CN116789528A - Bridged sesquiterpenoids, and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a bridged ring sesquiterpene compound, a preparation method and application thereof, belongs to the field of genetic engineering, and is named as Biposopenenoid A, biposopenenoid B, biposopenenoid C, biposopenenoid D, biposopenenoid E, biposopenenoid F, biposopenenoid G, biposopenenoid H, biposopenenoid I, biposopenenoid J and Biposopenenoid K respectively, and provides specific structures. The synthetic gene cluster of the bridgering sesquiterpenoids is derived from the helminth fungus 11134 (Bipolaris sorokiniana 11134), and the gene cluster contains 2 genes, namely a gene BsstB or a functional equivalent thereof for encoding sesquiterpene synthases and a gene BsstA or a functional equivalent thereof for encoding cytochrome P450 enzyme BsstA. Wherein the nucleotide sequence of BstA is shown as SEQ ID NO.1, and the nucleotide sequence of BstB is shown as SEQ ID NO. 2. The biposolophenoids gene cluster discovered by the invention catalyzes and generates new bridged ring sesquiterpenoids, and provides valuable compound resources for enriching the structural diversity of natural products and discovering drug lead molecules.
Description
Technical Field
The invention relates to the technical field of preparation of sesquiterpenoids, in particular to a bridged ring sesquiterpenoid, a preparation method and application thereof.
Background
Fungal natural products are an important source of drug development, receiving widespread attention in their abundant types, complex structures and good activity. With the development of gene sequencing technology and the reduction of cost, public databases are recording more and more genome information, but the acquisition difficulty of novel natural products is gradually increased, and how to acquire active natural products with novel structures is always a key problem of concern in the research field. Terpenes are a general term for all isoprene polymers and derivatives thereof, sesquiterpenes are one of the most widely studied species, and various compounds have activity, such as the antimalarial agents paclitaxel, phytohormone abscisic acid, etc., and are widely used in clinical, perfumery and other fine chemicals. In recent years, genome mining technology is used as a guide, potential biosynthesis gene clusters are screened from abundant genome resources, and a plurality of terpene compounds with activity are successfully separated by means of a gene activation means of heterologous expression. Through a synthetic biological strategy, metabolic pathways are remodeled in microorganisms, so that not only can the structural diversity of natural products be widened, but also a foundation is laid for researching more terpenoid drug lead molecules.
Biposopropenoids are a class of sesquiterpenoids with potential agricultural application value, which contain two framework types: the derivative of the sativene and the derivative of the longifolene. It is reported that some of the derivatives of the savine class have phytotoxic activity and growth-promoting activity, while longifolene itself has anti-inflammatory, antibacterial and antioxidant properties, and is widely used in the perfume industry due to its unique woody fragrance. To date, nearly 60 biposolopenoids have been found, and have received extensive attention due to the rare bridged ring structure and wide activity, and have better research value.
Therefore, there is a need to develop more biposolopenoids with novel structures and corresponding preparation methods.
Disclosure of Invention
Based on the demand of developing more Biposoropenoids compounds with novel structures and corresponding preparation methods in the prior art, the invention provides various bridged ring sesquiterpenoids compounds, and a preparation method and application thereof.
The invention discovers the biosynthesis gene cluster for producing the bridged sesquiterpenes compound for the first time, and the Biposoropenoids compound with more active frameworks is efficiently obtained through the heterologous system expression of aspergillus oryzae.
The aim of the invention can be achieved by the following technical scheme:
it is a first object of the present invention to provide a class of bridged sesquiterpenoids Biposoropenoids A-K.
It is a second object of the present invention to provide a biosynthetic gene cluster for the synthesis of a variety of bridged sesquiterpenoids Biposoropenoids A-K.
The third purpose of the invention is to provide a preparation method of a bridged sesquiterpene compound Biposoropenoids A-K.
The fourth object of the invention is to provide application of a bridged sesquiterpene compound Biposoropenoids A-K.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a rare bridged sesquiterpene compound which is named as BiPosoropenoid A, biPosoropenoid B, biPosoropenoid C, biPosoropenoid D, biPosoropenoid E, biPosoropenoid F, biPosoropenoid G, biPosoropenoid H, biPosoropenoid I, biPosoropenoid J and BiPosoropenoid K, and the structural formula is shown as follows:
in a second aspect, the present invention provides a biosynthetic gene cluster for synthesizing a plurality of bridged sesquiterpenoids Biposoropenoids A-K, said gene cluster comprising 2 genes, bsstA or a functional equivalent thereof, encoding a cytochrome P450 enzyme BsstA, and BsstA or a functional equivalent thereof encoding a sesquiterpene synthase BsstA, respectively, wherein the nucleotide sequence of BsstA is shown in SEQ ID No.1 and the nucleotide sequence of BsstA is shown in SEQ ID No. 2.
In one embodiment of the invention, the functional equivalent of gene BsstA means that the gene nucleotide sequence is a DNA coding sequence corresponding to greater than 80% identity to the amino acid sequence encoding cytochrome P450 enzyme BsstA.
In one embodiment of the invention, the functional equivalent of gene BsstB means that the nucleotide sequence of the gene is a DNA coding sequence corresponding to greater than 80% identity to the amino acid sequence encoding the sesquiterpene synthase BsstB.
The third aspect of the invention provides a preparation method of the bridged sesquiterpenes Biposoropenoids A-K, wherein Biposoropenoids A-K is obtained by expressing genes in a biosynthesis gene cluster for synthesizing various bridged sesquiterpenes Biposoropenoids A-K in the vermiculella umbilicifolia by using an Aspergillus oryzae Aspergillus oryzae NSAR1 heterologous expression method.
In one embodiment of the invention, the preparation method of the bridged sesquiterpene compound Biposoropenoids A-K comprises the following steps:
(1) Taking the genome of B.sorokiniana 11134 as a template, and respectively carrying out PCR amplification on cytochrome P450 enzyme genes bsstA and sesquiterpene synthase genes bstB by using primers bstA-F/bstA-R and bstB-F/bstB-R to obtain PCR products of the genes bstA and bstB; then constructing a coexpression vector pUARA 4-bstA of bstA and a coexpression vector pUARA 4-bstAB of bstB by taking an Aspergillus oryzae A.oryzae expression vector pUARA4 as a vector, wherein the nucleotide sequences of the primers are respectively as follows:
bsstA-F：AAGCTCCGAATTCGAGCTCGATGGGACATTCTGCCAAAGAC
bsstA-R：GAGCTACTACAGATCCCCGGCTAGCCTGCAAACACTTCCT
bsstB-F：CCCCACAGCAAGCTCCGTTAATGGAGATCCTCAACAACAAAAC
bsstB-R：GTGCATATGATTTAAATTTATCATTTAGTAGCCAACCGGG；
(2) Under the mediation of PEG solvent, the expression vector pUARA 4-bstA or the co-expression vector pUARA 4-bstAB is transformed into protoplast of high-yield host Aspergillus oryzae A.oryzae NSAR1 (niaD-, sC-, delta argB, adeA-) which is easy to express terpene synthase genes to obtain Aspergillus oryzae transformant AO-bstA or AO-bstAB which can produce Biposoropenoids A-K;
(3) Inoculating mycelia of Aspergillus oryzae transformant AO-bsstAB into MPY culture medium to culture as seed solution, inoculating seed solution into MPY fermentation culture medium, and fermenting to obtain fermentation broth containing AO-bsstAB of Biposoropenoids A-K.
