CN112725227B

CN112725227B - Sponge-derived streptomyces and preparation and application of active substance for inhibiting hexokinase II

Info

Publication number: CN112725227B
Application number: CN202011604925.9A
Authority: CN
Inventors: 潘华奇; 白岩; 胡江春; 姚雪春
Original assignee: Institute of Applied Ecology of CAS
Current assignee: Institute of Applied Ecology of CAS
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-07-05
Anticipated expiration: 2040-12-30
Also published as: CN112725227A

Abstract

The invention relates to the field of microbial medicines, in particular to sponge-derived streptomyces with a hexokinase II inhibition effect and preparation and application of a hexokinase II inhibition active substance thereof. The marine Streptomyces is Streptomyces sp.18A01, is preserved in China general microbiological culture collection management center at 12.15.2020, and has a preservation number: CGMCC 21351. The strain can also be used for preparing compounds such as alpha-pyrones, ferulic acid, alpha-ribazone, N-acetyltryptophan and the like by fermentation, and particularly preparing a novel alpha-pyrone compound germicidins P-S (1-4). The prepared alpha-pyrone substance is used as an inhibitor of hexokinase II and has application prospects in development of candidate drugs of antitumor drugs and the like.

Description

Sponge-derived streptomyces and preparation and application of active substance for inhibiting hexokinase II

Technical Field

The invention relates to the field of microbial medicines, in particular to sponge-derived streptomyces with a hexokinase II inhibition effect and preparation and application of a hexokinase II inhibition active substance thereof.

Background

Actinomycetes are widely distributed in nature and they produce metabolites with a variety of biological functions. About 50% of the natural products of microbial origin that have been found to date originate from actinomycetes, whereas streptomyces contributes about 70% of their active substances. In recent years, the number of new compounds discovered from marine streptomyces has been on an increasing trend year by year, and about 67% of natural products of marine streptomyces exhibit cytotoxic, bacteriostatic, antimalarial and antiparasitic activities (marine science, 2016,51: 86-124).

Streptomyces antifocyanicus and Streptomyces humifusus have been reported, secondary metabolic activity products in Streptomyces tricolor have not been reported, Streptomyces coelsescens can prevent and treat aphid disease (CN106967629-A), and also can produce a series of glycerolipids with stain removing effect (biosci. Biotech. Bioch.,2012,76: 1746-. However, in general, the bioactive secondary metabolites of the streptomyces group with the highest sequence homology of 16S rDNA are less reported and need to be deeply and widely excavated.

Hexokinase (Hexokinase II, HK2), an enzyme that catalyzes the phosphorylation of hexose sugars, is expressed in small amounts in normal tissues. It is the first enzyme of the glycolytic pathway and also the rate-limiting enzyme of the glycolytic pathway (nat. Commun.2018,9,446). Glycolysis often occurs when cells are hypoxic and the division of tumor cells is vigorous, requiring enormous energy. It has been reported that tumor tissue acquires energy mainly by glycolysis even under conditions where oxygen is sufficiently supplied. The increase of the expression amount and activity of hexokinase in tumor tissue ensures that the tumor tissue can still ensure enough energy under the condition of lacking oxygen, and many intermediates of glycolysis can be utilized by tumor cells to synthesize protein, nucleic acid, lipid and the like, thereby providing necessary material basis for the growth and proliferation of the tumor cells (J.Nucl.Med.2002,43, 173-. Researches find that the characteristics of high sugar metabolism of tumors are as follows: approximately 60% of the ATP in tumor cells is derived from the glycolytic pathway, whose hexokinase content has been shown to be elevated in resected lung, gastrointestinal and breast malignant tissues, and in breast cancer it has been found that hexokinase activity is higher when the lesions metastasize (Eur. J. biochem. 2000,267, 6067-6073). It has also been reported that hexokinase activity is increased in renal tumor tissue (biochem. mol. Med.1995,54: 53-58).

Therefore, if an effective hexokinase inhibitor can be found and the activity of the hexokinase inhibitor is inhibited, glycolysis of tumor cells is inhibited, so that the tumor cells cannot obtain enough glucose-6-phosphate, ATP cannot be converted into ADP to provide energy for various activities of the cells, and the tumor cells are apoptotic. The invention provides a marine streptomyces with hexokinase II inhibition effect, and a novel alpha-pyrone hexokinase inhibitor and other active products can be prepared by utilizing the marine streptomyces.

