CN114940982A - Apigenin prepared from genetically engineered bacteria and application thereof in polyester fiber manufacturing - Google Patents
Apigenin prepared from genetically engineered bacteria and application thereof in polyester fiber manufacturing Download PDFInfo
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Abstract
Apigenin prepared from genetically engineered bacteria and its application in polyester fiber production are provided. The invention provides an optimized flavone synthase and a coding gene thereof, the flavone synthase is modified and optimized according to an escherichia coli expression system, and a recombinant escherichia coli engineering bacterium is obtained, compared with the recombinant escherichia coli obtained without gene modification, the yield and the conversion rate of apigenin production are obviously improved, the production cost of polyester fiber manufacturing is greatly reduced, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of metabolic engineering and synthetic biology, and relates to a genetically engineered bacterium for producing apigenin and application thereof.
Background
Apigenin (AP) is a natural plant flavonoid with chemical structure of 4', 5, 7-trihydroxyflavone, also called apigenin and apigenin, and molecular formula C 15 H 10 O 6 Is low molecular weight flavone (relative molecular mass 270.24), and melting point 347.5 deg.C; insoluble in water, moderately soluble in hot ethanol, soluble in diluted KOH and dimethyl sulfoxide (DMSO). Apigenin is mainly found in vegetables and fruits, such as celery, tea, onion, etc. The content of celery root is about 75 mg/kg. It can be isolated and identified by spectroscopy, chromatography, mass spectrometry, etc. The apigenin structure is shown as follows:
at present, the method for obtaining apigenin mainly adopts a plant extraction method and a chemical synthesis method, wherein the plant extraction method adopts a proper solvent or means and takes plants (all or a part of the plants) as raw materials for extraction or processing to obtain the apigenin. Taking apigenin extracted from celery as an example, picking a proper amount of celery leaves, drying at low temperature for 24h, crushing, extracting with 70% ethanol under reflux for 2h according to a material-liquid ratio of 1: 10, performing rotary evaporation and concentration on an extracting solution to obtain an extract, and separating and purifying the extract by sequentially passing through a macroporous resin (XDA-1), a polyamide resin and a sephadex LH-20 chromatographic column to obtain three flavone compound monomers, namely apigenin, apigenin and chrysoeriol-7-0-glucose-2-0-apioside. Huangfeijian et al extract apigenin from celery with 70% ethanol, and find that when the proportion of feed liquid is 1: 4, the extraction time is 3 hours, and when the reflux extraction is carried out twice, the content of apigenin in the obtained celery extract is 1.41 percent. Although apigenin is widely distributed in most plants, the content of apigenin in the plants is not very high, the traditional extraction method is simple to operate, but needs long time, needs a large amount of plant materials, is limited by factors such as the growing season and the growing region of the plants, is long in extraction time, high in cost, needs to improve the extraction rate and the purity, needs to consume a large amount of organic reagents, and has serious damage to human bodies and the environment. In recent years, extraction methods such as the method of cracking celery by using a combination of ultrasonic waves and glucosidase to improve the extraction efficiency of apigenin have been improved and optimized, however, the yield is still not high, and special instruments and equipment are required. The method of plant extraction is not suitable for the mass acquisition of apigenin. The chemical synthesis method basically adopts a semi-synthesis method using natural plant monomers as raw materials. For example, the xiao Min uses naringin as raw material, adds 1, 4-dioxane solvent, adds oxydichlordicyanobenzoquinone (DDQ) as oxidant at 95 deg.c, stirs and reacts for 8h, stands for 12h at room temperature, produces apigenin glucoside crude product, then uses 4% sodium hydroxide solution to dissolve apigenin glucoside crude product, adds alcohol to adjust pH to 5-6 (temperature should be kept at 70 deg.c to 75 deg.c before adding acid), stands to separate out crystal, uses methanol/water mixed solution to recrystallize for 2 times to obtain pure apigenin, adds 6% hydrochloric acid solution with 7 times volume to stir for 3.5h at 95 deg.c to 100 deg.c, stops stirring after reaction, adjusts pH to 5-6, stands for 4h, after precipitation, liquid is in clear liquid state, suction filters to obtain apigenin crude product, then uses 4% sodium hydroxide solution to dissolve, adds methanol, adjusting pH value to 6 with hydrochloric acid, precipitating crystal, and filtering. Then refluxing and purifying by ethanol to obtain the purer apigenin. The naringin used as the substrate in the method is also a dihydroflavonoids compound. Xiaojinxia et al chemically synthesize apigenin, mainly involving two steps: using p-hydroxyacetophenone as a substrate to prepare p-hydroxyacetoacetate and prepare apigenin through cyclization reaction. The steps involved in synthesizing the flavonoid compound by the chemical method are multiple, the process is complex, the yield is not high, the extremely harsh reaction conditions are also limiting factors, more toxic reagents are used in the synthesis process, and the purification difficulty of the required compound is increased by more byproducts.