The strain B.sorokiniana 11134, also called as the wheat root rot helminth fungus (Bipolaris sorokiniana) BS11134, is a mature strain which has been preserved in China general microbiological culture Collection center (CGMCC, address: north Chen West Lu No.1, institute of microbiology, university of China, code 100101) at 30 th month 04 in 2019, and has a preservation number of CGMCC No.17767, and is described in patent CN110343618B, CN115404229A, CN 110272345B.
Among them, the Aspergillus oryzae A.oryzae expression vector pUARA4 is a known biological material, pUARA2 is disclosed in the article "Norditerpenoids biosynthesized by variediene synthase-associated P450 machinery along with modifications by the host cell Aspergillus oryzae", pUARA2 is two restriction sites, pUAR4 means that modification is made on this plasmid, 4 is modified from two restriction sites, and the obtaining of the Aspergillus oryzae A.oryzae expression vector pUARA4 is a routine technical means for those skilled in the art.
Among them, aspergillus oryzae NSAR1 is known, which is a commonly used biological material, disclosed in the article "Norditerpenoids biosynthesized by variediene synthase-associated P450 machinery along with modifications by the host cell Aspergillus oryzae", niaD-, sC-, ΔargB, adeA-respectively represents: nitrate reductase auxotrophs; methionine auxotroph; arginine auxotroph; adenine auxotroph; the four defective gene screening markers in Aspergillus oryzae are shown.
In one embodiment of the invention, the MPY medium used to obtain the seed solution contains 0.01% adenine and 0.15% methionine.
In one embodiment of the present invention, when the seed liquid is obtained, the conditions for the culture are: the cells were incubated at 30℃and 220rpm for 2 days.
In one embodiment of the present invention, the seed solution was inoculated in an amount of 1.5% to the MPY fermentation medium, followed by inoculation in the MPY fermentation medium, and cultivation was performed at 30℃and 220rpm for 5 days to produce Biposoropenoids A-K.
In one embodiment of the present invention, the method for obtaining Biposoropenoids A-K by separating a fermentation broth containing AO-bsstAB of Biposoropenoids A-K comprises:
Adding the fermentation liquor of AO-bstAB into ethyl acetate with the same volume for extraction for 3 times, and evaporating to dryness to obtain a crude extract; crude separation is carried out on the crude extract by a decompression reversed phase chromatographic silica gel column, methanol and water are used as mobile phases for elution and target components Fr.4, fr.5 and Fr.6 are enriched, the three fractions are combined and separated by an LH-20 gel chromatographic column, and finally, the Biposopropenoid J is obtained from the Fr.456.14 gel component by eluting with an ACE 5C18-PFP (250 mm multiplied by 10 mm) chromatographic column and acetonitrile/0.1 percent formic acid water volume ratio of 40:60 as mobile phases; the crude fermentation extract was separated by a reduced pressure reverse phase silica gel column with the target components concentrated in fr.5, fr.6, fr.7 and fr.8.Fr.5 was further gel separated to obtain fr.5.6, fr.5.7, fr.5.8 and fr.5.9, fr.5.8, fr.5.9 and fr.6 were combined and then gel column separated, from fr.56. (8-12) further chiral column was used with CHIRALPAK IC (250 mm x 10 mm) at n-hexane/ethanol volume ratio 96:4 obtaining Biposopropenoid G and Biposopropenoid D for mobile phase; fr.7, performing pressurized normal phase silica gel column separation, wherein Fr.7.1 utilizes an ACE 5C18-PFP chromatographic column, acetonitrile/0.1% formic acid water volume ratio of 45:55 is used as a mobile phase to obtain biposopenoid H and biposopenoid I, fr.7.2 utilizes an ACE 5C18-PFP chromatographic column, acetonitrile/0.1% formic acid water volume ratio of 40:60 is used as a mobile phase to obtain biposopenoid F, and Fr.7.4 utilizes an ACE 5C18-PFP chromatographic column, acetonitrile/0.1% formic acid water volume ratio of 35:65 is used as a mobile phase to obtain biposopenoid E and biposopenoid K, fr.5.7 is subjected to crude separation under the conditions of 0-35min,23-40% ACN and 5C18-PFP, and then further uses Chiralpak IA to obtain a total of 96:4 is a mobile phase to obtain biposopenoid A, fr.8 is subjected to gel separation by LH-20, fr.8 (19-21) is separated by ACE 5C18-PFP under the condition of 70:30 flow equality elution of methanol/0.1% formic acid water volume ratio to obtain biposopenoid B and biposopenoid C.
In a third aspect of the invention there is provided a method of preparing said bridged sesquiterpenes Biposoropenoids A-K based on biological methods. In addition, the bridged ring sesquiterpenoids Biposoropenoids A-K can also be prepared by chemical synthesis based on the structure of the bridged ring sesquiterpenoids Biposoropenoids A-K.
In a fourth aspect of the present invention, there is further provided an engineering bacterium which is obtained by transforming an expression vector pUARA4-bsstA or a co-expression vector pUARA4-bsstAB into a strain to produce Biposoropenoids A-K. The strain selects Aspergillus oryzae A.oryzae NSAR1; the expression vector pUARA 4-bstA or the co-expression vector pUARA 4-bstAB is a bstA expression vector pUARA 4-bstA or a bstA 4-bstAB co-expression vector constructed by taking the Aspergillus oryzae A.oryzae expression vector pUARA4 as a vector.
In one embodiment of the present invention, the engineering bacteria are engineering bacteria capable of producing Biposoropenoids A-K, which are obtained by transforming protoplasts of an expression vector pUARA 4-bstA or a co-expression vector pUARA 4-bstAB into a high-yield host Aspergillus oryzae A.oryzae NSAR1 (niaD-, sC-, ΔargB, adeA-), and are also called Aspergillus oryzae transformant AO-bstA or AO-bstAB.
In a fifth aspect the invention provides the use of said bridged sesquiterpenoids Biposoropenoids A-K, any one of said bridged sesquiterpenoids Biposoropenoids A-K for the biosynthesis of sesquiterpenes.
Compared with the prior art, the invention has the advantages and beneficial effects that:
the invention provides rare bridged ring sesquiterpenoids, and a preparation method and application thereof. The present invention discovers a novel Biposoropenoids A-K biosynthetic gene cluster from Helminthosporum umbilicus (Bipolaris sorokiniana 11134) by means of genome mining strategies. The expression of the genes in the Biposoropenoids A-K biosynthesis gene cluster in Aspergillus oryzae is realized by means of heterologous expression. Finally, 11 brand-new bridged sesquiterpenoids, namely Biposoropenoids A-K, are obtained through separation and purification.
