力的方法及乙二醛酶SPG突变体。

Description

乙二醛酶SPG的功能及应用 技术领域
本发明属于植物学以及分子生物学领域，更具体地，本发明涉及乙二醛酶SPG或其同源物的功能及应用。
背景技术
植物中存在很多芳香化的化合物，包括芳香族氨基酸、黄酮类化合物和一些生物碱等，这些芳香化的化合物大多从芳香族氨基酸得到苯环，或者由聚酮合酶PKS催化而来。酶促芳香化反应形成苯环，对于芳香化化学物质的合成具有重要应用价值。棉酚是一种倍半萜类化合物，是棉属植物中具有芳香化苯环结构的植保素，在抵御病菌、害虫和食草动物侵食等方面发挥重要作用。棉酚的生物合成途径研究一直为人们所重视，但由于缺乏相关的转录组数据，该途径进展较为缓慢。截止到目前，已经有一些棉酚生物合成途径的基因被分离鉴定。杜松烯合酶CDN是一个倍半萜环化酶，催化FPP形成杜松烯。三个细胞色素P450单加氧酶CYP706B1、CYP82D113和CYP71BE79分别在杜松烯的骨架上进行羟化反应；脱氢酶DH1催化产生7-羰基-杜松烯[1]。棉酚是如何被芳构化的，还是一个未解之谜。
甲基乙二醛(Methyglyoxal，MG)，也称为丙酮醛，含有两个活性羰基，是一种对细胞具有毒性的化合物，是糖酵解过程中产生的固有的毒性副产物，对所有以糖为能量来源的生物都是不可避免的[2]。生物体在长期的进化过程中产生了MG解毒系统，名为乙二醛酶系统。乙二醛酶系统由两个酶组成，乙二醛酶I(Glyoxalase I，GLO1，GLXI，EC 4.4.1.5)和乙二醛酶II(Glyoxalase II，GLO2，GLXII，EC 3.1.2.6)。MG和还原性谷胱甘肽GSH通过非酶促反应生成硫代半缩醛，经GLXI催化生成S-D-乳酰谷胱甘肽，随后GLXII催化S-D-乳酰谷胱甘肽生成无毒的D-乳酸，并释放还原性谷胱甘肽以重复利用，从而达到解毒的作用[3]。其中GLXI是反应中的限速酶，在MG的代谢解毒方面起到重要作用，植物中GLXI家族成员众多，是否存在其它的催化功能还是未知。
真菌毒素是由镰刀菌、青霉菌和交链孢菌等感染植物和农作物形成的真菌次生代谢产物，污染粮油食品、畜牧饲料和水果蔬菜等，严重影响食品安全和人畜健康。脱氧雪腐镰刀菌烯醇(DON)及其酰基化衍生物3ADON和15ADON是最主要的真菌毒素，对谷物污染十分严重。其性质稳定，毒性高，被国际癌症研究机构IARC列为第三类致癌物，已被联合国粮农组织FAO和世界卫生组织WHO确定为最危险的天然食品污染物之一。采用生物学脱毒技术降解真菌毒素，具有绿色高效的优势，受到国际高度重视，但是此类研究目前进展缓慢。
综上，本领域中，以上各个方面的研究亟待进一步优化和提高。
发明内容
本发明的目的在于提供乙二醛酶SPG或其同源物的功能及应用。
在本发明的第一方面，提供乙二醛酶SPG或其同源物的用途，用于催化具有邻位羟基酮结构的环状化合物发生芳香化反应、异构化反应、脱水反应和/或环氧化反应；或用于制备催化具有邻位羟基酮结构的环状化合物发生芳香化反应、异构化反应、脱水反应和/或环氧化反应的制剂。
在一个优选例中，所述的环状化合物化合物为单环化合物或多环化合物(包括双环化合物，如包含2～10个环)。
在另一优选例中，所述的邻位羟基酮结构包括：
Figure PCTCN2020108157-appb-000001
其中X为环结构，如4～7元环，更佳地5～6元环。
在另一优选例中，所述的芳香化反应或异构化反应包括：形成苯环、增加环结构中的不饱和键数量、改变环结构中的不饱和键的位置、或改变环结构或其上基团的光学构象或手性。
在另一优选例中，所述的具有邻位羟基酮结构的环状化合物包括：3-羟基-2-羰基-呋喃卡拉曼或其类似物，其经乙二醛酶SPG或其同源物催化发生芳香化反应和/或异构化反应，生成脱氧半棉酚(deoxyhemigossypol，dHG)或其类似物；或，8,11-二羟基-7-羰基-杜松烯或其类似物，其经乙二醛酶SPG或其同源物催化发生异构化反应、脱水反应和/或环氧化反应，生成呋喃卡拉曼(furocalamen)或其类似物；或乙酰基脱氧雪腐镰刀菌烯醇(3A-DON)或其类似物，其经乙二醛酶SPG催化发生异构化反应，生成异构化乙酰基脱氧雪腐镰刀菌烯醇或其类似物。
在另一优选例中，所述的3-羟基-2-羰基-呋喃卡拉曼和8,11-二羟基-7-羰基-杜松烯为参与棉酚生物合成的中间物质，从而，所述的乙二醛酶SPG或其同源物被用于棉酚生物合成中。
在另一优选例中，所述乙二醛酶SPG包括：(a)如SEQ ID NO:2所示氨基酸序列的多肽；或(b)将SEQ ID NO:2所示氨基酸序列经过一个或多个(如1-20个；较佳地1-10个；更佳地1-5个)氨基酸残基的取代、缺失或添加而形成的，且具有(a)多肽功能的由(a)衍生的多肽；或(c)氨基酸序列与(a)限定的氨基酸序列有80％以上(较佳地85％以上；更佳地90％以上；更佳地95％以上；如98％以上或99％以上)相同性且具有(a)多肽功能的多肽；或(d)具有(a)多肽功能的SEQ ID NO:2的片段；较佳地，所述乙二醛酶SPG的编码基因的ID包括：Gh_A03G0247，Gh_A03G0248，Gh_D03G1320，Gh_D03G1321，Gh_D03G1322，Gorai.003G145000.1，Gorai.003G145100.1， Gorai.003G145200.1，Ga_01G2453，Ga_01G2453，GB_A03G0384，GB_A03G0385，GB_A03G0386，GB_D03G1602，GB_D03G1603，GB_D03G1604。
在另一优选例中，所述乙二醛酶SPG的同源物包括：乙二醛酶I(GLXI)，乙二醛酶GLO1；较佳地，所述的乙二醛酶I包括GhGLXI，CrGLXI，SmGLXI，OSGLXI，AtGLXI，DzGLXI，DmGLXI，DrGLXI；较佳地，所述的乙二醛酶GLO1包括HsGLO1。
在另一优选例中，所述的乙二醛酶SPG的同源物还包括所述乙二醛酶I或乙二醛酶GLO1的变体，如与其任一的氨基酸序列有80％以上(较佳地85％以上；更佳地90％以上；更佳地95％以上；如98％以上或99％以上)相同性且具有它们的功能的多肽。
在本发明的另一方面，提供乙二醛酶SPG的用途，用于降低或解除毒素脱氧雪腐镰刀菌醇(DON)的酰基化衍生物的毒性。
在本发明的另一方面，提供一种催化具有邻位羟基酮结构的环状化合物发生芳香化反应、异构化反应、脱水反应和/或环氧化反应的方法，包括：以乙二醛酶SPG或其同源物处理所述具有邻位羟基酮结构的环状化合物。
在一个优选例中，所述的环状化合物化合物为单环化合物或多环化合物(包括双环化合物，如包含2～10个环)；或，所述的邻位羟基酮结构包括：
