CN105462868B - A method of improving output of pyruvic acid and production intensity - Google Patents
A method of improving output of pyruvic acid and production intensity Download PDFInfo
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- CN105462868B CN105462868B CN201510907318.2A CN201510907318A CN105462868B CN 105462868 B CN105462868 B CN 105462868B CN 201510907318 A CN201510907318 A CN 201510907318A CN 105462868 B CN105462868 B CN 105462868B
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Abstract
The invention discloses a kind of raising output of pyruvic acid and the method for producing intensity, belong to fermentation engineering field.The present invention in Torulopsis glabrata strain by being overexpressed mitochondrial pyruvate acid vectors albumen, reinforce pyruvic acid and enters mitochondria, further strengthen the growth of thallus, to improve the yield and production intensity of growth coupling type product acetone acid, by technique for gene engineering, TgU is expressed in by having crossed from the MPC1 gene of saccharomyces cerevisiae‑In, obtain the engineered strain TgU that a plant height produces pyruvic acid‑(pY26-MPC1), relative to control strain TgU‑(pY26), engineering bacteria TgU‑(pY26-MPC1) dry cell weight, glucose consumption rate, the yield of pyruvic acid and Pyruvate production intensity is relative to control bacterium (TgU‑(pY26)) it has been respectively increased 24.9%, 28.2%, 91.9% and 94.1%.
Description
Technical field
The present invention relates to a kind of raising output of pyruvic acid and the method for producing intensity, belong to fermentation engineering field.
Background technique
Pyruvic acid (pyruvate) is the intermediate product of biological metabolism, the intermediate link in each metabolic pathway, such as: being sugar
The final product of glycolysis；Initial substance acetyl-the CoA of TCA circulation is converted by dehydrogenation decarboxylic reaction；Make in pyruvate carboxylase
With the intermediate product oxaloacetic acid of lower generation TCA circulation；Alanine etc. is generated by transamination, while being also extremely important
One of ketone acid.Since pyruvic acid is the synthesis precursor of many kinds of substance, present society increasingly increases the demand of pyruvic acid,
It is widely used in daily use chemicals, food, medical treatment, the industries such as agricultural.
The production method of pyruvic acid mainly has chemical synthesis and microbe fermentation method two major classes.Chemical synthesis mainly has
Winestone acid system, the cruel air oxidation process of lactic acid second, through benzylacetone method, electrochemical synthesis, lactic acid catalytic oxidation.Pyruvic acid at present
Production just pervious chemical industry synthesis be diverted through microbial fermentation preparation.Microbe fermentation method has more relative to chemical industry synthesis
The advantages that more advantages, such as the high conversion rate of raw material, pollution is small, at low cost.
It is ground both at home and abroad currently, Production by Microorganism Fermentation pyruvic acid becomes as a kind of alternative environmentally protective method
The hot topic studied carefully.And high yield pyruvic acid is obtained by microbial fermentation, wherein most critical is that have plant height production and high production
The pyruvic acid bacterial strain of intensity.The transformation of high yield pyruvic acid bacterial strain is concentrated mainly on to certain key enzymes in metabolic pathway at present
Be overexpressed and some alternative pathways are knocked out.
Summary of the invention
To solve the above-mentioned problems, the present invention provides one kind by being overexpressed mitochondrial pyruvate acid vectors albumen to enhance third
The method of the production intensity and yield of ketone acid fermentation.
The first purpose of the invention is to provide a kind of raising output of pyruvic acid and the method for producing intensity, the method is
Mitochondrial pyruvate acid vectors albumen is overexpressed in Pyruvate production bacterial strain.
In one embodiment of the invention, the method is the mitochondria for expressing Gene ID:852800 on NCBI
Pyruvic acid carrier protein MPC1.
In one embodiment of the invention, the production bacterial strain is the torulopsis glabrata to have lacked Ura gene
Obtained from MPC1 of the T.glabrata CCTCC M202019 for Gene ID:852800 on host expresses NCBI.
In one embodiment of the invention, the overexpression is (precious public purchased from moral with expression plasmid of yeast pY26
Department) it is carrier.
A second object of the present invention is to provide the recombinant bacterium that a kind of output of pyruvic acid and production intensity improve, the recombinations
Bacterium is overexpressed mitochondrial pyruvate acid vectors albumen MPC1.