The invention constructs the self-sufficient engineering bacteria for producing the novel compound Biposoropenoids A-K by utilizing a genetic engineering method. The whole operation process is simple, the process is mature, the cost is low, no harmful impurities are contained, the method is nontoxic, and the method is very friendly to the environment. The obtained product Biposoropenoids A-K provides a new resource for the biosynthesis of sesquiterpene compounds, provides a choice for the types of the compounds, and provides valuable lead compound resources for enriching a natural product compound library and discovering new antibiotics.
Drawings
FIG. 1 is an ultraviolet absorption spectrum of the compound Biposoropenoids A-K of the present invention.
FIG. 2 is a HR-ESI-MS spectrum of the compound Biposopropenoid A of the present invention.
FIG. 3 is a HR-ESI-MS spectrum of the compound Biposopropenoid B of the present invention.
FIG. 4 is a HR-ESI-MS spectrum of the compound Biposopropenoid C of the present invention.
FIG. 5 is a HR-ESI-MS spectrum of the compound Biposopropenoid D of the present invention.
FIG. 6 is a HR-ESI-MS spectrum of the compound Biposopropenoid E of the present invention.
FIG. 7 is a HR-ESI-MS spectrum of the compound Biposopropenoid F of the present invention.
FIG. 8 is a HR-ESI-MS spectrum of the compound Biposopropenoid G of the present invention.
FIG. 9 is a HR-ESI-MS spectrum of the compound Biposopropenoid H of the present invention.
FIG. 10 is a HR-ESI-MS spectrum of the compound of the present invention Biposopropenoid I.
FIG. 11 is a HR-ESI-MS spectrum of the compound of the present invention Biposopropenoid J.
FIG. 12 is a HR-ESI-MS spectrum of the compound Biposopropenoid K of the present invention.
FIG. 13 shows the dissolution of the compound of the invention Biposopropenoid A in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 14 shows the dissolution of the compound of the invention, biposopropenoid B, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 15 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 16 shows the dissolution of the compound of the invention, biposopropenoid D, in pyridine-D 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 17 shows the dissolution of the compound of the invention Biposopropenoid E in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 18 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 19 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 20 shows the dissolution of the compound of the invention Biposopropenoid H in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 21 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 22 shows the dissolution of the compound of the invention, biposopropenoid J, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 23 shows the dissolution of the compound of the invention, biposopropenoid K, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum.
FIG. 24 shows the dissolution of the compound of the invention Biposopropenoid A in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 25 shows the dissolution of the compound of the invention, biposopropenoid B, in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 26 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 27 shows the dissolution of the compound of the invention Biposopropenoid D in pyridine-D 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 28 shows the dissolution of the compound of the invention, biposopropenoid E, in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 29 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 30 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 31 shows the dissolution of the compound of the invention Biposopropenoid H in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 32 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 33 shows the dissolution of the compound of the invention Biposopropenoid J in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 34 shows the dissolution of the compound of the invention Biposopropenoid K in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum.
FIG. 35 shows the dissolution of the compound of the invention Biposopropenoid A in pyridine-d 5 The HSQC spectrum of (C).
FIG. 36 shows the dissolution of the compound of the invention, biposopropenoid B, in pyridine-d 5 The HSQC spectrum of (C).
FIG. 37 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 The HSQC spectrum of (C).
FIG. 38 shows the dissolution of the compound of the invention, biposopropenoid D, in pyridine-D 5 The HSQC spectrum of (C).
FIG. 39 shows the dissolution of the compound of the invention, biposopropenoid E, in pyridine-d 5 The HSQC spectrum of (C).
FIG. 40 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 The HSQC spectrum of (C).
FIG. 41 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 The HSQC spectrum of (C).
FIG. 42 shows the dissolution of the compound of the invention Biposopropenoid H in pyridine-d 5 The HSQC spectrum of (C).
FIG. 43 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 The HSQC spectrum of (C).
FIG. 44 shows the dissolution of the compound of the invention Biposopropenoid J in pyridine-d 5 The HSQC spectrum of (C).
FIG. 45 shows the dissolution of the compound of the invention Biposopropenoid K in pyridine-d 5 The HSQC spectrum of (C).
FIG. 46 shows the dissolution of the compound of the invention, biposopropenoid A, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 47 shows the dissolution of the compound of the invention, biposopropenoid B, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 48 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 49 shows the dissolution of the compound of the invention, biposopropenoid D, in pyridine-D 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 50 shows the dissolution of the compound of the invention, biposopropenoid E, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 51 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 52 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 53 shows the dissolution of the compound of the invention, biposopropenoid H, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 54 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 55 shows the dissolution of the compound of the invention, biposopropenoid J, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 56 shows the dissolution of the compound of the invention, biposopropenoid K, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 57 shows the dissolution of the compound of the invention, biposopropenoid A, in pyridine-d 5 HMBC spectra of (a).
FIG. 58 shows the dissolution of the compound of the invention, biposopropenoid B, in pyridine-d 5 HMBC spectra of (a).
FIG. 59 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 HMBC spectra of (a).
FIG. 60 shows the dissolution of the compound of the invention Biposopropenoid D in pyridine-D 5 HMBC spectra of (a).
FIG. 61 shows the dissolution of the compound of the invention, biposopropenoid E, in pyridine-d 5 HMBC spectra of (a).
FIG. 62 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 HMBC spectra of (a).
FIG. 63 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 HMBC spectra of (a).
FIG. 64 shows the dissolution of the compound of the invention Biposopropenoid H in pyridine-d 5 HMBC spectra of (a).
FIG. 65 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 HMBC spectra of (a).
FIG. 66 shows the dissolution of the compound of the invention, biposopropenoid J, in pyridine-d 5 HMBC spectra of (a).
FIG. 67 shows the dissolution of the compound of the invention Biposopropenoid K in pyridine-d 5 HMBC spectra of (a).
FIG. 68 shows the solubility of Biposopropenoid A in pyridine-d 5 NOESY spectrum of (B).
FIG. 69 shows the solubility of Biposopropenoid B in pyridine-d 5 NOESY spectrum of (B).
FIG. 70 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 NOESY spectrum of (B).
FIG. 71 shows the dissolution of the compound of the invention, biposopropenoid D, in pyridine-D 5 NOESY spectrum of (C)A drawing.
FIG. 72 shows the dissolution of the compound of the invention, biposopropenoid E, in pyridine-d 5 NOESY spectrum of (B).
FIG. 73 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 NOESY spectrum of (B).
FIG. 74 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 NOESY spectrum of (B).
FIG. 75 shows the dissolution of the compound of the invention Biposopropenoid H in pyridine-d 5 NOESY spectrum of (B).
FIG. 76 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 NOESY spectrum of (B).
FIG. 77 shows the dissolution of the compound of the invention, biposopropenoid J, in pyridine-d 5 NOESY spectrum of (B).