Figure PCTCN2020108157-appb-000002
其中X为环结构(如4～7元环，更佳地5～6元环)。
在另一优选例中，所述的芳香化反应或异构化反应包括：形成苯环、增加环结构中的不饱和键数量、改变环结构中的不饱和键的位置、或改变环结构或其上基团的光学构象或手性。
在另一优选例中，所述的具有邻位羟基酮结构的环状化合物包括：3-羟基-2-羰基-呋喃卡拉曼，其经催化发生芳香化反应和/或异构化反应，生成脱氧半棉酚(deoxyhemigossypol，dHG)；或，8,11-二羟基-7-羰基-杜松烯，其经催化发生异构化反应、脱水反应和/或环氧化反应，生成呋喃卡拉曼(furocalamen)；或乙酰基脱氧雪腐镰刀菌烯醇(3A-DON)，其经催化发生异构化反应，生成异构化乙酰基脱氧雪腐镰刀菌烯醇。
在本发明的另一方面，提供一种降低或解除脱氧雪腐镰刀菌醇(DON)或其酰基化衍生物的毒性的方法，包括以乙二醛酶SPG来处理。
在本发明的另一方面，提供一种提高乙二醛酶SPG在降低或解除甲基乙二醛(MG)毒性方面的能力的方法，包括对乙二醛酶SPG进行序列改造，使得其发生M69F/F101N突变、F101N/I121R突变或M69F/F101N/I121R，且其Loop13中增加4个氨基酸GKMK(对应SEQ ID NO:2序列，在其153～154位之间插入GKMK。
在本发明的另一方面，提供一种乙二醛酶SPG突变体，其具有降低或解除甲基 乙二醛(MG)毒性方面的能力，其对应SEQ ID NO:2序列，发生M69F/F101N突变、F101N/I121R突变或M69F/F101N/I121R，且其Loop13中增加4个氨基酸GKMK。
在本发明的另一方面，提供一种合成棉酚的方法，包括：
(1)以法尼基二磷酸(FPP)为底物，以杜松烯合成酶(CDN)催化，获得(+)-δ-杜松烯；
(2)将(1)的产物，以P450单加氧酶CYP706B1催化，获得7-羟基-(+)-δ-杜松烯；
(3)将(2)的产物，以醇脱氢酶-1(DH1)催化，获得7-羰基-δ-杜松烯；
(4)将(3)的产物，以P450单加氧酶CYP82D113催化，获得8-羟基-7-羰基-δ-杜松烯；
(5)将(4)的产物，以P450单加氧酶CYP71BE79催化，获得8,11-二羟基-7-羰基-杜松烯；
(6)将(5)的产物，以乙二醛酶SPG进行催化，获得呋喃卡拉曼；
(7)将(6)的产物，以P450单加氧酶CYP736A196催化，获得2-羟基-呋喃卡拉曼；
(8)将(7)的产物，以醇脱氢酶-1(DH1)催化，获得呋喃卡拉曼-2-酮；
(9)将(8)的产物，以2-酮戊二酸/Fe(ii)-依赖的双加氧酶-1催化，获得3-羟基-2-羰基-呋喃卡拉曼；
(10)将(9)的产物，以乙二醛酶SPG进行催化，获得脱氧半棉酚；
(11)将(10)的产物，自发反应获得半棉酚；
(12)将(11)的产物，以漆酶或过氧化物酶催化，获得棉酚。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、通过转录组分析及VIGS筛选到芳香化酶SPG。
a，SPG的表达与棉酚的积累呈正相关。SPG在无腺体棉和PGF抑制植株叶片中几乎不表达，而且能够受到激发子VdNEP的诱导。
b，SPG与棉酚途径其它已知基因的表达特征的相关性分析。
c，SPG在VIGS植株中的表达受到抑制。
d-i，SPG抑制植株叶片中半棉酚酮(d)、棉酚(e)、杀夜蛾素H1～H4(f-j)含量显著下降。
图2、SPG和其它乙二醛酶I催化活性羰基醛的芳香化反应。
a，棉酚生物合成途径。SPG催化的步骤用浅色阴影表示。CDN：杜松烯合成酶；DH1：醇脱氢酶-1；2-ODD-1：2-酮戊二酸/Fe(ii)-依赖的双加氧酶-1。
b-d，LC-MS检测SPG和GLXI的酶活产物。测定了陆地棉(G.hirstum)的乙二醛酶I GhGLXI、SPG和人乙二醛酶HsGLO1的活性。
e，酶学动力学测定。变性的蛋白质进行酶活实验作为实验的对照。
图3、SPG能够以3A-DON为底物进行催化反应。
a，SPG催化3A-DON(15)异构化反应的高效液相色谱图。
b，SPG催化3A-DON产生分子量为304的化合物。进行了三次生物学重复，得到相类似的结果。
c、不同浓度3A-DON和iso-3A-DON对小鼠成纤维细胞的毒性比较。每个值都是五个独立实验的结果。
d，S-D-乳糖谷胱甘肽的形成涉及到C2羰基转移到C3。
e，SPG催化3A-DON转化将双键从C9-C10转移到C8-C9，将C8羰基转移到C7。
f，化合物12到13的异构化涉及到羰基从C-2到C-3的转移，迫使C1-C11双键转移到C1-C2，然后C-3羰基的烯醇化提供了环芳构化。
g，化合物5到9的转化涉及到两轮异构化转变，即C7羰基到C11，C6-C7双键到C7-C8，这可能涉及到活性袋中中间体的反弹，以重新与锌离子协调。然后，中间体的C8羟基攻击C11羰基，在C8和C11之间形成半缩醛，经过两次连续脱水，最终得到芳香环B。
图4、通过NMR对呋喃卡拉曼(化合物9)的结构进行鉴定。
图5、通过NMR对iso-3A-DON(化合物16)的结构进行鉴定。
图6、SPG丢失了GSH结合功能。
a，SPG与经典乙二醛酶I的序列比对。绿色表示的是SPG的蛋白序列，蓝色表示的陆地棉中乙二醛酶I GhGLXI的蛋白序列，其它黑色表示的是其它物种中的乙二醛酶I。
b，Docking实验表明SPG结合GSH的位点发生了改变。以结合了S-(N-羟基-N-P-碘代苯甲酰-Bamoyl)谷胱甘肽(HIPC-GSH)底物的人GLO1蛋白结构(PDB ID:1QIN)为模板。
c-d，SPG对MG-GSH加合物(半硫代乙缩醛)失去了活性。基于图b对陆地棉乙二醛酶I GhGLXI(c)和SPG(d)相应位点进行互变，图c显示了GhGLXI突变体的活性出现不同程度的丧失，而图d显示SPG突变体部分恢复了对半硫代乙缩醛的活性。活动(单位/L)表示为平均值±SE，n＝3。
e，SPG和GhGLXI基因的染色体定位。每个亚基因组在13号染色体上有一个GLXI，在3号染色体上有两个(A亚基因组)和四个(D亚基因组)SPG，这表明在祖先出生后有局部扩增。
f，卡通图显示SPG结构域丢失，叶绿体转运肽(绿色)的丢失和谷胱甘肽结合位点(红色)的替换。
图7、SPG定位于细胞质。
a-b，陆地棉GhGLXI的可变剪切模型。
c，相比于GhGLXI来说SPG丢失了N端的质体定位信号肽。
d，SPG的亚细胞定位，将SPG的C端融合YFP，将农杆菌注射烟草叶片，FM-64用来标注细胞膜。
具体实施方式
