CN105018511B - A kind of method of external enzyme reaction method for synthesizing phloroglucinol and application - Google Patents
A kind of method of external enzyme reaction method for synthesizing phloroglucinol and application Download PDFInfo
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Abstract
The invention discloses a kind of method of external enzyme reaction method for synthesizing phloroglucinol and applications, belong to technical field of bioengineering.Method provided by the present invention is that structure contains acetyl-CoA-synthetase ACS genes respectively, acetyl-CoA carboxylase subunit gene accA, acetyl-CoA carboxylase subunit gene accB, acetyl-CoA carboxylase subunit gene accC, the recombinant vector of acetyl-CoA carboxylase subunit gene accD and polyketide synthases gene phlD, overexpression after recombinant vector being transformed into respectively in host strain, utilize ultrasonic disruption thalline, enzyme solution is obtained after isolating and purifying, and passes through external enzyme reaction method for synthesizing phloroglucinol after mixed enzyme solution and reaction substrate in proportion.The present invention is realized through external enzyme reaction method for synthesizing phloroglucinol, and can be with each enzyme of accuracy controlling and the dosage of confactor.
Description
Technical field
The present invention relates to a kind of method of external enzyme reaction method for synthesizing phloroglucinol and applications, belong to biotechnology neck
Domain.
Background technology
Phloroglucin also known as phloroglucinol are a kind of important fine chemical products, are synthesis flavones, isoflavones
The intermediate of class drug.Flavone compound has the function of extensive anticancer, anti-cardiovascular disease, increasingly by materia medica
The attention of family.The anti-immunity defective virus new drug synthesized by phloroglucin, belongs to second generation non-nucleoside reverse transcriptase inhibitor；
The Euglobals analogs synthesized by phloroglucin can effectively inhibit Epstein-Barr viral, have significant antitumaous effect.
The method of industrialized production phloroglucin is chemical synthesis at present, but in chemical synthesis process, because oxidation and acid were decomposed
A large amount of by-product is generated in journey so that the separating-purifying of final products is relatively difficult, very with simple physics and chemical method
Seldom arrive high purity product.
The various drawbacks of chemical method can be improved using bioanalysis method for synthesizing phloroglucinol, including raw material sources are difficult, by-product
Object is more, separating-purifying is difficult etc..With the development of biology, it is feasible that researcher starts discussion bioanalysis method for synthesizing phloroglucinol
Property.In bacterium circle, pseudomonad (Pseudomonas sp.) is the source of phloroglucinol derivatives compound.Achkar etc. is to fluorescence
2, the 4-DAPG synthetic genes cluster of pseudomonad Pf-5 further investigations have shown that, product outside 2,4-DAPG and MAPG,
It also being capable of method for synthesizing phloroglucinol.Under the conditions of phlD genes are complete, it is mutated phlA, phlB and phlC respectively, is as a result all both
It cannot detect final product 2,4-DAPG can not detect intermediate product MAPG, but can detect phloroglucin.According to
The above experimental result speculates that PhlD albumen has played key effect in being catalyzed phloroglucin building-up process, and PhlD can be catalyzed small point
Sub- substrate phosphinylidyne CoA polyketone is condensed to form long-chain and long-chain cyclisation, the series reaction in phenyl ring acetylation, is between synthesis
The key gene of benzenetriol.2011, Cao etc. constructed the engineering colon bacillus of phloroglucin synthesis, using glucose as substrate
By glycolytic pathway to acetyl coenzyme A, it is auxiliary that acetyl coenzyme A generates malonyl- under the action of acetyl-CoA carboxylase
Then enzyme A generates phloroglucin by the condensation of polyketide synthase.And by strengthening multiple resistance activation in Escherichia coli body
The expression of factor marA genes and acetyl-coA carboxylase gene cluster makes phloroglucin synthesizing in Escherichia coli
To larger raising, 3.8g/L is reached.
Although the research of forefathers has made it possible that the biosynthesis of phloroglucin, current fermentation method produce isophthalic
Triphenol, since phloroglucin itself is used as a kind of fungicide, normal growth and metabolism to Escherichia coli have serious inhibition
Effect, keeps the fermentation concentration of phloroglucin also relatively low.This research team takes metabolic engineering means and strengthens phloroglucin resistance
The expression of gene improves E. coli resistance ability, so that the Escherichia coli is increased to about the resistance of phloroglucin by 2g/L
3.5g/L, and the extracting process of online Integrated process is used, phloroglucin concentration is increased to about 4.5g/L in zymotic fluid, but
From commercial production level, there is also certain gaps.Generation and bacterial strain itself additionally, due to by-products such as acetic acid in fermentation process
The metabolic demand etc. of growth, the yield of biological fermentation process method for synthesizing phloroglucinol is also relatively low, constrains the reduction of production cost.It is another
It is needed using derivant and antibiotic etc. during engineering bacteria production phloroglucin of the aspect based on traditional metabolic engineering thinking,
Increase production process cost.
