CN100587060C - Construction of recombination bacterial strain for producing pyruvic acid and method for improving production strength of pyruvic acid - Google Patents
Construction of recombination bacterial strain for producing pyruvic acid and method for improving production strength of pyruvic acid Download PDFInfo
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- CN100587060C CN100587060C CN200810019988A CN200810019988A CN100587060C CN 100587060 C CN100587060 C CN 100587060C CN 200810019988 A CN200810019988 A CN 200810019988A CN 200810019988 A CN200810019988 A CN 200810019988A CN 100587060 C CN100587060 C CN 100587060C
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- pyruvic acid
- fermentation
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- pyruvate
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Abstract
The invention relates to a construction of a recombinant bacterium for producing pyruvate and a method for improving the production strength of pyruvate by using the recombinant bacterium, which pertains to the technical filed of auxiliary factor metabolic regulation and control strategic optimized fermentation process. The method regulates and controls the carbon metabolic flow to strengthen theproduction strength of the pyruvate by changing an auxiliary factor NADH and adopts molecular approach to allow the NADH oxidase noxE gene which is coded into water and is derived from L. lactis to beover expressed in an industrial bacterial strain Torulosis glabrata CCTCC NO: M 202019 for producing the pyruvate by the fermentation method, so as to obtain an NADH oxidase over expressed recombinant bacterium PdnoxE CCTCC NO: M 208022; compared with the starting bacterial strain, the bacterial dry weight, the glucose consumption rate and the pyruvate production strength thereof are respectivelyimproved by 168 percent, 44.9 percent and 12 percent. The method has universally applicable significance for the improvement of a plurality of important fermentation products (such as, organic acidsand amino acids etc.).
Description
Technical field
One strain is produced the structure of pyruvic acid reorganization bacterium and with its method that improves the pyruvic acid production intensity, is related to a kind of production intensity by cofactor NADH regulation and control of carbon metabolism flow reinforcement pyruvic acid, belongs to cofactor metabolic regulation policy optimization fermenting process technical field.By the influence of research cofactor NADH, realize strengthening the pyruvic acid production intensity to the metabolism stream flow direction and corresponding metabolic flux.
Background technology
Pyruvic acid claims 2-oxo propionic acid (2-Oxopropanic acid), α-Tong Jibingsuan (α-Ketopropionicacid) or ethanoyl formic acid (Acetylformic acid) again; for colourless to weak yellow liquid; being acetic acid fragrance and happy tart flavour, is one of most important alpha-oxo-carboxyl acid.
Pyruvic acid is the important mesostate of organism, because it is widely used in industries such as chemical industry, pharmacy, agrochemicals and dietary supplements, therefore in the world its business demand is constantly increased in recent years.
The production method of pyruvic acid mainly contains chemical synthesis and microbe fermentation method two big classes.Chemical synthesis mainly contains: winestone acid system, ethyl lactate air oxidation process, pyruvic alcohol method, electrochemical synthesis method, lactic acid catalytic oxidation.It is that Japanese researchist in 1989 selects the torulopsis bacterial strain that a series of output of pyruvic acid surpass 50g/L that the fermentative Production pyruvic acid really makes a breakthrough.The auxotroph T.glabrata CCTCC NO:M202019 of the nicotinic acid of particularly Chinese Southern Yangtze University seed selection, VitB1, pyridoxol and 4 kinds of VITAMIN of vitamin H, it is the first-selected bacterial strain of fermentative Production pyruvic acid, this bacterial strain has been applied for Chinese patent, and the patent No. is ZL 02113142.2.
The involved cofactor of cofactor engineering mainly contains: ATP/ADP/AMP, NADH/NAD
+, NADPH/NADP
+, coenzyme A and derivative, VITAMIN and trace element.Research at present mainly concentrates on regulates ATP/ADP/AMP, NADH/NAD
+, NADPH/NADP
+With cofactors such as coenzyme A and derivative thereof in the influence of intracellular form and content to pathways metabolism and metabolic flux.
1998, Holland Delft Hugenholtz J research group of university overexpression in Lactococcus lactis (Streptococcuslactis) derives from the nox-2 gene of Streptococcus mutans (Streptococcus mutans) coding nadh oxidase, and the bacterium glucose metabolism that causes recombinating speeds up and the redistribution of key node carbon metabolism flow; Heux S etc. are in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), and overexpression derives from milk-acid bacteria (Lactococcus lactis) coding and produces H
2The oxidasic noxE gene of O-NADH causes NADH and NADH/NAD in the born of the same parents
+Descend 5 and 6 times respectively, and glucose consumption increases by 10%.
