CN106544285B - A kind of reinforcing torulopsis glabrata synthesis Pyruvate Method - Google Patents

A kind of reinforcing torulopsis glabrata synthesis Pyruvate Method Download PDF

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CN106544285B
CN106544285B CN201611112789.5A CN201611112789A CN106544285B CN 106544285 B CN106544285 B CN 106544285B CN 201611112789 A CN201611112789 A CN 201611112789A CN 106544285 B CN106544285 B CN 106544285B
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pyruvic acid
pyc2
pck1
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CN106544285A (en
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周景文
陈坚
罗正山
堵国成
刘松
方芳
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Jiangnan University
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Abstract

The invention discloses a kind of reinforcing torulopsis glabratas to synthesize Pyruvate Method, belongs to fermentation engineering field.The present invention introduces ATP inefficient cycle by being overexpressed phosphoric acid enol pyruvic acid carboxylase and pyruvate carboxylase in Torulopsis glabrata strain, it is horizontal to reduce ATP intracellular, to enhance the yield and production intensity of Torulopsis glabrata strain pyruvic acid, by technique for gene engineering, will be overexpressed from the PYC2 gene of saccharomyces cerevisiae and PCK1 Gene Fusion in TgU‑In, obtain the engineered strain TgU that a plant height produces pyruvic acid and high pyruvate fermentation intensity‑(pY26-PYC2-PCK1), relative to control strain, engineering bacteria TgU‑(pY26-PYC2-PCK1) yield, the substrate transformation rate and the Pyruvate production intensity of glucose consumption rate, pyruvic acid have been respectively increased 15.6%, 50.0%, 54.5% and 81.5%.