Invention content
To solve the above problems, the present invention provides a kind of method of external enzyme reaction method for synthesizing phloroglucinol, taken
Technical solution is as follows：
The purpose of the present invention is to provide a kind of methods of external enzyme reaction method for synthesizing phloroglucinol.This method is to build respectively
Contain acetyl-CoA-synthetase ACS genes, acetyl-CoA carboxylase subunit gene accA, acetyl-CoA carboxylase subunit base
Because of accB, acetyl-CoA carboxylase subunit gene accC, acetyl-CoA carboxylase subunit gene accD and polyketide synthases base
Because of the recombinant vector of phlD, overexpression after recombinant vector being transformed into respectively in host strain utilizes ultrasonic disruption thalline, separation
Enzyme solution is obtained after purification, passes through external enzyme reaction method for synthesizing phloroglucinol after mixed enzyme solution and reaction substrate in proportion.
The step of the method, is as follows：
1) respectively by acetyl-CoA-synthetase ACS genes, acetyl-CoA carboxylase subunit gene accA, acetyl coenzyme A
Carboxylase subunit gene accB, acetyl-CoA carboxylase subunit gene accC, acetyl-CoA carboxylase subunit gene accD and
Polyketide synthases gene phlD is connected on plasmid vector, obtains recombinant vector；
2) recombinant vector obtained by step 1) is converted in host strain and is overexpressed respectively, obtain culture solution；
3) thalline in ultrasonic disruption culture solution, centrifugation are recycled after pretreatment through step 2) gained culture solution
Separation retains supernatant after being crushed bacterium solution, recycles ni-sepharose purification supernatant, obtains enzyme solution；
4) sealing and standing is reacted after mixing enzyme solution obtained by step 3) with reaction substrate by a certain percentage.
Preferably, the step 1) plasmid vector is plasmid pET-28a (+).
Preferably, the step 2) host strain is e. coli bl21 (DE3).
Preferably, the step 3) pretreatment is by culture solution at 4 DEG C, after centrifuging 5min under 6000rpm, uses 50mM
After the phosphate buffer solution of pH7.4 washes twice, then with 5mL phosphate buffers be resuspended；The ultrasonication, be
20min is handled under 20KHz, 60W；The centrifugation is that 20min is centrifuged under the conditions of 13000rpm at 4 DEG C.
Preferably, the step 4) enzyme solution mixes in proportion, and four subunits of acetyl-CoA carboxylase are according to subunit
accA：Subunit accB：Subunit accC：Subunit accD=(0.9-1.1)：(0.918-1.122)：(1.494-1.826)：
The ratio of (0.972-1.188) mixes；Acetyl-CoA-synthetase, acetyl-CoA carboxylase and polyketide synthases press acetylcoenzyme
A synzyme：Acetyl-CoA carboxylase：Polyketide synthases=3~7：3~7：3~7 ratio mixing, and acetyl coenzyme A synthesizes
Enzyme, acetyl-CoA carboxylase and a concentration of 60-140 μ g/L of polyketide synthases；Step 4) the reaction substrate, 1mL reaction systems
The type and concentration of reaction substrate be：100mM Tris-HCl buffer, 20mM potassium acetates, 30mM NaHCO3, 10mM
MgCl2, 10mM KCl, 1mM biotins, 20mM ATP, 1mM CoA, 1mM dithiothreitol (DTT)s.
The method is as follows：
1) respectively by acetyl-CoA-synthetase ACS genes, acetyl-CoA carboxylase subunit gene accA, acetyl coenzyme A
Carboxylase subunit gene accB, acetyl-CoA carboxylase subunit gene accC, acetyl-CoA carboxylase subunit gene accD and
Polyketide synthases gene phlD is connected on plasmid vector pET-28a (+), obtains recombinant vector；
2) recombinant vector obtained by step 1) is transformed into respectively in e. coli bl21 (DE3) and is obtained, recombinate large intestine bar
Bacterium after the IPTG of final concentration of 0.25mmol is added, cultivates 12-18h at 20 DEG C, obtains culture solution；
3) after the culture solution obtained by step 2) being centrifuged 5min under 4 DEG C, 6000rpm, with the phosphate of 50mM pH7.4
After buffer solution washes twice, then with 5mL phosphate buffers be resuspended, using ultrasonoscope under conditions of 20KHz, 60W
20min is managed, then at 4 DEG C, 20min is centrifuged under the conditions of 13000rpm, retains supernatant, after using supernatant obtained by ni-sepharose purification
Obtain purifying enzyme solution；
4) by four subunits of acetyl-CoA carboxylase in the purifying enzyme solution obtained by step 3) according to subunit accA：Subunit
accB：Subunit accC：Subunit accD=1:1.02:1.66:1.08 ratio mixing, then by acetyl-CoA-synthetase, acetyl is auxiliary
Enzyme A carboxylases and polyketide synthases are mixed according to equal proportion, and a concentration of 100 μ g/L of each enzyme are reaction substrate after mixing
It is mixed, ultimately forms every milliliter of following reaction system：100mM Tris-HCl buffer, 20mM potassium acetates, 30mM
NaHCO3, 10mM MgCl2, 10mM KCl, 1mM biotins, 20mM ATP, 1mM CoA, 1mM dithiothreitol (DTT)s are quiet at room temperature
Set reaction 5h.
