CN104805112A - Construction method of 3-hydroxy propionate high-yielding strain recombinant plasmids - Google Patents

Construction method of 3-hydroxy propionate high-yielding strain recombinant plasmids Download PDF

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CN104805112A
CN104805112A CN201510188882.3A CN201510188882A CN104805112A CN 104805112 A CN104805112 A CN 104805112A CN 201510188882 A CN201510188882 A CN 201510188882A CN 104805112 A CN104805112 A CN 104805112A
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puuc
hydroxy
gene
enzyme
plasmid
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CN104805112B (en
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田平芳
陈柳霓
李映
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Beijing University of Chemical Technology
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Beijing University of Chemical Technology
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Abstract

The invention discloses a construction method of 3-hydroxy propionate high-yielding strain recombinant plasmids and belongs to the production field of biochemical industry. In view of the fact that most conventional yields of 3-hydroxy propionate engineering bacteria are comparatively low, an effective tac promoter applicable to klebsiella pneumonia and puuc aldehyde dehydrogenase capable of converting 3-hydroxy propionaldehyde into 3-hydroxy propionate are discovered to construct and recombine the engineering bacteria. After the sequence of the encoding gene of the enzyme is analyzed, the gene is chemically synthesized; after sequencing confirmation, the promoter and enzyme are overexpressed in klebsiella pneumonia to obtain recombined klebsiella pneumonia pET (ptac-puuc) and the catalytic ability of the enzyme for 3-hydroxy propionaldehyde in vivo and in vitro is measured, wherein in vivo, the enzyme activity of the enzyme for 3-hydroxy propionaldehyde reaches 26.31 U/mg and is 6.9 times of the enzyme activity of the enzyme for original klebsiella pneumonia (3.82 U/mg). 48 hours after optimizing the medium components and IPTG concentration condition, the yield of 3-hydroxy propionate in the engineering bacteria cultivated and reformed in a 5L fermentation tank reaches 73 g/L.

