CN106350564A - Synthesis method of terpenoid and glycosylation products thereof in synthesis route of mogrol - Google Patents
Synthesis method of terpenoid and glycosylation products thereof in synthesis route of mogrol Download PDFInfo
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- CN106350564A CN106350564A CN201510425384.6A CN201510425384A CN106350564A CN 106350564 A CN106350564 A CN 106350564A CN 201510425384 A CN201510425384 A CN 201510425384A CN 106350564 A CN106350564 A CN 106350564A
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Abstract
The invention discloses a microbial fermentation method for synthesizing triterpene compounds, namely a distillers yeast recombination strain SY2 containing cucurbitane dienol synthetase SgCbQ, P450 CYP87D18 and cytochrome reductase CPR, in the metabolism route of mogrol, which takes dextrose as a substrate to synthesize three new products including 11-oxygen-cucurbitane dienol, 11-hydroxy-cucurbitane dienol and 11-oxygen-24, 25-epoxy-cucurbitane dienol. The invention further discloses a metabolism route for synthesizing mogrol through cucurbitane dienol, namely the cucurbitane dienol is catalyzed through P450 CYP87D18 and synthesized into 11-oxygen-cucurbitane dienol, 11-oxygen-cucurbitane dienol is catalyzed through enzyme and synthesized into 11-hydroxy-cucurbitane dienol and 11-oxygen-24, 25-epoxy-cucurbitane dienol, and 11-hydroxy-cucurbitane dienol and 11-oxygen-24, 25-epoxy-cucurbitane dienol can be synthesized into the mogrol through other P450 or epoxide hydrolase. The invention further discloses a novel thought for obtaining more glycosylation products by catalyzing new products through glycosyl transferase. The method has significance for determining the synthesis route of mogrol and synthesizing relevant intermediate metabolites and glycosylation products through the microbial fermentation method.
Description
Technical field
The present invention relates to biological technical field and in particular in momordica grosvenori alcohol synthesis path three kinds of terpenoids and its glycation product synthetic method.Use the recombinant Saccharomyces cerevisiae bacterial strain containing cucurbitane dienol synzyme and cytochrome p450, three kinds of terpenoids are synthesized for substrate with glucose, these three terpenoids are respectively 11- hydroxyl-cucurbitane dienol through mass spectrum and nuclear-magnetism identification, 11- oxygen-cucurbitane dienol, 11- oxygen -24,25- epoxy-cucurbitane dienol；These three terpenoids are combined to multiple glycation products through glycosyl further.
Background technology
Fructus Momordicae (siraitia grosvenorii) it is precious medicinal plants specific to China, it is distributed mainly on China Guangxi province, there is relieving cough and moistening lung, the function such as resolving sputum is enriched blood, loosening bowel to relieve constipation, blood stasis dispelling (indra prakash and venkata
sai prakash chaturvedula. additional new minor cucurbitane
glycosides from siraitia grosvenorii.
Molecules. 2014.19:3669-3680), first " integration of edible and medicinal herbs kind list " is listed in by Ministry of Public Health and the State Administration of Traditional Chinese Medicines.Fructus Momordicae extract main component is Momordica-Glycosides, it is a kind of high sugariness, low-energy natural sweetener, there is the effect such as blood sugar lowering, antioxidation, anticancer, antiviral simultaneously and can be widely applied to (chun-hui chiu in the industries such as nutrient and healthcare products, food, medicine, reuben wang, cho-ching lee, yi-chen lo, ting-jang lu. biotransformation of mogrosides from siraitia grosvenorii swingle by saccharomyces cerevisiae. journal
of agricultural and food chemistry. 2013.61:7127-7134).In Momordica-Glycosides, four kinds of triterpenoid compound contents are more, respectively mogroside v, mogroside, siamenoside i, 11- oxygen-mogroside；Additionally, also having some triterpenoid compound to have critically important function.
Momordica-Glycosides mainly extract from Fructus Momordicae at present, are affected by planting conditions and low extraction ratio, Momordica-Glycosides reach far away the market demand.With the development of metabolic engineering and synthetic biology technology, microbe fermentation method synthesis terpenoid is widely studied, some compounds such as arteannuin, and paclitaxel can be combined to by microbial fermentation mode scale.Biosynthetic pathway for Momordica-Glycosides, existing document report cucurbitane dienol synthesis enzyme catalysiss triterpene skeleton 2,3 oxidosqualene synthesis cucurbitane dienols, the latter obtains momordica grosvenori alcohol through three step p450 oxidations, obtain Momordica-Glycosides (qi tang, xiaojun through c-3 and c-24 glycosylation further
ma, changming mo, iain w wilson, cai
song, huan zhao, yanfang
yang, wei fu, deyou qiu. an efficient approach to finding siraitia
grosvenorii triterpene
biosynthetic genes by rna-seq and digital gene
Expression analysis. bmc genomics 2011.12:343).At present through 2,3- oxidosqualene synthesis cucurbitane dienol and to be combined to this two parts synthesis path ratio of Momordica-Glycosides through momordica grosvenori alcohol glycosyl more visible, relevant enzyme has been reported, for example with the glycosyl transferase catalysis momordica grosvenori alcohol synthesis Momordica-Glycosides i(dai l from arabidopsiss, liu c
zhu y, et al. functional characterization of cucurbitadienol
synthase and triterpene glycosyltransferase involved in biosynthesis of mogrosides from siraitia grosvenorii. plant and cell physiology. 2015. yaoquan liu, jung yeop lee,
monika khare. methods and materials for enzymatic
Synthesis of mogroside compounds. us 20140308698 a1.)；But catalysis cucurbitane dienol synthesizes the cytochrome p450 report of momordica grosvenori alcohol seldom, comparative structure finds, cucurbitane dienol needs through c-11 hydroxylating or oxidation, c-24 and c-25 hydroxylating just can obtain momordica grosvenori alcohol, have been reported that proposing cucurbitane dienol synthesizes the possible synthetic route of momordica grosvenori alcohol, but do not carry out accurate validation (yaoquan liu
jorgen hansen, jens houghton-larsen, muthuswamy panchapagesa murali, sathish kumar, nina nicoline rasmussen. methods and materials for biosynthesis
Of mogroside compounds. us 20150064743 a1).
