CN104164398A - Escherichia coli respiratory chain single-gene inactivated strain and applications thereof - Google Patents
Escherichia coli respiratory chain single-gene inactivated strain and applications thereof Download PDFInfo
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- CN104164398A CN104164398A CN201410309523.4A CN201410309523A CN104164398A CN 104164398 A CN104164398 A CN 104164398A CN 201410309523 A CN201410309523 A CN 201410309523A CN 104164398 A CN104164398 A CN 104164398A
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Abstract
The invention discloses an escherichia coli respiratory chain single-gene inactivated strain and applications thereof. The inactivated strain and the applications thereof are characterized in that: the inactivated strain is constructed by a process of inactivating an NADH dehydrogenase II coding gene of escherichia coli and then introducing a plasmid pBHR68 to obtain the inactivated strain; or the inactivated strain is constructed by a process of introducing the plasmid pBHR68 into escherichia coli and then inactivating the NADH dehydrogenase II coding gene of the escherichia coli to obtain the inactivated strain; and the NADH dehydrogenase II coding gene is shown as SEQ ID No.9. Experiments show that: by subjecting escherichia coli to genetic engineering transformation, glucose or xylose can be effectively utilized to produce poly(3-hydroxybutyrate), 6.16 g/L of the PHB can be accumulated by utilization of 20 g/L of the glucose, 3.5 g/L of the PHB can be accumulated by utilization of 20 g/L of the xylose, and the production cost of a biological product is largely reduced.
Description
Technical field
The invention belongs to biotechnology and Application Areas, relate to particularly the inactivation strain of intestinal bacteria respiratory chain single-gene and application.
Background technology
Polyhydroxyalkanoate (Polyhydroxyalkanoates, PHA) is that a class is extensively present in the high-molecular biologic polyester in microorganism cells, the general reserve substance mainly as carbon source and energy.The day by day high pollution exhausted and environment of petroleum resources is two large core restraining factors of current social development, is day by day subject to showing great attention to of national governments.Therefore, there is the features such as the petroleum of not relying on, environmental protection low-carbon (LC) and recyclability, the chance that biological manufacturing industry is developed rapidly.PHA has and traditional petrochemical industry plastics materialogy character as similar in polyethylene, polypropylene etc., PHA can be synthetic by renewable resourcess such as carbohydrate, lipid acid simultaneously, and can degradablely enter nature ecological circulation in environment, therefore be considered to a kind of eco-friendly " green plastic ", there is the not prospect of degradative plastics of the tradition of substituting.
Although PHA industrial chain development is swift and violent nearly ten years, the complicacy of PHA manufacturing processed makes its manufacturing cost high, cannot with take oil as basic traditional material competition.Major cause comprises material cost, energy consumption, downstream processing cost etc.So attempt at present to take a lot of methods to reduce the production cost of PHA, such as, by genetic engineering modified or pathways metabolism transformation, carry out the superior strain that acquired character is good, explore simple and effective downstream extraction purification process, utilize cheap starting material etc.
Poly 3-hydroxy butyrate (Polyhydroxybutyrate, PHB) be structure the simplest, be found PHA member the earliest, it is representative that its biosynthetic pathway be take the true bacteria Ralstonia of Roche eutropha.Carbohydrate carbon source generates acetyl-CoA through glycolytic pathway, 2 acetyl-CoA condensations of beta-keto thiolase PhaA catalysis form acetoacetyl-CoA, then under the effect of the Acetoacetyl-CoA reductase PhaB relying at NADPH, be reduced to (R)-3-maloyl group coenzyme A, finally by PHA synthase PhaC, aggregate into PHB.Because reduced coenzyme NADPH has participated in PHB building-up reactions, in born of the same parents, available reducing power level is considered to affect the synthetic important factor of PHB.
In intestinal bacteria, the enzyme that relates to the reaction of oxidation-respiration chain the first step enzymaticization mainly comprises two kinds of nadh dehydrogenases, be respectively nadh dehydrogenase I and nadh dehydrogenase II, wherein nadh dehydrogenase I pumps 4 protons in the process of transmitting pair of electrons are, and nadh dehydrogenase II does not have pumping of proton in the process of transmitting pair of electrons are.The transfer efficiency of respiratory chain pumps efficiency with proton and is associated, and it is higher that proton pumps efficiency, and the transfer efficiency of respiratory chain is also just higher, and the NADH of saving is also just more.Knock out inefficient respiratory chain NADH dehydrogenase II, can effectively save NADH, by the PHB building-up reactions that provides more reduced coenzyme NADPH to participate in.