FIG. 78 shows the dissolution of the compound of the invention, biposopropenoid K, in pyridine-d 5 NOESY spectrum of (B).
FIG. 79 is an X-ray single crystal diffraction pattern of the compound of the invention Biposopropenoid G.
FIG. 80 is an X-ray single crystal diffraction pattern of the compound Biposopropenoid I of the present invention.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The following examples are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and any simple modification, equivalent changes and modification made to the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
The basic molecular biology experimental techniques such as PCR amplification, plasmid extraction, transformation, etc., used in the examples of the present invention are usually carried out according to conventional methods unless otherwise specified, and can be specifically carried out according to the instructions provided by the relevant manufacturers (third edition of the guidelines for molecular cloning experiments, sambrook J, russell DW, janssen K, argentine J. Huang Peitang, et al, 2002, beijing: scientific Press).
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The synthetic gene cluster of the bridged sesquiterpenes compound Biposopropenoids is cloned from the helminth fungus 11134, and contains 2 genes, namely a gene BstA or a functional equivalent thereof for encoding cytochrome P450 enzyme BstA and a gene BstB or a functional equivalent thereof for encoding sesquiterpene synthase. Wherein the nucleotide sequence of BstA is shown as SEQ ID NO.1, and the nucleotide sequence of BstB is shown as SEQ ID NO. 2. Or the nucleotide sequence of the gene is a DNA coding sequence corresponding to the amino acid sequence of the coded proteins bsstA and bstB with the identity of more than 80 percent respectively.
Example 1
Heterologous expression of a synthetic gene of the bridged sesquiterpenes Biposoropenoids and structural identification of sesquiterpene skeleton compounds.
By utilizing a heterologous expression method, a Biposoropenoids A-K biosynthesis gene cluster in the strain body of the helminth fungus 11134 is transferred into a host Aspergillus oryzae by constructing an expression plasmid, and the production condition of a heterologous expression strain product is detected. The medium formulations used in this example are shown in Table 1.
Table 1 Medium and reagent formulations used in the examples
Construction of 1.Biposoropenoids A-K Gene cluster heterologous expression vector
(1) PCR amplification was performed on the cytochrome P450 enzyme gene bstA, the sesquiterpene synthase gene bstB with the genome of B.sorokiniana 11134 as a template and with the primers bstA-F/bstA-R, bstB-F/bstB-R, respectively.
(2) After the PCR product of the gene bstB is purified by a nucleic acid purification kit, the bstB is integrated into a linear vector pUARA4 digested by KpnI enzyme digestion by using an Ezmax recombination kit, and the ligation product is transformed into escherichia coli DH10B, and positive transformants are screened by ampicillin. Liquid culturing positive transformant, extracting plasmid PCR verification, obtaining pUARA 4-bstB plasmid; on this basis, the bsstA was integrated into the PacI digested linear vector pUARA4-bsstB using Ezmax recombination kit, the ligation product was transformed into E.coli DH10B, and positive transformants were selected by ampicillin. The positive transformants were liquid cultured, and the plasmid was extracted and verified by PCR to obtain pUARA 4-bstAB plasmid.
Table 2 primer sequences used in the examples
2. Transformation of protoplasts
(1) Aspergillus oryzae A.oryzae NSAR1 was plated on PDA plates containing 0.01% Ade and incubated for 7d at 30 ℃.
(2) Spores were collected in 10mL of 0.1% Tween-80 (1 plate of spores were typically required to be collected) and counted using a hemocytometer. Inoculation of 1mL spore solution (about 1X 10) 7 The spores) were cultured in 50mL of DPY medium at 30℃and 220rpm for 2-3d.
(3) 100mg of Yatalase (an enzyme that lyses cells) was weighed, and 30mL solution 0,solution was sterilized by filtration through a 0.22 μm filter in advance, and added to a 50ml centrifuge tube.
(4) And collecting the bacterial cells. Pouring 50ml of cultured fungus balls into a P250 glass filter, removing the culture medium, cleaning for 3-5 times by using 0.8M NaCl, squeezing out water by using a sterilizing medicine spoon, and then adding the squeezed fungus balls into Yatalase lyase liquid. Shake culturing at 30deg.C and 200rpm for 2-3 hr until the spherical mycelium disappears to make clear the dirt.
(5) The digested bacterial liquid was filtered through a Miracloth filter cloth, and protoplasts were collected and transferred to a new 50ml centrifuge tube and centrifuged at 4℃for 800g and 5 min.
(6) The supernatant was removed, washed by adding 20ml,0.8M NaCl, resuspended and centrifuged (washed twice) at 4℃under 800g for 5 min. The supernatant was removed and 10ml of 0.8M NaCl was added. The number of protoplasts was counted under a microscope with a bacterial counter. Number of protoplasts = total count/80 x 400ml x 10 4 X dilution factor。
(7) The protoplast concentration was adjusted to 2X 10 8 cell/ml. (solution 2/solution 3=4/1, volume ratio, the viability of protoplast is maintained well under the condition of the ratio), and 0.5ml-2ml protoplast can be harvested according to the growth condition of the bacteria.
(8) 200. Mu.l of the protoplast solution was transferred to a new 50ml centrifuge tube, and 10. Mu.g of the expression plasmids pUARA 4-bstB and pUARA 4-bstAB were added, respectively, and gently mixed. The mixture was allowed to stand on ice for 20 minutes, during which time the sterilized Top agar (soft agar medium, formulation and screening solid medium in Table 1 were substantially identical except for the amount of agar; soft agar in Top agar: 0.5%) was incubated in a water bath at 60 ℃.
(9) To the suspension of (8), 1ml of solution 3 was added, and the mixture was gently mixed with a gun head. Standing at room temperature for 20min. 10ml of solution 2 was added and gently mixed.
(10) Centrifugation at 4 ℃,800g,10min, removal of supernatant, addition of 1ml of solution 2, gentle suspension with a pipette, addition of 200 μl to the center of the solid screening medium (x 3 plate). 5ml of top agar incubated at 60℃was rapidly added around the dish and mixed rapidly. After the surface of the plate was sufficiently dried, it was wrapped with Parafilm, covered downward, and incubated at 30℃for 3-7 days.
(11) 2-3 clones were picked per plate, 8 total. And extracting gDNA from the grown transformant, and carrying out PCR verification, wherein the positive transformant is the Biposoropenoids A-K gene cluster heterologous expression strain AO-bstAB.
That is, in the present application, the recombinant product is first amplified in DH10B, the plasmid is then extracted, and the recombinant plasmid is then PEG/CaCl treated 2 Mediating transformation of Aspergillus oryzae protoplasts.
3. Isolation and purification of the expression product of the heterologous expression Strain AO-bsstAB
Inoculating the heterologous expression strain AO-bsstAB mycelium was inoculated in MPY medium containing 0.01% adenine and 0.15% methionine, cultured at 30℃and 220rpm for 2 days as seed liquid, and then inoculated in 1.5% inoculum size in a plurality of 1L MPY fermentation media, cultured at 30℃and 220rpm for 5 days.