本发明揭示了一种乙二醛酶I(SPG)，其是一种能够催化具有邻位羟基酮结构的化合物异构、脱水、环氧化的酶。本发明的研究证明，SPG能够催化天然产物的芳香化反应、异构化反应、脱水反应和/或环氧化反应。同时，乙二醛酶I(SPG)的同源物，包括乙二醛酶I(GLXI)家族蛋白，乙二醛酶GLO1，也能够不依赖GSH而进行催化反应。所述SPG还可以利用3A-DON为底物进行催化反应，生成分子量相同的低毒产物iso-3A-DON。因此，SPG在生物毒素降解、抗病作物工程和芳香剂设计方面具有广阔的应用前景。
如本文所用，所述的“乙二醛酶I(SPG)”、“乙二醛酶SPG”或“SPG”可互换使用，为具体SEQ ID NO:2所示氨基酸序列或该序列其同源性较高(如80％以上；较佳地85％以上；更佳地90％以上；更佳地95％以上；如98％以上或99％以上)的多肽(蛋白)。
如本文所用，所述的“乙二醛酶SPG同源物”是指与乙二醛酶SPG来自不同的物种，但是具有序列同源性(如40％以上；较佳地50％以上；更佳地55％以上)的多肽(蛋白)。所述“同源物”包括在多个物种中乙二醛酶SPG的同源多肽，例如但不限于：乙二醛酶I(GLXI)，乙二醛酶GLO1。
乙二醛酶SPG及其同源物
本发明中，所述的乙二醛酶SPG可以是SEQ ID NO:2所示氨基酸序列的多肽。同时，也可以是来自同物种的同功能多肽，包括但不限于由下述基因ID所编码的多肽：Gh_A03G0247，Gh_A03G0248，Gh_D03G1320，Gh_D03G1321，Gh_D03G1322，Gorai.003G145000.1，Gorai.003G145100.1，Gorai.003G145200.1，Ga_01G2453，Ga_01G2453，GB_A03G0384，GB_A03G0385，GB_A03G0386，GB_D03G1602，GB_D03G1603，GB_D03G1604。
本发明中，所述的乙二醛酶SPG同源物可以包括包括乙二醛酶I(GLXI)，乙二醛 酶GLO1；更具体地可以包括：GhGLXI，CrGLXI，SmGLXI，OSGLXI，AtGLXI，DzGLXI，DmGLXI，DrGLXI；HsGLO1。但是，由于乙二醛酶I在许多物种中均保守性地存在，因此应理解，本发明中并不仅限于上述具体列举的乙二醛酶SPG同源物。
本发明中还包括具有与上述多肽具有相同功能的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。本发明还提供所述多肽的类似物。这些类似物与天然多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些多肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
本发明中，也包括编码上述多肽的多核苷酸。编码乙二醛酶SPG或其同源物的多核苷酸可以是DNA形式或RNA形式。编码乙二醛酶SPG或其同源物的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
本发明中，也包括含有上述多核苷酸的表达载体以及宿主细胞，利用它们可以通过重组表达的方式获得活性的多肽。
功能和应用
在本发明的具体实施例中，本发明人通过生物信息学分析及病毒诱导的转基因沉默技术(VIGS)，筛选到本发明所述的乙二醛酶I(SPG)，其能够参与到棉酚的生物合成中。通过VIGS本发明人发现SPG抑制植株中棉酚、半棉酚酮及杀夜蛾素的含量急剧下降，证明SPG在棉花体内参与到了棉酚等萜类化合物的生物合成。通过体外酶活实验，本发明人发现SPG可以在不依赖于GSH的前提下，催化棉花中两个具有邻位羟基酮结构的化合物(3-羟基-2-羰基-呋喃卡拉曼和8,11-二羟基-7-羰基-杜松烯)的芳香化，从而参与到了棉酚的生物合成过程中。本发明人首次发现，植物和动物，包括人类中的GLXI都能催化棉花中天然产物的芳香化，而且也是不依赖于GSH的。
基于本发明人的新发现，本发明提供了乙二醛酶SPG或其同源物的用途，用于催化具有邻位羟基酮结构的环状化合物发生芳香化反应、异构化反应、脱水反应和/ 或环氧化反应。同时，所述乙二醛酶SPG或其同源物也可被用于制备催化具有邻位羟基酮结构的环状化合物发生芳香化反应、异构化反应、脱水反应和/或环氧化反应的制剂。
在包含有邻位羟基酮结构
Figure PCTCN2020108157-appb-000003
的情况下，所述的环状化合物所包含的环的数量没有特别的限制，可以为单环化合物或多环化合物，如包含2～10个环，更具体地如3、4、5、6个环。所述的
Figure PCTCN2020108157-appb-000004
结构中，X环可以为4～7元环，更佳地5～6元环。
所述的芳香化反应又称为“芳构化反应”，主要指由烷烃或环烷烃转变为芳香烃或类似化合物的反应。
所述的异构化反应，是指化合物经由本发明中的乙二醛酶SPG或其同源物催化后，其立体结构(构型)发生了变化。
本发明中，所述的芳香化反应或异构化反应包括：形成苯环、增加环结构不饱和键数量、改变环结构中的不饱和键的位置、或改变环结构或其上基团的光学构象或手性。
在本发明的具体实施例中，论证了具有邻位羟基酮结构的环状化合物发生了所述的芳香化反应、异构化反应、脱水反应和/或环氧化反应，包括：以3-羟基-2-羰基-呋喃卡拉曼为底物，其经乙二醛酶SPG催化发生芳香化反应和/或异构化反应，生成脱氧半棉酚(deoxylhemigossypol，dHG)；以8,11-二羟基-7-羰基-杜松烯为底物，其经乙二醛酶SPG催化发生异构化反应、脱水反应和/或环氧化反应，生成呋喃卡拉曼；或以乙酰基脱氧雪腐镰刀菌烯醇为底物，其经催化发生异构化反应，生成异构化乙酰基脱氧雪腐镰刀菌烯醇。应理解，上述实施例中论证的底物的类似物、衍生物或与这些底物具有相同母环结构的化合物，也可以被乙二醛酶SPG催化发生反应，也包含在本发明的范围内。
上述的底物中，所述的3-羟基-2-羰基-呋喃卡拉曼和8,11-二羟基-7-羰基-杜松烯为参与棉酚生物合成的中间物质。因此，所述的乙二醛酶SPG在棉酚生物合成中发挥催化作用。因此，乙二醛酶SPG或其同源物可被应用于棉酚的生物合成，包括体外合成或人工合成。
乙二醛酶SPG或其同源物催化含有邻位羟基酮结构的天然产物形成苯环的的芳香化反应，对芳香化化学物质的人工合成具有重要的应用价值。同时，其能够催化底物发生异构化反应、脱水反应和/或环氧化反应，也具有显著的应用价值。