Intestinal bacteria are as a kind of important type strain, its physio-biochemical characteristics and genetic background have been had to more deep understanding, relevant molecular biology method and gene manipulation techniques be comparative maturity all, is conducive to improve bacterial classification by the appropriate design of metabolic engineering and synthetic biology.
Summary of the invention
An object of the present invention is to overcome the deficiencies in the prior art, the strain of a kind of intestinal bacteria respiratory chain single-gene inactivation is provided.
Second object of the present invention is to provide the application of a kind of intestinal bacteria respiratory chain single-gene inactivation strain.
Technical scheme of the present invention is summarized as follows:
The strain of intestinal bacteria respiratory chain single-gene inactivation, is characterized in that building by following method, the colibacillary nadh dehydrogenase II of inactivation encoding gene, then import plasmid pBHR68, obtain the strain of intestinal bacteria respiratory chain single-gene inactivation; Or in intestinal bacteria, import plasmid pBHR68, the more colibacillary nadh dehydrogenase II of inactivation encoding gene, the strain of intestinal bacteria respiratory chain single-gene inactivation obtained; Described nadh dehydrogenase II encoding gene is with shown in SEQ ID No.9.
Intestinal bacteria are preferably E.coli JM109.
The strain of above-mentioned intestinal bacteria respiratory chain single-gene inactivation is in the application that utilizes glucose or xylose production poly 3-hydroxy butyrate.
Advantage of the present invention:
Experiment showed, that the present invention is genetic engineering modified by intestinal bacteria are carried out, can utilize efficiently glucose or xylose production poly 3-hydroxy butyrate, utilize 20g/L glucose can accumulate 6.16g/L PHB, utilize 20g/L wood sugar can accumulate 3.5g/L PHB; Significantly reduced the production cost of biological products.
Accompanying drawing explanation
Fig. 1 is electrophoresis proof diagram.
Fig. 2 is E.coli JM109 (pBHR68) and in shaking flask, take the variation of glucose dry cell weight, PHB output, PHB content in substrate containing pBHR68LJ03 bacterial strain.
Fig. 3 is E.coli JM109 (pBHR68) and in shaking flask, take the variation of wood sugar dry cell weight, PHB output, PHB content in substrate containing pBHR68LJ03 bacterial strain.
Embodiment
Term used herein " ndh " refers to nadh dehydrogenase II encoding gene.
" knock out gene " and refer to by by the encoder block Delete All of this gene, reach the object of this gene of inactivation.
" inactivation gene " refers to by deleting all or part of encoder block, and some other mode, make this gene can not expression product or expression product there is no function.
Dry cell weight used herein (CDW, g/L) is the ratio of the quality of freeze-drying thalline and tunning volume.
PHB is poly 3-hydroxy butyrate (Polyhydroxybutyrate).
The quality that PHB content used herein (wt%) is PHB and the per-cent that participates in the freeze-dried vaccine weight of esterification.
Below in conjunction with embodiment, the present invention is carried out to more concrete description.
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The present invention original strain E.coli used JM109 buys from Beijing Quanshijin Biotechnology Co., Ltd
(http://www.transgen.com.cn/)。
Original plasmid pTKRED and pTKS/CS source for Addgene (Addgene,
https: //www.addgene.org/).
Archaeal dna polymerase equimolecular biological reagent used from thermo company buy (
http:// www.thermoscientificbio.com/fermentas).
Other biochemical reagents used from raw work biotechnology (Shanghai) limited-liability company buy (
http:// www.sangon.com/).
Embodiment 1
The structure of intestinal bacteria respiratory chain single-gene inactivation strain
The first step: knock out intestinal bacteria respiratory chain single-gene
(1), utilize Δ ndh U_F (SEQ ID NO.1), Δ ndh U_R (SEQ ID NO.2) and Δ ndh L_F (SEQ ID NO.3), two pairs of primers of Δ ndh L_R (SEQ ID NO.4), the genome of E.coli JM109 of take is masterplate, uses the amplification of KOD-plus high-fidelity DNA polymerase to obtain respectively upstream homology arm Δ ndh-U and the downstream homology arm Δ ndh-L that size is 270bp and 245bp.