Description

A kind of construction process of 3-hydroxy-propionic acid Producing Strain recombinant plasmid
Technical field
The invention belongs to biochemical industry production field, relate to a kind of construction process of high yield 3-hydroxy-propionic acid genetic engineering bacterium recombinant plasmid.
Background technology
3-hydroxy-propionic acid (3-HP) has very high commercial value in industrial application, but its chemical synthesis process is comparatively complicated.In recent years, along with synthetic biology becomes the study hotspot of biochemical industry, Microbe synthesis method is produced 3-hydroxy-propionic acid and is urgently broken through.Klepsiella pneumoniae (Klebsiella pneumoniae) has stronger glycerol metabolism ability, is well suited for making expressive host from the angle of biomass accumulation.Klepsiella pneumoniae utilizes glycerine to generate intermediate product 3-HPA, and then 3-HPA is generated 3-hydroxy-propionic acid by aldehyde dehydrogenase catalysis.
Be that Host Strains is produced in the method for 3-hydroxy-propionic acid with klepsiella pneumoniae, engineering bacteria promotor used mostly is the pk promotor coming from dhaB gene self.It is weak that this promotor has startup ability, thus make the shortcomings such as expressing quantity is little, have impact on the output of 3-hydroxy-propionic acid.Therefore, the aldehyde dehydrogenase finding a kind of potent promotor and product 3-hydroxy-propionic acid that are suitable for klepsiella pneumoniae builds the breach that engineering bacteria becomes high yield 3-hydroxy-propionic acid.
Through a large amount of screening, find that tac promotor starts ability strong, and be applicable to klepsiella pneumoniae.So, founded a kind of method building 3-hydroxy-propionic acid high production bacteria, after tac promotor, connected aldehyde dehydrogenase gene puuC, determine expressing quantity and the inside and outside enzymic activity of aldehyde dehydrogenase puuC.Result shows, and aldehyde dehydrogenase obtains overexpression under the effect of tac promotor.On this basis, determine 3-hydroxy-propionic acid output, obtain high yield 3-hydroxy-propionic acid engineering bacteria.
Summary of the invention
In view of existing product 3-hydroxy-propionic acid engineering bacteria output is mostly lower, the present invention is intended to a kind of method founding 3-hydroxy-propionic acid high production bacteria, and determines the optimal culture condition of this project bacterium fermentation.Technical scheme of the present invention is summarized as follows:
Produce a recombinant plasmid for 3-hydroxy-propionic acid bacterial strain, it is characterized in that: import tac promotor and puuC aldehyde dehydrogenase gene.
Further, described recombinant plasmid is directed in Klebsiella pneumonia.
Build a method for described recombinant plasmid, comprise the following steps:
1) by promotor tac and aldehyde dehydrogenase gene puuC insertion vector plasmid, construction recombination plasmid pET (ptac-puuC);
2) by constructed recombinant plasmid transformed in Klebsiella pneumonia.
Further, it is characterized in that described construction recombination plasmid comprises:
1) pcr amplification of the DNA fragmentation containing puuC gene;
2) restriction enzyme cut vector plasmid and gene puuC, ptac;
3) puuC gene and promotor ptac are inserted in described vector plasmid.
Produce a method for 3-hydroxy-propionic acid, be carried out fermentation culture to having imported the Klebsiella pneumonia containing the pET-28a recombinant plasmid of tac promotor and aldehyde dehydrogenase gene puuC, and obtain 3-HP in fermented liquid.
Further, described fermented liquid is: K 2hPO 43H 2o 4-6g/L, KH 2pO 41.5-2.4g/L, (NH 4) 2sO 46.4-9.6g/L, MgSO 47H 2o 0.1-0.2g/L, yeast powder 3.2-4.8g/L, IPTG concentration are that 0.015-0.025mM and glycerol content are maintained to more than 30g/L.
Further, described fermentation culture is feeding culture mode, and make pH be constant between 6.5-7.5, air flow remains on 1.5vvm, and mixing speed is 400rpm.
Ptac-puuC gene order has the gene order as NO.1.
Through a large amount of screening, find to be applicable to the efficient tac promotor of klepsiella pneumoniae and the puuc aldehyde dehydrogenase that 3-HPA is 3-hydroxy-propionic acid can have been transformed, and having built recombinant bacterial strain with this.After the sequence of this enzyme coding gene being analyzed, chemosynthesis is carried out to this gene.After sequence verification by this promotor and enzyme overexpression in klepsiella pneumoniae, obtain restructuring klepsiella pneumoniae pET (ptac-puuc), measure in its body and the external catalytic capability to 3-HPA.In vitro, this enzyme is lived to the enzyme of 3-HPA and is reached 26.31U/mg, is 6.9 times of primary klepsiella pneumoniae (3.82U/mg).In 5L fermentor tank, cultivate improved engineering bacteria, after medium component, IPTG concentration conditions are optimized, after its 48h, 3-hydroxy-propionic acid output is up to 73g/L.
Accompanying drawing explanation
Fig. 1 recombinates the SDS-PAGE analysis chart of klepsiella pneumoniae pET (ptac-puuC).
M-marker；1-K.pneumoniae(ptac)；2-K.pneumoniae(ptac-puuC)
Fig. 2 different concns medium component is on the impact of 3-hydroxy-propionic acid and biomass.
A:K 2hPO 43H 2o; B:KH 2pO 4; C:(NH 4) 2sO 4; D:MgSO 47H 2o; E:Yeast extract (yeast powder) .Biomass: biomass; 3-HP:3-hydroxy-propionic acid; Lactic acid: lactic acid; Acetic acid: acetic acid