Although the existing biosynthesis method for producing the apigenin through genetic engineering exists, the existing biosynthesis still remains at the level of using natural products, and the satisfactory effect cannot be achieved in both yield and substrate conversion rate, so that the production cost is high. Therefore, there is an urgent need to develop a production method capable of obtaining apigenin at an excessively high yield.
Disclosure of Invention
In order to fill the blank of the prior art, the invention provides optimized flavone synthase (FNS-I) and a coding gene thereof, and the apigenin conversion rate of recombinant escherichia coli is remarkably improved by constructing a genetic engineering strain. In order to achieve the technical effects, the invention provides the following technical scheme:
in the first aspect of the invention, an optimized flavone synthase is provided, and the coding gene of the flavone synthase is shown in any one of SEQ ID NO. 2-4.
In a second aspect of the present invention, there is provided a recombinant host cell expressing the gene encoding flavone synthase described above.
In one embodiment, the recombinant host cell is E.coli.
In a third aspect of the invention, the application of the flavone synthase or the recombinant host cell in the production of apigenin and downstream products thereof is provided.
In one embodiment, the downstream product comprises a biofabric material comprising apigenin as described above.
In a fourth aspect of the present invention, there is provided a method for efficiently producing apigenin, which comprises transforming the recombinant host cell described above in a substrate containing naringenin.
Compared with the prior art, the invention achieves the following remarkable improvements:
the invention modifies and optimizes the existing flavone synthase aiming at an escherichia coli expression system, and obtains the recombinant escherichia coli, compared with the recombinant escherichia coli obtained without gene modification, the yield and the conversion rate of the apigenin are obviously improved, and the production cost is greatly reduced.
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The invention is described in further detail below with reference to the attached drawing figures:
FIG. 1 shows the synthesis route of apigenin.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 FNS-I sequence optimization
1. Experimental materials
pETDuet-1 vector: purchased from Youbao organisms; plasmid extraction kit, gel recovery kit: purchased from OMEGA; restriction enzymes were purchased from NEB.
The standard compounds apigenin (CAS number: 520-36-5) and naringenin (CAS number: 480-41-1) are purchased from Shanghai leaf Biotech limited, and have purity of more than 98%. Other reagents are domestic analytical pure or chromatographic pure reagents and are purchased from national pharmaceutical group chemical reagent limited.
Clone competent cell DH5 α, expression competent cell Rosetta (DE 3): purchased from holo-gold.
Liquid phase detection conditions: phase A: 0.1% formic acid water, phase B: acetonitrile; separation conditions are as follows: 0-20min 20% B phase-55% B phase, 20-22min 55% B phase-100% B phase, 22-27min 100% B phase, 27-35min 100% B phase-20% B phase, 35-40min, 20% B phase; detection wavelength: 340nm, column temperature: at 30 ℃. A chromatographic column: thermo syncronis C18 reversed phase column (250 mm. times.4.6 mm, 5 μm).