Cucurbitane dienol generates c-11- hydroxyl-cucurbitane dienol after p450 mono-oxygenase catalysis c-11 hydroxylating, and so this compound can be modified through glycosylase and obtain c-3 or c-11 glycation product.By that analogy, during cucurbitane diene alcohol catalysis obtain momordica grosvenori alcohol, produced hydroxyl all may be combined to new product through glycosyl in theory.Therefore probe into and determine that cucurbitane dienol synthesizes the mesostate in momordica grosvenori alcohol path, and its glycosylation is obtained with the central concept that new product is the present invention.
Content of the invention
An object of the present invention is to provide the synthetic method being synthesized three kinds of triterpenoid compound in momordica grosvenori alcohol path by cucurbitane dienol.Specifically will derive fromsiraitia grosvenoriiCytochrome p450 gene cyp87d18, cucurbitane dienol synzyme cbq and derive fromarabidopsis thalianaCytochrome reductase (cpr) enzyme gene be introduced in saccharomyces cerevisiae by4741, obtain recombinant Saccharomyces cerevisiae bacterial strain sy2；Gained recombinant bacterial strain sy2 synthesizes three kinds of triterpenoid compound with glucose for substrate, and these three compounds are defined as 11- oxygen-cucurbitane dienol, 11- hydroxyl-cucurbitane dienol, 11- oxygen -24,25- epoxy-cucurbitane dienol through mass spectrum and nuclear-magnetism checking.
The second object of the present invention is to propose the metabolic pathway being synthesized momordica grosvenori alcohol by cucurbitane dienol.Specifically cucurbitane dienol synthesizes 11- oxygen-cucurbitane dienol first through cytochrome p450 cyp87d18 catalysis, the latter catalyzes and synthesizes 11- hydroxyl-cucurbitane dienol and 11- oxygen -24 further through p450 enzyme cyp87d18,25- epoxy-cucurbitane dienol, 11- hydroxyl-cucurbitane dienol can be throughsiraitia grosvenoriiIn other kinds of p450 is oxidase catalyzed is hydroxylated synthesis momordica grosvenori alcohol respectively in c-24 and c-25；11- oxygen -24,25- epoxy-cucurbitane dienol can be throughsiraitia grosvenoriiIn other kinds of epoxide hydrolase (epoxide hydrolase) synthesize momordica grosvenori alcohol.Being proposed for of this synthesis path accurately determines that cucurbitane dienol synthesis momordica grosvenori alcohol synthesis path and the related intermediate product of microbial fermentation processes synthesis are significant.
The third object of the present invention be triterpenoid compound 11- oxygen-cucurbitane dienol is proposed, 11- hydroxyl-cucurbitane dienol, 11- oxygen -24,25- epoxy-cucurbitane dienol catalyze and synthesize the thinking of new product through glycosyl transferase.Specifically include following several aspect:
First, can be catalyzed through glycosyl transferase through triterpeness product 11- hydroxyl-cucurbitane dienol that recombinant Saccharomyces cerevisiae bacterial strain sy2 fermentation obtains and obtain c-3 glycosylation, c-11 glycosylation, or c-3 and c-11 glycosylated product simultaneously, glycosylated glucose can be one or more, as shown in Figure 1.
Secondly, can be catalyzed through glycosyl transferase through triterpeness product 11- oxygen-cucurbitane dienol that recombinant Saccharomyces cerevisiae bacterial strain sy2 fermentation obtains and obtain c-3 glycation product, glycosylated glucose can be one or more, as shown in Figure 1.
Again, triterpeness product 11- oxygen -24 obtaining through recombinant Saccharomyces cerevisiae bacterial strain sy2 fermentation, 25- epoxy-cucurbitane dienol can be catalyzed through glycosyl transferase and obtain c-3 glycation product, and glycosylated glucose can be one or more, as shown in Figure 1.
The proposition of this thinking contributes to developing more calabash alkanes glycation products, and these products have important biological activity and physiological function；Meanwhile, function is improved by glycosylation modified its solubility that increases.
Brief description
The synthesis strategy of Fig. 1 calabash alkanes glycation product
Fig. 2 shows the physical map of recombinant yeast expression vector pesc-leu-cbq-cyp87d18 and pyes2-cpr
(a is the fermentation gas chromatogram of saccharomyces cerevisiae recombinant bacterial strain sy1 to tri- kinds of terpenoid gas chromatograms of Fig. 3；B is the fermentation gas chromatogram of saccharomyces cerevisiae recombinant bacterial strain sy2).
(a is product 11- oxygen-cucurbitane dienol to the mass spectrum of tri- kinds of terpenoids of Fig. 4, and corresponding to No. 1 product peak of gas chromatogram in figure, appearance time is 20.27min；B is product 11- hydroxyl-cucurbitane dienol, corresponding No. 2 product peaks of gas chromatogram in figure, and appearance time is 21.31 min；C is 11- oxygen -24,25- epoxy-cucurbitane dienol, corresponding No. 3 product peaks of gas chromatogram in figure, and appearance time is 23.72 min).
(a is the external enzyme reaction of saccharomyces cerevisiae recombinant bacterial strain sy1 gained microsome to Fig. 5 vitro enzyme reaction gas chromatography figure；B is the external enzyme reaction of saccharomyces cerevisiae recombinant bacterial strain sy2 gained microsome；C is 11- hydroxyl-cucurbitane dienol mark product；D is 11- oxygen-cucurbitane dienol mark product).
Specific embodiment
Below in conjunction with the accompanying drawings and be embodied as case the specific embodiment of the present invention is done with detailed narration.Implementation below is merely to illustrate the present invention, but does not limit the range of the present invention.