(2), utilize Δ ndh T_F (SEQ ID NO.5), T2 (SEQ ID NO.6) and T1 (SEQ ID NO.7), Δ ndh T_R (SEQ ID NO.8) to take plasmid pTKS/CS as masterplate, use the amplification of KOD-plus high-fidelity DNA polymerase to obtain respectively tsiklomitsin upstream fragment Δ ndh-T2 and the tsiklomitsin downstream fragment Δ ndh-T1 that size is 1081bp and 876bp.
(3) fragment Δ ndh-U and Δ ndh-T2 that, amplification obtains, utilize primer Δ ndh U_F (SEQ ID NO.1), T2 (SEQ ID NO.6), use KOD-plus high-fidelity DNA polymerase to merge pcr amplification, obtain the splicing product Δ ndh-UT of upstream homology arm and tsiklomitsin upstream, size is 1321bp; Fragment Δ ndh-T1 and Δ ndh-L that amplification obtains, utilize primer T1 (SEQ ID NO.7), Δ ndh L_R (SEQ ID NO.4), the same KOD-plus high-fidelity DNA polymerase that uses merges pcr amplification, obtain tsiklomitsin downstream and downstream homology arm splicing product Δ ndh-TL, size is 1090bp.
(4), two fragments of Δ ndh-UT and Δ ndh-TL are utilized primer Δ ndh U_F (SEQ ID NO.1), Δ ndh L_R (SEQ ID NO.4) through cutting after glue reclaims, the same KOD-plus high-fidelity DNA polymerase that uses merges pcr amplification, obtains size and knocks out fragment Δ ndh-tet for 1833bp upstream and downstream homology arm and tsiklomitsin PCR.
(5), plasmid pTKRED is proceeded in E.coli JM109 bacterial strain and be made into electricity and turn competence; Fragment Δ ndh-tet is proceeded to electricity and turn competence, use tsiklomitsin screening positive clone, and verify with bacterium colony PCR, the primer of selecting is Δ ndh U_F (SEQ ID NO.1) and T2 (SEQ ID NO.6), and clip size is 1321bp (Figure 1A).Tetracycline resistance gene is by being deleted with the expression of pectinose induction playback endonuclease I-sceI, and verify with bacterium colony PCR, the primer of selecting is Δ ndh U_F (SEQ ID NO.1) and Δ ndh L_R (SEQ ID NO.4), clip size is 485bp (Figure 1B), thereby obtains metabolic engineering bacterial strain LJ03 bacterial strain.
Second step: import plasmid pBHR68
By plasmid pBHR68 (according to document (Spiekermann, P., et al., A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoicacids and other lipid storage compounds.Arch Microbiol, 1999.171 (2): p.73-80) described method builds) method transforming by electricity forwards in the above-mentioned metabolic engineering bacterial strain LJ03 building, obtain intestinal bacteria respiratory chain single-gene inactivation strain (called after is containing pBHR68LJ03 bacterial strain), also plasmid pBHR68 electricity is gone in control strain E.coli JM109 simultaneously, obtain E.coli JM109 (pBHR68).
Embodiment 2
The structure of intestinal bacteria respiratory chain single-gene inactivation strain
First in intestinal bacteria, import plasmid pBHR68, the more colibacillary nadh dehydrogenase II of inactivation encoding gene, the strain of intestinal bacteria respiratory chain single-gene inactivation obtained;
The first step: import plasmid pBHR68 in intestinal bacteria
By plasmid pBHR68 (according to document (Spiekermann, P., et al., A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoicacids and other lipid storage compounds.Arch Microbiol, 1999.171 (2): the method p.73-80) described method structure) transforming by electricity forwards in E.coli JM109;
Second step:
Press the method for embodiment 1 the first step (1)-(5), the E.coli JM109 that contains plasmid pBHR68 is carried out to large intestine bar
The operation of the nadh dehydrogenase II encoding gene inactivation of bacterium.