FNS-I sequence modification:
based on the codon preference of escherichia coli, GC content, codon balance and the like are comprehensively considered, an original FNS-I sequence (SEQ ID NO.1 is optimized to obtain 3 optimized sequences FNS-IM1, FNS-IM2 and FNS-IM3, and the nucleotide sequences are respectively shown in SEQ ID NO. 2-4.
Example 2 construction of the engineered bacteria
1. Respectively adding NcoI and BamHI at two ends of the three optimized FNS-I gene sequences and the original FNS-I gene sequence, synthesizing the optimized FNS-I gene by using a gene synthesis technology, and constructing cloning vectors pUC57FNS-IM1, pUC57FNS-IM2, pUC57FNS-IM3 and pUC57 FNS-I;
2. constructing recombinant expression vector, double-digesting pETDuet-1 plasmid and obtained pUC57FNS-IM1, pUC57FNS-IM2, pUC57FNS-IM3 and pUC57FNS-I plasmid with NcoI and BamHI, glue recovering to obtain pETDuet-1 and FNS-IM1, FNS-IM2, FNS-IM3 and pUC57FNS-I fragments, connecting pETDuet-1 with FNS-IM1, FNS-IM2, FNS-IM3 and pUC57FNS-I respectively with T4DNA library, transforming 5 alpha competent cell with the connection product, coating ampicillin resistance plate, inverting overnight culture at 37 deg.C, screening to obtain positive recombinant, extracting plasmid to obtain pETDue-pEIM 1 plasmid, TDS-IM 2, FNS-IM3, FNS-3-IM plasmid;
3. respectively transforming the pETDue-FNS-IM1 plasmid, pETDue-FNS-IM2 plasmid, pETDue-FNS-IM3 plasmid and pETDue-FNS-I plasmid obtained in the above steps into escherichia coli Rosetta (DE3), coating an ampicillin resistance plate, inverting overnight at 37 ℃, screening to obtain positive recombinants, namely obtaining FNS-IM1, FNS-IM2, FNS-IM3 and FNS-I engineering bacteria respectively, and preserving the licorice root tube.
Example 3 production of apigenin by fermentation of engineering bacteria
FNS-IM1, FNS-IM2, FNS-IM3 and FNS-I engineering bacteria are respectively subjected to amplification culture according to the following methods:
(1) under the aseptic condition, taking a ring strain from a glycerol tube by using an inoculating ring, inoculating the ring strain into a primary seed culture medium, and culturing at 37 ℃ and 240rpm until the OD600 is between 3.0 and 4.0 to obtain primary seed bacterial liquid;
(2) transferring the primary seed bacterial liquid into a secondary seed culture medium according to the inoculation amount of 6-10%, and culturing at 37 ℃ and 240rpm for 3-5 h to obtain secondary seed bacterial liquid;
(3) inoculating the cultured secondary seed bacterial liquid into a fermentation tank according to the inoculation amount of 6-10%, wherein the initial fermentation parameters are as follows: the temperature is 37 ℃, the rotating speed is 200rpm, the pH value is 7.0, the ventilation volume is 50L/h, and the tank pressure is 0.05 MPa; along with the prolonging of the fermentation time, the bacteria concentration is gradually increased, and the dissolved oxygen is controlled to be 20-40% by adjusting the rotating speed and the ventilation quantity; when the dissolved oxygen and the pH value are simultaneously and rapidly increased, feeding materials in a flowing manner is started; and when the OD600 reaches 20-25 ℃, reducing the temperature to 30 ℃, adding 0.2mM inducer, continuing to ferment for 8-12 h, putting the mixture into a tank, collecting the thalli through low-temperature centrifugation, and refrigerating the thalli.
2. Identifying the production capacity of the strain on apigenin:
(1) after FNS-IM1, FNS-IM2, FNS-IM3 and FNS-I engineering bacteria are washed by potassium phosphate buffer solution with pH of 6, cells are respectively placed in the following transformation systems for apigenin transformation (three groups are repeated): the transformation system comprises:
5% of wet bacteria of engineering bacteria, 0.1M potassium phosphate buffer, 2.0M M naringenin (apigenin synthesis substrate, figure 1), 5mM ascorbate and 10 μ MFeSO 4 The pH was adjusted to 6 with 2mol/L NaOH solution and the conversion was carried out at a temperature of 35 ℃ and 150rpm for 10 hours.