In following embodiments, method therefor is conventional method if no special instructions, concrete steps can be found in: " molecular cloning:a laboratory manual " (sambrook, j., russell, david w., molecular cloning:a laboratory
Manual, 3rd edition, 2001, ny, cold spring harbor).
In each embodiment, the material of same names used or reagent are identical if no special instructions.The acquirement approach of the various biomaterials described in embodiment is only to provide a kind of approach of experiment acquisition to reach specifically disclosed purpose, should not become the restriction to biological material source when implementing the present invention.It is true that the source of used biomaterial is extensive, any biomaterial that can obtain with moral ethics that keeps on the right side of the law can be replaced according to the prompting in embodiment and use.
In the present invention, the primer is synthesized by Jiangsu Jin Weizhi Bioisystech Co., Ltd, and gene chemical synthesis are synthesized by the Dracocephalum moldabium bio tech ltd in Wuxi.
Embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and specific operating process, and embodiment will be helpful to understand the present invention, but protection scope of the present invention is not limited to following embodiments.Embodiment 1, the structure of saccharomyces cerevisiae recombinant bacterial strain sy2
The structure of saccharomyces cerevisiae recombinant bacterial strain sy2, comprises the following steps:
1st, build saccharomyces cerevisiae recombiant plasmid pesc-leu-cbq
Early stage gene sgcbq(genebank id no:
Hq128567) have been found to as cucurbitane dienol synzyme.By analyzing the use Preference to codon for the saccharomyces cerevisiae, sgcbq gene is carried out after codon optimization, transfer to the Dracocephalum moldabium bio tech ltd in Wuxi to synthesize, the sgcbq gene fragment order such as seq id of synthesis
Shown in no.1；Then by the sgcbq gene of synthesis through xhoi and nhei double digestion, it is attached with the same pesc-leu plasmid through xhoi and nhei double digestion, obtains recombinant expression plasmid pesc-leu-cbq, recombinant plasmid physical map is shown in accompanying drawing 2.Carry out sequence verification by building the carrier pesc-leu-cbq obtaining, related examining order is completed by Jiangsu Jin Weizhi Bioisystech Co., Ltd, after checking is correct, carrier pesc-leu-cbq is placed in -20 preservations.
2nd, build saccharomyces cerevisiae recombiant plasmid pesc-leu-cbq-cyp87d18
According to transcript profile and express spectra data analysiss, excavate from Fructus Momordicae transcript profile data base and obtain annotating the gene (genbank id no. hq128571.1) for cytochrome p450, be named as cyp87d18.Carry out codon optimization for this enzyme gene and transfer to the Dracocephalum moldabium bio tech ltd in Wuxi to synthesize, the cyp87d18 gene fragment order of synthesis is as shown in seq id no.2；The cyp87d18 genetic fragment being obtained, through restriction enzyme site noti and saci, is connected in recombiant plasmid pesc-leu-cbq, obtains recombiant plasmid pesc-leu-cbq-cyp87d18, and its physical map is shown in accompanying drawing 2.Carry out sequence verification by building the carrier pesc-leu-cbq-cyp87d18 obtaining, related examining order is completed by Jiangsu Jin Weizhi Bioisystech Co., Ltd, after checking is correct, carrier pesc-leu-cbq-cyp87d18 is placed in -20 preservations.
3rd, build saccharomyces cerevisiae recombiant plasmid pyes2-cpr
Additionally, will come fromarabidopsis
thalianaCytochrome reductase (cpr) gene (ncbi:nm_118585.3) transfer to after codon optimization the Dracocephalum moldabium bio tech ltd in Wuxi synthesize, the cpr gene fragment order such as seq id no.3 of synthesis, obtained cpr genetic fragment is connected in carrier pyes2 through restriction enzyme site kpni and noti, obtain recombiant plasmid pyes2-cpr, its physical map is shown in that accompanying drawing 2. carries out sequence verification by building the carrier pyes2-cpr obtaining, related examining order is completed by Jiangsu Jin Weizhi Bioisystech Co., Ltd, after checking is correct, carrier pyes2-cpr is placed in -20 preservations.
4th, build saccharomyces cerevisiae recombinant bacterial strain sy2
Above-mentioned recombiant plasmid pyes2-cpr and pesc-leu-cbq-cyp87d18 successfully constructing is transferred in saccharomyces cerevisiae by4741 by lithium acetate transformation method, saccharomyces cerevisiae by4741 after conversion is coated and lacks on uracil and leucic auxotroph screening flat board, be positioned in 30 DEG C of incubators and cultivate 2-3 days.Select the positive colony in screening flat board, it is inoculated in 20 ml sd culture medium, cultivates 24 hours, extract yeast plasmid pcr checking, thus confirming that recombiant plasmid pyes2-cpr and pesc-leu-cbq-cyp87d18 has proceeded in saccharomyces cerevisiae by4741, obtain saccharomyces cerevisiae recombinant bacterial strain sy2.
For obtaining comparison saccharomyces cerevisiae recombinant bacterial strain sy1, the recombiant plasmid successfully constructing pyes2-cpr and pesc-leu-cbq is proceeded in saccharomyces cerevisiae by4741 by lithium acetate transformation method, screening technique is similar with recombinant bacterial strain sy2, verify through pcr and confirm that recombiant plasmid pyes2-cpr and pesc-leu-cbq proceeds in saccharomyces cerevisiae by4741, obtain saccharomyces cerevisiae recombinant bacterial strain sy1.
