CN105462868A - Method for improving yield and production intensity of pyruvic acid - Google Patents
Method for improving yield and production intensity of pyruvic acid Download PDFInfo
- Publication number
- CN105462868A CN105462868A CN201510907318.2A CN201510907318A CN105462868A CN 105462868 A CN105462868 A CN 105462868A CN 201510907318 A CN201510907318 A CN 201510907318A CN 105462868 A CN105462868 A CN 105462868A
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- mpc1
- pyruvic acid
- recombinant bacterium
- acid
- tgu
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
- C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
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- 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
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
Abstract
The invention discloses a method for improving the yield and production intensity of pyruvic acid, and belongs to the technical field of fermentation engineering. According to the method, pyruvic acid carrier proteins of mitochondria are over-expressed in a torulopsis glabrata strain, and enhanced pyruvic acid enters the mitochondria to further enhance the growth of the torulopsis glabrata, so that the yield and production intensity of a growth-related type product, namely, pyruvic acid are improved; MPC1 genes from saccharomyces cerevisiae are over-expressed in TgU- through a genetic engineering technology to obtain an engineering strain TgU-(pY26-MPC1) highly producing pyruvic acid; and compared with a reference strain TgU-(pY26), the dry cell weight, glucose consumption rate, pyruvic acid yield and pyruvic acid production intensity of the engineering strain TgU-(pY26-MPC1) are improved by 24.9%, 28.2%, 91.9% and 94.1% respectively.
Description
Technical field
The present invention relates to a kind of method improving output of pyruvic acid and production intensity, belong to fermentation engineering field.
Background technology
Pyruvic acid (pyruvate) is the intermediate product of biological metabolism, is in the middle-chain of each pathways metabolism, as: be glucolytic end product; Initial substance acetyl-the CoA of TCA circulation is converted into by dehydrogenation decarboxylic reaction; The intermediate product oxaloacetic acid of TCA circulation is generated under pyruvate carboxylase effect; Generate L-Ala etc. by transamination, be also one of very important ketone acid simultaneously.Due to the synthesis precursor that pyruvic acid is many kinds of substance, the demand of present society to pyruvic acid increases day by day, and it is widely used in daily use chemicals, food, medical treatment, the industries such as agricultural.
The production method of pyruvic acid mainly contains chemical synthesis and the large class of microbe fermentation method two.Chemical synthesis mainly contain winestone acid system, the cruel air oxidation process of lactic acid second, through benzylacetone method, electrochemical synthesis, lactic acid catalytic oxidation.The just former chemical industry synthesis of the production of current pyruvic acid turns to be prepared by fermentable.Microbe fermentation method has more advantage relative to chemical industry synthesis, and the transformation efficiency as raw material is high, pollutes little, low cost and other advantages.
At present, Production by Microorganism Fermentation pyruvic acid becomes the hot topic of research both at home and abroad as a kind of substituting environmental protection method.And high yield pyruvic acid to be obtained by fermentable, wherein most critical to have a plant height to produce and the pyruvic acid bacterial strain of high production intensity.At present the transformation of high yield pyruvic acid bacterial strain is mainly concentrated on and process LAN is carried out to some key enzyme in pathways metabolism and some alternative pathways are pounded out.
Summary of the invention
In order to solve the problem, the invention provides and a kind ofly strengthen the production intensity of pyruvate fermentation and the method for output by process LAN mitochondrial pyruvate acid vectors albumen.
First object of the present invention is to provide a kind of method improving output of pyruvic acid and production intensity, and described method is process LAN mitochondrial pyruvate acid vectors albumen in Pyruvate production bacterial strain.
In one embodiment of the invention, described method is the mitochondrial pyruvate acid vectors albumen MPC1 expressing GeneID:852800 on NCBI.
In one embodiment of the invention, described production bacterial strain is that the torulopsis glabrata T.glabrataCCTCCM202019 having lacked Ura gene obtains for the MPC1 of GeneID:852800 on host expresses NCBI.
In one embodiment of the invention, described process LAN is for carrier with expression plasmid of yeast pY26 (purchased from moral precious company).
Second object of the present invention is to provide the recombinant bacterium of a kind of output of pyruvic acid and production intensity raising, described recombinant bacterium process LAN mitochondrial pyruvate acid vectors albumen MPC1.
In one embodiment of the invention, described recombinant bacterium is the MPC1 that the T.glabrataCCTCCM202019 having lacked Ura gene is host, expression plasmid of yeast pY26 is GeneID:852800 on vector expression NCBI.
In one embodiment of the invention, the structure of described recombinant bacterium: by object fragment MPC1 after restriction enzyme EcoRI and XamI is double digested, and MPC1 is cloned in the double digested expression plasmid pY26 of EcoRI/XamI, obtain expression of recombinant yeast plasmid pY26-MPC1, recombinant plasmid is transferred to recipient bacterium T.glabrataCCTCCM202019 (TgU by electroporated method
-), screen in not containing the substratum of uridylic, obtain the recombinant bacterium TgU of mitochondrial pyruvate acid vectors albumen overexpression
-(pY26-MPC1).