Adding the fermentation liquor of AO-bsstAB into ethyl acetate with the same volume for extraction for 3 times, and evaporating to dryness to obtain a crude extract. The crude extract was subjected to crude separation using a reduced pressure reverse phase chromatography silica gel column, eluting with methanol and water as mobile phases and enriching the target components fr.4, fr.5 and fr.6, combining the three fractions and subjecting to LH-20 gel chromatography column separation, and finally obtaining biposopenoid J from the fr.456.14 gel components using an ACE 5C18-PFP (250 mm×10 mm) column, eluting with acetonitrile/0.1% formic acid water at a volume ratio of 40:60 as mobile phase. The crude fermentation extract was separated by a reduced pressure reverse phase silica gel column with the target components concentrated in fr.5, fr.6, fr.7 and fr.8.Fr.5 is further subjected to gel separation to obtain Fr.5.6, fr.5.7, fr.5.8 and Fr.5.9 (the preparation of the compound is carried out in a segmentation way, fr refers to the sequence of different fractions, fr.6 refers to the separation of the silica gel component of the fifth part by a gel column, and the target component is in the sixth fraction, so that the expression modes of Fr.5.6 and other Fr.5.7, fr.5.8 and the like are similar). Fr.5.8, fr.5.9 and Fr.6 were combined and subjected to gel column separation, further using CHIRALPAK IC (250 mm. Times.10 mm) chiral column from Fr.56. (8-12) at a volume ratio of n-hexane/ethanol of 96:4 obtaining Biposopropenoid G and Biposopropenoid D for mobile phase. Fr.7, performing pressure normal phase silica gel column separation, wherein Fr.7.1 uses an ACE 5C18-PFP chromatographic column, and acetonitrile/0.1% formic acid water volume ratio of 45:55 is used as mobile phase elution to obtain biposolopenid H and biposolopenid I. Fr.7.2 Biposopropenoid F was obtained by eluting with ACE 5C18-PFP column with a mobile phase of acetonitrile/0.1% formic acid water volume ratio 40:60. And eluting the mobile phase by using an ACE 5C18-PFP chromatographic column in Fr.7.4, wherein the volume ratio of acetonitrile/0.1% formic acid is 35:65 to obtain the Biposopropenoid E and the Biposopropenoid K. Fr.5.7 crude separation at 0-35min,23-40% ACN, ACE 5C18-PFP followed by further Chiralpak IA with n-hexane/isopropanol volume ratio 96:4 is the mobile phase to obtain biposopenoid A. Fr.8 gel separation with LH-20, fr.8. (19-21) separation with ACE 5C18-PFP under methanol/0.1% formic acid aqueous 70:30 flow equality elution to give biposolopenid B and biposolopenid C.
NMR measurement of the separated bridged sesquiterpenes was performed using a Bruker 600MHz NMR spectrometer 1 H 600MHz； 13 C150 MHz), the solvent being deuterated pyridine。
4. Identification of bridged sesquiterpenoids Biposoropenoids A-K
Identifying the bridged sesquiterpenes Biposoropenoids A-K obtained above:
(1) Appearance: biposopropenoid B, biposopropenoid C, biposopropenoid E, biposopropenoid F, biposopropenoid H, biposopropenoid J, biposopropenoid K are colorless oils; biposoropenoid G and Biposoropenoid I are needle-shaped crystals; biposoropenoid D and Biposoropenoid e are white solids;
(2) Solubility: is easily dissolved in methanol and acetonitrile and is difficult to dissolve in water.
(3) Ultraviolet spectrum: the ultraviolet spectrum of the compound Biposoropenoids A-K methanol solution has a maximum absorption peak at about 210nm, the ultraviolet spectrum is shown in figure 1, and the figure 1 is the ultraviolet spectrum of the compound Biposoropenoids A-K. The ultraviolet spectrum test instrument is SPD-M40 PHOTO DIODE ARRAY DETECTOR.
(4) Mass spectrometry: FIG. 2 is a HR-ESI-MS spectrum of the compound Biposopropenoid A of the present invention showing [ M+H ]] + The peak was m/z 255.1964, suggesting that its most probable molecular formula was C 15 H 26 O 3 . FIG. 3 is a HR-ESI-MS spectrum of the compound Biposopropenoid B of the present invention showing [ M+H ] ] + The peak was m/z 281.2104, suggesting that its most probable molecular formula was C 17 H 28 O 3 . FIG. 4 is a HR-ESI-MS spectrum of the compound Biposopropenoid C of the present invention, showing [ M+H ]] + The peak was m/z 281.2106, suggesting that its most probable molecular formula was C 17 H 28 O 3 . FIG. 5 is a HR-ESI-MS spectrum of the compound Biposopropenoid D of the present invention, showing [ M-H ] 2 O+H] + The peak was m/z 219.1742, suggesting that its most probable molecular formula was C 15 H 24 O 2 . FIG. 6 is a HR-ESI-MS spectrum of the compound Biposopropenoid E of the present invention showing [ M+H ]] + The peak was m/z 237.1849, suggesting that its most probable molecular formula was C 15 H 24 O 2 . FIG. 7 is a HR-ESI-MS spectrum of the compound Biposopropenoid F of the present invention showing [ M-H ] 2 O+H] + The peak was m/z 219.1752, suggesting that its most probable molecular formula was C 15 H 24 O 2 . FIG. 8 is a HR-ESI-MS spectrum of the compound Biposopropenoid G of the present invention showing [ M-H ] 2 O+H] + The peak was m/z 219.1742, suggesting that its most probable molecular formula was C 15 H 24 O 2 . FIG. 9 is a HR-ESI-MS spectrum of the compound Biposopropenoid H of the present invention showing [ M+H ]] + The peak was m/z 237.1858, suggesting that its most probable molecular formula was C 15 H 24 O 2 . FIG. 10 is a HR-ESI-MS spectrum of the compound Biposopropenoid I of the present invention, showing [ M+H ]] + The peak was m/z 235.1701, suggesting that its most probable molecular formula was C 15 H 22 O 2 . FIG. 11 is a HR-ESI-MS spectrum of the compound Biposopropenoid J of the present invention showing [ M+H ] ] + The peak was m/z 239.2012, suggesting that its most probable molecular formula was C 15 H 26 O 2 . FIG. 12 is a HR-ESI-MS spectrum of the compound Biposopropenoid K of the present invention showing [ M-H ] 2 O+H] + The peak was m/z 235.1692, suggesting that its most probable molecular formula was C 15 H 24 O 3 。
The HR-ESI-MS spectrum test adopts Thermal Fisher Orbitrap Q Exactive mass spectrometer and takes methanol as a solvent.