在本发明的具体实施例中，本发明人还通过寻找底物类似物，发现禾谷镰孢菌产 生的毒素脱氧雪腐镰刀菌醇(DON)及其酰基化衍生物具有类似的邻位羟基酮的结构，体外酶活实验表明SPG可以利用3A-DON为底物进行催化反应，生成分子量相同的产物iso-3A-DON，还能够产生另外一个分子量为304但是结构未知的化合物(预测可能为不稳定反化合物可)。动物细胞培养的毒性实验表明，iso-3A-DON毒性较3A-DON大大降低。进一步研究发现，其它物种中的GLXI并不能催化3A-DON的转化，因而这个活性可能是SPG所独有的。
因此，本发明还提供了乙二醛酶SPG的用途，用于降低或解除毒素脱氧雪腐镰刀菌醇或其酰基化衍生物的毒性。SPG可以作为解毒制剂应用到食品真菌毒素的生物解毒，也可以用来培育具有禾谷镰孢菌抗性或抑制真菌毒素积累的农作物。
经典的GLXI在发挥功能时，都需要有GSH的参与，通过蛋白序列比对及蛋白质模拟。在本发明的具体实施例中，本发明人发现，SPG丢失了GSH的结合位点，同时SPG也丢失了对MG的解毒活性。此外，与棉花中的GLXI不同，SPG丢失了质体定位的信号肽，亚细胞定位实验表明SPG定位于细胞质中，与棉酚生物合成在细胞质中进行是相吻合的。本发明人还对SPG进行了改造，预测其一些可能与解毒功能相关的位点，并进行一一突变和验证，结果发现SPG丢失了对MG的解毒能力，但是将GSH结合位点进行置换后，某些突变体部分恢复了对MG的解毒能力。
因此，本发明还提供了一种提高乙二醛酶SPG在降低或解除甲基乙二醛(MG)毒性方面的能力的方法，包括对乙二醛酶SPG进行序列改造，使得其发生M69F/F101N突变、F101N/I121R突变或M69F/F101N/I121R，且其Loop13中增加4个氨基酸GKMK(在其153～154位之间)。本发明还提供了所制备的突变体多肽。本发明的这一发现，为改变乙二醛酶SPG的生物活性，用于进行MG解毒提供了新的途径。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、SPG参与棉酚的生物合成
a：SPG基因序列及蛋白序列
SPG基因ID：由于棉花在进化过程中发生过多次基因组加倍事件，因而SPG同源基因有很多，在陆地棉(Gossypium hirsutum)中SPG的基因ID为：A亚基因组的Gh_A03G0247和Gh_A03G0248，D亚基因组的Gh_D03G1320，Gh_D03G1321和Gh_D03G1322。在雷蒙德氏棉中的ID为：Gorai.003G145000.1、Gorai.003G145100.1、 Gorai.003G145200.1。在亚洲棉中的基因ID为：Ga_01G2453和Ga_01G2453。在海岛棉中的基因ID为：GB_A03G0384、GB_A03G0385、GB_A03G0386、GB_D03G1602、GB_D03G1603和GB_D03G1604。根据本领域公知知识，棉属植物中与SPG序列同源性在90％以上，较佳地95％以上的蛋白被认为与SPG功能相同。
SPG(以Gh_D03G1322为例)核苷酸序列cDNA(SEQ ID NO:1)：
Figure PCTCN2020108157-appb-000005
SPG(以Gh_D03G1322为例)编码蛋白序列(SEQ ID NO:2)：
Figure PCTCN2020108157-appb-000006
b：转录组分析发现SPG的表达与棉酚的积累呈正相关
棉酚基因的表达与棉酚的积累呈现正相关的特征，通过比较有腺体棉叶片(含有棉酚)和无腺体棉叶片(没有棉酚)的转录组，本发明人发现SPG同棉酚途径已知基因类似，都在无腺体棉叶片中几乎不表达。PGF是正调控棉花腺体发育和棉酚生物合成的bHLH类的转录因子[Ma，D.et al.Nat Commun 7，10456(2016)]，PGF抑制植株叶片中没有腺体，叶片中也不含有棉酚，本发明人发现SPG在PGF抑制植株叶片中也不表达。棉酚作为棉花最重要的植保素，能够受到真菌激发子VdNEP的诱导，本发明人发现SPG同棉酚途径其它已知基因类似，也能够受到真菌激发子的诱导。如图1a所示。
通过相关性分析，本发明人发现SPG的表达特征与棉酚途径已知基因的表达特征的相关性非常高，如图1b所示，提示SPG参与棉酚生物合成。
c：构建SPG基因的病毒诱导的转基因沉默(VIGS)载体
PCR扩增300～500bp的基因特异片段(正向引物：GAGTAAGGTTACCGAATTCTCTAGATGCCAATAACCCAGGCCTTC(SEQ ID NO:7)，反向引物：CGCGTGAGCTCGGTACCGGATCCAACTCGGGATCGTTTTCGGT(SEQ ID NO:8))，正向引物引入BamHI酶切位点，反向引物引入XbaI酶切位点装入pTRV2载体，利用同源重组的方法将SPG基因连接到pTRV2载体(Biovector)上，将测序正确的载体转入农杆菌GV3101，28℃倒置培养2～3天。
d：将SPG基因VIGS载体转染棉花子叶
将测序正确的质粒转入农杆菌GV3101感受态细胞，同时将包含pTRV1载体的农杆菌划线(后续需要将装有基因片段的pTRV2载体农杆菌与pTRV1农杆菌1/1混合后注射棉花子叶)，同时装有PDS基因(该基因为正对照)的pTRV2农杆菌(SU)划线(SU与pTRV1农杆菌1/1混合注射棉花子叶后，可使叶片变黄，作为正对照)。挑取单克隆进行菌落PCR验证，将菌落PCR阳性的农杆菌克隆挑取至2mL选择抗性的液体LB培养基中，28℃培养过夜，至OD值为2.5。同时将pTRV1和SU农杆菌小摇。将小摇的农杆菌，按照1:100的比例，转接到50mL的选择抗性LB液体培养基中，继续在28℃震荡培养过夜。室温，8,000g离心10min，用等体积的重悬液(10mM MES，10mM MgCl 2，150μM acetosyringone，pH＝5.8)重悬，OD值调至1左右，室温放置至少3h，使菌体活化。将转有不同质粒的农杆菌重悬液与转有pTRV1载体的农杆菌重悬液以1：1(V/V)混合，用1mL注射器从棉花子叶背面注射进行转染。注射2～3周后观察正对照是否黄化，采取第二片真叶，液氮速冻，保存于-70℃。将SPG抑制植株的第二片真叶的RNA，进行qRT-PCR分析(表1)，发现SPG在VIGS植株叶片中表达量确实下调非常明显，如图1c所示。
表1、qRT-PCR所用引物
  序列(5’-3’) 序列编号
His3-qPCR-F GAAGCCTCATCGATACCGTC SEQ ID NO:3
His3-qPCR-R CTACCACTACCATCATGG SEQ ID NO:4
GhSPG-qPCR-F ACGATCCCGAGTTCAAAGGA SEQ ID NO:5
GhSPG-qPCR-R CAAGTCATCCACGGTAAGGC SEQ ID NO:6
e：HPLC检测SPG抑制植株叶片中棉酚及相关萜类化合物的含量