Embodiment 2, saccharomyces cerevisiae recombinant bacterial strain sy2 synthesize three kinds of cucurbitane type triterpenoid compound with glucose for substrate
Recombinant Saccharomyces cerevisiae sy2 contained in vivo with itself 2,3- oxidosqualene is substrate, obtain cucurbitane dienol through cucurbitane dienol synzyme, the latter catalyzes and synthesizes new product through cytochrome p450 cyp87d18 further, the determination for new product includes procedure below:
1st, ferment wine brewing yeast recombinant strain strain sy2 and sy1
Single bacterium colony is chosen from the flat board containing saccharomyces cerevisiae recombinant bacterial strain sy2 it is inoculated in 20 ml and lack in uracil and leucic yeast synthetic medium (supplement with the addition of 20 g/l glucoses and 13 mg/l chlorhematins), in 30 DEG C, cultivate 48 h under the conditions of 200rpm, so that yeast cells is bred in a large number；Collect cell the deionized water wash three times with sterilizing；It is subsequently transferred to lack in uracil and leucic yeast synthetic medium (supplement with the addition of 20 g/l galactose and 13 mg/l chlorhematins), in 30 DEG C, under the conditions of 200rpm, cultivate 24 h, the expression of induction sgcbq, cyp87d18 and cpr enzyme gene；Collect cell again, with the deionized water wash cell three times of sterilizing；The yeast body weight of collection is hanged to 20
Ml contains in 3% glucose and the yeast synthetic medium of 13 mg/l chlorhematins, in 30 DEG C, continues culture 48 h under the conditions of 200rpm.
New product can be generated for checking saccharomyces cerevisiae recombinant bacterial strain sy2, saccharomyces cerevisiae recombinant bacterial strain sy1 also will adopt the fermentation process of similar recombinant bacterial strain sy2,
2nd, extractive fermentation product
When fermentation terminates, yeast cells are collected by centrifugation, with 5 ml 40%
Saponification 10 min under 85 DEG C of water bath condition of koh/50% ethanol (v/v:1:1), is then extracted 3 times to saponification resultant with the normal hexane of 5 ml, merges n-hexane extraction product, and carry out concentrate drying by vacuum rotary steam to product；Finally the product being dried is dissolved in 500 μ l normal hexane, is analyzed using gc-ms after processing through 0.22 μm of membrane filtration.
Gas chromatograph is Agilent 7890, and mass spectrograph is Agilent q-tof-ms, and analytical column is hp-5ms(30 m × 0.25 mm × 0.5 μm), mobile phase is helium, and split ratio is 100:1, and sample size is 1 μ l, and column oven heating schedule is: 80 DEG C, 1min；20 DEG C/min rises to 300 DEG C；300 DEG C of maintenance 15 min.Mass spectrum is esi source, using positive ion mode.
As gas chromatograph results show (Fig. 3), compared with saccharomyces cerevisiae recombinant bacterial strain sy1, saccharomyces cerevisiae recombinant bacterial strain sy2 extract respectively 20.27min, 21.31
Generate new product peak at min and 23.72 min, 440(m/z be respectively according to the molecular weight that mass spectral results show three new products), 442(m/z), 456(m/z).By analyzing molecular ion peak and the major cleavage rule of new product, determine that wherein three products are possible to as product 11- oxygen-cucurbitane dienol, corresponding to No. 1 product peak of gas chromatogram in figure, appearance time is 20.27 min；Product 11- hydroxyl-cucurbitane dienol, corresponding No. 2 product peaks of gas chromatogram in figure, appearance time is 21.31 min；Product 11- oxygen -24,25- epoxy-cucurbitane dienol, corresponding No. 3 product peaks of gas chromatogram in figure, appearance time is 23.72 min.
3rd, the separation of new product and identification
For further confirming that these three products respectively 11- oxygen-cucurbitane dienol, 11- hydroxyl-cucurbitane dienol, 11- oxygen -24,25- epoxy-cucurbitane dienol, isolation identification need to be carried out to above-mentioned three kinds of products, method particularly includes:
Extraction product carries out separating through thin layer chromatography (tlc), wherein developing solvent is n-hexane-ethyl acetate (1:3), developer is the concentrated sulphuric acid-ethanol solution containing 0.5% vanillin (10% concentrated sulphuric acid), after in hair-dryer heating colour developing, new product region is scraped from silica gel plate, using n-hexane-ethyl acetate (1:1) extraction.
Agilent 1260 hplc system and bruker-microtof-ii electrospray mass spectrometer are adopted using liquid chromatography mass spectrometric nuclear-magnetism technology used in conjunction lc-ms-nmr analysis new product: lc-ms analysis.In efficient liquid phase using anti-phase c18 post (4.6 × 250 mm, 5 μm,
Welch, Shanghai), condition is flow velocity 0.5 ml/min, and ultraviolet detection wavelength is 210 nm.Mobile phase a is water (0.1% formic acid), and mobile phase b is acetonitrile (0.1% formic acid), using linear gradient elution method: 0-20 min, 50-100% b；20-50
