CN104531786A - Method for synthesizing poly 3-hydroxypropionic acid by using acetyl coenzyme A - Google Patents
Method for synthesizing poly 3-hydroxypropionic acid by using acetyl coenzyme A Download PDFInfo
- Publication number
- CN104531786A CN104531786A CN201410763819.3A CN201410763819A CN104531786A CN 104531786 A CN104531786 A CN 104531786A CN 201410763819 A CN201410763819 A CN 201410763819A CN 104531786 A CN104531786 A CN 104531786A
- Authority
- CN
- China
- Prior art keywords
- poly
- coa
- acetyl
- hydroxy
- propionic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
Abstract
The invention discloses a method for synthesizing a poly 3-hydroxypropionic acid by using acetyl coenzyme A, which comprises the following steps: (1) building a recombinant host cell for commonly overexpressing endogenous or exogenous enzyme genes; (2) culturing and fermenting the recombinant host cell in an appropriate culture medium, and adding an expression inducer; and (3) collecting thalluses, carrying out centrifugalizing, water scrubbing and freeze-drying on the thalluses, and carrying out chloroform extraction. According to the invention, glucose is used as an original material, an intermediate product acetyl coenzyme A produced by the degradation of the glucose is adopted for biosynthesizing a poly 3-hydroxypropionic acid, and the acetyl coenzyme A is used as a midbody of glycometabolism, therefore, the method is wider in raw material source, lower in cost and simple in production process, and the cost is obviously decreased.
Description
Technical field
The present invention relates to field of biomedicine technology, particularly relate to a kind of method applying the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis.
Background technology
3-hydroxy-propionic acid (3-hydroxypropionic acid, is abbreviated as 3-HP) molecular formula C
3h
60
3, a kind of syrupy shape material, soluble in water, ethanol, ether, be generally used for organic synthesis.Chemically structure is seen, 3-HP and lactic acid isomers each other, this molecule, with Liang Ge functional group hydroxyl and carboxyl, is the precursor of a lot of optically active substance.As the monomer of biogenic polymkeric substance, and the concern of various countries scientist can be received.Industrial, 3-HP is a kind of important chemical intermediate of rising in recent years, as 3-HP can be used as the raw material of synthesising biological biodegradable thermoplastic polyester (PHA), and the monomer of the poly-3-hydroxy-propionic acid of synthesis and sterilant.Also can be used for producing coating, sizing agent, Water Treatment Chemicals and personal-care supplies.In addition, generate ester with alcohol esterification, oxidation generates propanedioic acid, and dehydration generates vinylformic acid, also generates 1,3-PD etc. by reductive action.Wherein 1,3-PD can be used as the monomer of solvent, antifreezing agent or protective material and new polyester-Poly(Trimethylene Terephthalate) (PTT).This polyester has excellent characteristic, as uvioresistant, and the absorption of anti-internal stress, low water, low electrostatic and biodegradable, reusable edible.Based on these good characteristics of 3-HP, it commercially has great Development volue.The USDOE in August, 2004 reports 3-HP one of Chemicals being classified as 12 kinds of most potentialities to be exploited on our times.
At present, the synthetic method in the world with the 3-HP of prospects for commercial application mainly contains chemical synthesis and microbe fermentation method.Chemical synthesis is produced 3-HP and is mainly contained vinylformic acid hydration method, 3-HPA oxidation style, 3-hydroxypropionitrile hydrolysis method and this base of thunder formal (Reformatsky) reaction.
The representative of microbe fermentation method is that method is company that U.S.'s card gill (Cargi l l) company and Code mono-kind apply the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis coacts and have studied the new process for fermenting that " with cereals carbohydrate " produces 3-HP, has applied for patent in 2002.Two kinds of methods of producing 3-HP are discussed: one builds genetic engineering bacterium, obtains 3-HP after fermentation in born of the same parents or outside born of the same parents in patent; Another kind directly utilizes enzyme to produce 3-HP in cell-free system.
Though microbe fermentation method environmental protection, equipment and production process are complicated and domestic fermentation manufacturing technique is immature, and fermentation time is longer, and due to bacterial classification, the purification of products process produced is loaded down with trivial details, and productive rate is also low, and in obtained aqueous solution, the content of 3-HP is lower.In addition Biological preparation obtains that 3-HP is expensive and source is not enough, and can not meet the demand in market, only have and synthesize for laboratory on a small quantity, application prospect is limited.