At regulating and controlling microbial cellular NAD H and NAD
+Concentration and ratio thereof are to having carried out more research work on carbon metabolism group and the flux group.But to NADH/NAD in the regulation and control microorganism cells
+Strengthen the research of the production intensity of target meta-bolites, then rarely seen bibliographical information.
Summary of the invention
The purpose of this invention is to provide a kind of method of strengthening the pyruvic acid production intensity by cofactor NADH regulation and control of carbon metabolism flow.By the influence of research cofactor NADH, reach the purpose of strengthening the pyruvic acid production intensity to the metabolism stream flow direction and corresponding metabolic flux.
Technical scheme of the present invention:
Pyruvic acid reorganization bacterium is produced in one strain, and its classification called after torulopsis glabrata Pdnox E has been preserved in Chinese typical culture collection center, and deposit number is CCTCC NO:M 208022.
The construction process of a kind of torulopsis glabrata PdnoxE CCTCC NO:M 208022, utilization molecule means, with Lactococcus lactis NZ 9000 genomes is template, pcr amplification obtains goal gene noxE fragment, goal gene noxE is after restriction enzyme EcoRI and the digestion of BamH I double digestion, concentrated and purified, noxE gene directed cloning in expression plasmid pYX212, is obtained the expression of recombinant yeast plasmid; With the method for expression of recombinant yeast plasmid with the electric shock conversion, change recipient bacterium Torulopsis glabrata CCTCC NO:M202019 over to, in the MM substratum, screen, obtain the reorganization bacterium PdnoxE CCTCC NO:M 208022 of nadh oxidase overexpression.The overexpression of nadh oxidase turns to the nadh oxidase approach that forms water with the NADH oxidative pathway from the oxidative phosphorylation approach of a large amount of formation ATP, has guaranteed the high NAD that high glycolysis speed is required
+Level and low ATP level.Thereby removed NAD
+The concentration deficiency is to the restriction of glycolytic pathway key enzyme activity and alleviated high-level ATP the allosteric of glycolysis-key enzyme activity is suppressed, and has strengthened the production intensity of pyruvic acid.
A kind of method by cofactor NADH regulation and control of carbon metabolism flow reinforcement pyruvic acid production intensity, adopting torulopsis glabrata PdnoxE CCTCC NO:M 208022 is starting strain, produces pyruvic acid through seed culture and liquid fermenting;
Seed culture: seed culture medium is with g/L Meter: glucose 30, peptone 10, KH
2PO
41, MgSO
47H
2O 0.5, transfers pH 5.5; Culture condition: 30 ℃ of temperature, shaking speed 200rpm, incubation time are 20-24h;
Liquid fermentation and culture: fermention medium is with g/L Meter: glucose 100, sodium acetate 6, NH
4Cl 7, KH
2PO
45, MgSO
47H
2O 0.8, CaCO
340, liquid microelement 10mL, VITAMIN liquid 10mL, pH5.0; Fermentation condition: change seed liquor over to fermentation culture respectively with 10% inoculum size and cultivate based under 30 ℃, 200rpm condition, fermentation proceeds to the 36h glucose consumption and finishes, and adds 50g/L glucose and continues fermentation 20h, and pyruvic acid concentration is improved;
Used liquid microelement is: CaCl
22H
2O 2g, FeSO
47H
2O 2g, ZnCl
20.5g, MnCl
24H
2O 12g, CuSO
45H
2O 0.05g is settled to 1L after the 2mol/L HCl dissolving;
Used VITAMIN liquid is: nicotinic acid 80mg, and VitB1 0.15mg, pyridoxol 40mg, vitamin H 4mg, riboflavin 10mg, tap water is settled to 1L.
The nadh oxidase activity of CCTCC NO:M 208022 bacterial strains is 34.8U/mg protein, compare with bacterial strain Torulopsis glabrata CCTCC NO:M202019, dry cell weight, glucose consumption speed and pyruvic acid production intensity have improved 168%, 44.9% and 12% respectively; Fermentation proceeds to the 36h glucose consumption and finishes, and adds 50g/L glucose and continues fermentation 20h, makes pyruvic acid concentration bring up to 67.2g/L.
Bacterial classification and plasmid: torulopsis glabrata (Torulopsis glabrata) CCTCC NO:M202019, it is nicotinic acid, vitamin H, VitB1,4 kinds of vitamin deficient strain of pyridoxine hydrochloride, and the active composing type of pyruvic carboxylase reduces, be mainly used in fermentation production of acetone acid, carried out preservation.Expression plasmid of yeast pYX212 is the shuttle vectors between intestinal bacteria-yeast, selective marker is ampr in intestinal bacteria, and selective marker is the complementation of uridylic defective type in yeast, expression plasmid of yeast pYX212 is at [Chinese biological engineering magazine 2006,26 (1): 38～41] open, give by teacher Shen Wei of Southern Yangtze University.