The substrate conversion rate is calculated by the substrate content existing in a conversion system, the naringenin content is calculated by preparing naringenin standard products with different concentration gradients (0, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15 and 20g/L), measuring an A240 value and drawing a standard curve.
(2) And (3) conversion result:
TABLE 1
| Apigenin product concentration (mg/L) | Conversion (%) | |
| FNS-IM1 | 108.55±2.67 | 87 |
| FNS-IM2 | 121.34±1.89 | 98 |
| FNS-IM3 | 101.46±2.21 | 82 |
| FNS-I (control group) | 88.67±1.87 | 72 |
The results in the table 1 show that the optimized flavone synthases FNS-IM1, FNS-IM2 and FNS-IM3 have conversion rates superior to those of the original sequence FNS-I, wherein FNS-IM2 has the optimal conversion capacity, and the conversion rate can reach 98%.
EXAMPLE 4 preparation of Terylene macrobio fiber containing apigenin component
Large biological fiber: bioactive molecules are added into fibers (cotton, hemp, wool, silk, viscose, polyester, nitrile and brocade) for modification, so as to produce the active fiber with biological functions. The polyester macrobiotic fiber containing apigenin component prepared by the invention is active fiber with the functions of antibiosis, antivirus and the like, which is produced by adding apigenin component in the production process of polyester fiber.
1. Preparation of SiO by sol-gel method 2 Nano-microspheres:
weighing 0.5g of CTAB, dissolving in 1000mL of deionized water, stirring at 40 ℃, dropwise adding a NaOH solution after the solution is clarified, and adjusting the pH to 11.8; the temperature was then raised to 80 ℃; after 30min, 2.5mL TEOS is added, and stirring is continued for 2h at 80 ℃; standing the obtained white suspension, cooling to room temperature, centrifuging at a rotating speed of 9000r/min, repeatedly washing with deionized water and absolute ethyl alcohol for 3 times, drying the obtained white gel in a vacuum drying oven for 12 hours, placing the dried sample in a muffle furnace, and calcining at 550 ℃ for 6 hours; cooling to obtain white solid powder which is mesoporous SiO 2 And (3) nanoparticles. Grinding and crushing for later use to prepare the mesoporous SiO 2 The granularity of the nano particles is 100 +/-10 nm, and the aperture is 2.5 +/-0.5 nm; 2. concentrating and purifying the apigenin produced in the example 3 according to a conventional method in the field to obtain a pure apigenin product (the purity is more than or equal to 95%), ultrasonically dissolving the pure apigenin product by using a 95% ethanol solution to obtain a 30% apigenin suspension, and weighing 10g of mesoporous SiO prepared in the step 1 2 Sequentially adding the nano powder and 0.02g of dispersant XD-5040 into 500mL of deionized water, and shearing and dispersing by using a magnetic stirrer for 250 r/min; obtaining SiO containing apigenin component 2 Nano composite dispersion liquid, volatilizing the solvent of the dispersion liquid to obtain mesoporous SiO containing apigenin component 2 Grinding and crushing molecular nest (the particle size is 9000 meshes) for later use;
firstly, the mesoporous SiO containing apigenin component prepared in step 2 2 Vacuum drying the molecular nest and the polyester chip at 180 ℃ for 24 h; then, according to the following steps of 1: 10, 0.1 percent of dispersant XD-5040, 0.5 percent of antioxidant 1098 and 0.2 percent of coupling agent KH-550 are added into a double-screw extruder for mixing and granulation, the melting temperature is controlled at 260-280 ℃, and the screw rotating speed is controlled at 260 r/min; obtaining a master batch containing apigenin; and 3, mixing the master batch containing the apigenin component with common polyester chips according to the weight ratio of 1: ratio of 20Adding the obtained mixture together with 0.2% of dispersant PT-200E, 0.2% of antioxidant KB-6 and 0.15% of coupling agent Z-6020 into a double-screw extruder for mixing and spinning, wherein the spinning speed is 1700m/min, the stretching temperature is 80 ℃, the side-blowing cooling air temperature is 30 ℃, the side-blowing air speed is 0.5m/s, the total drafting multiplying factor is 5 times, and the winding speed is 1050m/min, so as to prepare the polyester macrobio-fiber containing apigenin component.