(5) Nuclear magnetic resonance spectroscopy: FIG. 13 shows the dissolution of the compound of the invention Biposopropenoid A in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum. FIG. 14 shows the dissolution of the compound of the invention, biposopropenoid B, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum. FIG. 15 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum. FIG. 16 shows the dissolution of the compound of the invention, biposopropenoid D, in pyridine-D 5 In (a) and (b) 1 H-NMR spectrum. FIG. 17 shows the dissolution of the compound of the invention Biposopropenoid E in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum. FIG. 18 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum. FIG. 19 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum. FIG. 20 shows the dissolution of the compound of the invention Biposopropenoid H in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum. FIG. 21 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum. FIG. 22 shows the dissolution of the compound of the invention, biposopropenoid J, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum. FIG. 23 shows the dissolution of the compound of the invention, biposopropenoid K, in pyridine-d 5 In (a) and (b) 1 H-NMR spectrum. FIG. 24 shows the dissolution of the compound of the invention Biposopropenoid A in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum. FIG. 25 shows the dissolution of the compound of the invention, biposopropenoid B, in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum. FIG. 26 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum. FIG. 27 shows the dissolution of the compound of the invention Biposopropenoid D in pyridine-D 5 In (a) and (b) 13 C-NMR spectrum. FIG. 28 shows the dissolution of the compound of the invention, biposopropenoid E, in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum. FIG. 29 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum. FIG. 30 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum. FIG. 31 shows the dissolution of the compound of the invention Biposopropenoid H in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum. FIG. 32 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum. FIG. 33 shows the dissolution of the compound of the invention Biposopropenoid J in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum. FIG. 34 shows the dissolution of the compound of the invention Biposopropenoid K in pyridine-d 5 In (a) and (b) 13 C-NMR spectrum. FIG. 35 shows the dissolution of the compound of the invention Biposopropenoid A in pyridine-d 5 The HSQC spectrum of (C). FIG. 36 shows the dissolution of the compound of the invention, biposopropenoid B, in pyridine-d 5 The HSQC spectrum of (C). FIG. 37 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 The HSQC spectrum of (C). FIG. 38 shows the dissolution of the compound of the invention, biposopropenoid D, in pyridine-D 5 The HSQC spectrum of (C). FIG. 39 shows the dissolution of the compound of the invention, biposopropenoid E, in pyridine-d 5 The HSQC spectrum of (C). FIG. 40 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 The HSQC spectrum of (C). FIG. 41 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 The HSQC spectrum of (C). FIG. 42 is a diagram of a compound of the present invention, biposoropenoidH is dissolved in pyridine-d 5 The HSQC spectrum of (C). FIG. 43 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 The HSQC spectrum of (C). FIG. 44 shows the dissolution of the compound of the invention Biposopropenoid J in pyridine-d 5 The HSQC spectrum of (C). FIG. 45 shows the dissolution of the compound of the invention Biposopropenoid K in pyridine-d 5 The HSQC spectrum of (C). FIG. 46 shows the dissolution of the compound of the invention, biposopropenoid A, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile. FIG. 47 shows the dissolution of the compound of the invention, biposopropenoid B, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile. FIG. 48 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile. FIG. 49 shows the dissolution of the compound of the invention, biposopropenoid D, in pyridine-D 5 In (a) and (b) 1 H- 1 H COSY profile. FIG. 50 shows the dissolution of the compound of the invention, biposopropenoid E, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile. FIG. 51 Biposopropenoid F of the invention in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile. FIG. 52 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile. FIG. 53 shows the dissolution of the compound of the invention, biposopropenoid H, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile. FIG. 54 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile. FIG. 55 shows the dissolution of the compound of the invention, biposopropenoid J, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile. FIG. 56 shows the dissolution of the compound of the invention, biposopropenoid K, in pyridine-d 5 In (a) and (b) 1 H- 1 H COSY profile.
FIG. 57 shows the dissolution of the compound of the invention, biposopropenoid A, in pyridine-d 5 HMBC spectra of (a). FIG. 58 shows the dissolution of the compound of the invention, biposopropenoid B, in pyridine-d 5 HMBC spectra of (a). FIG. 59 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 HMBC spectra of (a). FIG. 60 shows the dissolution of the compound of the invention Biposopropenoid D in pyridine-D 5 HMBC spectra of (a). FIG. 61 shows the dissolution of the compound of the invention, biposopropenoid E, in pyridine-d 5 HMBC spectra of (a). FIG. 62 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 HMBC spectra of (a). FIG. 63 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 HMBC spectra of (a). FIG. 64 shows the dissolution of the compound of the invention Biposopropenoid H in pyridine-d 5 HMBC spectra of (a). FIG. 65 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 HMBC spectra of (a). FIG. 66 shows the dissolution of the compound of the invention Biposopropenoid J in pyridine-d 5 HMBC spectra of (a). FIG. 67 shows the dissolution of the compound of the invention Biposopropenoid K in pyridine-d 5 HMBC spectra of (a). FIG. 68 shows the solubility of Biposopropenoid A in pyridine-d 5 NOESY spectrum of (B). FIG. 69 shows the solubility of Biposopropenoid B in pyridine-d 5 NOESY spectrum of (B). FIG. 70 shows the dissolution of the compound of the invention, biposopropenoid C, in pyridine-d 5 NOESY spectrum of (B). FIG. 71 shows the dissolution of the compound of the invention, biposopropenoid D, in pyridine-D 5 NOESY spectrum of (B). FIG. 72 shows the dissolution of the compound of the invention, biposopropenoid E, in pyridine-d 5 NOESY spectrum of (B). FIG. 73 shows the dissolution of the compound of the invention, biposopropenoid F, in pyridine-d 5 NOESY spectrum of (B). FIG. 74 shows the dissolution of the compound of the invention, biposopropenoid G, in pyridine-d 5 NOESY spectrum of (B). FIG. 75 shows the dissolution of the compound of the invention Biposopropenoid H in pyridine-d 5 NOESY spectrum of (B). FIG. 76 shows the dissolution of the compound of the invention Biposopropenoid I in pyridine-d 5 NOESY spectrum of (B). FIG. 77 shows the dissolution of the compound of the invention, biposopropenoid J, in pyridine-d 5 NOESY spectrum of (B). FIG. 78 shows the dissolution of the compound of the invention, biposopropenoid K, in pyridine-d 5 NOESY spectrum of (B).
(6) Compound structure X-ray single crystal diffraction pattern:
FIG. 79 is an X-ray single crystal diffraction pattern of the compound of the invention Biposopropenoid G. FIG. 80 is an X-ray single crystal diffraction pattern of the compound Biposopropenoid I of the present invention. The final structural formula is determined as follows:
TABLE 3 Biposopropenoid A-C 1 H and 13 assignment of peaks in the C-NMR spectrum
TABLE 4 Biposopropenoid D-F 1 H and 13 assignment of peaks in the C-NMR spectrum
TABLE 5 Biposopropenoid G-I 1 H and 13 assignment of peaks in the C-NMR spectrum
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TABLE 6 Biposopropenoid J-K 1 H and 13 assignment of peaks in the C-NMR spectrum
NMR of Compound Biposoropenoids A-K was performed using Bruker 600MHz @ 1 H 600MHz； 13 C150 MHz). The solvent for compound Biposoropenoids J-K is pyridine-d 5 。
5. Bridged sesquiterpenoid Biposopropenoid Activity test
The cells were tested for human malignant melanoma cells (A-375), human liver cancer cells (HepG 2), human breast cancer cells (MCF 7), human esophageal cancer cells (TE-1), and human gastric cancer cells (MKN-45). In the experiment, doxorubicin hydrochloride (Dox) is used as a positive medicament, and DMSO is used as a negative control.