Summary of the invention
The object of the invention is the technological deficiency for existing in prior art, and provide a kind of productive rate higher, prepare the method for the shorter poly-3-hydroxy-propionic acid of application acetyl-CoA synthesis of required time.
The technical scheme adopted for realizing object of the present invention is: a kind of method applying the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis, comprises the steps:
(1) acetyl-coA carboxylase gene (acc) and the propionyl-CoA synthetase gene (prpE) of the endogenous or external source of common overexpression is built, and the malonyl-CoA reductase gene (mcr) of external source and the recombinant host cell of poly-hydroxy fatty acid synthase gene (phaC);
(2) step (1) is built the recombinant host cell cultivation and fermentation in suitable substratum obtained, add induced expression agent induction reasonable time;
(3) thalline is collected, centrifugal, washing, after freeze-drying, chloroform extraction, the poly-3-hydroxy-propionic acid product obtained.
Described host cell is intestinal bacteria or Klebsiella pneumonia.
The method of structure recombinant host cell used is molecular cloning method, under being building up to the control of inducible promoter by the various enzymes described in step (1).
Induced expression agent in described step (2) is isopropylthiogalactoside (IPTG).
Described substratum is the substratum being suitable for intestinal bacteria or Klebsiella pneumonia growth in prior art; The carbon source of described substratum is the glucose that cost is low.
The acetyl-coA carboxylase gene that described acetyl-coA carboxylase gene (acc) is Salmonellas.
The propionyl-CoA synthetase gene that described propionyl-CoA synthetase gene (prpE) is Escherichia coli.
The malonyl-CoA reductase gene that described malonyl-CoA reductase gene (mcr) is Chloroflexus aurantiacus.
The poly-hydroxy fatty acid synthase gene that described poly-hydroxy fatty acid synthase gene (phaC) is Alkaligenes Ralstonia eutropha.
Principle of work of the present invention and beneficial effect are: using glucose as starting materials, the biosynthesizing of glucose degradation intermediate product acetyl-CoA is utilized to gather 3-hydroxy-propionic acid, common molecular recombinant clone technology is utilized to be connected with suitable expression controlling elements operability by the nucleotide sequence of several genes or operability connects each other, then be cloned in suitable Bacillus coli cells, and screening is comprised poly-hydroxy fatty acid synthetic enzyme simultaneously, propionyl-CoA synthetase, the recombinant escherichia coli strain of malonyl CoA and these four kinds of enzymic activitys of acetyl-CoA carboxylase.Compared with prior art, acetyl-CoA is as glycometabolic intermediate, and raw material sources are more extensive, and cost is lower; In addition, from the process of the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis, production technique is simple, with low cost, has broad application prospects.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment:
Apply a method for the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis, comprise the steps:
(1) acetyl-coA carboxylase gene (acc) and the propionyl-CoA synthetase gene (prpE) of the endogenous or external source of common overexpression is built, and the malonyl-CoA reductase gene (mcr) of external source and the recombinant host cell of poly-hydroxy fatty acid synthase gene (phaC);
(2) step (1) is built the recombinant host cell cultivation and fermentation in suitable substratum obtained, add induced expression agent induction reasonable time;
(3) thalline is collected, centrifugal, washing, after freeze-drying, chloroform extraction, the poly-3-hydroxy-propionic acid product obtained.
Acetyl-CoA is the important mesostate of energy substance metabolism, is the material of a pivotability in vivo in energy substance metabolism.Sugar, fat, protein three major nutrient pool common metabolic pathway---tricarboxylic acid cycle and an oxidative phosphorylation by acetyl-CoA, generate carbonic acid gas and water, release energy in order to the synthesis of ATP through this path exhaustive oxidation.Acetyl-CoA is the precursor substance of synthetic fatty acid, ketoboidies equal energy source material, is also the precursor substance of the physiologically active substance such as synthetic cholesterol and derivative thereof.The aerobic oxidation of glucose can change into pyruvic acid, then changes into acetyl-CoA, is finally decomposed into CO
2+ H
2o, this process can only carried out in the cell of mitochondrial, and must have oxygen supply.
Described host cell is intestinal bacteria or Klebsiella pneumonia.
The method of structure recombinant host cell used is molecular cloning method, under being building up to the control of inducible promoter by the various enzymes described in step (1).
Induced expression agent in described step (2) is isopropylthiogalactoside (IPTG).
Described substratum is the substratum being suitable for intestinal bacteria or Klebsiella pneumonia growth in prior art; The carbon source of described substratum is preferably the low glucose of cost or glycerine.