Inclined-plane and seed culture medium: glucose 30g, peptone 10g, KH
2PO
41g, MgSO
47H
2O 0.5g, agar 20g (inclined-plane is used), pH5.5, tap water is settled to 1L.
Minimum medium (MM): glucose 100g, NH
4Cl 7g, KH
2PO
45g, MgSO
47H
2O 0.8g, CaCO
340g (shake bottle time add), liquid microelement 10mL, VITAMIN liquid 10mL, pH5.0, tap water is settled to 1L.Add the 6g sodium acetate and be fermention medium in the MM substratum, adding the 60mg uridylic is supplemental medium (CM), adds 0.5g 5-fluororotic acid (5-FOA) and be the 5-FOA substratum in the CM substratum.
The mensuration of dry cell weight: get a certain amount of bacteria suspension and place the 10mL volumetric flask, add the lime carbonate in the 2mL dissolving with hydrochloric acid bacteria suspension, add deionized water and be settled to 10mL, shake up, with UV 7500 type visible spectrophotometers, survey the OD value in 660nm place colorimetric, with the dry cell weight typical curve calculate dry cell weight.
The mensuration of pyruvic acid and α-Tong Wuersuan concentration: high performance liquid chromatography (HPLC).
Instrument: Agilent 1100 high performance liquid chromatographs (joining UV, visible light detector, parallax refraction detector and workstation).
Chromatographic condition: chromatographic column: C18 post, 5 μ m, 4.6mm * 250mm; Moving phase: 0.1%H
3PO
4Flow velocity: 1mL/min; Column temperature: 28 ℃; Sample size: 10 μ L; UV-detector wavelength: 215nm.
Specimen preparation: the 5mL fermented liquid is centrifugal 10min under 10000rpm, gets to prepare against when surveying pyruvic acid and residual sugar in the supernatant liquor immigration test tube and uses.When surveying pyruvic acid, get the 1mL supernatant liquor and move in the 50mL volumetric flask, deionized water is settled to groove, and behind 0.45 μ m membrane filtration, filtrate feed flow analysis of hplc is used.
Beneficial effect of the present invention: obtained strain nadh oxidase activity and be the reorganization bacterium CCTCC NO:M 208022 of 34.8U/mg protein, compare with starting strain CCTCC NO:M202019, dry cell weight, glucose consumption speed and pyruvic acid production intensity have improved 168%, 44.9% and 12% respectively.Fermentation proceeds to the 36h glucose consumption and finishes, and adds 50g/L glucose continuation fermentation 20h and then makes pyruvic acid concentration bring up to 67.2g/L.
The biological material specimens preservation
Pyruvic acid reorganization bacterium is produced in one strain, and its classification called after torulopsis glabrata Pdnox E has been preserved in Chinese typical culture collection center, is called for short CCTCC, and preservation date on January 27th, 2008, deposit number is CCTCC NO:M 208022.
Embodiment
The amplification of embodiment 1 Lactococcus lactis noxE gene and the structure of expression plasmid
With Lactococcus lactis NZ9000 genome is template, and pcr amplification obtains goal gene noxE fragment, and agarose electrophoresis obtains the specific band of about 1.5kb.Goal gene noxE is after restriction enzyme EcoR I and the digestion of BamH I double digestion, and is concentrated and purified, and noxE gene directed cloning in expression plasmid pYX212, is obtained the expression of recombinant yeast plasmid.The LB flat board that expression of recombinant yeast plasmid transformed competence colibacillus cell JM109 and coating contain penbritin increases.
Structure and the evaluation of embodiment 2 reorganization bacterium
Owing to have the Ura3 gene on the recombinant plasmid, electric shock transformation receptor bacterium T.glabrata (Ura
-), obtain can be on the basic medium that does not add uridylic the reorganization bacterium PdnoxE CCTCC NO:M208022 of normal growth.Find that in enzyme activity determination the ratio vigor of nadh oxidase is a 34.8U/mg albumen in the reorganization bacterium, and is difficult to detect the activity of nadh oxidase in starting strain CCTCC NO:M202019.