Cells were prepared as single cell suspensions with 10% fetal bovine serum in culture medium and 90 μl of 5×10 cells were inoculated per well in 96 well plates 4 Wall-attached cells/mL and 9X 10 4 Suspension cells per mL at 5% CO 2 Pre-culturing for 24 hours at 37 ℃, and then adding a sample solution to be tested: 10 mu L of sample solution is added into each hole, the concentration of an active primary screening sample is set to be 10 mu M, 3 compound holes are arranged, the mixture is placed in an incubator for culturing for 48 hours, and a blank group, a control group and a drug group are arranged in an experiment. The adherent cells aspirate the old medium and drug solution (suspension cells were directly added to 10. Mu.L of CCK-8 stock solution), 100. Mu.L of CCK-8 solution diluted ten times per well was added at 37℃with 5% CO 2 Culturing for 1-4h (light-shielding operation, real-time observation), measuring absorbance at 450nm with enzyme-labeled instrument, and recording original data.
The experimental results are shown in the following table:
TABLE 7 results of cytotoxic Activity test of the Compound Biposopropenoids
The cytotoxic activity assay shows that the isolated compounds of the invention exhibit weak cytotoxic activity.
Sequence information according to the invention
SEQ ID NO.1: nucleotide sequence of bsstA.
atggagatcctcaacaacaaaaccctacccgagttggcctggcttctcctcgggcctttggtactcttttatgtcttcaagctgttcatctacaacgtatacttccatcccctgcgcaagttccctggcccttggataaacaagattagcatcgtatgcaccatctttcagagctctgcttgactcaaacactgacgcctgtagatcccccatctgtacacggttttccagggtaaacaatcttatgaactcctcaagcttcaccgcaaatatggtaagtgcctcatggtctccaaaatagcacaaccacagccagcactactaacttgtctgtgaacctcaggccacatcgttcgctacggaccaaatgaactcagcttcagctcagcccgcgcttggaaagacatctacggctcccgcccgggccaccaaaccttcgtcaaaggcacctggtacgatggtctaagcatctttgcagcccaagatgtacgctccatcatcacggagcgtgaccccacgaaacacgctgccattgctcgggtattcggcggcgccttctcacgttccttcctgaacgagatggagcccatgatcaacgactacatcgaccgctttatcgagcacgtcaagaccaagacagccaacggcggcgtcgtggacttaacattcggctacagctccatgaccttcgacatcatcggcgacctggcttttggacaagactttggcgccatcgggagggaaaccacccacccattcatcctcgaactcaacgagtctttgacgttcaccagcttccacgaggccattcagcaatttccggctttgggccccattgctcgcttcttcttccgggaaaaggttaacaagctcgaagagaccgcgcggaagggaggcgaattcgccctccaggtcatgaggaagcgcgttgcggagcaggacacgacgtcgcgcaaggacttcttgactaaggtattagagcagcgcgccagttccaaggtacagatgtctgagattcagctcgccgcgcagtcatgggactttatcggcgcaggcacagagacaacggcttcggtgatgacatccacgacttactacctactgcgagacaaaaagcttctggccgaactcactgccgagattcgcgcggctttccctaacgctgacgcgattacaaatgcctccaccgaaaagctagaacttctccatcgcgtgtgtcttgagggcttgcgtctcccgacgggagcaccacccatcctacctcgtctagttcccaaaggcggcgacaccgtcgatgggcatttcattccgggaggcactccagttaccatcgctcctatggtcgcagcactcgacccgctcaatttcaaagatcccttggagttcaagccggagcggtggttgggcaagagcggtgacattttggaagcgagccagcccttttcgtacggtacccgcggatgtgcaggaaaagcgtaagttccatacccttgtacccccttgagctacatatacagagaaagtccgactaacgacttgttccaatcttagcatcgcgttgatggaagtgcgtgtgacaatcgccaagatgctctacacgttcgacatggaactggagaatcctgatttggactggaccggcaacgatttcaacaacctgctacagttcggtctgtgggtcaggccactgctgaacgtcagggcccggttggctactaaatga
SEQ ID NO.2: nucleotide sequence of bsstB.
atgggacattctgccaaagacgaggcccccgtcgtggccctccccaagttgggctccattttcaccaaactcctccgcgatctcaaatatcgaacgcctcagcataaagatacacgccctgccctcgaagctgctatgctcgaatatgccgtccgctcgggggctccatacgagtctgaatatgcccagcggtacttcgatgttggacttaccctcgcttgtgtatgtcttcttctatgactccagtactatctgcctcgagttcgacgtatacttagacttctactaactacgcgagttaaggcctattatcctactcattcttttgctgtgaaattacacatagctatctactcctggctagccatctacattgatgacgatgacgacggaaacgaggaccttatcggtttccaagagcgcttccagaagggggagcctcagccatctgctctcttgcagcgcttcgcagagaacctccaagaaatgtcgatccatttcgaacccctcgtagccaacttcatcgtgctttcgtcgctacaattcgtcgccgccaccctgctagagagacgcagcgagctccacagcctgcagcactgcaaagaagcaaaaaggtggcctgactatgtccgcgacaggagcggagtgcctgaggccttcgcgtacttcatcttcccgagagacgagtgcccggatataggagcgtatatgcaaggcataccggacatgatgacttacattaactacgcaaatgatattctgtcgtacgtatggacctgttcctggcagcctatgacgttctcattgtacctgcaacgcttgctaaccaatcatcgttgtcacaggttccataaggagaccctcgctggcgagacggacaactacatcaatacccgagccgtctgtgagcaacgcgagccttttgccatgctcgaaattgtcattgccgagacaatcgcggcaaattcgcgcgtggttgggcttctggacaccagatctgaccccgtgtacgctcgcaagtggaacgaattcttcaacggctacatcttcttccacgtcacagctcggcggtacaagctgcacgtgtataccgggctcgcagatgtgggaaccaaatggcaggaagtgtttgcaggctag
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. The bridge sesquiterpenoids are respectively named as Bipoisosopenoid A, bipoisosopenoid B, bipoisosopenoid C, bipoisosopenoid D, bipoisosopenoid E, bipoisosopenoid F, bipoisosopenoid G, bipoisosopenoid H, bipoisosopenoid I, bipoisosopenoid J and Bipoisosopenoid K, and the structural formulas are shown as follows:
2. a biosynthetic gene cluster for the synthesis of a bridged sesquiterpenoid according to claim 1, wherein said gene cluster comprises 2 genes, bsstA or a functional equivalent thereof, encoding the cytochrome P450 enzyme BsstA, and BsstA or a functional equivalent thereof, encoding the sesquiterpene synthase BsstA, wherein the nucleotide sequence of BsstA is shown in SEQ ID No.1 and the nucleotide sequence of BsstA is shown in SEQ ID No. 2.