棉花叶片用液氮磨碎后每100mg材料加1ml叶片提取液，叶片提取液成分：乙腈:水:磷酸＝80:20:0.1。浸泡1小时，离心，上清用0.22μm滤头过滤，进行HPLC检测。
HPLC分析采用Agilent 1200系统，Agilent ZORBAX Eclipse XDB-C18 analytical column(150mm×4.6mm，5μm)反向C18分析柱。质谱检测采用安捷伦6120四级杆检测器，API-ES离子源，正离子模式，碎裂电压70V。HPLC流动相：乙醇:甲醇:异丙醇:乙腈:水:乙酸乙酯:DMF:磷酸＝16.7:4.6:12.1:20.2:37.4:3.8:5.1:0.1。流动相流速为1mL/min，进样量为10μL，柱温40℃，检测时间50min。
经过HPLC的检测，发现在VIGS抑制SPG表达的株系中，棉酚、半棉酚酮及杀夜蛾素的含量均显著下降，结果如图一d-i所示。说明SPG参与了棉花体内棉酚的生物合成。
实施例2、SPG基因克隆及原核表达载体构建和蛋白表达纯化
a：棉花总RNA的提取和cDNA反转录制备
取100mg棉花叶片在液氮中迅速研磨成粉末，加入1mL在65℃预热的RNA提取试剂，立即涡旋剧烈震荡混匀，65℃放置30min。13,500g离心2min。将上清液转移至新的2mL管中，加入0.6mL氯仿，13,500g，离心5min，小心吸取上清至新的RNase-Free的离心管中。重复步骤3。上清转移至新的2mL管中，加入LiCl至终浓度为2M，-20℃放置3h。13,500g离心10min，倒掉废液，沉淀用70％的乙醇清洗两次，室温晾干后加入RNase-Free ddH 2O。
RNA提取试剂：0.2M Tris，50mM EDTA，1M NaCl，1％CTAB，1％β-巯基乙醇，pH 8.0。
b：PCR扩增目的基因SPG及原核表达载体构建
用高保真酶
Figure PCTCN2020108157-appb-000007
HS DNA Polymerase扩增SPG的全长cDNA片段(549bp)。PCR反应条件为：98℃，变性1分钟；98℃，变性10秒钟；57℃，复性10秒；72℃，延伸40秒钟；72℃，5分钟；4℃保温。将pET-32a载体用BamHI/SacI双酶切后，将SPG利用同源重组的方法连分别连接到pET-32a原核表达载体中。
c：原核表达
将测序正确的SPG的原核表达载体质粒转入BL21菌株，涂布于Amp抗性的LB培养基上，37℃，倒置培养过夜。在96孔板中加入Amp抗性的LB，挑取单克隆进行菌落PCR。PCR鉴定阳性的克隆加入到2mL抗性的液体LB(100μg/mL Ampr)中，37℃培养过夜。按照1:100的比例进行转接大摇，大摇体积可视情况而定，可定为100mL，37℃培养至OD600≈0.6(3h左右)。当OD600到达0.6左右时，加入IPTG到终浓度为0.5mM/L，转至18℃诱导蛋白表达，培养20h左右(培养箱需提前预冷)。吸取6mL过夜培养的菌液，13,500g离心5min，沉淀悬浮于3mL buffer[25mM Mopso，pH 7.0，5mM DTT，和10％甘油，5mM MgCl 2]中，利用one shot Cell Disrupter System进行破碎，破碎压力20～25kpsi。4℃，13,500g，离心，取上清进行SDS-PAGE电泳检测。
d：原核表达蛋白纯化
将100mL培养过夜的菌液分为2份，分装到2个50mL管中，13,500g离心5min，收集肠杆菌细胞，分别重悬于5mL的Lysis buffer中，利用高压破碎仪进行低温破碎，将上清转入8mL管中。4℃，13,500g离心20min。此时可以装Ni-NAT agrose的小 柱，一般100mL菌液可以使用1mL的Ni柱，用10mL的Lysis buffer清洗Ni柱。将步骤2中的上清，过装好1mL Ni-NAT agrose的小柱。使用10mL的Wash buffer清洗Ni-NATagrose。使用2mL洗脱缓冲液(Elution buffer)将SPG蛋白从镍柱中洗脱，使用Bradford法，以牛血清蛋白作参照，对SPG蛋白的浓度进行定量。
实施例3、SPG和同源乙二醛酶I的体外酶活实验和酶动力学分析
a：利用LC-MS检测SPG酶活体系的反应产物
SPG酶活体系：25mM HEPES，100μM ZnCl 2，5μg蛋白，100μM棉酚途径中间体底物，包括3-羟基-2-羰基-呋喃卡拉曼(3-hydro-furocalamen-2-one)和8,11-二羟基-7-羰基-杜松烯(8,11-dihydroxy-7-keto-(δ)-cadinene)。30℃反应两小时，利用乙酸乙酯进行萃取，抽干后溶于乙腈，过滤后进行HPLC-MS检测。当底物是乙酰基脱氧雪腐镰刀菌烯醇(3A-DON，详见实施例4)时，利用20μL甲醇将蛋白酶活体系中的蛋白去除，离心后取上清，过滤之后进行LC-MS分析。
以3-羟基-2-羰基-呋喃卡拉曼或3A-DON为底物时的检测条件：HPLC分析采用Agilent 1100系统，Agilent Eclipse XDB-C18 semi-preparative column(250mm×9.4mm，5μm)反向C18分析柱。流动相为乙腈(B)和甲酸溶液(A)，甲酸浓度为0.1％，流动相流速为1mL/min，进样量为10μl。梯度洗脱条件：0-3min，20-70％B；3-5min，70-80％B；5-7min，80-84％B；7-8min，84-100％B；8-10min，100-20％B。
以3-羟基-2-羰基-呋喃卡拉曼为底物进行SPG体外酶活反应，HPLC分析结果发现了两个新的产物峰，一个为脱氧半棉酚(图2b所示)，一个是半棉酚(图2c所示)。至此，本发明人解析了完整的棉酚生物合成途径，如图2a所示。
以8,11-二羟基-7-羰基-杜松烯为底物时的检测条件：HPLC分析采用Agilent 1100系统，Agilent Eclipse XDB-C18 semi-preparative column(250mm×9.4mm，5μm)反向C18分析柱。流动相为乙腈(B)和甲酸溶液(A)，甲酸浓度为0.1％，流动相流速为1mL/min，进样量为10μl。梯度洗脱条件：0-6min，20-70％B；6-10min，70-80％B；10-14min，80-84％B；14-16min，84-100％B；16-18.5min，100％B；18.5-19min，100-20％B；19-21min，20％B。体外酶活反应的产物检测峰如图2d所示，产物峰为呋喃卡拉曼。
