CN103740630A - Engineering bacterium containing NADH (nicotinamide adenine dinucleotide) kinase gene and application thereof - Google Patents
Engineering bacterium containing NADH (nicotinamide adenine dinucleotide) kinase gene and application thereof Download PDFInfo
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Abstract
The invention discloses an engineering bacterium containing an NADH (nicotinamide adenine dinucleotide) kinase gene and an application thereof. The invention provides a method for constructing a recombinant bacterium. The method is characterized by guiding an NADH kinase coding gene pos5 and a PHB (poly-beta-hydroxybutyrate) synthetic gene phbCAB into a target bacterium to obtain the recombinant bacterium. Experiments prove that the NADH kinase gene in the recombinant bacterium can promote transformation of NADH in cells to NADPH (nicotinamide adenine dinucleotide phosphate), thus improving synthesis of NADPH-dependent poly-3-hydroxybutyrate. Compared with former strains, the engineering bacterium has higher PHB production efficiency.
Description
Technical field
The present invention relates to biological technical field, relate in particular to engineering bacteria of a kind of NADH of containing kinase gene and uses thereof.
Background technology
NAD (H) and NADP (H) are the important coenzyme in organism, and they participate in various redox reactions mainly as electron carrier.The reaction that coenzyme relates to is very extensive, and the change of its concentration can cause the variation of intracellular matter and energy metabolism, thereby affects the synthetic of many meta-bolitess.By intracellular coenzyme concentration and redox state ratio etc. are regulated and controled to improving biosynthetic efficiency to receive increasing concern.NADH kinases can utilize ATP etc. as the donor of phosphate group, and the phosphorylation of catalyzing N ADH forms NADPH, has played very crucial effect in coenzyme metabolism in vivo.NADH kinase gene in Saccharomyces Cerevisiae in S accharomyces cerevisiae has three, be respectively UTR1, YEF1 and POS5(Genomic characterization of POS5, the Saccharomyces cerevisiae mitochondrial NADH kinase, Mitochondrion, 2006, 6 (2): 94-101), the reaction that is wherein positioned at the catalysis of Intramitochondrial Pos5 institute is considered to the main source of plastosome reduced coenzyme NADPH, in a plurality of physiological functions such as anti-oxidant, play important effect (Mitochondrial NADH kinase, Pos5p, is required for efficient iron-sulfur cluster biogenesis in Saccharomyces cerevisiae, Journal of Biological Chemistry, 2010, 285 (50): 39409-39424).
Studied definite NADH kinases also has the Ndk1(Deletion of the mitochondrial NADH kinase increases mitochondrial DNA stability and life span in the filamentous fungus Podospora anserine in Podospora anserina at present, Experimental Gerontology, 2010, 45 (7-8): Nadhk(Biochemical and functional characterization of novel NADH kinase in the enteric protozoan parasite Entamoeba histolytica 543-549) and in Entamoeba histolytica, Biochimie, 2013, 95 (2): 309-319) etc.
Poly--3-hydroxybutyrate ester (poly-3-hydroxybutyrates, be called for short PHB) be many microorganisms a kind of high molecular polymers at thin intracellular accumulation under specific growth conditions, be mainly used to the reserve substance as carbon source and energy, when nutrition is deficient, again decompose utilization, give cell various physiological potential.PHB has and similar plastics (polyethylene, polypropylene etc.) materialogy character, different from the plastic material in oil source is, PHB can be by renewable physical resources (starch, Mierocrystalline cellulose, lipid acid etc.) method by microbial transformation obtains, after discarding in physical environment, PHB can be degradable faster by microorganism is carbonic acid gas and water, have biological renewable, the characteristics such as biodegradable, can be used as supplementing of traditional nondegradable oil source plastic material, thereby be subject to extensive concern (Increased diversification of polyhydroxyalkanoates by modification reactions for industrial and medical applications, Applied Microbiology and Biotechnology, 2007, 74 (1): 1-12).In most of microbe, PHB is that the acetyl-CoA being produced by mass degradations such as carbohydrates is that precursor synthesizes, under the effect of the beta-keto thiolase of phaA genes encoding, two acetyl-CoA molecule condensations generate acetoacetyl-CoA, next the Acetoacetyl-CoA reductase catalysis acetoacetyl-CoA that the NADPH of phaB genes encoding relies on is reduced to (R)-3-maloyl group coenzyme A (3HB-CoA), finally by PHA polysaccharase catalyzed polymerization, becomes PHB.Reduced coenzyme NADPH has participated in the building-up reactions of PHB, and in born of the same parents, available reducing power level is to be considered to affect the synthetic important factor of PHB.The key issue of restriction PHB large-scale industrial production is the much higher production cost of its relative oil source plastic material at present.The biosynthetic process of PHB in microorganism is carried out to comprehensive and systematic research, to producing bacterial classification, carry out genetic modification targetedly, improve the synthetic efficiency of PHB, will contribute to reduce production costs, advance the industrialization process of PHB.
