Method for producing C-glycoside compound

Abstract

The invention discloses a method for producing a C-glycoside compound, which comprises the following steps: constructing a gene G and a gene K into a pCDFDuet-1 plasmid vector with double T7 promoters to obtain a recombinant plasmid pCDFDuet-1-GK1, and transforming the recombinant plasmid into an escherichia coli BL21 (DE3) competent cell to obtain recombinant escherichia coli; the method comprises the following steps: inducing recombinant Escherichia coli protein expression by isopropyl thiogalactoside to obtain a strain for producing a compound B with a structure shown in a formula II, adding 3, 5-dihydroxybenzyl alcohol or a compound A with a structure shown in a formula I as a substrate, and culturing to obtain the compound B. According to the invention, a recombinant plasmid is constructed by using a pET-28a (+) vector and a gene G, and a strain for producing the compound A is further constructed. The invention provides methods for heterologous biosynthesis of compounds A and B for the first time. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology and industrial biotechnology, and relates to a method for constructing an Escherichia coli producing a C-glycoside compound to produce a C-glycoside compound, and specifically relates to a method for constructing an Escherichia coli producing a C-glycoside compound A to produce a C-glycoside compound A, and a method for constructing an Escherichia coli producing a C-glycoside compound B by using a double promoter vector to produce a C-glycoside compound B. Background Art

[0002] Natural products with complex and diverse structures are an important source of innovative drugs and play an important role in new drug discovery.

[0003] In recent years, C-glycoside compounds have become important components of many natural alkaloids and pharmaceutically active drug molecules. Compound A and Compound B both belong to C-glycoside compounds and are important precursors of antifungal compounds. The structures of Compound A and Compound B are shown in Formula I and II, respectively:

[0004]

[0005] Both contain a C-glycosyl bond formed by D-glucose and resorcinol, and compound B contains a unique spiro structure connected to the aromatic ring through a CO bond and a CC bond. Its complex structure has attracted many scientific researchers to explore chemical synthesis.

[0006] The chemical synthesis methods of C-glycoside compounds mainly include: synthesis of racemic-spirone units through hetero-Diels-Alder reaction and dihydroxylation of 5-aryl-2-vinylfuran, followed by Achmatowicz rearrangement; condensation of functionalized organic lithium reagents with cyclic or acyclic derivatives of d-gluconolactone. These methods provide a way to prepare the spirocyclic structure in compound B, but the yield is low. There is also a method of obtaining aryl-pyranoside by nucleophilic addition of lithiated hexylpyranose to functionalized quinone, and then preparing compound B through palladium-catalyzed cross-coupling reaction of protected (tributyl)-hexynyl sugar with aryl bromide, but this method requires excessive toxic tin reagents due to the dimerization of the donor.

[0007] At present, there are no reports on the biosynthesis of the above two C-glycoside compounds. Compared with chemical synthesis methods, biosynthesis methods have the advantages of strong selectivity, high catalytic efficiency, mild reaction conditions, environmental friendliness, low catalyst manufacturing cost, and wide reaction range. With the development of synthetic biology, more and more natural products can be artificially prepared through biological cell factories, thus providing new and complementary synthetic strategies.

[0008] Escherichia coli has become an excellent industrial model microorganism due to its long research history, clear genetic background, fast anaerobic growth, ability to use simple inorganic salt culture medium and a variety of substrates (glucose, xylose, glycerol), etc. Metabolic engineering of Escherichia coli can be used to produce a variety of products such as organic alcohols, amino acids, organic acids, organic amines, vitamins, natural products and polyhydroxyalkanoates (PHA), and is applied to the green biosynthesis of bulk chemicals such as L-alanine, L-lysine, L-threonine, 1,3-propylene glycol, D-lactic acid, succinic acid, and pentamethylenediamine.