Description

A kind of reinforcing torulopsis glabrata synthesis Pyruvate Method
Technical field
The present invention relates to a kind of reinforcing torulopsis glabratas to synthesize Pyruvate Method, belongs to fermentation engineering field.
Background technique
Pyruvic acid is the intermediate product of microbial metabolism, the intermediate link in a metabolic pathway, such as: being the end of glycolysis Product；The initial substance acetyl-CoA of TCA circulation is converted by a step dehydrogenation decarboxylation；It can also be generated by carboxylation reaction The intermediate product oxaloacetic acid etc. of TCA circulation is also that can be used as many high added value substance synthesis to plant important ketone acid Precursor, such as: levodopa；Ethyl pyruvate etc., thus daily use chemicals are widely used in, 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, ethyl lactate air oxidation process, hydroxypropanone- method, electrochemical synthesis, lactic acid catalytic oxidation.Pyruvic acid at present Production by pervious chemical industry synthesis be diverted through microbial fermentation preparation.Microbe fermentation method has 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 will by microbial fermentation obtain high yield pyruvic acid, wherein it is crucial that need to construct a plant height produce and The bacterial strain of high production intensity.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of by fusion overexpression phosphoric acid enol pyruvic acid carboxylase ATP inefficient cycle is constructed with pyruvate carboxylase to reduce ATP concentration intracellular, thus to enhance the production intensity of pyruvate fermentation With the method for yield.
The first purpose of the invention is to provide the recombinant bacterium that a kind of output of pyruvic acid and production intensity improve, the recombinations The recombination torulopsis glabrata of bacterium be amalgamation and expression pyruvate carboxylase and phosphoric acid enol pyruvic acid carboxylase.
In one embodiment of the invention, the recombinant bacterium is the T.glabrata to have lacked URA3 gene CCTCC M202019 is host, using pY26-TEF-GPD as carrier, co-expresses the gene PYC2 and Gene of Gene ID:852519 The gene PCK1 of ID:853972.
It is with torulopsis glabrata for host a second object of the present invention is to provide the construction method of the recombinant bacterium, Using pY26-TEF-GPD as carrier, the gene PYC2 and PCK1 of Saccharomyces cerevisiae are co-expressed.
In one embodiment of the invention, PYC2 the and PCK1 gene source is in Saccharomyces Cerevisiae in S 288c genome.
In one embodiment of the invention, the method is by the gene PYC2 and Gene of Gene ID:852519 The gene PCK1 of ID:853972 is connect with carrier pY26-TEF-GPD, then in the torulopsis glabrata for having lacked URA3 gene It is expressed in T.glabrata CCTCC M 202019.
In one embodiment of the invention, the method specifically comprises the following steps: (1) from Saccharomyces Cerevisiae in S 288c Genome on PCR amplification target gene PYC2 and PCK1；(2) target fragment PYC2 is connected with PCK1 with link peptide GGGS； (3) the PYC2-PCK1 segment for obtaining fusion is after restriction enzyme Not I and Pac I are double digested, directed cloning Into expression plasmid pY26-TEF-GPD, expression of recombinant yeast plasmid pY26-PYC2-PCK1 is obtained；(4) recombinant plasmid pY26- PYC2-PCK1 electrotransformation is screened in the culture medium without uracil to uracil auxotrophy recipient bacterium, obtains weight Group bacterium TgU-(pY26-PYC2-PCK1)。
Third object of the present invention is to provide a kind of raising output of pyruvic acid and the method for producing intensity, the method is ATP inefficient cycle is constructed in torulopsis glabrata；The building ATP inefficient cycle is fusion and overexpression phosphoenolpyruvate Pyruvate carboxylase and pyruvate carboxylase.
In one embodiment of the invention, the phosphoric acid enol pyruvic acid carboxylase is by Gene ID:852519's PYC2 gene coding；The pyruvate carboxylase is encoded by the PCK1 gene of Gene ID:853972.
Fourth object of the present invention is to provide a kind of method using the recombinant bacterium fermentation production of acetone acid, is that will weigh Group bacterium is seeded in fermentation medium, ferments in 28-30 DEG C, 400-600rpm, 1-3vvm.
In one embodiment of the invention, the method is will to be seeded to after recombinant bacterium activation equipped with fermentation training The 3L-NBS fermentor for supporting base, carries out fermented and cultured under the conditions of 28-30 DEG C, 400-600rpm, 1-3vvm.
In one embodiment of the invention, the fermentation medium (/L): glucose 50-120g, urea 0-5g, MgSO4.7H2O 0-1g, KH2PO40-5g, CH3COONa 0-5g, liquid microelement 10ml, vitamin liquid 10ml, initial pH= 5.5；The every L of liquid microelement contains: MnCl2·4H2O 0-12g, FeSO4·7H2O 0-2g, CaCl2·2H2O0-2g, CuSO4·5H2O 0-2g, ZnCl20-0.1g；The every L of vitamin liquid contains: biotin 0-0.01g, thiamine 0-2mg, pyrrole Tremble alcohol 0-0.2g, niacin 0-2g.
In one embodiment of the invention, the liquid microelement is by MnCl2·4H2O 0-12g, FeSO4· 7H2O 0-2g, CaCl2·2H2O 0-2g, CuSO4·5H2O 0-2g and ZnCl2It is fixed after the 0-0.1g HCl dissolution of 2mol/L Hold to 1L；The vitamin liquid is to use biotin 0-0.01g, thiamine 0-2mg, pyridoxol 0-0.2g and niacin 0-2g 1L is settled to after the HCl dissolution of 2mol/L.
In one embodiment of the invention, the inoculum concentration of the inoculation is 5-10% by volume.
The present invention also provides the recombinant bacterium food, chemical industry, field of medicaments application.
In one embodiment of the invention, the application is to prepare the product containing pyruvic acid.
The utility model has the advantages that the method for the present invention can be improved output of pyruvic acid and produce intensity；Relative to control strain TgU- (pY26), the recombinant bacterial strain TgU that the present invention constructs-The yield of glucose consumption rate, pyruvic acid (pY26-PYC2-PCK1), The substrate transformation rate and Pyruvate production intensity have been respectively increased 15.6%, 50.0%, 54.5% and 81.5%.
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 URA3 gene is knocked out on plinth).Shuttle vector of the expression plasmid of yeast pY26-TEF-GPD between E. coli-Yeast, Selected marker is amp in Escherichia colir, 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.
Seed culture medium (g/L): glucose 15-30g, phytone 5-10g, potassium dihydrogen phosphate 0.5-2g, seven water sulphur Sour magnesium 0-1g, solid medium add 20g agar.115 DEG C of sterilizing 15min.
Fermentation medium (g/L): glucose 50-120g, urea 0-5g, MgSO4.7H2O 0-1g, KH2PO40-5g, CH3COONa 0-5g, liquid microelement (filtration sterilization) 10ml, vitamin liquid (filtration sterilization) 10ml sterilize at 115 DEG C 15min.The calcium carbonate (individually sterilizing) of 40g/L is added for adjusting pH.Liquid microelement: MnCl2·4H2O 0-12g, FeSO4·7H2O 0-2g, CaCl2·2H2O 0-2g, CuSO4·5H2O 0-0.1g, ZnCl20-1g, it is molten with the HCl of 2mol/L 1L is settled to after solution.Vitamin liquid: biotin 0-0.01g, thiamine 0-2mg, pyridoxol 0-0.2g, niacin 0-2g use 2mol/ 1L is settled to after the HCl dissolution of L.
The amplification and the building for the expression plasmid for introducing ATP inefficient cycle of 1 saccharomyces cerevisiae PYC2 and PCK1 gene of embodiment
Using saccharomyces cerevisiae genome as template, expanded using designed primer from PCR on the genome of Saccharomyces Cerevisiae in S 288c Increase target gene PYC2 and PCK1 out, PYC2 and PCK1 target fragment is respectively obtained by recycling；It will using the method for fusion DNA vaccine 2 segment connection fusions get up to obtain PYC2-PCK1 segment；The PYC2-PCK1 sequence that fusion is obtained is by restriction enzyme After enzyme Not I and Pac I are double digested, directed cloning obtains expression of recombinant yeast into expression plasmid pY26-TEF-GPD Plasmid pY26-PYC2-PCK1；Above-mentioned recombinant expression plasmid is transformed into competent cell JM109 and is coated on green containing ammonia benzyl It is screened on the LB plate of mycin, obtains correct clone.
1 primer of table
The building and identification of 2 recombinant bacterium of embodiment
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(URA3-) (having lacked the T.glabrata CCTCC M202019 of URA3 gene), obtain be not added urine it is phonetic The recombinant bacterium TgU of normal growth on the basal medium of pyridine-(pY26-PYC2-PCK1)。
3 recombinant bacterium of embodiment is tested with bacterium control fermentation is compareed
Picking recombinant bacterium TgU-(pY26-PYC2-PCK1) and the T.glabrata containing empty plasmid pY26-TEF-GPD (URA3-) (as control bacterium) single colonie be seeded to activation culture 18-24h in seed culture medium respectively, body (is pressed with 5-10% Product ratio) inoculum concentration, the good seed liquor of above-mentioned activation culture is inoculated into respectively in the 3L fermentor equipped with fermented and cultured, and is added Add the calcium carbonate of 40g/L to carry out the pH during buffered fermentation, ferments under the conditions of 28-30 DEG C, 400-600rpm.Fermentation knot Fruit is as shown in table 1, and the fermentation period of recombinant bacterium shortens 12 hours, shortens 16.7% compared with compareing bacterium；Glucose consumption Rate, the yield of pyruvic acid, the substrate transformation rate and Pyruvate production intensity are relative to control bacterium (TgU-(pY26)) it is respectively increased 15.6%, 50.0%, 54.5% and 81.5%.
1 recombinant bacterium TgU of table-(pY26-PYC2-PCK1) with the 3L ferment tank Comparative result that compares bacterium
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.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of reinforcing torulopsis glabrata synthesizes Pyruvate Method
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Claims (10)