The acetyl-coA carboxylase gene that described acetyl-coA carboxylase gene (acc) is Salmonellas.
The propionyl-CoA synthetase gene that described propionyl-CoA synthetase gene (prpE) is Escherichia coli.
The malonyl-CoA reductase gene that described malonyl-CoA reductase gene (mcr) is Chloroflexus aurantiacus.
The poly-hydroxy fatty acid synthase gene that described poly-hydroxy fatty acid synthase gene (phaC) is Alkaligenes Ralstonia eutropha.
The present invention is using glucose as starting materials, the biosynthesizing of glucose degradation intermediate product acetyl-CoA is utilized to gather 3-hydroxy-propionic acid, common molecular recombinant clone technology is utilized to be connected with suitable expression controlling elements operability by the nucleotide sequence of several genes or operability connects each other, then be cloned in suitable Bacillus coli cells, and screening is comprised the recombinant escherichia coli strain of these four kinds of enzymic activitys of poly-hydroxy fatty acid synthetic enzyme, propionyl-CoA synthetase, malonyl CoA and acetyl-CoA carboxylase simultaneously.Compared with prior art, acetyl-CoA is as glycometabolic intermediate, and raw material sources are more extensive, and cost is lower; In addition, from the process of the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis, production technique is simple, with low cost, has broad application prospects.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. apply a method for the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis, it is characterized in that: comprise the steps:
(1) acetyl-coA carboxylase gene (acc) and the propionyl-CoA synthetase gene (prpE) of the endogenous or external source of common overexpression is built, and the malonyl-CoA reductase gene (mcr) of external source and the recombinant host cell of poly-hydroxy fatty acid synthase gene (phaC);
(2) step (1) is built the recombinant host cell cultivation and fermentation in suitable substratum obtained, add induced expression agent induction reasonable time;
(3) thalline is collected, centrifugal, washing, after freeze-drying, chloroform extraction, the poly-3-hydroxy-propionic acid product obtained.
2. a kind of method applying the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis according to claim 1, is characterized in that described host cell is intestinal bacteria or Klebsiella pneumonia.
3. a kind of method applying the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis according to claim 1, it is characterized in that the method for structure recombinant host cell used is molecular cloning method, under being building up to the control of inducible promoter by the various enzymes described in step (1).
4. a kind of method applying the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis according to claim 1, is characterized in that the induced expression agent in described step (2) is isopropylthiogalactoside (IPTG).
5. a kind of method applying the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis according to claim 1, is characterized in that described substratum is the substratum being suitable for intestinal bacteria or Klebsiella pneumonia growth; The carbon source of described substratum is glucose.
6. a kind of method applying the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis according to claim 1, is characterized in that the acetyl-coA carboxylase gene that described acetyl-coA carboxylase gene (acc) is Salmonellas.
7. a kind of method applying the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis according to claim 1, is characterized in that the propionyl-CoA synthetase gene that described propionyl-CoA synthetase gene (prpE) is Escherichia coli.
8. a kind of method applying the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis according to claim 1, is characterized in that the malonyl-CoA reductase gene that described malonyl-CoA reductase gene (mcr) is Chloroflexus aurantiacus.
9. a kind of method applying the poly-3-hydroxy-propionic acid of acetyl-CoA synthesis according to claim 1, is characterized in that the poly-hydroxy fatty acid synthase gene that described poly-hydroxy fatty acid synthase gene (phaC) is Alkaligenes Ralstonia eutropha.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410763819.3A CN104531786A (en) | 2014-12-10 | 2014-12-10 | Method for synthesizing poly 3-hydroxypropionic acid by using acetyl coenzyme A |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410763819.3A CN104531786A (en) | 2014-12-10 | 2014-12-10 | Method for synthesizing poly 3-hydroxypropionic acid by using acetyl coenzyme A |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104531786A true CN104531786A (en) | 2015-04-22 |
Family
ID=52847408