[0009] The technology of constructing E. coli cell factory mainly lies in three aspects: pathway design, synthetic pathway creation and optimization, and cell global optimization. In terms of pathway design, it is mainly to optimize the metabolic pathway of E. coli itself, introduce exogenous metabolic pathways, and create new pathways that do not exist in nature. In terms of synthetic pathway creation, DNA fragment assembly technology, genome editing technology and gene expression regulation technology are needed. Among them, single gene regulation, multi-gene regulation and gene dynamic regulation are essential to improve the efficiency of metabolic pathways and are also important technical barriers to constructing efficient E. coli cell factories. In terms of cell global optimization, the most important factor is to consider how to achieve cofactor balance. For this reason, it is often necessary to change the preference of cofactors or the regeneration pathway of cofactors according to the characteristics of the enzymes used in the pathway. On this basis, through adaptive evolution, the production efficiency of the target product is continuously improved. If the above strategies can be used to construct E. coli that produces C-glycoside compounds A and B, and realize efficient heterologous biosynthesis of complex natural products, it will be of great significance to promote the industrial production and clinical application of related compounds. Summary of the invention

[0010] The present invention aims to provide a method for constructing Escherichia coli for producing C-glycoside compound A, and a method for producing C-glycoside compound A based on Escherichia coli producing C-glycoside compound A; and a method for constructing Escherichia coli for producing C-glycoside compound B using a dual promoter vector, by adding a biosynthetic precursor 3,5-dihydroxybenzyl alcohol or compound A to the culture medium, thereby achieving heterologous biosynthesis of compound B.

[0011] The objective of the present invention is achieved through the following technical solutions:

[0012] A method for producing C-glycoside compounds comprises the following steps:

[0013] Step (b1), constructing the gene G with a nucleotide sequence as shown in SEQ ID NO.1 and the gene K with a nucleotide sequence as shown in SEQ ID NO.2 into a pCDFDuet-1 plasmid vector having a double T7 promoter to obtain a recombinant plasmid pCDFDuet-1-GK1, and transforming the recombinant plasmid pCDFDuet-1-GK1 into Escherichia coli (E. coli) BL21 (DE3) competent cells to obtain recombinant E. coli;

[0014] Step (b2), inoculating the recombinant Escherichia coli described in step (b1) in a culture medium containing spectinomycin, adding isopropyl thiogalactoside (isopropyl-β-D-thiogalactopyranoside, IPTG) to induce the expression of the recombinant Escherichia coli protein to obtain a strain producing compound B with a structure as shown in formula II, and then adding 3,5-dihydroxybenzyl alcohol or compound A with a structure as shown in formula I as a substrate to the culture medium to obtain compound B;

[0015]

[0016] In step (b2), the recombinant Escherichia coli is grown in LB liquid culture medium containing 50 μg / mL spectinomycin to an OD600 of 0.8, isopropylthiogalactoside is added to induce the expression of recombinant Escherichia coli protein, and then 3,5-dihydroxybenzyl alcohol or compound A is added to the culture medium as a substrate for cultivation, the cells are collected, extracted and separated to obtain compound B.

[0017] Preferably, the recombinant E. coli is grown in LB liquid medium containing 50 μg / mL spectinomycin at a temperature of 37° C. to an OD600 of 0.8.

[0018] Preferably, the temperature for inducing the expression of the recombinant E. coli protein is 16-20° C. and the time is 4-8 hours.

[0019] Preferably, the final concentration of isopropylthiogalactoside is 100 μM.

[0020] Preferably, the culture medium is LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, natural pH.

[0021] Preferably, 0.01 to 0.1 g of 3,5-dihydroxybenzyl alcohol is added to every 1 L of culture medium.

[0022] The extraction and separation method of the compound B is as follows: after the cultivation is completed, the culture is centrifuged, the bacteria are collected, the bacteria are resuspended in methanol, ultrasonic extraction is performed 2 to 4 times, the extracts are combined, the extracts are concentrated under reduced pressure to remove methanol, and a crude extract is obtained. The crude extract is applied to an MCI chromatographic column, and a methanol-water system is selected for gradient elution to obtain 5 fractions FrB.1-FrB.5; fraction FrB.1 is taken and subjected to Sephadex LH-20 gel column chromatography, and isocratic elution is performed with pure water as the mobile phase to obtain 4 subfractions FrB.9.1-FrB.9.4; subfraction Fr.9.4 is taken and subjected to semi-preparative RP-HPLC (reverse-phase high performance liquid chromatography) to obtain compound B.

[0023] The centrifugal temperature is 4°C, the centrifugal speed is 5000 rpm, and the centrifugal time is 20 minutes.

[0024] During each ultrasonic extraction, the volume ratio of the bacteria to methanol is 1:1 to 1:1.5. The ultrasonic power of the ultrasonic extraction is 500W; each ultrasonic extraction lasts 20 to 30 minutes.

[0025] The filler of the MCI chromatographic column is HP20; the methanol-water system is water, 30% methanol, 60% methanol, 90% methanol, and 100% methanol.