In one embodiment of the invention, described MPC1 gene with Wine brewing yeast strain N85 genome for template amplification obtains.
In one embodiment of the invention, described expression plasmid of yeast pY26 is the shuttle vectors between E. coli-Yeast, and in intestinal bacteria, selective marker is amp
r, and selective marker is that uracil-deficient type is complementary in yeast.
3rd object of the present invention is to provide a kind of method utilizing the acid of described recombinant bacterium fermentation production of acetone, is to be seeded to fermention medium after being activated by recombinant bacterium, at 30 DEG C, carries out fermentation culture under 220rpm condition.
In one embodiment of the invention, described fermention medium (/L): glucose 120g, urea 3.86g, MgSO
4.7H
2o0.8g, KH
2pO
42g, CH
3cOONa3g, liquid microelement 10ml, VITAMIN liquid 10ml, initial pH=5.5; Described liquid microelement: MnCl
24H
2o12g, FeSO
47H
2o2g, CaCl
22H
2o2g, CuSO
45H
2o0.05g, ZnCl
20.5g, is settled to 1L after dissolving with the HCl of 2mol/L; Described VITAMIN liquid: vitamin H 0.004g, VitB1 0.75mg, pyridoxol 0.04g, nicotinic acid 0.8g, be settled to 1L after dissolving with the HCl of 2mol/L.
In one embodiment of the invention, the inoculum size of described inoculation is 10%.
Beneficial effect:
The inventive method can improve output of pyruvic acid and production intensity; Relative to control strain (TgU
-(pY26)), the recombinant bacterial strain TgU of the present invention's structure
-(pY26-MPC1) output of dry cell weight, glucose consumption rate, pyruvic acid and Pyruvate production intensity improve 24.9%, 28.2%, 91.9% and 94.1% respectively.
Embodiment
Bacterial strain and plasmid: torulopsis glabrata (T.glabrataCCTCCM202019, TgU
-) it is nicotinic acid, vitamin H, VitB1, pyridoxine hydrochloride, uridylic 5 kinds of auxotrophic strains (namely having knocked out Ura gene on the basis of T.glabrataCCTCCM202019).Expression plasmid of yeast pY26 is the shuttle vectors between E. coli-Yeast, and in intestinal bacteria, selective marker is amp
r, and selective marker is that uracil-deficient type is complementary in yeast.
The mensuration of dry cell weight: get a certain amount of bacteria suspension in 10mL volumetric flask, add 2mL hydrochloric acid (2mol/L) and dissolve calcium carbonate in suspension, add deionized water constant volume to 10mL, abundant mixing, with UV7500 type visible spectrophotometer, measure OD value in 660nm place, utilize dry cell weight typical curve to calculate dry cell weight.
The mensuration of pyruvic acid and glucose: high performance liquid chromatography (HPLC).Instrument: Agilent1260 high performance liquid chromatograph (joining UV-vis detector, differential refraction detector and workstation), chromatographic condition: chromatographic column: AminexHPX-87Hionexchangecolumn, moving phase: 5mMH
2sO
4flow velocity: 0.6mL/min, column temperature: 40 DEG C, sample size: 10 μ L, UV-detector wavelength: 210nm (detection pyruvic acid), differential refraction detector: detect glucose, sample preparation: 1mL fermented liquid is in 12, centrifugal 5min under 000rpm, supernatant, through suitable dilution process with after 0.22 μ L membrane filtration, carries out efficient liquid phase chromatographic analysis.
Substratum: seed culture medium (g/L): glucose 30g, phytone 10g, potassium primary phosphate 1.0g, magnesium sulfate heptahydrate 0.5g, solid medium adds 20g agar.115 DEG C of sterilizing 15min.Fermention medium (g/L): glucose 120g, urea 3.86g, MgSO
4.7H2O0.8g, KH
2pO
42g, CH
3cOONa3g, liquid microelement (filtration sterilization) 10ml, VITAMIN liquid (filtration sterilization) 10ml, sterilizing 15min at 115 DEG C.The calcium carbonate (independent sterilizing) of 40g/L is added for regulating pH.Liquid microelement: MnCl
24H
2o12g, FeSO
47H
2o2g, CaCl
22H
2o2g, CuSO
45H
2o0.05g, ZnCl
20.5g, is settled to 1L after dissolving with the HCl of 2mol/L.VITAMIN liquid: vitamin H 0.004g, VitB1 0.75mg, pyridoxol 0.04g, nicotinic acid 0.8g, be settled to 1L after dissolving with the HCl of 2mol/L.
Embodiment 1: the amplification of yeast saccharomyces cerevisiae MPC1 and the structure of expression plasmid
With genes of brewing yeast group for template, pcr amplification obtains goal gene MPC1 fragment, the specific fragment of about 400bp is obtained by agarose gel electrophoresis, goal gene MPC1 is after restriction enzyme EcoRI and XamI is double digested, concentrated and purified, by MPC1, MPC2 gene directed cloning in expression plasmid pY26, obtain expression of recombinant yeast plasmid pY26-MPC1, above-mentioned recombinant expression plasmid is transformed into competent cell JM109 and is coated on the LB flat board containing penbritin and increase.