The either method is for producing phloroglucin and using phloroglucin as the chemicals of intermediate product.
What the present invention obtained has the beneficial effect that：
1. the method that the present invention is built can convert acetic acid to phloroglucin using enzyme reaction in vitro；
2. utilizing the method for the present invention, the dosage of each enzyme of accuracy controlling and confactor, it is determined that synthesize way in phloroglucin
Acetyl-CoA-synthetase in diameter and acetyl-CoA carboxylase；
3. building a controllable adjustment and control system, the synthesis of phloroglucin is controlled, while closing for intracellular phloroglucin
Foundation is provided at metabolic regulation.
4. for solving the problems, such as that phloroglucin provides the inhibition of cell new resolving ideas in full cell synthetic system.
Description of the drawings
Fig. 1 is phloroglucin chromatograms.
Relationships of the Fig. 2 between enzyme solution concentration and phloroglucin.
Specific implementation mode
With reference to specific embodiment, the present invention will be further described, but the present invention should not be limited by the examples.
Agents useful for same, material, instrument and method in following embodiment, without Special Statement, be this field conventional reagent,
Material, instrument and method can be obtained by commercial channel.
Each subunit of acetyl-CoA-synthetase used in embodiment, acetyl-CoA carboxylase, polyketide synthases gene point
It is not connected on coli expression carrier on pET-28a (+) and converts to e. coli bl21 (DE3)；Obtained positive colony
It cultivates to OD600It is 0.6~0.8, the final concentration of 0.25mmol of IPTG, 20 DEG C of cultures is added to obtain for 12~18 hours.
Embodiment 1
The vitro reactions of phloroglucin anabolism relevant enzyme, are as follows：
(1) large intestine of 100ml expression acetyl-CoA-synthetase, 4 subunits of acetyl-CoA carboxylase, polyketide synthases is taken
Bacillus culture solution 6000rpm, 4 DEG C centrifuge 5min, abandon supernatant, are washed twice with 7.4 phosphate buffer of 50mM, PH, finally
It is resuspended using 5ml phosphate buffer；Then it is crushed (20kHz, 60w, 20min) with Ultrasonic Cell Disruptor, 13000rpm after being crushed
4 DEG C of centrifugation 20min, supernatant is detached with precipitation, retains supernatant, is contained using Coomassie brilliant blue combined techniques kit measurement albumen
Amount.Using the ni-sepharose purification enzyme solution of Novagen companies, destination protein is collected, and measure protein content.The acetyl for purifying gained is auxiliary
The quality proportioning of enzyme A four subunits of carboxylase is according to accA:accB:accC:AccD=1:1.02:1.66:1.08 ratio
It is compound.
(2) acetyl-CoA-synthetase, acetyl-CoA carboxylase and each 100 μ g/L of polyketide synthases is made, and it is added
Its reactive material, final system are：100mM pH7.4Tris-HCl buffer, 20mM potassium acetates, 30mM NaHCO3, 10mM
MgCl2, 10mM KCl, 1mM biotins, 20mM ATP, 1mM CoA, 1mM dithiothreitol (DTT)s.
(3) step (2) mixed liquor is added in 10ml anaerobic reaction bottles, designs 1ml reaction systems, with water polishing, use is close
Blocking seals, and is stored at room temperature reaction 5h.Liquid chromatographic detection phloroglucin, as shown in Figure 1.