[0026] The semi-preparative RP-HPLC conditions were: Capcell Pak MGⅡC18 column (5 μm; 4.6×250 mm), mobile phase: 1.5% MeOH-H2O, isocratic elution, flow rate 3 mL·min -1 .

[0027] A method for producing C-glycoside compounds comprises the following steps:

[0028] Step (a1), constructing the gene G with the nucleotide sequence as shown in SEQ ID NO.1 into the pET-28a(+) plasmid vector to obtain the recombinant plasmid pET-28a(+)-1, and transforming the recombinant plasmid pET-28a(+)-1 into Escherichia coli (E. coli) BL21(DE3) competent cells to obtain recombinant E. coli;

[0029] Step (a2), inoculating the recombinant Escherichia coli described in step (a1) in a culture medium containing kanamycin sulfate, adding isopropylthiogalactoside to induce the expression of the recombinant Escherichia coli protein to obtain a strain producing compound A with a structure as shown in formula I, and then adding 3,5-dihydroxybenzyl alcohol as a substrate to the culture medium to obtain compound A;

[0030]

[0031] In step (a2), the recombinant Escherichia coli is grown in a culture medium containing 50 μg / mL kanamycin sulfate to an OD600 of 0.8, isopropylthiogalactoside is added to induce the expression of the recombinant Escherichia coli protein, and then 3,5-dihydroxybenzyl alcohol is added to the culture medium for cultivation, the cells are collected, and the cells are extracted and separated to obtain compound A.

[0032] The temperature for inducing the expression of the recombinant Escherichia coli protein is 16-20° C. and the time is 4-8 hours.

[0033] In the culture medium, the final concentration of isopropyl thiogalactoside is 100 μM.

[0034] The culture medium is LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, natural pH.

[0035] Add 0.01-0.1 g of 3,5-dihydroxybenzyl alcohol to every 1 L of culture medium.

[0036] The extraction and separation method of the compound A is as follows: after the cultivation is completed, the culture is centrifuged, the bacterial cells are collected, the bacterial cells are resuspended in methanol, ultrasonic extraction is performed 2 to 4 times, the extracts are combined, the extracts are concentrated under reduced pressure to remove methanol, and a crude extract is obtained. The crude extract is applied to an MCI chromatographic column, and a methanol-water system is selected for gradient elution to obtain 5 fractions FrA.1-FrA.5; fraction FrA.1 is taken and subjected to Sephadex LH-20 gel column chromatography, and isocratic elution is performed with pure water as the mobile phase to obtain 4 subfractions FrA.9.1-FrA.9.4; subfraction Fr.9.2 is taken and subjected to semi-preparative RP-HPLC to obtain compound A.

[0037] The centrifugal temperature is 4°C, the centrifugal speed is 5000 rpm, and the centrifugal time is 20 minutes.

[0038] During each ultrasonic extraction, the volume ratio of the bacteria to methanol is 1:1 to 1:1.5.

[0039] The ultrasonic power of the ultrasonic extraction is 500W; each ultrasonic extraction lasts 20 to 30 minutes.

[0040] The filler of the MCI chromatographic column is HP20; the methanol-water system is water, 30% methanol, 60% methanol, 90% methanol, and 100% methanol.

[0041] The semi-preparative RP-HPLC conditions were: Capcell Pak MGⅡC18 column (5 μm; 4.6×250 mm), mobile phase: 1.0% MeOH-H2O, isocratic elution, flow rate 3 mL·min -1 .

[0042] Another object of the present invention is to provide a gene G, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0043] Another object of the present invention is to provide a gene K, the nucleotide sequence of gene K is shown in SEQ ID NO.2.

[0044] Another object of the present invention is to provide a biomaterial, wherein the biomaterial is a biomaterial comprising the gene G and / or gene K, and the biomaterial is an expression cassette, a plasmid vector, an engineered bacterium or a transgenic cell line.

[0045] Preferably, the biological material is a biological material comprising the gene G or a biological material comprising gene G and gene K.