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 amalgamation and expression The recombination torulopsis glabrata of pyruvate carboxylase and phosphoric acid enol pyruvic acid carboxylase.
2. recombinant bacterium according to claim 1, which is characterized in that the recombinant bacterium is to have lacked URA3 gene T.glabrata CCTCC M 202019 is host, using pY26-TEF-GPD as carrier, co-expresses the base of Gene ID:852519 Because of the gene PCK1 of PYC2 and Gene ID:853972.
3. the construction method of recombinant bacterium described in claim 1, which is characterized in that with torulopsis glabrata be host, with pY26- TEF-GPD is carrier, co-expresses the gene PYC2 and PCK1 of Saccharomyces cerevisiae.
4. according to the method described in claim 3, it is characterized in that, the method is by the gene PYC2 of Gene ID:852519 It is connect with the gene PCK1 of Gene ID:853972 with carrier pY26-TEF-GPD, then quasi- in the smooth ball for having lacked URA3 gene It is expressed in yeast T.glabrata CCTCC M 202019.
5. a kind of method for improving output of pyruvic acid and producing intensity, which is characterized in that the method is in torulopsis glabrata Middle building ATP inefficient cycle；The building ATP inefficient cycle be fusion and overexpression phosphoric acid enol pyruvic acid carboxylase and Pyruvate carboxylase.
6. according to the method described in claim 5, it is characterized in that, the phosphoric acid enol pyruvic acid carboxylase is by Gene ID: 852519 PYC2 gene coding；The pyruvate carboxylase is encoded by the PCK1 gene of Gene ID:853972.
7. a kind of method using recombinant bacterium fermentation production of acetone acid described in claim 1, which is characterized in that recombinant bacterium to be inoculated with Into fermentation medium, ferment in 28-30 DEG C, 400-600rpm, 1-3vvm.
8. the method according to the description of claim 7 is characterized in that the every L of the fermentation medium contains: glucose 50-120g, Urea 0-5g, MgSO4·7H2O 0-1g, KH2PO40-5g, CH3COONa 0-5g, liquid microelement 10ml, vitamin liquid 10ml, initial pH=5.5；The every L of liquid microelement contains: MnCl2·4H2O 0-12g, FeSO4·7H2O 0-2g, CaCl2·2H2O 0-2g, CuSO4·5H2O 0-2g, ZnCl20-0.1g；The every L of vitamin liquid contains: biotin 0- 0.01g, thiamine 0-2mg, pyridoxol 0-0.2g, niacin 0-2g.
9. the method according to the description of claim 7 is characterized in that it is described inoculation be with volume ratio for 5-10% inoculum concentration into Row inoculation.
10. any recombinant bacterium of claim 1-4 is in food, chemical industry, medicine, product of the agriculture field preparation containing pyruvic acid Application.
CN201611112789.5A 2016-12-07 2016-12-07 A kind of reinforcing torulopsis glabrata synthesis Pyruvate Method Active CN106544285B (en)