Disclosure of Invention

The invention aims to provide marine streptomyces with a hexokinase II inhibition effect and preparation and application of a hexokinase II inhibition active substance thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a Streptomyces spongiensis, Streptomyces sp.18A01, is preserved in China general microbiological culture collection management center at 12.10.12.2020 with the preservation number: CGMCC 21351.

The marine streptomyces contains an 8.76Mbp genome; the genome contains clusters of genes that synthetically produce Desferrioxamine B, Geosmin, Isorenieratene, Hopen, Albaflavone, Germidin, SapB (lanthionide), Spore fragment, Calcium-dependent antibody (CDA), Coelimycin P1, Undeclyprodigoside, Coelicidin, Melanin, Coelictin and Actinorhoddin backbone compounds.

An application of marine streptomyces, namely an application of a metabolic product of the marine streptomyces 18A01 as a hexokinase II inhibitor or an anti-tumor candidate drug.

An application of marine streptomycete, namely an application of a metabolic product of the marine streptomycete 18A01 in preparing any one of compounds of alpha-pyrone germicidins, ferulic acid, alpha-ribazole and N-acetyltryptophan.

The preparation of the marine streptomyces 18A01 metabolite comprises the following steps:

1) inoculating the marine streptomyces 18A01 on an ISP3 solid culture medium for 2-5d activation, then inoculating on an ISP3 liquid culture medium containing artificial sea salt, and culturing at 28 ℃ for 2-4d to be used as seed liquid; inoculating the fermented seed liquid 2-10% by volume to ISP3 liquid culture medium containing artificial sea salt, and performing shaking culture at 28 deg.C for 5-10 d;

2) centrifuging the fermentation liquor to remove thallus to obtain fermentation supernatant, and adsorbing active ingredients in the fermentation liquor by using macroporous adsorption resin XAD16 solid phase, wherein the volume percentage of the resin to the fermentation liquor is 3-6%; after adsorption is completed for 2h-4h, the resin is washed by distilled water, then desorbed by methanol, and a crude extract A obtained after the methanol solvent is recovered is the metabolic product of the marine streptomyces 18A 01.

Further purifying and preparing the crude extract A to obtain germicidins, ferulic acid, alpha-ribazole and N-acetyltryptophan: separating the crude extract A by adopting a flash silica gel column chromatography, and performing separation according to a dichloromethane ratio: gradient eluting with methanol (v/v) ratio of 100:0-0:100 to obtain 4 fractions (F)_A-F_D). Fraction F_BThe compounds germicidin P (1), germicidin Q (2), germicidin R (3), germicidin S (4), germicidin L (11), germicidin M (12) and (1S,2S) -germicidin N (13) are obtained by adopting semi-preparative reverse phase HPLC and eluting with 35% methanol aqueous solution at the flow rate of 2.5 mL/min. Fraction F_ARemoving impurities by gel LH20, adopting semi-preparative reverse phase HPLC, eluting by 40% -55% methanol water solution with the flow rate of 2.5mL/min to obtain compounds germicidin A (5), germicidin B (6), isogermicidin B (7), germicidin C (8), germicidin D (9), germicidin H (10) and ferulic acid (14), and mixing the fractions F_CRemoving impurities by gel LH20, eluting with 25% methanol water solution by semi-preparative reverse phase HPLC at flow rate of 2.5mL/min to obtain compound alpha-ribavirin (15), and collecting fraction F_DRemoving impurities by gel LH20, adopting semi-preparative reverse phase HPLC, eluting with 30% methanol water solution at flow rate of 2.5mL/min to obtain compound N-acetyltryptophan (16).

A new alpha-pyrone (germicidin) compound is generated from marine streptomyces, the alpha-pyrone (germicidin P, germicidin Q, germicidin R and germicidin S) compound is shown as (I),

the application of alpha-pyrone (germicidin) compounds, wherein the compounds germicidins P (1), Q (2), R (3), S (4), D (9), H (10), L (11), M (12) and (1S,2S) -germicidin N (13) can be used as hexokinase II inhibitors.