进一步研究SPG和同源乙二醛酶I的体外酶活，发现多个物种包括棉花的乙二醛酶I GhGLXI和人的乙二醛酶HsGLO1都能以3-羟基-2-羰基-呋喃卡拉曼和8,11-二羟基-7-羰基-杜松烯为底物，催化其活性羰基醛结构的异构、脱水形成苯环的芳香化反应(图2b，c，d；表2)。通过酶动力学分析，本发明人发现SPG的催化能力远远高于GhGLXI。如图2e所示。SPG催化底物3-羟基-2-羰基-呋喃卡拉曼和8,11-二羟基-7- 羰基-杜松烯的反应机制如图3f和图3g所示。
表2、多个物种中乙二醛酶I的催化活性
Figure PCTCN2020108157-appb-000008
活性1：底物为3-羟基-2-羰基-呋喃卡拉曼；
活性2：底物为8,11-二羟基-7-羰基-杜松烯。
实施例4、SPG能够以3A-DON为底物进行催化反应降低其毒性
通过寻找底物类似物，本发明人发现禾谷镰孢菌产生的毒素脱氧雪腐镰刀菌醇(DON)及其酰基化衍生物具有类似的邻位羟基酮的结构，体外酶活实验表明SPG可以利用乙酰基脱氧雪腐镰刀菌烯醇(3A-DON)为底物进行催化反应，经过LC-MS分析，发现SPG催化3A-DON产生了2个产物，第一个产物iso-3A-DON的分子量和3A-DON相同，皆为338(图3a)，另一个分子量是304，结构未知(图3b)。进一步比较发现，其它物种如人和棉花中的GLXI并不能催化3A-DON的转化(图3a，b)，因而这个活性可能是SPG所独有的。
利用不同浓度的3A-DON和iso-3A-DON对小鼠成纤维细胞进行了毒性比较，表明iso-3A-DON毒性较3A-DON低(图3c)。由此，发现SPG可以利用3A-DON为底物进行催化反应，降低其毒性。SPG催化3A-DON的机制如图3e所示。
进一步以TMS为内标，在Bruker AVANCEIIITM 500核磁共振仪上检测了SPG酶活产物的结构。分析1H，13C NMR和2D NMR谱，分别对呋喃卡拉曼(化合物9)和iso-3A-DON(化合物16)的结构进行了鉴定。如图4和图5所示。
实施例5、通过序列比对及蛋白质模拟发现SPG的GSH结合位点发生改变
a：利用人乙二醛酶I蛋白GLO1的晶体结构进行模拟和底物docking实验
通过序列比对本发明人得知SPG与人乙二醛酶I(GLO1)的蛋白相似性为59％，说明SPG的序列是很保守的。本发明人将SPG与人乙二醛酶I的晶体结构进行比较， 发现与GSH结合相关的4个位点发生了改变。
根据序列比对结果，SPG蛋白69位的氨基酸变成了M，而对应的GhGLXI和其它物种的GLXI在此位置的氨基酸是F；SPG在101位置的氨基酸是F，而GhGLXI和其它物种的GLXI在此位置是N；SPG在121位置的氨基酸是I，而GhGLXI和其它物种的GLXI在此位置是R，另外SPG缺失了Loop13中4个氨基酸(其153～154位之间缺失GKMK)，GhGLXI在此处是GKMK。这些位点可能与GSH的binding密切相关，如图6b所示。本发明人根据这些位点的特征，对SPG和GhGLXI进行了组合突变，将SPG相应的点突变成GhGLXI对应的点，相反，把GhGLXI相应的点突变成SPG对应的点。如图6c中F119M代表将GhGLXI 119位的F变成M；再如图6d中SPG突变体M69F/F101N/I121R代表将SPG这三个位点的氨基酸置换成GhGLXI对应位置的氨基酸。
b：SPG和GhGLXI乙二醛酶活性测定
用水溶液配置100mM GSH和MG，将1420μL 0.1M磷酸氢二钠溶液(PH＝7.0)，40μL 100mM GSH和40μL 100mM MG混合，在室温孵育15min，使MG和GSH自发反应成加合物。用磷酸氢二钠缓冲液将SPG稀释至0.4ng/μL，将50μL和150μL加合物混匀，测定240nm处的吸光度变化。根据吸光度的变化，测定SPG的乙二醛酶I活力。1个单位(units/L)的乙二醛酶I指的是能够在25℃，pH 7.0的溶液中，每分钟生成1μM产物的蛋白量。经过测定，本发明人发现，陆地棉乙二醛酶I GhGLXI具有MG解毒活性，而其突变体不同程度地丧失了对MG的解毒能力，如图6c所示。相反，SPG丢失了对MG的解毒能力，但是将GSH结合位点进行置换后，某些突变体部分恢复了对MG的解毒能力，包括M69F/F101N/GKMK、F101N/I121R/GKMK、M69F/F101N/I121R/GKMK，如图6d所示。
c：陆地棉中乙二醛酶I GhGLXI和特化的乙二醛酶I SPG的染色体分布和亚细胞定位
通过分析，本发明人得知SPG在染色体呈现串联重复，A亚基因组中的Gh_A03G0247和Gh_A03G0248，D亚基因组中的Gh_D03G1320、Gh_D03G1321、Gh_D03G1322、Gh_D03G1323。其中Gh_D03G1323在各个组织中几乎不表达，猜测是一个假基因。陆地棉乙二醛酶I GhGLXI位于13号染色体上，分别是A13染色体的Gh_A13G2029和D13染色体的Gh_D13G2432，如图6e所示。拟南芥只有一个锌离子依赖的乙二醛酶I，具有可变剪切，而GhGLXI也具有可变剪切，如图7a，b所示。与GhGLXI相比，SPG都缺失了N端的质体定位的信号肽，如图6f，图7c，d所示，这与倍半萜类化合物的生物合成位于细胞质中也是相吻合的。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独 引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
主要参考文献
1.Tian，X.et al.Characterization of gossypol biosynthetic pathway.Proceedings of the National Academy of Sciences of the United States of America 115，E5410-e5418(2018).
2.Richard，J.P.Mechanism for the formation of methylglyoxal from triosephosphates.Biochemical Society transactions 21，549-553(1993).
3.Sousa Silva，M.，Gomes，R.A.，Ferreira，A.E.，Ponces Freire，A.&Cordeiro，C.The glyoxalase pathway:the first hundred years...and beyond.Biochem J 453，1-15，doi:10.1042/BJ20121743(2013).