The IPTG of Fig. 3 different concns is on the impact of recombinant bacterium
Biomass: biomass; 3-HP:3-hydroxy-propionic acid; Lactic acid: lactic acid; Acetic acid: acetic acid
The upper tank fermentograph of Fig. 4 recombinant bacterium K.pneumoniae (ptac-puuC)
3-HP:3-hydroxy-propionic acid; Lactic acid: lactic acid; Acetic acid: acetic acid;
1,3-PD:1, ammediol; 1,3-PD:2,3-butyleneglycol; Biomass: biomass
Embodiment
Concrete grammar below can make the present invention of those skilled in the art's comprehend, but does not limit the present invention in any way.
The concrete steps of the inventive method comprise:
1. the clone of tac promotor and aldehyde dehydrogenase puuc in klepsiella pneumoniae
Bacterial strain and plasmid: intestinal bacteria (E.coli) top10 is purchased from Beijing Bo Maide company, klepsiella pneumoniae (Klebsiella pneumoniae) DSM2026 buys from German DSM, and expression vector pET-28a buys from Novozymes Company.
Substratum: LB substratum (g/L) peptone 10, yeast powder 5, NaCl 10, adds the agar of 1.5% (adding 1.5g agar in 100ml liquid nutrient medium) in solid medium.Resistance culture base need add 50 μ L/mL sulphuric acid kanamycins.
Klepsiella pneumoniae fermention medium (g/L): K 2hPO 43H 2o, 3.4; KH 2pO 4, 1.3; (NH 4) 2sO 4, 4; MgSO 47H 2o, 0.5; CaCO 3, 0.1; Yeast powder, 3; Glycerine, 40; Trace element, 1.25mL/L (adding 1.25mL trace element in 1L substratum).
Trace element solution (g/L) FeSO 47H 2o, 1; ZnCl 2, 0.07; CuCl 22H 2o, 0.02; MnCl 24H 2o, 0.1; NiCl 26H 2o, 0.025; H 3bO 3, 0.06; Na 2mO 42H 2o, 0.035; CoCl 22H 2o, 0.2; Saturated HCl, 4mL.
Molecular cloning and expression: carrying out chemosynthesis by screening the gene order obtained, being connected in carrier pET-28a, obtaining recombinant plasmid pET (ptac-puuC), proceed in intestinal bacteria top10 and carry out sequence verification.Afterwards the plasmid electroporation of extraction be converted into klepsiella pneumoniae and verify.The result display tac promotor is connected correctly with aldehyde dehydrogenase gene puuC.
2. the expression of aldehyde dehydrogenase
Cultivate recombinant bacterium in the fermentation medium, take out fermented liquid at 12h, the centrifugal 10min of 12000rpm, removes supernatant.By the PBS damping fluid (KH of thalline 50mM collected 2pO 4, 0.27g/L; Na 2hPO 4, 1.42g/L; NaCl, 8g/L; KCl, 0.2g/L) resuspended.Be placed on ice by thalline subsequently, sonicated cells, power is 100W, and work 3s, stops 2s, totally 50 times.After fragmentation, appropriate broken liquid being joined enzyme lives in reaction system (50mM PBS damping fluid, 0.2mM 3-HPA, 0.4mM aminoantipyrene, 7mM phenol, 7U catalase), 37 DEG C carry out reaction 5min after under 500nm, measure absorbancy.A unit of activity is defined as under this condition, and 1min catalysis generates the enzyme amount required for 1 μm of ol hydrogen peroxide.
Measure the SDS-PAGE protein electrophoresis of puuC aldehyde dehydrogenase.
Experimental result shows, aldehyde oxidase reaches 26.31U/mg to the activity of 3-HPA in vitro, is 5.9 times of control group.
3. the determination of optimal culture condition
The optimal culture condition of 3-hydroxy-propionic acid is produced according to shake flask fermentation determination recombinant bacterium.
In 250ml shaking flask, load 100ml fermention medium, be placed in shaking table, 37 DEG C and carry out shake flask fermentation under 150rpm condition.The mensuration that one time fermentation liquid carries out 3-hydroxy-propionic acid output is taken out every 3h.Analytical procedure is as follows: the product quality concentration in fermented liquid adopts Shimadzu high performance liquid chromatography SPD-20A to measure, and chromatographic column is C18 post, column temperature 30 DEG C, and moving phase is 0.05% phosphoric acid, and working flow rate is 0.8mL/min, and detector adopts UV-detector.Get the centrifugal 10min of 1mL fermented liquid 12000rpm, get supernatant liquor and detect for high performance liquid chromatography, through 0.22 μm of micro-filtrate membrane filtration before sample analysis.The measuring method of glycerine uses sodium periodate oxidation.
The determination of medium component:
Show according to conditions of flask fermentation Fig. 2, obtain optimal medium composition dosage range:
Table 1 optimal medium composition
The determination of IPTG concentration:
Reach a conclusion according to IPTG concentration gradient fermentation of biomass output in Fig. 3, determining to ferment be the best IPTG concentration of recombinant bacterium is 0.015-0.025mM.
4. the upper tank fermentation of recombinant bacterium K.pneumoniae (ptac-puuc)
Upper tank fermentation completes in 5L fermentor tank (Shanghai is protected emerging), and liquid amount is 3L, carries out at 37 DEG C, pH maintains 6.5-7.5 automatically, air flow 1.5vvm, and mixing speed is 400rpm, every 3h sampling and measuring, makes the content of glycerine maintain more than 30g/L.The same shake flask fermentation of analytical procedure.
Experimental result shows, in upper tank fermenting experiment, during 48h, 3-hydroxy-propionic acid output reaches 73g/L (Fig. 4).The result of shake flask fermentation and the fermentation of upper tank shows, tac promotor ensure that the high expression of aldehyde dehydrogenase puuC in Klebsiella Pneumoniae body, improve the output of 3-hydroxy-propionic acid, successfully construct the Producing Strain K.pneumoniae (ptac-puuC) of a strain 3-hydroxy-propionic acid.