The alpha-pyrone (germicidin) compound is applied to serving as an anti-tumor candidate drug.

Furthermore, the compound germicidin R (3) can be used as an anti-tumor candidate drug; wherein the tumor is colon cancer HCT 116.

The invention has the advantages that:

1. the obtained strain 18A01 has uniqueness, the living environment is specially from sponge in the sea area of Yongxing island, and the strain has the potential of generating various active substances. Several strains with the same 16S rDNA sequence have not been reported to be capable of producing compounds such as alpha-pyrones, ferulic acid, alpha-ribazole and N-acetyltryptophan by fermentation, and the genome ANI is lower than 18A01, showing that the sequence is obviously different from 18A 01.

2. The strain 18A01 can simultaneously prepare alpha-pyrones, ferulic acid, alpha-ribazole and N-acetyltryptophan type compounds, and is the first invention; among them, alpha-pyrone germicidins A-C has been reported to have a hexokinase II inhibitory effect, but germicidins D, H, L-N, P-S are invented for the first time as hexokinase II inhibitors.

3. Four novel alpha-pyrone germicidins P-S compounds are invented for the first time.

Drawings

FIG. 1 is a colony morphology diagram of marine Streptomyces provided by the invention.

FIG. 2 shows the taxonomic status of 18A01 for the phylogenetic tree based on the 16S rDNA sequence provided by the present invention.

FIG. 3 shows the alpha-pyrone type secondary metabolites produced by strain 18A01 in ISP3 fermentation medium.

FIG. 4 is a chemical structural diagram of compounds 1-16 obtained by using the strain 18A01 according to the present invention.

FIG. 5 provides HRESIMS spectra of Germicidin P (1) obtained using strain 18A01 according to the invention.

FIG. 6 shows Germicidins P-S (1-4) obtained by using the strain 18A01 according to the present invention¹H-¹Important correlations between H COSY and HMBC.

FIG. 7 shows Germicidin P (1) in CH obtained from strain 18A01 according to the invention₃Experimental ECD spectra in OH, calculated ECD spectra.

FIG. 8 is a HRESIM plot of Germicidin Q (2) obtained using strain 18A01 according to the invention.

FIG. 9 shows Germicidin Q (2) in CH obtained from strain 18A01 according to the invention₃Experimental ECD spectra in OH, calculated ECD spectrogram.

FIG. 10 provides an HRESIMS spectrum of Germicidin R (3) obtained using strain 18A01 in accordance with the invention.

FIG. 11 provides an HRESIMS spectrum of Germicidin S (4) obtained using strain 18A01 in accordance with the invention.

Detailed Description

For a better understanding of the present invention, reference will now be made to the following examples, which are included to illustrate, but are not intended to limit the scope of the present invention.

Example 1 identification and characterization of Streptomyces spongiensis 18A01

The strain 18A01 is separated from sponge collected from sea area of Yongxing island of Xisha Genjima, and is preserved in China general microbiological culture collection management center in 12 months and 10 days in 2020 with the preservation number: CGMCC 21351. The strain grew well on TSB, Gao's No. 1, ISP3 medium, forming a round off-white colony without pigment production (FIG. 1).

Streptomyces 18A01 was inoculated into TSB liquid medium containing 2.5% artificial sea salt, cultured at 28 ℃ for 36 hours, and total DNA was extracted according to the conventional method. Then, genome determination and assembly are carried out by adopting a second-generation high-throughput sequencing technology Illumina HiSeq2500 to obtain a genome sketch of the strain 18A01, the genome size is about 8.76Mbp, the GC content is 72.17%, and 54 Scaffol are obtained by assemblyds, N50 size 385,800 bp. BLAST analysis of 16S rRNA gene revealed that it is different from the standard strain S.coelescens DSM 40421 of four different species^T、S.anthocyanicus NBRC 14892^T、 S.violaceoruber NBRC 12826^TTricolor NBRC 15461^TSequence homology is up to 100%. Phylogenetic trees further constructed based on the 16S rDNA sequences showed that these strains clustered in the same branch (fig. 2), showing that they have the closest genetic evolutionary relationship. Calculation of strains 18A01 and NBRC 15461^T(genome size about 8.83Mbp, GC content 72.48%) and results show that Kostas lab calculated ANI between them was 83.66% (int.J.Syst.Evol.Microbiol.,2007,57(Pt1):81-91) and Ezbiocool calculated ANI between them was 82.64% (Antonie van Leeuwenhoek, 2017, 110: 1281-1286). Strain 18a01 was suggested to be a different species from s.tricolor, as indicated by the identity of strain ANI greater than 95%. The summary information shows that 18A01 is taxonomically certain.