Claims (17)

  1. 乙二醛酶SPG或其同源物的用途，用于催化具有邻位羟基酮结构的环状化合物发生芳香化反应、异构化反应、脱水反应和/或环氧化反应；或
    用于制备催化具有邻位羟基酮结构的环状化合物发生芳香化反应、异构化反应、脱水反应和/或环氧化反应的制剂。
  2. 如权利要求1所述的用途，其特征在于，所述的环状化合物化合物为单环化合物或多环化合物。
  3. 如权利要求1所述的用途，其特征在于，所述的邻位羟基酮结构包括：
    Figure PCTCN2020108157-appb-100001
    其中X为环结构。
  4. 权利要求1所述的用途，其特征在于，所述的芳香化反应或异构化反应包括：形成苯环、增加环结构中的不饱和键数量、改变环结构中的不饱和键的位置、或改变环结构或其上基团的光学构象或手性。
  5. 如权利要求1所述的用途，其特征在于，所述的具有邻位羟基酮结构的环状化合物包括：
    3-羟基-2-羰基-呋喃卡拉曼或其类似物，其经乙二醛酶SPG或其同源物催化发生芳香化反应和/或异构化反应，生成脱氧半棉酚或其类似物；
    8,11-二羟基-7-羰基-杜松烯或其类似物，其经乙二醛酶SPG或其同源物催化发生异构化反应、脱水反应和/或环氧化反应，生成呋喃卡拉曼或其类似物；或
    乙酰基脱氧雪腐镰刀菌烯醇或其类似物，其经乙二醛酶SPG催化发生异构化反应，生成异构化乙酰基脱氧雪腐镰刀菌烯醇或其类似物。
  6. 如权利要求1所述的用途，其特征在于，所述的3-羟基-2-羰基-呋喃卡拉曼和8,11-二羟基-7-羰基-杜松烯为参与棉酚生物合成的中间物质，从而，所述的乙二醛酶SPG或其同源物被用于棉酚生物合成中。
  7. 如权利要求1～6任一所述的用途，其特征在于，所述乙二醛酶SPG包括：(a)如SEQ ID NO:2所示氨基酸序列的多肽；或(b)将SEQ ID NO:2所示氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有(a)多肽功能的由(a)衍生的多肽；或(c)氨基酸序列与(a)限定的氨基酸序列有80％以上相同性且具有(a)多肽 功能的多肽；或(d)具有(a)多肽功能的SEQ ID NO:2的片段；较佳地，所述乙二醛酶SPG的编码基因的ID包括：Gh_A03G0247，Gh_A03G0248，Gh_D03G1320，Gh_D03G1321，Gh_D03G1322，Gorai.003G145000.1，Gorai.003G145100.1，Gorai.003G145200.1，Ga_01G2453，Ga_01G2453，GB_A03G0384，GB_A03G0385，GB_A03G0386，GB_D03G1602，GB_D03G1603，GB_D03G1604。
  8. 如权利要求1～6任一所述的用途，其特征在于，所述乙二醛酶SPG的同源物包括：乙二醛酶I，乙二醛酶GLO1；较佳地，所述的乙二醛酶I包括GhGLXI，CrGLXI，SmGLXI，OSGLXI，AtGLXI，DzGLXI，DmGLXI，DrGLXI；较佳地，所述的乙二醛酶GLO1包括HsGLO1。
  9. 乙二醛酶SPG的用途，用于降低或解除毒素脱氧雪腐镰刀菌醇的酰基化衍生物的毒性。
  10. 一种催化具有邻位羟基酮结构的环状化合物发生芳香化反应、异构化反应、脱水反应和/或环氧化反应的方法，包括：以乙二醛酶SPG或其同源物处理所述具有邻位羟基酮结构的环状化合物。
  11. 如权利要求10所述的方法，其特征在于，所述的环状化合物化合物为单环化合物或多环化合物；或
    所述的邻位羟基酮结构包括：
    Figure PCTCN2020108157-appb-100002
    其中X为环结构。
  12. 如权利要求10所述的方法，其特征在于，所述的芳香化反应或异构化反应包括：形成苯环、增加环结构中的不饱和键数量、改变环结构中的不饱和键的位置、或改变环结构或其上基团的光学构象或手性。
  13. 如权利要求10所述的方法，其特征在于，所述的具有邻位羟基酮结构的环状化合物包括：
    3-羟基-2-羰基-呋喃卡拉曼，其经催化发生芳香化反应和/或异构化反应，生成脱氧半棉酚；或
    8,11-二羟基-7-羰基-杜松烯，其经催化发生异构化反应、脱水反应和/或环氧化反应，生成呋喃卡拉曼；或
    乙酰基脱氧雪腐镰刀菌烯醇，其经催化发生异构化反应，生成异构化乙酰基脱氧 雪腐镰刀菌烯醇。
  14. 一种降低或解除脱氧雪腐镰刀菌醇或其酰基化衍生物的毒性的方法，包括以乙二醛酶SPG来处理。
  15. 一种提高乙二醛酶SPG在降低或解除甲基乙二醛毒性方面的能力的方法，包括对乙二醛酶SPG进行序列改造，使得其发生M69F/F101N突变、F101N/I121R突变或M69F/F101N/I121R，且其Loop13中增加4个氨基酸GKMK；较佳地，对应SEQ ID NO:2序列，在其153～154位之间插入GKMK。
  16. 一种乙二醛酶SPG突变体，其具有降低或解除甲基乙二醛(MG)毒性方面的能力，其对应SEQ ID NO:2序列，发生M69F/F101N突变、F101N/I121R突变或M69F/F101N/I121R，且其Loop13中增加4个氨基酸GKMK。
  17. 一种合成棉酚的方法，其特征在于，包括：
    (1)以法尼基二磷酸为底物，以杜松烯合成酶催化，获得(+)-δ-杜松烯；
    (2)将(1)的产物，以P450单加氧酶CYP706B1催化，获得7-羟基-(+)-δ-杜松烯；
    (3)将(2)的产物，以醇脱氢酶-1催化，获得7-羰基-δ-杜松烯；
    (4)将(3)的产物，以P450单加氧酶CYP82D113催化，获得8-羟基-7-羰基-δ-杜松烯；
    (5)将(4)的产物，以P450单加氧酶CYP71BE79催化，获得8,11-二羟基-7-羰基-杜松烯；
    (6)将(5)的产物，以乙二醛酶SPG进行催化，获得呋喃卡拉曼；
    (7)将(6)的产物，以P450单加氧酶CYP736A196催化，获得2-羟基-呋喃卡拉曼；
    (8)将(7)的产物，以醇脱氢酶-1催化，获得呋喃卡拉曼-2-酮；
    (9)将(8)的产物，以2-酮戊二酸/Fe(ii)-依赖的双加氧酶-1催化，获得3-羟基-2-羰基-呋喃卡拉曼；
    (10)将(9)的产物，以乙二醛酶SPG进行催化，获得脱氧半棉酚；
    (11)将(10)的产物，自发反应获得半棉酚；
    (12)将(11)的产物，以漆酶或过氧化物酶催化，获得棉酚。
PCT/CN2020/108157 2019-08-14 2020-08-10 乙二醛酶spg的功能及应用 WO2021027771A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910748506.3A CN112391416B (zh) 2019-08-14 2019-08-14 乙二醛酶spg的功能及应用
CN201910748506.3 2019-08-14