In one embodiment of the invention, the recombinant bacterium is the T.glabrata to have lacked Ura gene
The MPC1 that CCTCC M202019 is host, expression plasmid of yeast pY26 is Gene ID:852800 on carrier expression NCBI.
In one embodiment of the invention, the building of the recombinant bacterium: by target fragment MPC1 by restricted interior
After enzyme cutting EcoR I and Xam I are double digested, and MPC1 is cloned into the double digested expression plasmid of EcoR I/Xam I
In pY26, expression of recombinant yeast plasmid pY26-MPC1 is obtained, recombinant plasmid is transferred to recipient bacterium by electroporated method
T.glabrata CCTCC M202019(TgU-), it is screened in the culture medium without uracil, obtains mitochondrial pyruvate
The recombinant bacterium TgU of acid vectors albumen overexpression-(pY26-MPC1)。
In one embodiment of the invention, the MPC1 gene is using Wine brewing yeast strain N85 genome as template
What amplification obtained.
In one embodiment of the invention, expression plasmid of yeast pY26 the wearing between E. coli-Yeast
Shuttle carrier, selected marker is amp in Escherichia colir, and selected marker is that uracil-deficient is complementary in yeast.
Third object of the present invention is to provide a kind of methods using the recombinant bacterium fermentation production of acetone acid, are that will weigh
It is seeded to fermentation medium after group bacterium activation, at 30 DEG C, fermented and cultured is carried out under the conditions of 220rpm.
In one embodiment of the invention, the fermentation medium (/L): glucose 120g, urea 3.86g,
MgSO4.7H2O 0.8g, KH2PO42g, CH3COONa 3g, liquid microelement 10ml, vitamin liquid 10ml, initial pH=5.5；Institute
State liquid microelement: MnCl2·4H2O 12g, FeSO4·7H2O 2g, CaCl2·2H2O 2g, CuSO4·5H2O 0.05g,
ZnCl20.5g is settled to 1L after being dissolved with the HCl of 2mol/L；The vitamin liquid: biotin 0.004g, thiamine
0.75mg, pyridoxol 0.04g, niacin 0.8g are settled to 1L after being dissolved with the HCl of 2mol/L.
In one embodiment of the invention, the inoculum concentration of the inoculation is 10%.
The utility model has the advantages that
The method of the present invention can be improved output of pyruvic acid and produce intensity；Relative to control strain (TgU-(pY26)), originally
Invent the recombinant bacterial strain TgU of building-(pY26-MPC1) dry cell weight, glucose consumption rate, the yield of pyruvic acid and acetone
Acid production intensity has been respectively increased 24.9%, 28.2%, 91.9% and 94.1%.
Specific embodiment
Bacterial strain and plasmid: torulopsis glabrata (T.glabrata CCTCC M202019, TgU-) it is niacin, biology
Element, thiamine, puridoxine hydrochloride, 5 kinds of auxotrophic strains of uracil are (i.e. in the base of T.glabrata CCTCC M202019
Ura gene is knocked out on plinth).Shuttle vector of the expression plasmid of yeast pY26 between E. coli-Yeast, in Escherichia coli
Selected marker is ampr, and selected marker is that uracil-deficient is complementary in yeast.
The measurement of dry cell weight: taking a certain amount of bacteria suspension in 10mL volumetric flask, and 2mL hydrochloric acid (2mol/L) dissolution is added
Calcium carbonate in suspension adds deionized water constant volume to 10mL, mixes well, with 7500 type visible spectrophotometer of UV, in
OD value is measured at 660nm, calculates dry cell weight using dry cell weight standard curve.
The measurement of pyruvic acid and glucose: high performance liquid chromatography (HPLC).Instrument: 1260 high performance liquid chromatography of Agilent
Instrument (matches UV-vis detector, differential refraction detector and work station), chromatographic condition: chromatographic column: Aminex HPX-87H
Ion exchange column, mobile phase: 5mM H2SO4, flow velocity: 0.6mL/min, column temperature: 40 DEG C, sample volume: 10 μ L, it is ultraviolet
Detector wavelength: 210nm (detection pyruvic acid), differential refraction detector: detection glucose, sample preparation: 1mL fermentation liquid in
5min is centrifuged under 12,000rpm, supernatant handles by dilution appropriate and after 0.22 μ L membrane filtration, carries out high-efficient liquid phase color
Spectrum analysis.