[0046] The biomaterial is at least one of (1) to (14):

[0047] (1) an expression cassette containing the gene G;

[0048] (2) a recombinant plasmid vector containing the gene G;

[0049] (3) a recombinant plasmid vector containing the expression cassette described in (1);

[0050] (4) an engineered bacterium containing the gene G;

[0051] (5) An engineered bacterium containing the expression cassette described in (1);

[0052] (6) an engineered bacterium containing the recombinant plasmid vector described in (2);

[0053] (7) An engineered bacterium containing the recombinant plasmid vector described in (3);

[0054] (8) an expression cassette containing the gene G and gene K;

[0055] (9) a recombinant plasmid vector containing the gene G and gene K;

[0056] (10) a recombinant plasmid vector containing the expression cassette described in (8);

[0057] (11) An engineered bacterium containing the gene G and gene K;

[0058] (12) An engineered bacterium containing the expression cassette described in (8);

[0059] (13) An engineered bacterium containing the recombinant plasmid vector described in (9);

[0060] (14) An engineered bacterium containing the recombinant plasmid vector described in (10).

[0061] Another object of the present invention is to provide the use of the gene G in the preparation of C-glycoside compound A or in the construction of a recombinant plasmid vector for producing C-glycoside compound A or in the construction of a recombinant Escherichia coli for producing C-glycoside compound A.

[0062] Another object of the present invention is to provide a method for constructing a recombinant plasmid vector for producing C-glycoside compound A, comprising: selecting a pET-28a(+) plasmid vector, constructing gene G into the pET-28a(+) plasmid vector, and obtaining a recombinant plasmid pET-28a(+)-1.

[0063] Another object of the present invention is to provide a method for constructing a recombinant Escherichia coli for producing C-glycoside compound A, comprising: selecting a pET-28a(+) plasmid vector, constructing gene G into the pET-28a(+) plasmid vector to obtain a recombinant plasmid pET-28a(+)-1, and transforming the recombinant plasmid pET-28a(+)-1 into Escherichia coli (E. coli) BL21(DE3) competent cells to obtain a recombinant Escherichia coli.

[0064] Another object of the present invention is to provide the use of the gene G and gene K in the preparation of C-glycoside compound B, or in the construction of a recombinant plasmid vector for producing C-glycoside compound B, or in the construction of a recombinant Escherichia coli for producing C-glycoside compound B.

[0065] Another object of the present invention is to provide a method for constructing a recombinant plasmid vector for producing C-glycoside compound B, comprising: selecting a pCDFDuet-1 plasmid vector with a double T7 promoter, constructing gene G and gene K into the pCDFDuet-1 plasmid vector, and obtaining a recombinant plasmid pCDFDuet-1-GK1.

[0066] Another object of the present invention is to provide a method for constructing a recombinant Escherichia coli for producing C-glycoside compound B, comprising: selecting a pCDFDuet-1 plasmid vector with a double T7 promoter, constructing gene G and gene K into the pCDFDuet-1 plasmid vector to obtain a recombinant plasmid pCDFDuet-1-GK1, and transforming the recombinant plasmid pCDFDuet-1-GK1 into Escherichia coli (E. coli) BL21 (DE3) competent cells to obtain recombinant Escherichia coli.

[0067] Beneficial effects of the present invention:

[0068] The present invention utilizes pET-28a(+) vector and gene G to construct a recombinant plasmid, and further constructs a compound A production strain.

[0069] The present invention selects a pCDFDuet-1 plasmid vector with a double T7 promoter, constructs a co-expression system with a gene G and a gene K after directed evolution, transforms them into E.coli BL21 (DE3) competent cells to obtain recombinant Escherichia coli, induces protein expression of the recombinant Escherichia coli by isopropylthiogalactoside, adds a biosynthetic precursor 3,5-dihydroxybenzyl alcohol or a compound A to an LB culture medium, and finally realizes the heterologous biosynthesis of the compound B.

[0070] The present invention provides for the first time a method for heterologous biosynthesis of C-glycoside compounds A and B, thereby improving production efficiency and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The HPLC analysis comparison chart of compound A produced by Escherichia coli; wherein, i) is the control group; ii) is the experimental group.

[0072] Figure 2 This is the high-resolution mass spectrum of compound A.

[0073] Figure 3 This is a comparative chart of HPLC analysis of compound B produced by Escherichia coli; wherein, i) is the control group; ii) is the experimental group.

[0074] Figure 4 This is the high-resolution mass spectrum of compound B. DETAILED DESCRIPTION

[0075] The following embodiments are intended to help those skilled in the art better understand the technical solutions of the present invention, but are not intended to limit the present invention in any way.

[0076] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, natural pH.