Embodiment 2: the Construction and identification of recombinant bacterium
Due on above-mentioned recombinant plasmid with Ura3 gene, above-mentioned recombination yeast plasmid is electroporated to recipient bacterium T.glabrata (Ura-) (namely having lacked the T.glabrataCCTCCM202019 of Ura gene) respectively, obtain the recombinant bacterium TgU of normal growth on the basic medium not adding uridylic
-(pY26-MPC1)
Embodiment 3: recombinant bacterium with contrast bacterium control fermentation and test
Picking recombinant bacterium TgU
-(pY26-MPC1) T.glabrata (Ura with containing empty plasmid pY26
-) single bacterium colony activation culture 18-24h on seed culture medium, with the inoculum size of 10%, the seed liquor that above-mentioned activation culture is good is inoculated in fermention medium, at 30 DEG C, under 220rpm condition, carries out fermentation culture.Fermentation results is as shown in table 1, and the output of the dry cell weight of recombinant bacterium, glucose consumption rate, pyruvic acid and Pyruvate production intensity are relative to contrast bacterium (TgU
-(pY26)) improve 24.9%, 28.2%, 91.9% and 94.1% respectively.
Table 1 recombinant bacterium TgU
-(pY26-MPC1) with contrast bacterium TgU
-(pY26) fermentation contrast
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. a recombinant bacterium for output of pyruvic acid and production intensity raising, is characterized in that, described recombinant bacterium process LAN mitochondrial pyruvate acid vectors albumen MPC1.
2. recombinant bacterium according to claim 1, is characterized in that, described recombinant bacterium has lacked the MPC1 that the torulopsis glabrata T.glabrataCCTCCM202019 of Ura gene is GeneID:852800 on host expresses NCBI.
3. recombinant bacterium according to claim 1, is characterized in that, described process LAN is for carrier with expression plasmid of yeast pY26.
4. recombinant bacterium according to claim 1, it is characterized in that, the structure of described recombinant bacterium: by object fragment MPC1 after restriction enzyme EcoRI and XamI is double digested, and by MPC1 directed cloning in expression plasmid pY26, obtain expression of recombinant yeast plasmid pY26-MPC1, recombinant plasmid is transferred in uracil auxotrophy recipient bacterium by electroporated method, screen in not containing the substratum of uridylic, obtain the recombinant bacterium TgU of mitochondrial pyruvate acid vectors albumen overexpression
-(pY26-MPC1).
5. improve a method for output of pyruvic acid and production intensity, it is characterized in that, described method is process LAN mitochondrial pyruvate acid vectors albumen in Pyruvate production bacterial strain.
6. method according to claim 5, is characterized in that, described method is the mitochondrial pyruvate acid vectors albumen MPC1 expressing GeneID:852800 on NCBI.
7. the application of the arbitrary described recombinant bacterium of claim 1-4 in fermentation production of acetone acid.
8. application according to claim 7, is characterized in that, described application is seeded to fermention medium after being activated by recombinant bacterium, at 30 DEG C, carries out fermentation culture under 220rpm condition.
9. application according to claim 8, is characterized in that, contains: glucose 120g, urea 3.86g, MgSO in described fermention medium 1L
4.7H
2o0.8g, KH
2pO
42g, CH
3cOONa3g, liquid microelement 10ml, VITAMIN liquid 10ml, initial pH=5.5; Described liquid microelement: MnCl
24H
2o12g, FeSO
47H
2o2g, CaCl
22H
2o2g, CuSO
45H
2o0.05g, ZnCl
20.5g, is settled to 1L after dissolving with the HCl of 2mol/L; Described VITAMIN liquid: vitamin H 0.004g, VitB1 0.75mg, pyridoxol 0.04g, nicotinic acid 0.8g, be settled to 1L after dissolving with the HCl of 2mol/L.
10. the pyruvic acid produced of the arbitrary described recombinant bacterium of claim 1-4 at daily use chemicals, food, prepare the application of medicine, agriculture field.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106544285A (en) * | 2016-12-07 | 2017-03-29 | 江南大学 | A kind of reinforcing torulopsis glabrata synthesis Pyruvate Method |
| CN106544286A (en) * | 2016-12-07 | 2017-03-29 | 江南大学 | It is a kind of to reduce the method that polysaccharide accumulation reinforcing torulopsis glabrata produces pyruvic acid |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106544285A (en) * | 2016-12-07 | 2017-03-29 | 江南大学 | A kind of reinforcing torulopsis glabrata synthesis Pyruvate Method |
| CN106544286A (en) * | 2016-12-07 | 2017-03-29 | 江南大学 | It is a kind of to reduce the method that polysaccharide accumulation reinforcing torulopsis glabrata produces pyruvic acid |
| CN106544286B (en) * | 2016-12-07 | 2019-05-10 | 江南大学 | A method of it reducing polysaccharide accumulation and strengthens torulopsis glabrata production pyruvic acid |
| CN106544285B (en) * | 2016-12-07 | 2019-07-02 | 江南大学 | A kind of reinforcing torulopsis glabrata synthesis Pyruvate Method |
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