Min, 100% b.Electric spray ion source (esi) parameter is: sweep limitss: negative ion mode, 100-1000 m/z；Spray voltage: 4500 v, capillary temperature: 400 ° of c, dry gas flow: 6 ml/min, baking temperature: 180 ° of c, atomization air pressure: 1 bar.Product is collected by mass signal, collects the product (Fig. 4) that m/z is 441.3727,443,3884 and 457.3676 respectively；It is dissolved in after drying in deuterated acetonitrile, do nmr analysis through bruker dmx-600 nuclear magnetic resonance spectrometer under 600 mhz.These three product nuclear magnetic datas are respectively as follows:
The c spectrum of table 1 11- oxygen-cucurbitane dienol and h modal data
| C number | δ13c | δ1h |
| 1 | 21.3 | ch2,1.19(m),1.47(m) |
| 2 | 29.4 | ch2,1.53(m),1.80(m) |
| 3 | 76.3 | ch,3.37(br,s) |
| 4 | 42.0 | |
| 5 | 141.5 | |
| 6 | 120.0 | ch,5.59(d, j=5.82hz) |
| 7 | 24.5 | ch2,1.92(m),2.33(m) |
| 8 | 44.7 | ch,1.93(m) |
| 9 | 49.1 | |
| 10 | 36.0 | ch,2.39(m) |
| 11 | 207.0 | |
| 12 | 49.3 | ch2, 2.30(d, j=14.2hz), 3.03(d, j=14.2hz) |
| 13 | 50.3 | |
| 14 | 49.5 | |
| 15 | 30.2 | ch2,1.28(m) |
| 16 | 28.3 | ch2,1.34(m),2.01(m) |
| 17 | 50.2 | ch,1.74(m) |
| 18 | 17.1 | ch3,0.71(s) |
| 19 | 18.6 | ch3,1.03(s) |
| 20 | 36.6 | ch,1.06(m),1.41(m) |
| 21 | 18.7 | ch3,0.90(d, j=6.45hz) |
| 22 | 35.0 | ch2,1.27(m),1.35(m) |
| 23 | 25.2 | ch2,1.88(m),2.04(m) |
| 24 | 125.9 | ch,5.10(br,s) |
| 25 | 126.0 | |
| 26 | 25.8 | ch3,1.66(s) |
| 27 | 17.7 | ch3,1.59(s) |
| 28 | 27.9 | ch3,0.99(s) |
| 29 | 26.0 | ch3,1.09(s) |
| 30 | 20.1 | ch3,1.04(s) |
The c spectrum of table 2 11- hydroxyl-cucurbitane dienol and h modal data
| c | δ13c | δ1h |
| 1 | 21.3 | ch2,0.99(m),1.49(m) |
| 2 | 25.9 | ch2,1.40(m),2.21(m) |
| 3 | 76.8 | ch,3.36(br,s) |
| 4 | 41.7 | |
| 5 | 142.9 | |
| 6 | 120.3 | ch,5.47(d,j=6.10hz) |
| 7 | 24.8 | ch2,1.77(m),2.39(m) |
| 8 | 43.1 | ch,1.35(m) |
| 9 | 56.1 | |
| 10 | 41.0 | ch,1.74(m) |
| 11 | 78.7 | ch,3.78(m) |
| 12 | 37.2 | ch2,1.03(m),1.40(m) |
| 13 | 49.5 | |
| 14 | 49.9 | |
| 15 | 28.6 | ch2,1.26(m),1.89(m) |
| 16 | 30.3 | ch2,1.47(m),1.92(m) |
| 17 | 51.0 | ch,1.57(m) |
| 18 | 19.6 | ch3,0.81(s) |
| 19 | 17.0 | ch3,0.87(s) |
| 20 | 36.6 | ch,1.44(m),2.45(m) |
| 21 | 18.8 | ch3,0.94(d,j=6.40hz) |
| 22 | 34.8 | ch2,1.09(m),1.15(m) |
| 23 | 25.4 | ch2,1.88(m),2.03(m) |
| 24 | 126.1 | ch,5.10(br,s) |
| 25 | 124.9 | |
| 26 | 25.7 | ch3,1.66(s) |
| 27 | 17.6 | ch3,1.59(s) |
| 28 | 27.0 | ch3,1.02(s) |
| 29 | 26.0 | ch3,1.09(s) |
| 30 | 25.9 | ch3,1.05(s) |
The c spectrum of table 3 11- oxygen -24,25- epoxy-cucurbitane dienol and h modal data
| c | δ13c | δ1h |
| 1 | 21.3 | ch2,1.20(m),1.49(m) |
| 2 | 29.4 | ch2,1.53(dd),1.94(m) |
| 3 | 76.3 | ch,3.37(br,s) |
| 4 | 41.2 | |
| 5 | 140.2 | |
| 6 | 120.1 | ch,5.59(d, j=5.89hz) |
| 7 | 23.9 | ch2,1.94(m),2.36(m) |
| 8 | 44.1 | ch,1.94(m) |
| 9 | 48.8 | |
| 10 | 35.9 | ch,2.36(m) |
| 11 | 207.0 | |
| 12 | 49.6 | ch2,2.37(d,j=14.5hz),3.04(d,j=14.4hz) |
| 13 | 48.9 | |
| 14 | 50.2 | |
| 15 | 31.6 | ch2, 1.31(m) |
| 16 | 22.4 | ch2,1.77(m),1.84(m) |
| 17 | 51.3 | ch,2.05(m) |
| 18 | 19.3 | ch3,0.85(s) |
| 19 | 18.8 | ch3,1.04(s) |
| 20 | 45.0 | ch,1.29(m),1.43(m) |
| 21 | 26.3 | ch3,1.20(s) |
| 22 | 34.6 | ch2,1.31(m),1.36(m) |
| 23 | 23.9 | ch2,1.94(m) |
| 24 | 125.6 | ch,5.10(br,s) |
| 25 | 131.2 | |
| 26 | 25.7 | ch3,1.66(s) |
| 27 | 17.7 | ch3,1.60(s) |
| 28 | 27.8 | ch3,0.99(s) |
| 29 | 25.9 | ch3,1.09(s) |
| 30 | 20.2 | ch3,1.03(s) |
Verify through nuclear-magnetism and confirm that these three products are respectively 11- oxygen-cucurbitane dienol, 11- hydroxyl-cucurbitane dienol, 11- oxygen -24,25- epoxy-cucurbitane dienol.The establishment of three kinds of products is significant for exploration and research cucurbitane dienol synthesis momordica grosvenori alcohol synthesis path, and this three kinds of products also can obtain multiple triterpenoid compound through glycosyl transferase is glycosylation modified simultaneously.
Embodiment 3, external p450 enzyme cyp87d18 reaction determine associated products synthesis order in momordica grosvenori alcohol synthesis path
Cytochrome p450 cyp87d18 is measured using microsome preparation method and is catalyzed the synthesis order that cucurbitane dienol obtains three kinds of products.Specific operation process is as follows:
1st, cytochrome p450 cyp87d18 in induction saccharomyces cerevisiae recombinant bacterial strain sy2
Similar with the cultural method of sy2 bacterial strain in embodiment 2 and the abductive approach of relevant enzyme.In order to contrast the external enzymic catalytic reaction of cytochrome p450 cyp87d18, saccharomyces cerevisiae recombinant bacterial strain sy1 also adopts as the yeast culture in embodiment 2 and enzyme induction method simultaneously.