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CN102286388A (en) * 2011-06-21 2011-12-21 江南大学 Construction method and use of malic acid producing candida glabrata engineering strain
CN102296082A (en) * 2011-07-18 2011-12-28 南京工业大学 Construction method of escherichia coli genetic engineering bacteria for producing succinic acid by utilizing xylose metabolism
CN105368884A (en) * 2015-12-10 2016-03-02 江南大学 Recombinant bacterium with function of pyruvate production intensity improvement and application of recombinant bacterium with function of pyruvate production intensity improvement
CN105462868A (en) * 2015-12-10 2016-04-06 江南大学 Method for improving yield and production intensity of pyruvic acid

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6455186A (en) * 1987-08-25 1989-03-02 Toray Industries Production of pyruvic acid by fermentation method
CN102286388A (en) * 2011-06-21 2011-12-21 江南大学 Construction method and use of malic acid producing candida glabrata engineering strain
CN102296082A (en) * 2011-07-18 2011-12-28 南京工业大学 Construction method of escherichia coli genetic engineering bacteria for producing succinic acid by utilizing xylose metabolism
CN105368884A (en) * 2015-12-10 2016-03-02 江南大学 Recombinant bacterium with function of pyruvate production intensity improvement and application of recombinant bacterium with function of pyruvate production intensity improvement
CN105462868A (en) * 2015-12-10 2016-04-06 江南大学 Method for improving yield and production intensity of pyruvic acid

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Title
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光滑球拟酵母发酵生产丙酮酸;刘立明，等;《精细与专用化学品》;20031231(第23期);第15-18页
生物技术法生产丙酮酸的研究进展;刘立明，等;《生物工程学报》;20021130;第18卷(第6期);第651-655页

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