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410763819.3A Pending CN104531786A (en) | 2014-12-10 | 2014-12-10 | Method for synthesizing poly 3-hydroxypropionic acid by using acetyl coenzyme A |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104531786A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107400651A (en) * | 2017-07-09 | 2017-11-28 | 东北林业大学 | A kind of dynamic promotes the biology sensor construction method of 3 hydracrylic acid high yields |
| CN109112156A (en) * | 2018-09-19 | 2019-01-01 | 江苏师范大学 | A kind of recombination pseudomonad producing 3- hydracrylic acid, its construction method and its application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307336A (en) * | 2008-05-04 | 2008-11-19 | 清华大学 | Method for fermentation co-production of PDO,BDO and PHP by constructing gene engineering strain |
| CN102690774A (en) * | 2011-03-24 | 2012-09-26 | 三星电子株式会社 | Method of producing 3-hydroxypropionic acid using malonic semialdehyde reducing pathway |
| CN103898034A (en) * | 2012-12-27 | 2014-07-02 | 中国科学院青岛生物能源与过程研究所 | Method for biologically synthesizing poly-3-hydroxypropionic acid |
-
2014
- 2014-12-10 CN CN201410763819.3A patent/CN104531786A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307336A (en) * | 2008-05-04 | 2008-11-19 | 清华大学 | Method for fermentation co-production of PDO,BDO and PHP by constructing gene engineering strain |
| CN102690774A (en) * | 2011-03-24 | 2012-09-26 | 三星电子株式会社 | Method of producing 3-hydroxypropionic acid using malonic semialdehyde reducing pathway |
| CN103898034A (en) * | 2012-12-27 | 2014-07-02 | 中国科学院青岛生物能源与过程研究所 | Method for biologically synthesizing poly-3-hydroxypropionic acid |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107400651A (en) * | 2017-07-09 | 2017-11-28 | 东北林业大学 | A kind of dynamic promotes the biology sensor construction method of 3 hydracrylic acid high yields |
| CN109112156A (en) * | 2018-09-19 | 2019-01-01 | 江苏师范大学 | A kind of recombination pseudomonad producing 3- hydracrylic acid, its construction method and its application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sun et al. | Efficient production of lactic acid from sugarcane molasses by a newly microbial consortium CEE-DL15 | |
| Dezam et al. | Microbial production of organic acids by endophytic fungi | |
| CN109136161A (en) | Synthesis gas or other gaseous carbon sources and methanol are converted into the method and organism of 1,3 butylene glycol | |
| Wang et al. | Enhanced co-production of hydrogen and poly-(R)-3-hydroxybutyrate by recombinant PHB producing E. coli over-expressing hydrogenase 3 and acetyl-CoA synthetase | |
| Maina et al. | Improvement on bioprocess economics for 2, 3-butanediol production from very high polarity cane sugar via optimisation of bioreactor operation | |
| CN107849522A (en) | For producing method and the microorganism of 1,3 butanediols | |
| Le Meur et al. | Poly (4-hydroxybutyrate)(P4HB) production in recombinant Escherichia coli: P4HB synthesis is uncoupled with cell growth | |
| CN104254612A (en) | A method of production of 2,4-dihydroxybutyric acid | |
| Shi et al. | Production of polyhydroxyalkanoate from acetate by metabolically engineered Aeromonas hydrophilia | |
| Tenhaef et al. | Production of D-xylonic acid using a non-recombinant Corynebacterium glutamicum strain | |
| CN107849521A (en) | For the process by carboxylic acid synthetic polymer precursor and microorganism | |
| CN105026548A (en) | D-glucaric acid-producing bacterium, and method for manufacturing d-glucaric acid | |
| CN101978052A (en) | Polypeptide having glyoxalase iii activity, polynucleotide encoding the same and uses thereof | |
| CN107429271A (en) | From one-carbon compound biosynthetic products | |
| Ma et al. | C2 feedstock-based biomanufacturing of value-added chemicals | |
| CN105039376B (en) | The recombinant bacterium and construction method of production 3- hydracrylic acids homopolymer/copolymer and application | |
| Lee et al. | Factors affecting the competitiveness of bacterial fermentation | |
| CN103497922B (en) | Recombination klebsiella pneumonia capable of co-producing 3-HP and P3HP, and preparation method and application thereof | |
| Tan et al. | Metabolic engineering of Halomonas bluephagenesis to metabolize xylose for poly-3-hydroxybutyrate production | |
| CN104531786A (en) | Method for synthesizing poly 3-hydroxypropionic acid by using acetyl coenzyme A | |
| CN108431208A (en) | Arginine is augmented to improve the efficiency of gaseous fermentation acetogen | |
| CN103890187B (en) | Use the manufacture method of the organic acid of CoA-transferase | |
| CN104870645A (en) | Method for producing butanediol | |
| CN107603998A (en) | Utilize the genetic engineering bacterium and its construction method of acetic acid production glycolic and application | |
| Zhao et al. | Role of microbubbles coupling fibrous-bed bioreactor in butyric acid production by Clostridium tyrobutyricum using Brewer’s spent grain as feedstock |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150422 |