Culture medium: seed culture medium (g/L): glucose 30g, phytone 10g, potassium dihydrogen phosphate 1.0g, seven water sulphur
Sour magnesium 0.5g, solid medium add 20g agar.115 DEG C of sterilizing 15min.Fermentation medium (g/L): glucose 120g, urea
3.86g MgSO4.7H2O 0.8g, KH2PO42g, CH3COONa 3g, liquid microelement (filtration sterilization) 10ml, vitamin liquid
(filtration sterilization) 10ml, sterilize 15min at 115 DEG C.The calcium carbonate (individually sterilizing) of 40g/L is added for adjusting pH.It is micro-
Secondary element liquid: MnCl2·4H2O 12g, FeSO4·7H2O 2g, CaCl2·2H2O 2g, CuSO4·5H2O 0.05g, ZnCl2
0.5g is settled to 1L after being dissolved with the HCl of 2mol/L.Vitamin liquid: biotin 0.004g, thiamine 0.75mg, pyridoxol
0.04g, niacin 0.8g are settled to 1L after being dissolved with the HCl of 2mol/L.
Embodiment 1: the amplification of saccharomyces cerevisiae MPC1 and the building of expression plasmid
Using saccharomyces cerevisiae genome as template, PCR amplification obtains target gene MPC1 segment, passes through agarose gel electrophoresis
Obtain the specific fragment of about 400bp, target gene MPC1 after restriction enzyme EcoR I and Xam I are double digested,
It concentrates and purifies, by MPC1, MPC2 gene directed cloning into expression plasmid pY26, obtains expression of recombinant yeast plasmid pY26-
Above-mentioned recombinant expression plasmid is transformed into competent cell JM109 and is coated on the LB plate containing ampicillin by MPC1
It is expanded.
Embodiment 2: the building and identification of recombinant bacterium
It is due to having Ura3 gene on above-mentioned recombinant plasmid, above-mentioned recombination yeast plasmid difference is electroporated to recipient bacterium
T.glabrata (Ura-) (has lacked the T.glabrata CCTCC M202019 of Ura gene), obtains that uracil is being not added
Basal medium on normal growth recombinant bacterium TgU-(pY26-MPC1)
Embodiment 3: recombinant bacterium is tested with bacterium control fermentation is compareed
Picking recombinant bacterium TgU-(pY26-MPC1) T.glabrata (Ura and containing empty plasmid pY26-) single bacterium fall within
The good seed liquor of above-mentioned activation culture is inoculated into fermentation with 10% inoculum concentration by activation culture 18-24h on seed culture medium
In culture medium, at 30 DEG C, fermented and cultured is carried out under the conditions of 220rpm.Fermentation results are as shown in table 1, the dry cell weight of recombinant bacterium,
Glucose consumption rate, the yield of pyruvic acid and Pyruvate production intensity are relative to control bacterium (TgU-(pY26)) it is respectively increased
24.9%, 28.2%, 91.9% and 94.1%.
1 recombinant bacterium TgU of table-(pY26-MPC1) with compare bacterium TgU-(pY26) fermentation comparison
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (13)
1. the recombinant bacterium that a kind of output of pyruvic acid and production intensity improve, which is characterized in that the recombinant bacterium is overexpressed mitochondria
Pyruvic acid carrier protein MPC1；The recombinant bacterium is to lackUraThe torulopsis glabrata of geneTorulopsis.glabrataCCTCC M202019 is Gene ID:852800 on host expresses NCBIMPC1Gene.
2. recombinant bacterium according to claim 1, which is characterized in that the overexpression is with expression plasmid of yeast pY26 for load
Body.
3. recombinant bacterium according to claim 1, which is characterized in that the building of the recombinant bacterium: by target fragmentMPC1Through
Cross restriction enzymeEcoR I andXamAfter I is double digested, and willMPC1Directed cloning is obtained into expression plasmid pY26
Expression of recombinant yeast plasmid pY26-MPC1, recombinant plasmid is transferred to uracil auxotrophy by electroporated method
In recipient bacterium, is screened in the culture medium without uracil, obtain the weight of mitochondrial pyruvate acid vectors albumen overexpression
Group bacteriumTgU - -pY26-MPC1。
4. a kind of method for improving output of pyruvic acid and producing intensity, which is characterized in that the method is using claim 1-3
Any recombinant bacterium.