Wherein the 16S rRNA gene sequence 16S rDNA of the streptomycete 18A01 is as follows:

>18A01 16S rDNA

ACGATGAACCACTTACGGTGGGGATTAGTGGCGAACGGGTGAGT AACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACG GGGTCTAATACCGGATACTGACCCTCGCAGGCATCTGCGAGGTTCGAA AGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTG AGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGG CGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGA GGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCG ACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGC AGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTA ACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCC GGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTG TGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGC TAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATG CGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCC GATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAKA TACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAA CATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTG GGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCC CGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAA CCTTACCAAGGCTTGACATACACCGGAAAGCATCAGAGATGGTGCCC CCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGT CGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCG TGTTGCCAGCAAGCCCTTCGGGGTGTTGGGGACTCACGGGAGACCGC CGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCC CCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGC TGCGATACCGCAAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTC GGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAAT CGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACA CCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCC AACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTGGGA CGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACC TCCTTT

then 18A01 was found to harbor more than 30 secondary metabolite gene clusters using anti SMASH analysis and manual correction, with the potential to produce Desferrioxamine B, Geosmin, Isorenieratene, Hopen, Albaflavone, Ectoine, Germicidin, SapB (lanthione), Spore fragment, Calcium-dependent inhibitory (CDA), Coelimycin P1, undercyclic prediiosin, Coelicidin, Melanin, Coelictin and Actinorhodorin backbone compounds, and the potential to synthesize various novel PKS and NRPS classes (Table 1). It was demonstrated that 18A01 is an excellent strain for producing a variety of bioactive lead compounds.

TABLE 1 potential of Strain 18A01 for the Synthesis of diverse Secondary metabolites

Example 2 production of various types of bioactive substances by Streptomyces marinus 18A01 and methods of preparation

Further using OSMAC strategy to ferment and improve the chemical diversity of the secondary metabolites produced by 18A01, and through activity screening and HPLC-DAD-MS analysis, the strain can produce a series of secondary metabolites of alpha-pyrone and other types when ISP3 (the components comprise 20 g of oat flour, 1mL of trace elements, 1L of tap water and pH 7.2) is used as a fermentation medium (figure 3); the method specifically comprises the following steps:

inoculating marine streptomyces 18A01 on ISP3 solid culture medium, culturing for 48h, activating, and inoculating on artificial sea saltCulturing the ISP3 liquid culture medium at 28 ℃ for 72h to obtain a seed solution; and then, mixing the fermented seed liquid according to the volume ratio of 1: 18 were inoculated into the same fermentation medium and cultured with shaking at 28 ℃ for 7 days. Centrifuging the fermentation liquor to remove thallus to obtain fermentation supernatant, and adsorbing active ingredients in the fermentation liquor by using macroporous adsorption resin XAD16 solid phase, wherein the volume ratio of the resin to the fermentation liquor is 1: 25; and after the adsorption is finished for 2h, washing the resin with distilled water, desorbing with methanol, and recovering the methanol solvent to obtain a crude extract A. Separating the crude extract A by adopting a flash silica gel column chromatography, and performing separation according to a dichloromethane ratio: gradient eluting with methanol (v/v) ratio of 100:0-0:100 to obtain 4 fractions (F)_A-F_D). Fraction F_ARemoving impurities by gel LH20, eluting with 40-55% methanol water solution by semi-preparative reverse phase HPLC at flow rate of 2.5mL/min to obtain germicidin A (6.0mg, t)_R:19.86min)，germicidin B(2.7mg,t_R:17.95 min)，isogermicidin B(0.8mg,t_R:18.20min)，germicidin C(1.6mg,t_R:18.22 min)，germicidin D(4.0mg,t_R:15.97min)，germicidin H(3.6mg,t_R16.33 min), ferulic acid (2.5mg, t)_R14.40 min). Fraction F_BEluting with 35% methanol water solution by semi-preparative reverse phase HPLC at flow rate of 2.5mL/min to obtain compound germicidin P (4.0 mg, t)_R:13.21min)，germicidin Q(4.0mg,t_R:13.69min)，germicidin R(5.2mg, t_R:14.04min)，germicidin S(4.3mg,t_R:14.35min)，germicidin L(9.7mg,t_R: 12.58min)，germicidin M(3.6mg,t_R:12.85min)，(1S,2S)-germicidin N(22.4 mg,t_R14.90 min). Fraction F_CRemoving impurities by gel LH20, eluting with 25% methanol water solution by semi-preparative reverse phase HPLC at flow rate of 2.5mL/min to obtain compound alpha-ribavirin (5.9 mg, t)_R10.53 min). Fraction F_DRemoving impurities by gel LH20, eluting with 30% methanol water solution by semi-preparative reverse phase HPLC at flow rate of 2.5mL/min to obtain compound N-acetyltryptophan (17.3mg, t)_R14.29 min). The structure of the compound is shown in figure 4.