2nd, MC preparation
Bacterium solution after 50ml is induced, is centrifuged 5 min collects thallines through 5000 rpm, and with distilled water wash twice；With 2 ml 0.25 m sucrose solution resuspended bacterium solution, add Phenylmethanesulfonyl fluoride (pmsf) to final concentration 0.67 m, add microballon to covering liquid level, vibrate 3 min that are vortexed；1000 rpm centrifugation 3min, go out unbroken cell and fragment；Take supernatant, 5000 rpm, it is centrifuged 10min, remove mitochondrion；Improve rotating speed to 9000 rpm, be centrifuged 10min, remove broken MF.Supernatant is taken to be transferred in new ep pipe, 10000 rpm are centrifuged 90 min；Remove supernatant, with 2 ml ice-cold 0.154
The resuspended precipitation of m kcl, 10000 rpm are centrifuged 30 min, remove supernatant；2 ml pde buffer (50mm kaliumphosphate buffer, ph 7.5 will be suspended in containing MC precipitation;
0.5% deoxycholic acid;1mm dtt and 1mm edta) in；The microsome preparing is directly used in external enzyme reaction.
3rd, external enzyme reaction
For determining the genesis sequence of cytochrome p450 cyp87d18 catalysis cucurbitane dienol three kinds of products of synthesis, 11- oxygen-cucurbitane dienol and 11- hydroxyl-cucurbitane dienol is added successively in vitro in enzyme reaction system, concrete vitro enzyme reaction system (800 μ l) is as follows: kaliumphosphate buffer (100mm, ph 7.5), microsome enzyme liquid containing p450 enzyme cyp87d18 (200 μ l), 11- oxygen-cucurbitane dienol or 11- hydroxyl-cucurbitane dienol (1 mg), nadph (1 mm), 30 DEG C of reaction 2h, react after terminating with n-hexane extraction twice, and carry out gc-ms analysis by obtaining sample.
With 11- oxygen-cucurbitane dienol for substrate carry out vitro reactions when, liquid phase it is found that generate 11- hydroxyl-cucurbitane dienol (Fig. 5)；Contrary with 11- hydroxyl-cucurbitane dienol for substrate carry out vitro reactions when, liquid phase result is not detected by 11- oxygen-cucurbitane dienol, and cytochrome p450 is described
Probably catalysis cucurbitane dienol first generates 11- oxygen-cucurbitane dienol to cyp87d18, the latter catalyzes and synthesizes 11- hydroxyl-cucurbitane dienol through p450 enzyme cyp87d18 further, due to 11- oxygen -24,25- epoxy-cucurbitane dienol is similar with 11- oxygen-cucurbitane dienol structure, speculate this product also very likely by cytochrome p450 cyp87d18 catalysis 11- oxygen-cucurbitane dienol synthesis, but because of 11- oxygen -24,25- epoxy-cucurbitane diene alcohol content is very low, and external enzyme reaction is not detected by.Obtained 11- hydroxyl-cucurbitane dienol can warp furthersiraitia grosvenoriiIn other p450 enzyme catalysiss c-24 and c-25 hydroxylating or epoxidation synthesis momordica grosvenori alcohol；Obtained 11- oxygen -24,25- epoxy-cucurbitane dienol can warp furthersiraitia grosvenoriiIn other epoxide enzyme catalysiss generate momordica grosvenori alcohol.
Sequence table
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>in momordica grosvenori alcohol synthesis path three kinds of terpenoids and its glycation product synthetic method
<130> 3
<160> 3
<170> patentin
version 3.3
<210> 1
<211> 2280
<212> dna
<213>cucurbitane dienol synzyme sgcbq gene order
<400> 1
atgtggagat tgaaggttgg tgctgaatct gttggtgaaa acgacgaaaa gtggttgaag 60
tctatctcta accacttggg tagacaagtt tgggaattct gtccagacgc tggtactcaa 120
caacaattgt tgcaagttca caaggctaga aaggctttcc acgacgacag attccacaga 180
aagcaatctt ctgacttgtt catcactatc caatacggta aggaagttga aaacggtggt 240
aagactgctg gtgttaagtt gaaggaaggt gaagaagtta gaaaggaagc tgttgaatct 300
tctttggaaa gagctttgtc tttctactct tctatccaaa cttctgacgg taactgggct 360
tctgacttgg gtggtccaat gttcttgttg ccaggtttgg ttatcgcttt gtacgttact 420
ggtgttttga actctgtttt gtctaagcac cacagacaag aaatgtgtag atacgtttac 480
aaccaccaaa acgaagacgg tggttggggt ttgcacatcg aaggtccatc tactatgttc 540
ggttctgctt tgaactacgt tgctttgaga ttgttgggtg aagacgctaa cgctggtgct 600
atgccaaagg ctagagcttg gatcttggac cacggtggtg ctactggtat cacttcttgg 660
ggtaagttgt ggttgtctgt tttgggtgtt tacgaatggt ctggtaacaa cccattgcca 720
ccagaattct ggttgttccc atacttcttg ccattccacc caggtagaat gtggtgtcac 780
tgtagaatgg tttacttgcc aatgtcttac ttgtacggta agagattcgt tggtccaatc 840
actccaatcg ttttgtcttt gagaaaggaa ttgtacgctg ttccatacca cgaaatcgac 900