5. application of the recombinant bacterium described in claim 1 in terms of fermentation production of acetone acid.
6. application of the recombinant bacterium described in claim 2 in terms of fermentation production of acetone acid.
7. application of the recombinant bacterium described in claim 3 in terms of fermentation production of acetone acid.
8. application according to claim 5, which is characterized in that the application is to be seeded to fermentation training after activating recombinant bacterium
It supports base and carries out fermented and cultured under the conditions of 220rpm at 30 DEG C.
9. application according to claim 6, which is characterized in that the application is to be seeded to fermentation training after activating recombinant bacterium
It supports base and carries out fermented and cultured under the conditions of 220rpm at 30 DEG C.
10. application according to claim 7, which is characterized in that the application is to be seeded to fermentation after activating recombinant bacterium
Culture medium carries out fermented and cultured at 30 DEG C under the conditions of 220rpm.
11. application according to claim 8, which is characterized in that contain in the fermentation medium 1L: glucose 120g,
Urea 3.86g, MgSO4·7H2O 0.8g, KH2PO42g, CH3COONa 3g, liquid microelement 10ml, vitamin liquid 10ml, just
Beginning pH=5.5；The liquid microelement: MnCl2·4H2O 12g, FeSO4·7H2O 2g, CaCl2·2H2O 2g, CuSO4·
5H2O 0.05g, ZnCl20.5g is settled to 1L after being dissolved with the HCl of 2mol/L；The vitamin liquid: biotin 0.004g, sulphur
Amine element 0.75mg, pyridoxol 0.04g, niacin 0.8g are settled to 1L after being dissolved with the HCl of 2mol/L.
12. application according to claim 9, which is characterized in that contain in the fermentation medium 1L: glucose 120g,
Urea 3.86g, MgSO4·7H2O 0.8g, KH2PO42g, CH3COONa 3g, liquid microelement 10ml, vitamin liquid 10ml, just
Beginning pH=5.5；The liquid microelement: MnCl2·4H2O 12g, FeSO4·7H2O 2g, CaCl2·2H2O 2g, CuSO4·
5H2O 0.05g, ZnCl20.5g is settled to 1L after being dissolved with the HCl of 2mol/L；The vitamin liquid: biotin 0.004g, sulphur
Amine element 0.75mg, pyridoxol 0.04g, niacin 0.8g are settled to 1L after being dissolved with the HCl of 2mol/L.
13. application according to claim 10, which is characterized in that contain in the fermentation medium 1L: glucose 120g,
Urea 3.86g, MgSO4·7H2O 0.8g, KH2PO42g, CH3COONa 3g, liquid microelement 10ml, vitamin liquid 10ml, just
Beginning pH=5.5；The liquid microelement: MnCl2·4H2O 12g, FeSO4·7H2O 2g, CaCl2·2H2O 2g, CuSO4·
5H2O 0.05g, ZnCl20.5g is settled to 1L after being dissolved with the HCl of 2mol/L；The vitamin liquid: biotin 0.004g, sulphur
Amine element 0.75mg, pyridoxol 0.04g, niacin 0.8g are settled to 1L after being dissolved with the HCl of 2mol/L.
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| CN106544285B (en) * | 2016-12-07 | 2019-07-02 | 江南大学 | A kind of reinforcing torulopsis glabrata synthesis Pyruvate Method |
| 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 |
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| CN100587060C (en) * | 2008-03-18 | 2010-02-03 | 江南大学 | Construction of recombination bacterial strain for producing pyruvic acid and method for improving production strength of pyruvic acid |
| CN101691545B (en) * | 2009-09-09 | 2011-10-05 | 江南大学 | Structuring of pyruvic acid-producing recombinant bacteria strain and method for enhancing synthesis rate of pyruvic acid by pyruvic acid-producing recombinant bacteria |
| KR20150034867A (en) * | 2013-09-25 | 2015-04-06 | 삼성전자주식회사 | Yeast cell with increased with pyruvate pool in the cytosol and a method of producing pyruvate based metabolites using the same |
| CN103710274B (en) * | 2013-12-24 | 2016-03-23 | 江南大学 | The genetic engineering bacterium that the outer output of pyruvic acid of a kind of born of the same parents improves and application thereof |
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