The structure of the compound obtained above was analyzed, and specifically, Compound 1 was a yellow oily substanceHRESIMS data show an M/z 199.0965 [ M + H ]]⁺Ion peak (FIG. 5), corresponding to molecular formula C₁₀H₁₄O₄The unsaturation degree is 4. The ultraviolet absorption at 290nm in the ultraviolet spectrum is the characteristic absorption of alpha-pyrone.¹H NMR(600MHz,DMSO-d₆) Showing the presence of an olefinic hydrogen proton delta_H6.01(s, H-5), two methylene protons, δ_H2.42(m, H-7) and 2.25(d, J ═ 14.7,7.3Hz, H-10), one methine proton δ_H3.89(m, H-8), one doublet methyl delta_H1.08(d, J ═ 6.2Hz, H-9) and one triplet methyl delta_H 0.94(t,J＝14.7,7.3Hz,H-11)。¹³C NMR (600MHz,DMSO-d₆) Shows 5 sp²Hybrid carbon and 5 aliphatic carbons. H-10 and C-4 (. delta.) in HMBC spectra_C164.7), 4-OH and C-3 (. delta.))_C102.8), H-11 and C-3 (. delta.))_C102.8) and H-5 and C-3 (. delta.))_C102.8) concluded the presence of a 4-hydroxy-3-ethyl-alpha-pyrone backbone (FIG. 6). Linkage of H-7, H-8 and H-9 in COSY spectra and H-7 with C-9 (. delta.) in HMBC spectra_C23.5) concluded that a 2-hydroxy-propyl group is present (FIG. 6). H-7 and C-5 (. delta.) in HMBC spectra_C100.9) and C-6 (. delta.))_C160.8) that the 2-hydroxy-propyl group is attached to C-6 (. delta.) of the 4-hydroxy-3-ethyl-. alpha. -pyrone skeleton_C160.8). Thus, the planar structure of compound 1 is 4-hydroxy-3-ethyl-6- (2-hydroxy-propyl) - α -pyrone. The absolute configuration of compound 1 obtained by calculation of ECD was 8R (fig. 7). Compound 1 was therefore designated as a novel compound germicidin P (figure 4).

Compound 2 was a yellow oil and HRESIMS data showed an M/z 199.09634 [ M + H ]]⁺Ion peak (FIG. 8) with molecular formula C₁₀H₁₄O₄The unsaturation degree is 4. The ultraviolet absorption at 290nm in the ultraviolet spectrum is the characteristic absorption of alpha-pyrones. The 1D NMR spectrum showed that Compound 2 was structurally similar to Compound 1 except for the substituent at the C-6 position. Linkage of H-7, H-8 and H-9 in COSY spectra and H-9 to C-8 (. delta.) in HMBC spectra_C63.5) concluded the presence of a 2-hydroxy-1-methyl-ethyl group (FIG. 6). H-7 and C-5 (. delta.) in HMBC spectra_C99.4) and C-6 (. delta.))_C164.8) can be connectedKnowing that the 2-hydroxy-1-methyl-ethyl group is attached to the C-6 (. delta.) -3-ethyl-. alpha. -pyrone skeleton_C160.8). Thus, the planar structure of compound 2 is 4-hydroxy-3-ethyl-6- (2-hydroxy-1-methyl-ethyl) - α -pyrone. The absolute configuration of compound 2 obtained by the calculation of ECD was 7S (fig. 9). Compound 2 was therefore named germicidin Q as a novel compound (figure 4).