[0077] Example 1 Construction of gene G and gene K protein expression plasmids

[0078] After bioinformatics analysis, the genome sequence of Apodospora peruviana strain CBS118394 (GenBank: JAUEDM010000005.1) in the NCBI (http: / / www.ncbi.nlm.nih.gov) database was used as a template to synthesize plasmids pET-28a(+)-GT1 and pET-28a(+)-KG. Primers were designed using the genome sequence of Apodospora peruviana strain CBS118394 as a template, and PCR amplification was performed to obtain a target fragment containing gene G (SEQ ID NO.1), and then the target fragment was constructed into the pET-28a(+) plasmid to obtain the recombinant plasmid pET-28a(+)-GT1; primers were designed using the genome sequence of Apodospora peruviana strain CBS118394 as a template, and PCR amplification was performed to obtain a target fragment containing gene K (SEQ ID NO.2), and then the target fragment was constructed into the pET-28a(+) plasmid to obtain the recombinant plasmid pET-28a(+)-KG; the above vector construction process was entrusted to Jiutian Gene Technology (Tianjin) Co., Ltd.

[0079] (1) Construction of gene G protein expression plasmid pET-28a(+)-1

[0080] Plasmid pET-28a(+)-GT1 was used as a template, and primers pET-28a(+)-GF and pET-28a(+)-GR were used to perform PCR amplification to obtain a 1435 bp gene G (SEQ ID NO.1). The NEBuilder HiFi DNA assembly cloning kit was used to assemble the PCR product of gene G and the pET-28a(+) plasmid vector after double digestion with NdeI and HindIII to obtain the recombinant plasmid pET-28a(+)-1. The recombinant plasmid pET-28a(+)-1 was transformed into the Escherichia coli DH5α strain by heat shock method, coated on a LB solid plate containing 50 μg / mL kanamycin sulfate, and cultured at 37°C for 12 hours. PCR verification was performed using primers pET-28a(+)-GF and pET-28a(+)-GR, and the correct transformant was able to obtain a 1435 bp band. The obtained recombinant plasmid pET-28a(+)-1 was confirmed by sequencing.

[0081] (2) Construction of dual promoter vector for gene G and gene K protein expression plasmid pCDFDuet-1-GK1

[0082] Using plasmid pET-28a(+)-GT1 as a template, primers pCDFDuet-GK1-F1 and pCDFDuet-GK1-R1 were used for PCR amplification to obtain gene G (SEQ ID NO.1). Using plasmid pCDFDuet-1 with a double T7 promoter as a template, primers pCDFDuet-GK1-F2 and pCDFDuet-GK1-R2 were used for PCR amplification to obtain a 148bp downstream fragment of the target gene G. Using plasmid pET-28a(+)-KG as a template, primers pCDFDuet-GK1-F3 and pCDFDuet-GK1-R3 were used for PCR amplification to obtain the target gene K (SEQ ID NO.2). The above three fragments were fused PCR amplified using primers pCDFDuet-GK1-F1 and primer pCDFDuet-GK1-R3 to obtain a 2705bp fusion fragment. The fusion fragment and the pCDFDuet-1 vector after double digestion with NcoI and AvrII were assembled using the NEBuilder HiFi DNA assembly cloning kit to obtain the recombinant plasmid pCDFDuet-1-GK1. The recombinant plasmid pCDFDuet-1-GK1 was transformed into the Escherichia coli DH5α strain by heat shock method, coated on a LB solid plate containing 50μg / mL spectinomycin, and cultured at 37°C for 12h. PCR verification was performed using primers pCDFDuet-GK1-F2 and pCDFDuet-GK1-R2, and the correct transformant was able to obtain a 148bp band. The obtained recombinant plasmid pCDFDuet-1-GK1 was confirmed by sequencing.

[0083] Table 1. Primer sequence list

[0084]

[0085] Example 2 Construction of expression strains for producing compounds A and B

[0086] (1) Construction of Escherichia coli producing compound A and preparation of compound A