Embodiment 3 reorganization bacterium and the experiment of contrast bacterium control fermentation
As shown in the table, two bacterium are compared: (1) reorganization bacterium PdnoxE CCTCC NO:M 208022 36h glucose consumptions finish, and starting strain CCTCC NO:M202019 remaining 5.34g/L glucose in the fermented liquid when proceeding to 48h; (2) spending rate of reorganization bacterium glucose is 2.57g/L/h, has improved 44.9% (1.78g/L/h) than starting strain; Output of pyruvic acid was 31.4g/L when (3) the reorganization bacterium was fermented 36h, this moment, the starting strain output of pyruvic acid only was 28.3g/L, continue to ferment to 48h, the starting strain output of pyruvic acid is increased to 37.3g/L, but the output of pyruvic acid of reorganization bacterium does not increase along with the prolongation of fermentation time; (4) the pyruvic acid production intensity of reorganization bacterium is 0.872g/L/h, is 1.12 times (0.778g/L/h) of starting strain; (5) reorganization bacterium fermentation period is 36h, and the starting strain fermentation period is 50h.
Embodiment 4 overexpression nadh oxidases are to the metabolic influence of NADH
As shown in the table, the overexpression of nadh oxidase makes NADH content reduce by 18.1% (comparing with control strain), NAD
+Content has increased by 11.1%, causes NADH/NAD
+Ratio has reduced by 26.3%, and ATP content descends 15.8% in the born of the same parents.
Claims (3)
1, pyruvic acid reorganization bacterium is produced in a strain, and its classification called after torulopsis glabrata Pdnox E has been preserved in Chinese typical culture collection center, and deposit number is CCTCC NO:M 208022.
2, a kind of method by cofactor NADH regulation and control of carbon metabolism flow reinforcement pyruvic acid production intensity is characterized in that adopting torulopsis glabrata PdnoxE CCTCC NO:M 208022 to be starting strain, produces pyruvic acid through seed culture and liquid fermenting;
Seed culture: seed culture medium is in g/L: glucose 30, peptone 10, KH
2PO
41, MgSO
47H
2O 0.5, transfers pH 5.5; Culture condition: 30 ℃ of temperature, shaking speed 200rpm, incubation time are 20-24h;
Liquid fermentation and culture: fermention medium is in g/L: glucose 100, sodium acetate 6, NH
4Cl 7, KH
2PO
45, MgSO
47H
2O 0.8, CaCO
340, liquid microelement 10mL, VITAMIN liquid 10mL, pH5.0; Fermentation condition: change seed liquor over to fermentation culture respectively with 10% inoculum size and cultivate based under 30 ℃, 200rpm condition, fermentation proceeds to the 36h glucose consumption and finishes, and adds 50g/L glucose and continues fermentation 20
H improves pyruvic acid concentration;
Used liquid microelement is: CaCl
22H
2O 2g, FeSO
47H
2O 2g, ZnCl
20.5g, MnCl
24H
2O 12g, CuSO
45H
2O 0.05g is settled to 1L after the 2mol/L HCl dissolving;
Used VITAMIN liquid is: nicotinic acid 80mg, and VitB1 0.15mg, pyridoxol 40mg, vitamin H 4mg, riboflavin 10mg, tap water is settled to 1L.
3, method according to claim 2, the nadh oxidase activity that it is characterized in that CCTCC NO:M 208022 bacterial strains is 34.8U/mg albumen, compare with industrial strain Torulopsis glabrata CCTCCNO:M202019, dry cell weight, glucose consumption speed and pyruvic acid production intensity have improved 168%, 44.9% and 12% respectively; Fermentation proceeds to the 36h glucose consumption and finishes, and adds 50g/L glucose and continues fermentation 20h, makes pyruvic acid concentration bring up to 67.2g/L.
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| 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 |
| CN102622533A (en) * | 2012-04-06 | 2012-08-01 | 江南大学 | Construction and application technology of Torulopsis glabrata genome metabolism model |
| CN105462868B (en) * | 2015-12-10 | 2019-02-22 | 江南大学 | A method of improving output of pyruvic acid and production intensity |
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Non-Patent Citations (4)
| Title |
|---|
| Cofactor engineering in Saccharomycescerevisiae:Expressionof a H2O-forming NADH oxidase andimpact on redoxmetabolism. S.Heux et al.Metabolic Engineering,No.8. 2006 |
| Cofactor engineering in Saccharomycescerevisiae:Expressionof a H2O-forming NADH oxidase andimpact on redoxmetabolism. S.Heux et al.Metabolic Engineering,No.8. 2006 * |
| 光滑球拟酵母中糖酵解效率与丙酮酸合成的调控研究. 刘立明.中国优秀博硕士学位论文全文数据库（博士）工程科技Ⅰ辑，第B018-6卷第2007年第02期. 2007 |
| 光滑球拟酵母中糖酵解效率与丙酮酸合成的调控研究. 刘立明.中国优秀博硕士学位论文全文数据库(博士)工程科技Ⅰ辑，第B018-6卷第02期. 2007 * |
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