tggaacaagt ctagaaacac ttgtgctaag gaagacttgt actacccaca cccaaagatg 960
caagacatct tgtggggttc tttgcaccac gtttacgaac cattgttcac tagatggcca 1020
gctaagagat tgagagaaaa ggctttgcaa actgctatgc aacacatcca ctacgaagac 1080
gaaaacacta gatacatctg tttgggtcca gttaacaagg ttttgaactt gttgtgttgt 1140
tgggttgaag acccatactc tgacgctttc aagttgcact tgcaaagagt tcacgactac 1200
ttgtgggttg ctgaagacgg tatgaagatg caaggttaca acggttctca attgtgggac 1260
actgctttct ctatccaagc tatcgtttct actaagttgg ttgacaacta cggtccaact 1320
ttgagaaagg ctcacgactt cgttaagtct tctcaaatcc aacaagactg tccaggtgac 1380
ccaaacgttt ggtacagaca catccacaag ggtgcttggc cattctctac tagagaccac 1440
ggttggttga tctctgactg tactgctgaa ggtttgaagg ctgctttgat gttgtctaag 1500
ttgccatctg aaactgttgg tgaatctttg gaaagaaaca gattgtgtga cgctgttaac 1560
gttttgttgt ctttgcaaaa cgacaacggt ggtttcgctt cttacgaatt gactagatct 1620
tacccatggt tggaattgat caacccagct gaaactttcg gtgacatcgt tatcgactac 1680
ccatacgttg aatgtacttc tgctactatg gaagctttga ctttgttcaa gaagttgcac 1740
ccaggtcaca gaactaagga aatcgacact gctatcgtta gagctgctaa cttcttggaa 1800
aacatgcaaa gaactgacgg ttcttggtac ggttgttggg gtgtttgttt cacttacgct 1860
ggttggttcg gtatcaaggg tttggttgct gctggtagaa cttacaacaa ctgtttggct 1920
atcagaaagg cttgtgactt cttgttgtct aaggaattgc caggtggtgg ttggggtgaa 1980
tcttacttgt cttgtcaaaa caaggtttac actaacttgg aaggtaacag accacacttg 2040
gttaacactg cttgggtttt gatggctttg atcgaagctg gtcaagctga aagagaccca 2100
actccattgc acagagctgc tagattgttg atcaactctc aattggaaaa cggtgacttc 2160
ccacaacaag aaatcatggg tgttttcaac aagaactgta tgatcactta cgctgcttac 2220
agaaacatct tcccaatctg ggctttgggt gaatactgtc acagagtttt gactgaataa 2280
<210> 2
<211> 1422
<212> dna
<213>cytochrome p450 cyp87d18 gene order
<400> 2
atgtggactg ttgttttggg tttggctact ttgttcgttg cttactacat ccactggatc 60
aacaagtgga gagactctaa gttcaacggt gttttgccac caggtactat gggtttgcca 120
ttgatcggtg aaactatcca attgtctaga ccatctgact ctttggacgt tcacccattc 180
atccaaaaga aggttgaaag atacggtcca atcttcaaga cttgtttggc tggtagacca 240
gttgttgttt ctgctgacgc tgaattcaac aactacatca tgttgcaaga aggtagagct 300
gttgaaatgt ggtacttgga cactttgtct aagttcttcg gtttggacac tgaatggttg 360
aaggctttgg gtttgatcca caagtacatc agatctatca ctttgaacca cttcggtgct 420
gaagctttga gagaaagatt cttgccattc atcgaagctt cttctatgga agctttgcac 480
tcttggtcta ctcaaccatc tgttgaagtt aagaacgctt ctgctttgat ggttttcaga 540
acttctgtta acaagatgtt cggtgaagac gctaagaagt tgtctggtaa catcccaggt 600
aagttcacta agttgttggg tggtttcttg tctttgccat tgaacttccc aggtactact 660
taccacaagt gtttgaagga catgaaggaa atccaaaaga agttgagaga agttgttgac 720
gacagattgg ctaacgttgg tccagacgtt gaagacttct tgggtcaagc tttgaaggac 780
aaggaatctg aaaagttcat ctctgaagaa ttcatcatcc aattgttgtt ctctatctct 840
ttcgcttctt tcgaatctat ctctactact ttgactttga tcttgaagtt gttggacgaa 900
cacccagaag ttgttaagga attggaagct gaacacgaag ctatcagaaa ggctagagct 960
gacccagacg gtccaatcac ttgggaagaa tacaagtcta tgactttcac tttgcaagtt 1020
atcaacgaaa ctttgagatt gggttctgtt actccagctt tgttgagaaa gactgttaag 1080
gacttgcaag ttaagggtta catcatccca gaaggttgga ctatcatgtt ggttactgct 1140
tctagacaca gagacccaaa ggtttacaag gacccacaca tcttcaaccc atggagatgg 1200
aaggacttgg actctatcac tatccaaaag aacttcatgc cattcggtgg tggtttgaga 1260
cactgtgctg gtgctgaata ctctaaggtt tacttgtgta ctttcttgca catcttgtgt 1320
actaagtaca gatggactaa gttgggtggt ggtactatcg ctagagcgca catcttgtct 1380
ttcgaagacg gtttgcacgt taagttcact ccaaaggaat aa
1422
<210> 3
<211> 2079
<212> dna
<213>cytochrome reductase cpr gene order
<400> 3
atgacttctg ctttgtatgc ttccgatttg tttaagcagc tcaagtcaat tatggggaca 60
gattcgttat ccgacgatgt tgtacttgtg attgcaacga cgtctttggc actagtagct 120
ggatttgtgg tgttgttatg gaagaaaacg acggcggatc ggagcgggga gctgaagcct 180
ttgatgatcc ctaagtctct tatggctaag gacgaggatg atgatttgga tttgggatcc 240
gggaagacta gagtctctat cttcttcggt acgcagactg gaacagctga gggatttgct 300
aaggcattat ccgaagaaat caaagcgaga tatgaaaaag cagcagtcaa agtcattgac 360
ttggatgact atgctgccga tgatgaccag tatgaagaga aattgaagaa ggaaactttg 420
gcatttttct gtgttgctac ttatggagat ggagagccta ctgacaatgc tgccagattt 480
tacaaatggt ttacggagga aaatgaacgg gatataaagc ttcaacaact agcatatggt 540
gtgtttgctc ttggtaatcg ccaatatgaa cattttaata agatcgggat agttcttgat 600
gaagagttat gtaagaaagg tgcaaagcgt cttattgaag tcggtctagg agatgatgat 660
cagagcattg aggatgattt taatgcctgg aaagaatcac tatggtctga gctagacaag 720
ctcctcaaag acgaggatga taaaagtgtg gcaactcctt atacagctgt tattcctgaa 780
taccgggtgg tgactcatga tcctcggttt acaactcaaa aatcaatgga atcaaatgtg 840