Publications (1)

Publication Number Publication Date
WO2021027771A1 true WO2021027771A1 (zh) 2021-02-18

Family

ID=74569267

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/108157 WO2021027771A1 (zh) 2019-08-14 2020-08-10 乙二醛酶spg的功能及应用

Country Status (2)

Country Link
CN (1) CN112391416B (zh)
WO (1) WO2021027771A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1059354A2 (en) * 1999-06-10 2000-12-13 Ceres Incorporated Sequence-determined DNA fragments and corresponding polypeptides encoded thereby
CN104818289A (zh) * 2015-05-20 2015-08-05 中国农业科学院油料作物研究所 耐高温甘蓝型油菜乙二醛酶基因和蛋白及其应用
CN108410905A (zh) * 2018-03-16 2018-08-17 广东溢达纺织有限公司 调节棉花的棉酚性状的基因以及调节方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001016334A2 (en) * 1999-09-01 2001-03-08 Incyte Genomics, Inc. Human hydrolytic enzymes
AU2001285370A1 (en) * 2000-08-31 2002-03-13 Millennium Pharmaceuticals, Inc. 50566, human glyoxalase ii related factor and uses thereof
ATE389014T1 (de) * 2001-01-31 2008-03-15 Vlaams Interuniv Inst Biotech Phosphorylierte glyoxalase i und deren verwendung
CN109251933B (zh) * 2017-07-13 2020-12-08 华中农业大学 一种与镰刀菌毒素及有毒醛类脱毒相关基因akr18a1及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1059354A2 (en) * 1999-06-10 2000-12-13 Ceres Incorporated Sequence-determined DNA fragments and corresponding polypeptides encoded thereby
CN104818289A (zh) * 2015-05-20 2015-08-05 中国农业科学院油料作物研究所 耐高温甘蓝型油菜乙二醛酶基因和蛋白及其应用
CN108410905A (zh) * 2018-03-16 2018-08-17 广东溢达纺织有限公司 调节棉花的棉酚性状的基因以及调节方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN GENPEPT; 12 September 2016 (2016-09-12), ANONYMOUS: "PREDICTED: lactoylglutathione lyase isoform X2 [Theobroma cacao]", XP055780428, Database accession no. XP_007016854 *
DATABASE PROTEIN GENPEPT; 22 May 2015 (2015-05-22), ANONYMOUS: "PREDICTED: lactoylglutathione lyase-like [Gossypium raimondii]", XP055780433, Database accession no. XP_012471607 *
HUANG JIN-QUAN; FANG XIN; TIAN XIU; CHEN PING; LIN JIA-LING; GUO XIAO-XIANG; LI JIAN-XU; FAN ZHEN; SONG WEI-MENG; CHEN FANG-YAN; A: "Aromatization of natural products by a specialized detoxification enzyme", NATURE CHEMICAL BIOLOGY, NATURE PUB. GROUP, NEW YORK, vol. 16, no. 3, 13 January 2020 (2020-01-13), New York, pages 250 - 256, XP037027534, ISSN: 1552-4450, DOI: 10.1038/s41589-019-0446-8 *
HUANG, JQ ET AL.: "RES transformation for biosynthesis and detoxification", SCIENCE CHINA-LIFE SCIENCES, vol. 63, no. 9, 8 June 2020 (2020-06-08), ISSN: 1674-733X *
LANDRO JAMES A., EDWARD J. BRUSH, JOHN W. KOZARICH: "Isomerization of (R)-and (S)-Glutathiolactaldehydes by Glyoxalase I: The Case for Dichotomous Stereochemical Behavior in a Single Active Sitet", BIOCHEMISTRY, vol. 31, no. 104, 1 January 1992 (1992-01-01), pages 6069 - 6077, XP055780436 *

Also Published As

Publication number Publication date
CN112391416B (zh) 2022-09-16
CN112391416A (zh) 2021-02-23

Similar Documents

Publication Publication Date Title
Barajas‐Lopez et al. Upstream kinases of plant Sn RK s are involved in salt stress tolerance
Catinot et al. Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana
US20150059018A1 (en) Methods and compositions for producing drimenol
US10370675B2 (en) Transgenic plants exhibiting enhanced phytochelatin-based heavy metal tolerance and methods of use thereof
US20170247713A1 (en) Trichome-specific transcription factor modulating terpene biosynthesis
Sobajima et al. Cloning and characterization of a jasmonic acid-responsive gene encoding 12-oxophytodienoic acid reductase in suspension-cultured rice cells
Yonamine et al. Overexpression of NtHAL3 genes confers increased levels of proline biosynthesis and the enhancement of salt tolerance in cultured tobacco cells
WO2008007031A1 (fr) Genes codant pour la cis-labda-12,14-dien-8 alpha-ol (cis-abienol) synthase et la syn-copalyl-8-ol diphosphate synthase et leurs utilisations
US11952582B2 (en) Herbicide-resistance gene and application thereof in plant breeding
US10550409B2 (en) Drimenol synthases III
WO2021027771A1 (zh) 乙二醛酶spg的功能及应用
US9926539B2 (en) Cytochrome P450 enzymes from sorghum bicolor
US7897840B2 (en) Salt responsive genes useful for generating salt resistant transgenic plants
CN115160422A (zh) 甘薯耐盐抗旱相关蛋白IbMYB44及其编码基因与应用
HU227706B1 (en) Methods and materials for making transgenic dicamba-degrading organisms
CN108410905B (zh) 调节棉花的棉酚性状的基因以及调节方法
CN110317823B (zh) 棉酚生物合成途径酶基因2-odd-1的功能鉴定和应用
US20150247161A1 (en) METHOD OF PRODUCING STRESS TOLERANT PLANTS OVER-EXPRESSING OsAlba1
EP3436573B1 (en) Nucleotide sequence encoding 9-lipoxygenase and recombinant constructs comprising the same
JP2000152791A (ja) ルマジンシンターゼおよびリボフラビンシンターゼ
Lokdarshi Calmodulin-like protein 38: a component of ribonucleoprotein particles during hypoxic stress responses in arabidopsis
Dreff-Kerwin The HOTHEAD Protein: Assessing Enzymatic Activity using Computational and Recombinant Protein Expression Approaches
CN116410278A (zh) Mekk1-mkk1/2-mpk4级联对抗stop1蛋白的磷酸化和稳定作用
CN117230030A (zh) 一种生物全合成紫杉醇前体物质巴卡亭iii的方法、生物材料及应用
WO2000000585A9 (en) MAIZE CYTOCHROME P450 MONOOXYGENASE cDNA (CYP71C3v2)

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: 20853140

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: 20853140

Country of ref document: EP

Kind code of ref document: A1