Embodiment 3
The application in preparation PHB containing pBHR68LJ03 bacterial strain of embodiment 1
1, culture medium prescription
Seed liquor activation and shake flask fermentation all adopt MM substratum.
In 50mL MM substratum, contain: 1mL component I, 1mL compositionⅱ, 1mL component III, final concentration is 1g/L yeast extract, with distilled water, prepares.
Component I (200mL): 10g ammonium sulfate, 2g anhydrous magnesium sulfate, is settled to 200mL with distilled water, 121 ℃ of sterilizings 20 minutes;
Compositionⅱ (200mL): 96.5g disodium hydrogen phosphate, 15g potassium primary phosphate, is settled to 200mL with distilled water, 121 ℃ of sterilizings 20 minutes;
Component III (200mL): 10mL50g/L ironic citrate aqueous ammonium, the 10mL29.8g/L calcium chloride hexahydrate aqueous solution, 4.17mL12mol/L concentrated hydrochloric acid, 1mL trace element solution, with distilled water, be settled to 200mL, then regulate pH to 4.5-5.5,121 ℃ of sterilizings 20 minutes; Wherein, trace element solution (1L): 1g Zinc Sulphate Heptahydrate, 0.3g tetrahydrate manganese chloride, 3g boric acid, 2g CoCL2 6H2O, 0.064g copper sulfate, 0.2g six water nickelous chlorides, 0.3g Sodium Molybdate Dihydrate, be settled to 1L with distilled water, 121 ℃ of sterilizings 20 minutes.
In actual culturing process, in above-mentioned substratum, add again certain density microbiotic to maintain the stability of plasmid, the present embodiment is 100 μ g/mL penbritins.
2. fermentation
1) seed activation is cultivated
The pBHR68LJ03 bacterial strain that contains of control strain E.coli JM109 (pBHR68) and embodiment 1 acquisition is inoculated in respectively to 50mL MM substratum, in 37 ℃, 220 revs/min shaking tables, cultivates 12h, obtain seed culture fluid.
2) shake flask fermentation
By step 1) control strain E.coli JM109 (pBHR68) seed culture fluid that obtains take initial OD and is inoculated into respectively in the 500mL triangular flask of the 50mL MM substratum that contains glucose and xylose as 0.04, and wherein the final concentration of glucose and xylose in substratum is 20g/L; By step 1) obtain containing pBHR68LJ03 bacterial strain seed culture fluid, take initial OD and be inoculated into respectively in the 500mL triangular flask of the 50mL MM substratum that contains glucose and xylose as 0.04, wherein the final concentration of glucose and xylose in substratum is 20g/L; Under 37 ℃, 220 revs/min conditions, shaking table is cultivated 48 hours, collects tunning.Each shaking flask is done three Duplicate Samples.
3) tunning analysis
(1) dry cell weight
Get 9000 revs/min of 40mL tunnings and within centrifugal 20 minutes, collect thalline, distilled water wash twice, lyophilize, obtains respectively E.coli JM109 (pBHR68) and does rear thalline containing pBHR68LJ03 bacterial strain ice, after ice is dry, thalline is weighed and is calculated dry cell weight (CDW), results averaged.
(2) output of PHB
The thalline of getting after 40-60mg step (1) ice is done carries out esterification, and gas-chromatography (GC) detects PHB content, results averaged; Specific as follows:
Esterification process is: gets 40-60mg ice dry mycelium in esterification pipe, adds respectively 2mL esterifying liquid, 2mL chloroform to mix, and covered and enclosed, in 100 ℃ of baking ovens, esterification is 4 hours.Be cooled to after room temperature, add 1mL distilled water, fully vibration mixes, stratification.After chloroform is completely separated with water, gets chloroform and carry out mutually gas chromatographic analysis.
500mL esterifying liquid collocation method: 0.5g phenylformic acid, the 15mL vitriol oil are dissolved in 485mL methyl alcohol, obtain esterifying liquid.The mark product of simultaneously getting the PHB of 10-20mg carry out esterification.
Gas-chromatography (GC) is analyzed: column temperature is 80 ℃, sampler temperature is 240 ℃, and detector temperature is 250 ℃, and temperature programming condition is: 80 ℃ keep 1.5 minutes, with the speed of 30 ℃/minute, be warming up to 140 ℃, then be warming up to 250 ℃ and keep 2 minutes in this temperature with the speed of 40 ℃/minute.Sample feeding amount is 2 μ L.