[0087] The recombinant plasmid pET-28a(+)-1 was transferred to Escherichia coli BL21 (DE3) by heat shock method to construct Escherichia coli producing compound A; the Escherichia coli producing compound A was grown in LB liquid culture medium containing 50 μg / mL kanamycin sulfate at 37°C to an OD600 of 0.8, and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the LB liquid culture medium containing 50 μg / mL kanamycin sulfate to make the final concentration of 100 μM, and the protein expression was induced at 18°C ​​for 6 hours, and then an experimental group and a control group were set up, and 3,5-dihydroxybenzyl alcohol was added to the LB liquid culture medium of the experimental group To a final concentration of 0.1 g / L, 3,5-dihydroxybenzyl alcohol was not added to the LB liquid culture medium of the control group, and the rest were the same as the experimental group. Both the treatment group and the control group were cultured at a temperature of 18°C ​​for 24 hours; at a temperature of 4°C, the cultures of the experimental group and the control group were centrifuged at a speed of 4500 rpm for 20 minutes, the cell pellets were taken, and methanol was added for extraction, and a total of 3 extractions were performed, each time methanol was added according to a volume ratio of cell pellet to methanol of 1:1.2, and ultrasonic extraction (ultrasonic power 500 W) was performed for 30 minutes, the extracts were combined, and the extracts were concentrated under reduced pressure to remove methanol to obtain a crude extract, 10 mL of pure water was added to the crude extract to dissolve, filtered with a 0.22 μm filter membrane, and analyzed by HPLC.

[0088] HPLC conditions: chromatographic column: Agilent Eclipse Plus C18 analytical column (5μm, 4.6×150mm), linear elution mobile phase: H2O(A):MeOH(B) (0-6min 1% B; 6-12min 3% B; 12-25min 95% B; 25-30min 95% B), detection wavelength: 280nm.

[0089] Results Figure 1 By comparing the peak area and retention time, it can be seen that: under the same culture time, the experimental group can produce compound A compared with the control group.

[0090] Isolation and purification of compound A: The crude extract was dissolved in an appropriate amount of pure water, and the sample was evenly loaded onto an MCI chromatographic column (filler: HP20). A methanol-water system was selected as the eluent, and gradient elution was performed according to a methanol content of 0%, 30%, 60%, 90%, and 100%. Each gradient elution was 3 column volumes, and each 100 mL was 1 fraction. Each fraction was analyzed by HPLC, and similar fractions were combined and evaporated to dryness under reduced pressure to finally obtain 5 fractions, recorded as FrA.1-FrA.5; FrA.1 was dissolved in pure water and subjected to Sephadex LH-20 gel column chromatography. Pure water was used as the mobile phase for isocratic elution, and 3 mL was 1 fraction. Similar fractions were combined according to the HPLC analysis results to finally obtain 4 sub-fractions, recorded as FrA.9.1-FrA.9.4; FrA.9.2 was subjected to semi-preparative RP-HPLC (reverse phase high performance liquid chromatography), and the chromatographic column was Capcell Pak MGⅡC18 (5μm; 4.6×250mm), mobile phase 1.0% MeOH-H2O, isocratic elution; flow rate: 3mL·min -1 , compound A was prepared, HRMS (ESI, M+H + )303.1077( Figure 2 ), C 13 H 18 O8 + .

[0091] (2) Construction of Escherichia coli producing compound B and preparation of compound B

[0092] The recombinant plasmid pCDFDuet-1-GK1 was transferred into Escherichia coli BL21 (DE3) to construct Escherichia coli producing compound B; the Escherichia coli producing compound B was grown in LB liquid culture medium containing 50 μg / mL spectinomycin at a temperature of 37°C to an OD600 of 0.8, and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the LB liquid culture medium containing 50 μg / mL spectinomycin to make the final concentration of 100 μM, and the protein expression was induced at a temperature of 18°C ​​for 6 h, and then an experimental group and a control group were set up, and 3,5-dihydroxybenzyl alcohol was added to the LB liquid culture medium of the experimental group to The final concentration was 0.1 g / L. 3,5-dihydroxybenzyl alcohol was not added to the LB liquid culture medium of the control group. The rest were the same as the treatment group. The experimental group and the control group were cultured at 18°C ​​for 24 hours. At 4°C, the cultures of the experimental group and the control group were centrifuged at 4500 rpm for 20 minutes, the cell pellets were taken, and methanol was added for extraction. The extraction was performed 3 times in total, and methanol was added each time according to the volume ratio of cell pellet to methanol of 1:1.2. Ultrasonic extraction (ultrasonic power 500 W) was performed for 30 minutes, the extracts were combined, and the extracts were concentrated under reduced pressure to remove methanol to obtain a crude extract. The crude extract was dissolved in 10 mL of pure water, filtered with a 0.22 μm filter membrane, and analyzed by HPLC.