The polyester macrobiotic fiber containing the apigenin component is made into a fiber fabric through a spinning production line, the bacteriostasis rate of the fiber fabric containing the apigenin component is tested according to GB/T20944.2-2007, and the bacteriostasis rate of the fiber fabric after 100 times of standard washing (GB/T8629-2017) is achieved. The results are shown in table 2 below.
TABLE 2
Through detection, the fabric obtained by spinning the polyester macrobio-fiber containing the apigenin component has obvious inhibition effects on staphylococcus aureus, escherichia coli and candida albicans on the surface of human skin, and still has excellent antibacterial property after being washed for many times.
Unless otherwise stated, the proportions described in the present examples are mass proportions, and the percentages described are mass percentages.
Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Baicao Bingzhi DaBiotech (Qingdao) Co., Ltd
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atttctttag ctggtatcga tgatgattcc aatggcagga gacctgaagt gtgtcgtaaa 180
atagtggaag ccttcgaaaa ctgggggatt ttccaggtag ttgatcatgg tattgacagc 240
ggtttgatcg cggaaatgtc tcgtctgtct cgtgaattct ttgctttgcc tgccgaagaa 300
aaacttcggt atgatattat tggtggaaaa agaagcggct tcattatctc cattcatctt 360
cagggtgacg atgtgaaaga ttggcgtgaa tttgttgttt atttttcgta cccagtcgat 420
gctcgggact actcgaaatg gcctgataaa ccagaaggat ggaagtctgt tatggaagtt 480
tatagtgaaa agttgatggc gctaggtgcc aaattattgg aaatgctatc agaagccatg 540
gggcttgaaa aaaaggctct tatagaagct tgtgtgaaca tggaacagaa agtgttgatt 600
aattattatc ctatatgtcc ccagccggac ttgacatttg gaatcagaaa gcatacggat 660
ccgggtatga ttatcatttt gcttcaggac atggttgggg ggttataggc tatcagggat 720
ggcggcagaa cttggattat tgttcagcct gtcgaaggaa cttttgtcgt cagtttgggt 780
gatcatggtc attatttgaa cagtggaaag ttcagaaatg ccgatcatca agcagtagtg 840
aattcagctt ctagcagatt gtctatcgca actttccaga acccggctca gaatgctata 900
gtgtatccat taaaaatcag ggaaggcgaa aaaccagttc ttgaagaagc catgacatat 960
gccgaaatgt ataaaaaaaa catgactaaa catattgaag tggctatcca gaaaaaattg 1020
gccagaaaga aaaaattgca gaacgaaaaa gccagactgg agacgaaatt ttag 1074
<210> 4
<211> 1074
<212> DNA
<213> Artificial Sequence
<400> 4
atggctccaa caactattac tgcattggcc aaggtaaaaa catttaactc tgattttgtc 60
cgggttgagg atgagcgtcc caaagttgcc tacaatcaat tcagcattga aattcccatt 120
atttctttaa ctggtatcga tgatgattcc aatggcaggt gacctgaggt gtgtcgtaaa 180
ataatggtgg ccttcgaaga ctgggggttt ttccaggtaa ttgatcatgg tattgacagc 240
ggtttgatcg cggtaatgtc tcgtctgtct cgtgaattct ttgctttgcc tgccgaggtg 300
aaacttcggt atgatactac tggtggtaag agaggcggct tcattatctc cattcatctt 360
cagggtgacg atgtgaaggt ttggcgtgag tttgttgttt atttttcgta cccagtcgat 420
gctcgggtct actcgagatg gcctgataag ccagagggtt ggtggtctgt tacggtggtt 480
tataatgaga agttgatggc gctaagtgcc aagttactgg aagtgctatc agaggccatg 540
gggcttgaaa aagaggctct tacagaggct tgtgtgaaca tggtacagaa agtgttgatt 600
aattactatc ctacatgtcc ccaaccggtc ttgacatttg gagtcagaag gcatacggtt 660