gccaatggaa atactactat tgacattcat catccctgca gagttgatgt tgctgtgcag 900
aaggagcttc acacacatga atctgatcgg tcttgcattc atctcgagtt cgacatatcc 960
aggacgggta ttacatatga aacaggtgac catgtaggtg tatatgctga aaatcatgtt 1020
gaaatagttg aagaagctgg aaaattgctt ggccactctt tagatttagt attttccata 1080
catgctgaca aggaagatgg ctccccattg gaaagcgcag tgccgcctcc tttccctggt 1140
ccatgcacac ttgggactgg tttggcaaga tacgcagacc ttttgaaccc tcctcgaaag 1200
tctgcgttag ttgccttggc ggcctatgcc actgaaccaa gtgaagccga gaaacttaag 1260
cacctgacat cacctgatgg aaaggatgag tactcacaat ggattgttgc aagtcagaga 1320
agtcttttag aggtgatggc tgcttttcca tctgcaaaac ccccactagg tgtatttttt 1380
gctgcaatag ctcctcgtct acaacctcgt tactactcca tctcatcctc gccaagattg 1440
gcgccaagta gagttcatgt tacatccgca ctagtatatg gtccaactcc tactggtaga 1500
atccacaagg gtgtgtgttc tacgtggatg aagaatgcag ttcctgcgga gaaaagtcat 1560
gaatgtagtg gagccccaat ctttattcga gcatctaatt tcaagttacc atccaaccct 1620
tcaactccaa tcgttatggt gggacctggg actgggctgg caccttttag aggttttctg 1680
caggaaagga tggcactaaa agaagatgga gaagaactag gttcatcttt gctcttcttt 1740
gggtgtagaa atcgacagat ggactttata tacgaggatg agctcaataa ttttgttgat 1800
caaggcgtaa tatctgagct catcatggca ttctcccgtg aaggagctca gaaggagtat 1860
gttcaacata agatgatgga gaaggcagca caagtttggg atctaataaa ggaagaagga 1920
tatctctatg tatgcggtga tgctaagggc atggcgaggg acgtccaccg aactctacac 1980
accattgttc aggagcagga aggtgtgagt tcgtcagagg cagaggctat agttaagaaa 2040
cttcaaaccg aaggaagata cctcagagat gtctggtga
2079
Claims (8)
1. a kind of fermentation method synthesis 11- oxygen-cucurbitane dienol, 11- hydroxyl-cucurbitane dienol, the method for 11- oxygen -24,25- epoxy-cucurbitane dienol.
2. it is characterized in that, saccharomyces cerevisiae recombinant bacterial strain sy2 containing cucurbitane dienol synzyme sgcbq, cytochrome p450 cyp87d18 and cytochrome reductase cpr, three kinds of triterpenoid compound can be synthesized for fermenting substrate with glucose, these three compounds are respectively 11- oxygen-cucurbitane dienol, 11- hydroxyl-cucurbitane dienol, 11- oxygen -24,25- epoxy-cucurbitane dienol through mass spectrum and nuclear-magnetism checking.
3. the 11- oxygen-cucurbitane dienol obtained by claim 1 can be catalyzed through glycosyl transferase and obtain c-3 position glycation product, and glycosylated glucose can be one or more.
4. the 11- hydroxyl-cucurbitane dienol obtained by claim 1 can be catalyzed through glycosyl transferase and obtain the glycosylation of c-3 position, the simultaneously glycosylated new product of the glycosylation of c-11 position or c-3 and c-11 position, and glycosylated glucose can be one or more.
5. 11- oxygen -24 obtained by claim 1,25- epoxy-cucurbitane dienol can be catalyzed through glycosyl transferase and obtain c-3 position glycation product, and glycosylated glucose can be one or more.
6. the metabolic pathway of momordica grosvenori alcohol is synthesized by cucurbitane dienol.
7. it is characterized by, cucurbitane dienol synthesizes 11- oxygen-cucurbitane dienol first through cytochrome p450 cyp87d18 catalysis, the latter catalyzes and synthesizes 11- hydroxyl-cucurbitane dienol and 11- oxygen -24,25- epoxy-two kinds of cucurbitane dienol product further through cytochrome p450 cyp87d18.
8.11- hydroxyl-cucurbitane dienol can be throughsiraitia
grosvenoriiIn other kinds of p450 oxidase c-24 and c-25 hydroxylating synthesis momordica grosvenori alcohol；11- oxygen -24,25- epoxy-cucurbitane dienol also can be throughsiraitia
grosvenoriiIn other kinds of epoxide hydrolase (epoxide hydrolase) synthesize momordica grosvenori alcohol.
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| CN110283224A (en) * | 2019-07-16 | 2019-09-27 | 湖南华诚生物资源股份有限公司 | A kind of semisynthesis of momordica glycoside V |
| CN113755355A (en) * | 2021-09-30 | 2021-12-07 | 河北维达康生物科技有限公司 | Engineering strain for biosynthesis of mogrol by taking glucose as substrate, construction and application thereof |
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| CN110283224A (en) * | 2019-07-16 | 2019-09-27 | 湖南华诚生物资源股份有限公司 | A kind of semisynthesis of momordica glycoside V |
| CN113755355A (en) * | 2021-09-30 | 2021-12-07 | 河北维达康生物科技有限公司 | Engineering strain for biosynthesis of mogrol by taking glucose as substrate, construction and application thereof |
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