Compound 3 was a yellow oil, with HRESIMS data showing an M/z 199.09636 [ M + H ]]⁺Ion peak (FIG. 10) with molecular formula C₁₀H₁₄O₄The unsaturation degree is 4. The ultraviolet absorption at 290nm in the ultraviolet spectrum is the characteristic absorption of alpha-pyrone. The 1D NMR spectrum shows that the compound 3 has a structure similar to that of the compound germicidin B7, and is composed of C-7 (delta)_C69.5) the difference is that the hydrogen in position 7 is replaced by a hydroxyl group. The planar structure of compound 3 was inferred to be 4-hydroxy-3-ethyl-6- (1-hydroxy-1-methyl-ethyl) - α -pyrone by the linkage of COSY spectrum and HMBC spectrum (fig. 6). Compound 3 was therefore named germicidin R as a novel compound (figure 4).

Compound 4 was a yellow oil and HRESIMS data showed an M/z 213.11220 [ M + H ]]⁺Ion peak (FIG. 11) with molecular formula C₁₁H₁₆O₄The unsaturation degree is 4. The ultraviolet absorption at 290nm in the ultraviolet spectrum is the characteristic absorption of alpha-pyrone. The 1D NMR spectrum showed that Compound 4 was structurally similar to Compound 1, consisting of C-9 and C-10 (. delta.) (delta.)_C29.4) the difference is that the hydrogen in position 8 is replaced by a methyl group. The planar structure of compound 4 was inferred to be 4-hydroxy-3-ethyl-6- (2-hydroxy-2-methyl-propyl) - α -pyrone by the linkage of COSY spectrum and HMBC spectrum (fig. 6). Compound 4 was therefore designated as a novel compound germicidin S (figure 4). The 1D-NMR data of the above 4 novel compounds are shown in tables 2 and 3.

TABLE 2 preparation of Compounds 1 to 4¹H NMR data (600MHz, DMSO-d)₆,δin ppm,J in Hz)

TABLE 3 Compounds1-4 of¹³C NMR data (150MHz, DMSO-d)₆)

Other known compounds, germicidin A (5), germicidin B (6), isogermicidin B (7), germicidin C (8), germicidin D (9), germicidin H (10), germicidin L (11), germicidin M (12), (1S,2S) -germicidin N (13), ferulic acid (14), alpha-ribazole (15) and N-acetyltryptophan (16) (FIG. 4), were determined from literature relevant to comparative reports of data such as 1D-NMR spectra and optical rotation.

Example 3 Streptomyces marinus 18A01 and alpha-pyrone Compounds have HK2 inhibitory Activity

HK2 is the rate-limiting enzyme in the first step of the glycolysis pathway and is expressed at higher levels in cancer cells than in normal cells. HK2 provides a new target for tumor therapy due to its key role in tumorigenesis and metastasis.

The extract of the marine streptomyces 18A01 and the compound prepared by separation are used for measuring the inhibition effect of the extract on HK2 by using an HK2 enzyme activity measuring kit. HK2 catalyzes the phosphorylation of glucose to glucose-6-phosphate, which is oxidized by glucose-6-phosphate dehydrogenase to form NADPH. The production rate of NADPH is related to glucose concentration, NADPH has specific absorption peak at 340nm, absorbance change is detected at 340nm, and IC of inhibitory activity of compound on HK2 is determined₅₀Values, each set of experiments was performed in triplicate. Test results show that the alpha-pyrone compounds germicidins obtained by separation exhibit inhibitory activity to HK2 to different degrees, and specifically comprise the following components:

the compounds germicidins P, Q, R and (1S,2S) -germicidin N have stronger inhibitory activity on HK2, and particularly the compounds germicidin Q and (1S,2S) -germicidin N inhibit IC of HK2₅₀Values of 5.13 and 5.30. mu.M, respectively, and positivityIC of control drug benserazide₅₀The values are approximately the same (IC)₅₀5.52 μ M), both of them are promising candidates for HK2 inhibitors.