[0093] HPLC conditions: chromatographic column: Agilent Eclipse Plus C18 analytical column (5μm, 4.6×150mm), linear elution mobile phase: H2O(A):MeOH(B) (0-6min 1% B; 6-12min 3% B; 12-25min 95% B; 25-30min 95% B), detection wavelength: 273nm.

[0094] Results Figure 3 By comparing the peak area and retention time, it can be seen that: under the same culture time, the experimental group can produce compound B (HRMS (ESI, M+H + )C 13 H 17 O8 + 301.0918).

[0095] Isolation and purification of compound B: The crude extract was dissolved in an appropriate amount of pure water, and the sample was evenly loaded onto an MCI chromatographic column (filler: HP20). A methanol-water system was selected as the eluent, and gradient elution was performed according to a methanol content of 0%, 30%, 60%, 90%, and 100%. Each gradient elution had 3 column volumes, and each 100 mL was 1 fraction. Each fraction was analyzed by HPLC, and similar fractions were combined and evaporated to dryness under reduced pressure to obtain 5 fractions, recorded as FrB.1-FrB.5; Fr.1 was dissolved in pure water and subjected to Sephadex LH-20 gel column chromatography. Pure water was used as the mobile phase for isocratic elution, and 3 mL was 1 fraction. Similar fractions were combined according to the HPLC analysis results to obtain 4 sub-fractions, recorded as FrB.9.1-FrB.9.4; FrB.9.4 was subjected to semi-preparative RP-HPLC, and the chromatographic column was Capcell PakMGⅡC18 (5μm; 4.6×250mm), mobile phase 1.5% MeOH-H2O, isocratic elution; flow rate: 3mL·min -1 Compound B was prepared, HRMS (ESI, M+H + )301.0926( Figure 4 ), C 13 H 16 O8.

Claims

1. A method for producing C-glycoside compounds, characterized in that: The following steps are involved: Step (b1), constructing the gene G with a nucleotide sequence as shown in SEQ ID NO.1 and the gene K with a nucleotide sequence as shown in SEQ ID NO.2 into a pCDFDuet-1 plasmid vector having a double T7 promoter to obtain a recombinant plasmid pCDFDuet-1-GK1, and transforming the recombinant plasmid pCDFDuet-1-GK1 into Escherichia coli BL21 (DE3) competent cells to obtain recombinant Escherichia coli; Step (b2), inoculating the recombinant Escherichia coli described in step (b1) in a culture medium containing spectinomycin, adding isopropylthiogalactoside to induce the expression of the recombinant Escherichia coli protein to obtain a strain that produces compound B with a structure as shown in formula II, and then adding 3,5-dihydroxybenzyl alcohol or compound A with a structure as shown in formula I as a substrate to the culture medium to obtain compound B; 2. The method for producing C-glycoside compounds according to claim 1, characterized in that: In step (b2), the culture medium is LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, natural pH; Add 0.01-0.1 g of 3,5-dihydroxybenzyl alcohol to every 1 L of culture medium.

3. The method for producing C-glycoside compounds according to claim 1, characterized in that: In step (b2), the extraction and separation method of compound B is as follows: after the culture is completed, the culture is centrifuged, the bacterial cells are collected, the bacterial cells are resuspended in methanol, ultrasonic extraction is performed 2 to 4 times, the extracts are combined, the extracts are concentrated under reduced pressure to remove methanol, and a crude extract is obtained. The crude extract is applied to an MCI chromatographic column, and a methanol-water system is selected for gradient elution to obtain 5 fractions FrB.1-FrB.5; fraction FrB.1 is taken and subjected to Sephadex LH-20 gel column chromatography, and isocratic elution is performed with pure water as the mobile phase to obtain 4 sub-fractions FrB.9.1-FrB.9.4; sub-fraction Fr.9.4 is taken and subjected to semi-preparative RP-HPLC to obtain compound B; Wherein, during each ultrasonic extraction, the volume ratio of the bacteria to methanol is 1:1 to 1:1.5; The filler of the MCI chromatographic column is HP20; the methanol-water system is water, 30% methanol, 60% methanol, 90% methanol, and 100% methanol; The semi-preparative RP-HPLC conditions were: Capcell Pak MGⅡC18 column (5 μm; 4.6×250 mm), mobile phase: 1.5% MeOH-H2O, isocratic elution, flow rate 3 mL·min -1 .