Embodiment 2
Using the vitro reactions of enzyme, determines the key enzyme in phloroglucin anabolism, be as follows：
(1) large intestine of 100ml expression acetyl-CoA-synthetase, 4 subunits of acetyl-CoA carboxylase, polyketide synthases is taken
Bacillus culture solution 6000rpm, 4 DEG C centrifuge 5min, abandon supernatant, are washed twice with 7.4 phosphate buffer of 50mM, PH, finally
It is resuspended using 5ml phosphate buffer；Then it is crushed (20kHz, 60w) with Ultrasonic Cell Disruptor, 13000rpm4 DEG C of centrifugation after being crushed
20min detaches supernatant with precipitation, retains supernatant, using Coomassie brilliant blue combined techniques kit measurement protein content.Using
The ni-sepharose purification enzyme solution of Novagen companies collects destination protein, and measures protein content.Purify the acetyl-coa carboxylase of gained
The quality proportioning of four subunits of enzyme is according to accA:accB:accC:AccD=1:1.02:1.66:1.08 ratio is compound.
(2) it is the action intensity for investigating each enzyme in catalysis is reacted, the concentration of obtained two enzymes of step (1) is fixed
For 100 μ g/L, only change the dosage of an enzyme, concentration is respectively 60 μ g/L, 80 μ g/L, 100 μ g/L, 120 μ g/L, 140 μ g/
L, does five reactions, and end reaction system is：100mM pH7.4Tris-HCl buffer, 20mM potassium acetates, 30mM
NaHCO3, 10mM MgCl2, 10mM KCl, 1mM biotins, 20mM ATP, 1mM CoA, 1mM dithiothreitol (DTT)s.
(3) step (2) mixed liquor is added in 10ml serum bottles, design 1ml reaction systems use sealing-plug with water polishing
Sealing is stored at room temperature reaction 5h.Liquid chromatographic detection phloroglucin, phloroglucin yield is in 0.02-0.1g/L.The result shows that
Three kinds of enzymes are mixed according to equal proportion, and concentration is the yield highest of phloroglucin under conditions of 100 μ g/L, are 0.1g/L.
(4) the result shows that in the case where phlD changes its concentration, phloroglucin change of production unobvious.And change ACS
And in the case of ACC concentration, clearly, with being continuously increased for concentration, phloroglucin yield goes out phloroglucin change of production
Now significantly increase.As shown in Figure 2.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this
The people of technology can do various changes and modification, therefore the protection of the present invention without departing from the spirit and scope of the present invention
Range should be subject to what claims were defined.
Claims (2)
1. a kind of method of external enzyme reaction method for synthesizing phloroglucinol, which is characterized in that be as follows：
1) respectively by acetyl-CoA-synthetase ACS genes, acetyl-CoA carboxylase subunit gene accA, acetyl-coa carboxylase
Enzyme subunit gene accB, acetyl-CoA carboxylase subunit gene accC, acetyl-CoA carboxylase subunit gene accD and polyketone
Synthase gene phlD is connected on plasmid vector pET-28a (+), obtains recombinant vector；
2) recombinant vector obtained by step 1) is transformed into respectively in e. coli bl21 (DE3) and obtains recombination bacillus coli, added
After the IPTG for entering final concentration of 0.25mmol, 12-18h is cultivated at 20 DEG C, obtains culture solution；
3) after the culture solution obtained by step 2) being centrifuged 5min under 4 DEG C, 6000rpm, with the phosphate-buffered of 50mM pH7.4
After solution washes twice, then with 5mL phosphate buffers be resuspended, handled under conditions of 20KHz, 60W using ultrasonoscope
20min centrifuges 20min then at 4 DEG C under the conditions of 13000rpm, retain supernatant, obtained after using supernatant obtained by ni-sepharose purification
Enzyme solution must be purified；
4) by four subunits of acetyl-CoA carboxylase in the purifying enzyme solution obtained by step 3) according to subunit accA：Subunit accB：
Subunit accC：Subunit accD=1:1.02:1.66:1.08 ratio mixing, then by acetyl-CoA-synthetase, acetyl coenzyme A carboxylic
Change enzyme and polyketide synthases are mixed according to equal proportion, a concentration of 100 μ g/L of each enzyme, then enzyme solution and reaction substrate are mixed
It closes, ultimately forms every milliliter of reaction system for containing following component：100mM Tris-HCl buffer, 20mM potassium acetates, 30mM
NaHCO3, 10mM MgCl2, 10mM KCl, 1mM biotins, 20mM ATP, 1mM CoA, 1mM dithiothreitol (DTT)s are quiet at room temperature
Set reaction 5h.
2. method described in claim 1, which is characterized in that for producing phloroglucin and using phloroglucin as intermediate product
Chemicals.
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| FR3068368A1 (en) * | 2017-06-30 | 2019-01-04 | Compagnie Generale Des Etablissements Michelin | USE OF TYPE III POLYKETIDE SYNTHASES OF BACTERIA AS PHLOROGLUCINOL SYNTHASES |
| CN110551769B (en) * | 2019-09-27 | 2021-09-21 | 北京理工大学 | Production method of phloroglucinol |
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