TABLE 4.18A 01 extracts and their alpha-pyrones prepared inhibit the activity of HK2

Compd.	IC₅₀	Compd.	IC₅₀ ^a
				germicidin P(1)	8.20±0.21	germicidin H(10)	39.51±1.34
germicidin Q(2)	5.13±0.53	germicidin L(11)	10.55±0.84
				germicidin R(3)	7.84±0.78	germicidin M(12)	11.03±0.67
germicidin S(4)	10.27±0.69	(1S,2S)-germicidin N(13)	5.30±0.64
				germicidin A(5)	19.33±0.74	benserazide^b	5.52±0.17
germicidin B(6)	25.16±1.10	18A01 extract^c	0.784μg/mL
				germicidin D(9)	32.38±0.44

^a IC50 values represent the means±SD of three parallel measurements,μM.

^b positive control.^c μg/mL.

Example 4 anti-tumor cell Activity of alpha-pyrone germicidins Compounds

In-vitro cell activity proliferation inhibition experiment, the effect of the compound on the inhibition rate of each cell is examined by adopting a CCK-8 detection method.

Digesting each cell in logarithmic growth phase with pancreatin, collecting, mixing, counting, and adjusting density to 1 × 10⁵Cells per mL were seeded at 100. mu.l/well in 96-well plates and cultured overnight (5% CO) at 37 ℃₂100% humidity); the stock culture was then discarded and given serum-free blank medium containing different final concentrations of germicidins (0-40. mu.M) for 72h (3 replicates per concentration). Then 10. mu.l of CCK-8 solution 5mg/mL are addedWells, after 2h incubation. Measuring its light absorbance at 570nm with microplate reader, and calculating half inhibition concentration IC of each cell after administration₅₀. Experimental results show that germicidin R has activity (IC) against colon cancer HCT116 cells₅₀＝15.35±1.56μM)。

Sequence listing

<110> Shenyang application ecological research institute of Chinese academy of sciences

<120> preparation and application of streptomyces spongiensis and substance for inhibiting hexokinase II activity

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1463

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

acgatgaacc acttacggtg gggattagtg gcgaacgggt gagtaacacg tgggcaatct 60

gcccttcact ctgggacaag ccctggaaac ggggtctaat accggatact gaccctcgca 120

ggcatctgcg aggttcgaaa gctccggcgg tgaaggatga gcccgcggcc tatcagcttg 180

ttggtgaggt aatggctcac caaggcgacg acgggtagcc ggcctgagag ggcgaccggc 240

cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg gaatattgca 300

caatgggcga aagcctgatg cagcgacgcc gcgtgaggga tgacggcctt cgggttgtaa 360

acctctttca gcagggaaga agcgaaagtg acggtacctg cagaagaagc gccggctaac 420

tacgtgccag cagccgcggt aatacgtagg gcgcaagcgt tgtccggaat tattgggcgt 480

aaagagctcg taggcggctt gtcacgtcgg ttgtgaaagc ccggggctta accccgggtc 540

tgcagtcgat acgggcaggc tagagttcgg taggggagat cggaattcct ggtgtagcgg 600

tgaaatgcgc agatatcagg aggaacaccg gtggcgaagg cggatctctg ggccgatact 660

gacgctgagg agcgaaagcg tggggagcga acaggattak ataccctggt agtccacgcc 720

gtaaacggtg ggcactaggt gtgggcaaca ttccacgttg tccgtgccgc agctaacgca 780

ttaagtgccc cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa ttgacggggg 840