4. A method for producing C-glycoside compounds, characterized in that: The following steps are involved: Step (a1), constructing the gene G with the nucleotide sequence as shown in SEQ ID NO.1 into the pET-28a(+) plasmid vector to obtain the recombinant plasmid pET-28a(+)-1, and transforming the recombinant plasmid pET-28a(+)-1 into Escherichia coli BL21(DE3) competent cells to obtain recombinant Escherichia coli; Step (a2), inoculating the recombinant Escherichia coli described in step (a1) in a culture medium containing kanamycin sulfate, adding isopropylthiogalactoside to induce the expression of the recombinant Escherichia coli protein to obtain a strain producing compound A with a structure as shown in formula I, and then adding 3,5-dihydroxybenzyl alcohol as a substrate to the culture medium to obtain compound A; 5. The method for producing C-glycoside compounds according to claim 4, characterized in that: In step (a2), the culture medium is LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, natural pH; Add 0.01-0.1 g of 3,5-dihydroxybenzyl alcohol to every 1 L of culture medium.

6. The method for producing C-glycoside compounds according to claim 4, characterized in that: The extraction and separation method of compound A is as follows: after the culture is completed, the culture is centrifuged, the bacterial cells are collected, the bacterial cells are resuspended in methanol, ultrasonic extraction is performed 2 to 4 times, the extracts are combined, the extracts are concentrated under reduced pressure to remove methanol, and a crude extract is obtained. The crude extract is applied to an MCI chromatographic column, and a methanol-water system is selected for gradient elution to obtain 5 fractions FrA.1-FrA.5; fraction FrA.1 is taken and subjected to Sephadex LH-20 gel column chromatography, and pure water is used as the mobile phase for isocratic elution to obtain 4 subfractions FrA.9.1-FrA.9.4; subfraction Fr.9.2 is taken and subjected to semi-preparative RP-HPLC to obtain compound A; Wherein, during each ultrasonic extraction, the volume ratio of the bacteria to methanol is 1:1 to 1:1.5; The filler of the MCI chromatographic column is HP20; the methanol-water system is water, 30% methanol, 60% methanol, 90% methanol, and 100% methanol; The semi-preparative RP-HPLC conditions were: Capcell Pak MGⅡC18 column (5 μm; 4.6×250 mm), mobile phase: 1.0% MeOH-H2O, isocratic elution, flow rate 3 mL·min -1 .

7. A gene G, characterized in that: The nucleotide sequence of gene G is shown in SEQ ID NO.

1.

8. A gene K, characterized in that: The nucleotide sequence of gene K is shown in SEQ ID NO.

2.

9. A biomaterial, characterized in that: The biological material is a biological material comprising the gene G according to claim 7 and / or the gene K according to claim 8, and the biological material is an expression cassette, a plasmid vector, an engineered bacterium or a transgenic cell line; preferably, the biological material is a biological material comprising the gene G according to claim 7, or a biological material comprising the gene G according to claim 7 and the gene K according to claim 8; more preferably, the biological material is at least one of (1) to (14): (1) an expression cassette containing the gene G; (2) a recombinant plasmid vector containing the gene G; (3) a recombinant plasmid vector containing the expression cassette described in (1); (4) an engineered bacterium containing the gene G; (5) An engineered bacterium containing the expression cassette described in (1); (6) an engineered bacterium containing the recombinant plasmid vector described in (2); (7) An engineered bacterium containing the recombinant plasmid vector described in (3); (8) an expression cassette containing the gene G and gene K; (9) a recombinant plasmid vector containing the gene G and gene K; (10) a recombinant plasmid vector containing the expression cassette described in (8); (11) An engineered bacterium containing the gene G and gene K; (12) An engineered bacterium containing the expression cassette described in (8); (13) An engineered bacterium containing the recombinant plasmid vector described in (9); (14) An engineered bacterium containing the recombinant plasmid vector described in (10).

10. Use of the gene G according to claim 7 in preparing a C-glycoside compound A with a structure as shown in formula I, or in constructing a recombinant plasmid vector for producing a C-glycoside compound A, or in constructing a recombinant Escherichia coli for producing a C-glycoside compound A; or use of the gene G according to claim 7 and the gene K according to claim 8 in preparing a C-glycoside compound B with a structure as shown in formula II, or in constructing a recombinant plasmid vector for producing a C-glycoside compound B, or in constructing a recombinant Escherichia coli for producing a C-glycoside compound B;

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