Claims (8)

1. produce a recombinant plasmid for 3-hydroxy-propionic acid bacterial strain, it is characterized in that: import tac promotor and puuC aldehyde dehydrogenase gene.
2. recombinant plasmid according to claim 1, is characterized in that: described recombinant plasmid is directed in Klebsiella pneumonia.
3. build a method for recombinant plasmid described in claim 2, comprise the following steps:
1) by promotor tac and aldehyde dehydrogenase gene puuC insertion vector plasmid, construction recombination plasmid pET (ptac-puuC);
2) by constructed recombinant plasmid transformed in Klebsiella pneumonia.
4. method according to claim 3, is characterized in that described construction recombination plasmid comprises:
1) pcr amplification of the DNA fragmentation containing puuC gene;
2) restriction enzyme cut vector plasmid and gene puuC, ptac;
3) puuC gene and promotor ptac are inserted in described vector plasmid.
5. produce a method for 3-hydroxy-propionic acid, be carried out fermentation culture to having imported the Klebsiella pneumonia containing the pET-28a recombinant plasmid of tac promotor and aldehyde dehydrogenase gene puuC, and obtain 3-HP in fermented liquid.
6. method according to claim 5, is characterized in that: described fermented liquid is: K 2hPO 43H 2o 4-6g/L, KH 2pO 41.5-2.4g/L, (NH 4) 2sO 46.4-9.6g/L, MgSO 47H 2o 0.1-0.2g/L, yeast powder 3.2-4.8g/L, IPTG concentration are that 0.015-0.025mM and glycerol content are maintained to more than 30g/L.
7. method according to claim 5 or 6, is characterized in that: described fermentation culture is feeding culture mode, and make pH be constant between 6.5-7.5, air flow remains on 1.5vvm, and mixing speed is 400rpm.
8. recombinant plasmid according to claim 1, has the ptac-puuC gene order as NO.1.
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Cited By (3)

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Publication number Priority date Publication date Assignee Title
CN107119003A (en) * 2017-04-28 2017-09-01 中国科学院青岛生物能源与过程研究所 A kind of utilization glucan synthesizes recombinant bacterium and its construction method and the application of 3 hydracrylic acids
CN108949760A (en) * 2018-07-27 2018-12-07 北京化工大学 A kind of combined promoter and its application in the 3- hydracrylic acid yield for improving klepsiella pneumoniae
CN114806593A (en) * 2022-05-05 2022-07-29 北京化工大学 Preparation method of composite soil remediation agent containing 3-hydroxypropionic acid fermentation liquor

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107119003A (en) * 2017-04-28 2017-09-01 中国科学院青岛生物能源与过程研究所 A kind of utilization glucan synthesizes recombinant bacterium and its construction method and the application of 3 hydracrylic acids
CN107119003B (en) * 2017-04-28 2020-04-24 中国科学院青岛生物能源与过程研究所 Recombinant bacterium for synthesizing 3-hydroxypropionic acid by utilizing glucan and construction method and application thereof
CN108949760A (en) * 2018-07-27 2018-12-07 北京化工大学 A kind of combined promoter and its application in the 3- hydracrylic acid yield for improving klepsiella pneumoniae
CN108949760B (en) * 2018-07-27 2022-06-10 北京化工大学 Composite promoter and application thereof in increasing yield of 3-hydroxypropionic acid of klebsiella pneumoniae
CN114806593A (en) * 2022-05-05 2022-07-29 北京化工大学 Preparation method of composite soil remediation agent containing 3-hydroxypropionic acid fermentation liquor

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