cccgcacaag cggcggagca tgtggcttaa ttcgacgcaa cgcgaagaac cttaccaagg 900

cttgacatac accggaaagc atcagagatg gtgcccccct tgtggtcggt gtacaggtgg 960

tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1020

cccttgtccc gtgttgccag caagcccttc ggggtgttgg ggactcacgg gagaccgccg 1080

gggtcaactc ggaggaaggt ggggacgacg tcaagtcatc atgcccctta tgtcttgggc 1140

tgcacacgtg ctacaatggc cggtacaatg agctgcgata ccgcaaggtg gagcgaatct 1200

caaaaagccg gtctcagttc ggattggggt ctgcaactcg accccatgaa gtcggagtcg 1260

ctagtaatcg cagatcagca ttgctgcggt gaatacgttc ccgggccttg tacacaccgc 1320

ccgtcacgtc acgaaagtcg gtaacacccg aagccggtgg cccaacccct tgtgggaggg 1380

agctgtcgaa ggtgggactg gcgattggga cgaagtcgta acaaggtagc cgtaccggaa 1440

ggtgcggctg gatcacctcc ttt 1463

Claims

1. A Streptomyces spongiensis is characterized in that: the marine streptomyces isStreptomycessp.18A01, deposited in China general microbiological culture Collection center at 12 months and 10 days in 2020, with the deposit number: CGMCC 21351.

2. The Streptomyces spongiensis according to claim 1, wherein: the marine streptomyces contains an 8.76Mbp genome; the genome contains clusters of genes that synthetically produce Desferrioxamine B, Geosmin, Isorenieratene, Hopen, Albaflavone, Germidin, SapB (lanthionide), Spore fragment, Calcium-dependent antibody (CDA), Coelimycin P1, Undeclyprodigoside, Coelicidin, Melanin, Coelictin and Actinorhoddin backbone compounds.

3. Use of streptomyces spongiensis according to claim 1, characterized in that: the metabolic product of the marine streptomyces 18A01 is used as a hexokinase II inhibitor or an anti-tumor candidate drug.

4. Use of streptomyces spongiensis according to claim 1, characterized in that: the metabolic product of the marine streptomyces 18A01 is used for preparing alpha-pyrone germicidins, ferulic acid,α-ribazole andN-any compound of acetyltryptophan.

5. Use of streptomyces spongiensis according to claim 3 or 4, characterized in that: the preparation of the marine streptomyces 18A01 metabolite comprises the following steps:

1) inoculating the marine streptomyces 18A01 of claim 1 on an ISP3 solid culture medium for 2-5 days of activation, then inoculating on an ISP3 liquid culture medium containing artificial sea salt, and culturing at 28 ℃ for 2-4 days as a seed solution; inoculating the fermented seed liquid 2-10% by volume to ISP3 liquid culture medium containing artificial sea salt, and performing shaking culture at 28 deg.C for 5-10 d;

2) centrifuging the fermentation liquor to remove thallus to obtain fermentation supernatant, and adsorbing active ingredients in the fermentation liquor by using macroporous adsorption resin XAD16 solid phase, wherein the volume percentage of the resin to the fermentation liquor is 3% -6%; after adsorbing for 2h-4h, washing the resin with distilled water, desorbing with methanol, and recovering the methanol solvent to obtain crude extract A, namely the metabolic product of Streptomyces marinus 18A 01.

6. Use of Streptomyces spongiensis according to claim 5, characterized in that: further purifying and preparing the crude extract A to obtain germicidins, ferulic acid,α-ribazole andN-acetyltryptophan: separating the crude extract A by adopting a flash silica gel column chromatography, and performing separation according to a dichloromethane ratio: methanol (c)v/v) Gradient eluting at a ratio of 100:0-0:100 to obtain 4 fractions (F)_A-F_D) (ii) a Fraction F_BAdopting semi-preparative reverse phase HPLC, eluting with 35% methanol water solution at the flow rate of 2.5mL/min to obtain compounds germicidin P (1), germicidin Q (2), germicidin R (3), germicidin S (4), germicidin L (11), germicidin M (12), (1)S, 2S) -germicidin N (13); fraction F_ARemoving impurities by gel LH20, adopting semi-preparative reverse phase HPLC, eluting by 40-55% methanol water solution with the flow rate of 2.5mL/min, and obtaining compounds germicidin A (5), germicidin B (6), isogermicidin B (7), germicidin C (8), germicidin D (9), germicidin H (10) and ferulic acid (14); fraction F_CRemoving impurities by gel LH20, performing semi-preparative reverse phase HPLC, eluting with 25% methanol water solution at flow rate of 2.5mL/min to obtain compoundα-a ribazole (15); fraction F_DRemoving impurities by gel LH20, eluting with 30% methanol water solution by semi-preparative reverse phase HPLC at flow rate of 2.5mL/min to obtain compoundN-acetyltryptophan (16)。