3. The biosynthetic gene cluster of claim 2 wherein the functional equivalent of gene BsstA means that the gene nucleotide sequence is a DNA coding sequence corresponding to greater than 80% identity to the amino acid sequence encoding cytochrome P450 enzyme BsstA;
the functional equivalent of gene BsstB means that the nucleotide sequence of the gene is a DNA coding sequence corresponding to greater than 80% identity to the amino acid sequence encoding the sesquiterpene synthase BsstB.
4. The process for producing bridged sesquiterpenes according to claim 1, wherein the bridged sesquiterpenes according to claim 1 are obtained by expressing the genes in the biosynthetic gene cluster according to claim 2 in the genus helminthosporium by means of heterologous expression in Aspergillus oryzae Aspergillus oryzae NSAR.
5. The method for preparing bridged sesquiterpenes according to claim 4, wherein the method for preparing bridged sesquiterpenes Biposoropenoids A-K comprises the steps of:
(1) Taking the genome of B.sorokiniana 11134 as a template, and respectively carrying out PCR amplification on cytochrome P450 enzyme genes bsstA and sesquiterpene synthase genes bstB by using primers bstA-F/bstA-R and bstB-F/bstB-R to obtain PCR products of the genes bstA and bstB; then constructing a coexpression vector pUARA 4-bstA of bstA and a coexpression vector pUARA 4-bstAB of bstB by taking an Aspergillus oryzae A.oryzae expression vector pUARA4 as a vector, wherein the nucleotide sequences of the primers are respectively as follows:
bsstA-F：AAGCTCCGAATTCGAGCTCGATGGGACATTCTGCCAAAGAC
bsstA-R：GAGCTACTACAGATCCCCGGCTAGCCTGCAAACACTTCCT
bsstB-F：CCCCACAGCAAGCTCCGTTAATGGAGATCCTCAACAACAAAACbsstB-R：GTGCATATGATTTAAATTTATCATTTAGTAGCCAACCGGG；
(2) Under the mediation of PEG solvent, the expression vector pUARA 4-bstA or the co-expression vector pUARA 4-bstAB is transformed into protoplast of high-yield host Aspergillus oryzae A.oryzae NSAR1 (niaD-, sC-, delta argB, adeA-) which is easy to express terpene synthase genes to obtain Aspergillus oryzae transformant AO-bstA or AO-bstAB which can produce Biposoropenoids A-K;
(3) Inoculating mycelia of Aspergillus oryzae transformant AO-bsstAB into MPY culture medium to culture as seed solution, inoculating seed solution into MPY fermentation culture medium, and fermenting to obtain fermentation broth containing AO-bsstAB of Biposoropenoids A-K.
6. The method for producing bridged sesquiterpenes according to claim 5, wherein the MPY medium for obtaining the seed solution contains 0.01% adenine and 0.15% methionine;
when used for obtaining seed liquid, the culture conditions are as follows: culturing at 30 ℃ and 220rpm for 2 days;
the seed solution was inoculated in an amount of 1.5% to the MPY fermentation medium, and then inoculated in the MPY fermentation medium, and cultured at 30℃and 220rpm for 5 days to produce Biposoropenoids A-K.
7. The process for producing bridged sesquiterpenes according to claim 5, wherein the process for obtaining Biposoropenoids A-K by separating the fermentation broth containing AO-bstAB of Biposoropenoids A-K comprises:
adding the fermentation liquor of AO-bstAB into ethyl acetate with the same volume for extraction for 3 times, and evaporating to dryness to obtain a crude extract; crude separation is carried out on the crude extract by a decompression reversed phase chromatographic silica gel column, methanol and water are used as mobile phases for elution and target components Fr.4, fr.5 and Fr.6 are enriched, the three fractions are combined and separated by an LH-20 gel chromatographic column, and finally, the Biposopropenoid J is obtained from the Fr.456.14 gel component by eluting with an ACE 5C18-PFP (250 mm multiplied by 10 mm) chromatographic column and acetonitrile/0.1 percent formic acid water volume ratio of 40:60 as mobile phases; the crude fermentation extract was separated by a reduced pressure reverse phase silica gel column with the target components concentrated in fr.5, fr.6, fr.7 and fr.8.Fr.5 was further gel separated to obtain fr.5.6, fr.5.7, fr.5.8 and fr.5.9, fr.5.8, fr.5.9 and fr.6 were combined and then gel column separated, from fr.56. (8-12) further chiral column was used with CHIRALPAK IC (250 mm x 10 mm) at n-hexane/ethanol volume ratio 96:4 obtaining Biposopropenoid G and Biposopropenoid D for mobile phase; fr.7, performing pressurized normal phase silica gel column separation, wherein Fr.7.1 utilizes an ACE 5C18-PFP chromatographic column, acetonitrile/0.1% formic acid water volume ratio of 45:55 is used as a mobile phase to obtain biposopenoid H and biposopenoid I, fr.7.2 utilizes an ACE 5C18-PFP chromatographic column, acetonitrile/0.1% formic acid water volume ratio of 40:60 is used as a mobile phase to obtain biposopenoid F, and Fr.7.4 utilizes an ACE 5C18-PFP chromatographic column, acetonitrile/0.1% formic acid water volume ratio of 35:65 is used as a mobile phase to obtain biposopenoid E and biposopenoid K, fr.5.7 is subjected to crude separation under the conditions of 0-35min,23-40% ACN and 5C18-PFP, and then further uses Chiralpak IA to obtain a total of 96:4 is a mobile phase to obtain biposopenoid A, fr.8 is subjected to gel separation by LH-20, fr.8 (19-21) is separated by ACE 5C18-PFP under the condition of 70:30 flow equality elution of methanol/0.1% formic acid water volume ratio to obtain biposopenoid B and biposopenoid C.
8. An engineering bacterium which is obtained by transforming an expression vector pUARA4-bsstA or a co-expression vector pUARA4-bsstAB into a strain and can produce Biposoropenoids A-K.
9. An engineered bacterium according to claim 8, wherein the strain is selected from aspergillus oryzae a.oryzae NSAR1;
the expression vector pUARA 4-bstA or the co-expression vector pUARA 4-bstAB is a bstA expression vector pUARA 4-bstA or a bstA 4-bstAB co-expression vector constructed by taking the Aspergillus oryzae A.oryzae expression vector pUARA4 as a vector.
10. Use of a bridged sesquiterpenoid according to claim 1, wherein any one of the bridged sesquiterpenoids Biposoropenoids A-K is used for the biosynthesis of sesquiterpenes.
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