ccgggtacga ttaccatttt gcttcaggtc atggttgggg ggttacaggc taccagggtt 720
ggcggcaaaa cttggtttac tgttcagcct gtcgagggtg cttttgtcgt caatttgggt 780
gatcatggtc attatttgag caatggtagg ttcaagaatg ccgatcatca agcagtaatg 840
aattcaactt ctaacagatt gtctatcgca actttccaga acccggctca gaatgctata 900
gtgtatccat taaagatcag ggtgggcgag aagccaattc ttgaggtggc catgacatac 960
gccgagatgt ataagaaaaa catgactaaa catattgagg tggctaccca gaagaaattg 1020
gccaaggtga aaagattgca gaacgagaag gccaagctgg agacgaaatt ttaa 1074
Claims (7)
1. An optimized flavone synthase, wherein the coding gene of the flavone synthase is shown as any one of SEQ ID NO. 2-4.
2. A recombinant host cell expressing the gene encoding the flavone synthase of claim 1.
3. The recombinant host cell of claim 2, wherein the recombinant host cell is e.
4. Use of a flavone synthase as defined in claim 1 or a recombinant host cell as defined in any one of claims 2-3 for the production of apigenin and its downstream products.
5. The use as claimed in claim 4, wherein the downstream product is a biofiber material containing apigenin.
6. The use of claim 5, wherein the biofiber material is dacron fiber.
7. A method for efficiently producing apigenin, comprising transforming the recombinant host cell of claim 2 or 3 in a substrate comprising naringenin.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001005981A1 (en) * | 1999-07-19 | 2001-01-25 | Suntory Limited | Genes coding for flavone synthases |
| US20060019334A1 (en) * | 2004-07-10 | 2006-01-26 | Mattheos Koffas | Production of flavonoids by recombinant microorganisms |
| CN110616204A (en) * | 2019-09-27 | 2019-12-27 | 佛山市汇腾生物技术有限公司 | Flavone synthase and application thereof |
| CN110616162A (en) * | 2019-09-27 | 2019-12-27 | 佛山市汇腾生物技术有限公司 | Pichia pastoris for expressing flavone synthase |
| CN112725256A (en) * | 2021-02-22 | 2021-04-30 | 湖南省农产品加工研究所 | Recombinant escherichia coli and method for biosynthesizing diosmetin by using recombinant escherichia coli |
-
2022
- 2022-06-08 CN CN202210643356.1A patent/CN114940982B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001005981A1 (en) * | 1999-07-19 | 2001-01-25 | Suntory Limited | Genes coding for flavone synthases |
| US20060019334A1 (en) * | 2004-07-10 | 2006-01-26 | Mattheos Koffas | Production of flavonoids by recombinant microorganisms |
| CN110616204A (en) * | 2019-09-27 | 2019-12-27 | 佛山市汇腾生物技术有限公司 | Flavone synthase and application thereof |
| CN110616162A (en) * | 2019-09-27 | 2019-12-27 | 佛山市汇腾生物技术有限公司 | Pichia pastoris for expressing flavone synthase |
| CN112725256A (en) * | 2021-02-22 | 2021-04-30 | 湖南省农产品加工研究所 | Recombinant escherichia coli and method for biosynthesizing diosmetin by using recombinant escherichia coli |
Non-Patent Citations (2)
| Title |
|---|
| GENBANK: AY817675.1: "Daucus carota flavone synthase I mRNA, complete cds", 《GENBANK》 * |
| NGUYEN HUY THUAN 等: "Engineering co-culture system for production of apigetrin in Escherichia coli", 《J IND MICROBIOL BIOTECHNOL》 * |
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