The result of above-mentioned two experiments is as shown in table 1, table 2 and Fig. 2, Fig. 3.
Table 1 take for E.coli JM109 (pBHR68) with containing pBHR68LJ03 the experimental result that glucose produces PHB as single substrate
Table 2 take for E.coli JM109 (pBHR68) with containing pBHR68LJ03 the experimental result that wood sugar produces PHB as single substrate
From the above results, knocked out take in the fermention medium that glucose is single substrate containing pBHR68LJ03 of coding nadh dehydrogenase II gene ndh, the accumulation volume of PHB is 6.16g/L, compares and has improved 1.76 times with the PHB accumulation volume of E.coli JM109 (pBHR68) 2.23g/L; Same, take in the fermention medium that wood sugar is single substrate, containing pBHR68LJ03, also shown same advantage, the accumulation volume of its PHB is 4.43 times of E.coli JM109 (pBHR68) PHB accumulation volume.
The second application in preparation PHB containing pBHR68LJ03 bacterial strain that embodiment 4 embodiment 2 obtain
Step is with embodiment 3, the intestinal bacteria respiratory chain single-gene inactivation strain that embodiment 2 builds take in the fermention medium that glucose is single substrate, the accumulation volume of PHB is 6.16g/L, and in the fermention medium that the wood sugar of take is single substrate, the accumulation volume of PHB is 3.5g/L.
From the above results, the engineering strain that has knocked out coding nadh dehydrogenase II gene ndh is compared with reference strain E.coli JM109 (pBHR68) containing pBHR68LJ03, be no matter to take glucose in the fermention medium that single substrate or the wood sugar of take are single substrate, it is all being significantly improved aspect accumulation volume of dry cell weight, PHB and the content of PHB.
Claims (3)
1. intestinal bacteria respiratory chain single-gene inactivation strain, is characterized in that building by following method, the colibacillary nadh dehydrogenase II of inactivation encoding gene, then import plasmid pBHR68, obtain the strain of intestinal bacteria respiratory chain single-gene inactivation; Or in intestinal bacteria, import plasmid pBHR68, the more colibacillary nadh dehydrogenase II of inactivation encoding gene, the strain of intestinal bacteria respiratory chain single-gene inactivation obtained; Described nadh dehydrogenase II encoding gene is with shown in SEQ ID No.9.
2. intestinal bacteria respiratory chain single-gene inactivation according to claim 1 strain, is characterized in that described intestinal bacteria are E.coliJM109.
3. claim 1 or 2 intestinal bacteria respiratory chain single-gene inactivation strain are in the application that utilizes glucose or xylose production poly 3-hydroxy butyrate.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112575041A (en) * | 2019-09-30 | 2021-03-30 | 江南大学 | Engineering bacterium for producing PHB (polyhydroxybutyrate) by high-efficiency fermentation of mixed carbon source and application of engineering bacterium |
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| WO2013184602A2 (en) * | 2012-06-04 | 2013-12-12 | Genomatica, Inc. | Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013184602A2 (en) * | 2012-06-04 | 2013-12-12 | Genomatica, Inc. | Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds |
| WO2013184602A3 (en) * | 2012-06-04 | 2014-01-30 | Genomatica, Inc. | Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds |
Non-Patent Citations (4)
| Title |
|---|
| HSIEN-CHUNG TSENG,ET AL: "Metabolic Engineering of Escherichia coli for Enhanced Production of (R)- and (S)-3-Hydroxybutyrate", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| JONATHAN W. CHIN,ET AL: "Analysis of NADPH Supply During Xylitol Production by Engineered Escherichia coli", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
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| QIAO JIE,ET AL: "Improved poly(3-hydroxybutyrate) production in Escherichia coliby inactivation of cytochrome bd-II oxidase or/and NDH-IIdehydrogenase in low efficient respiratory chains", 《JOURNAL OF BIOTECHNOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112575041A (en) * | 2019-09-30 | 2021-03-30 | 江南大学 | Engineering bacterium for producing PHB (polyhydroxybutyrate) by high-efficiency fermentation of mixed carbon source and application of engineering bacterium |
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Application publication date: 20141126 |