Summary of the invention
An object of the present invention is to provide a kind of method that builds recombinant bacterium.
Method provided by the invention, for the gene phbCAB of NADH kinases encoding gene pos5 and synthetic gather-3-hydroxybutyrate ester is imported in object bacterium, obtains recombinant bacterium.
In aforesaid method, the gene phbCAB of described NADH kinases encoding gene pos5 and synthetic gather-3-hydroxybutyrate ester imports in object bacterium by recombinant vectors;
Described recombinant vectors is that NADH kinases encoding gene pos5 expression cassette is inserted in the expression vector of the gene phbCAB that contains synthetic gather-3-hydroxybutyrate ester, the recombinant vectors obtaining.
In aforesaid method, described NADH kinases encoding gene pos5 expression cassette comprises pyruvic carboxylase promotor, NADH kinases encoding gene pos5 and the terminator that driving N ADH kinases encoding gene pos5 expresses.
In aforesaid method, described NADH kinases encoding gene pos5 derives from Saccharomyces Cerevisiae in S accharomyces cerevisiae s288c; The kinase whose aminoacid sequence of described NADH is the sequence 3 in sequence table.
In aforesaid method, described in contain synthetic poly--3-hydroxybutyrate ester the expression vector of gene phbCAB be pBHR68;
The nucleotides sequence of described NADH kinases encoding gene pos5 is classified sequence 2 in sequence table as from 5 ' end 223-1416 position Nucleotide;
The nucleotides sequence of the gene phbCAB of described synthetic gather-3-hydroxybutyrate ester is classified the sequence 4 in sequence table as;
The nucleotides sequence of described NADH kinases encoding gene pos5 expression cassette is classified the sequence 2 in sequence table as;
The described bacterium that sets out is intestinal bacteria, can, for intestinal bacteria wild-type or its common bacterial strain, can be K12, BW25113, MG1655, JM109, DH5 α, XL1-Blue etc.In an embodiment of the present invention, described intestinal bacteria are specially E.coli JM109.
In an embodiment of the present invention, described recombinant vectors is the carrier obtaining between the AvrII of the pos5 expression casette insertion expression vector pBHR68 shown in sequence 2 and NheI restriction enzyme site.
The recombinant bacterium being built by above-mentioned method is also the scope of protection of the invention.
Another object of the present invention is to provide a kind of method of producing poly--3-hydroxybutyrate ester.
Method provided by the invention, comprises the steps: the above-mentioned recombinant bacterium that ferments, and collects tunning, gathered-3-hydroxybutyrate ester.
In aforesaid method, described leavening temperature is 30 ℃-40 ℃; Described fermentation time is 40-50h; Described leavening temperature is specially 37 ℃; Described fermentation time is specially 48h.
The 3rd object of the present invention is to provide a kind of recombinant vectors of preparing poly--3-hydroxybutyrate ester.
Recombinant vectors provided by the invention, for NADH kinases encoding gene pos5 expression cassette being inserted in the expression vector of the gene phbCAB that contains synthetic gather-3-hydroxybutyrate ester, the recombinant vectors obtaining;
Described NADH kinases encoding gene pos5 expression cassette specifically comprises pyruvic carboxylase promotor, NADH kinases encoding gene pos5 and the terminator that driving N ADH kinases encoding gene pos5 expresses;
The nucleotide sequence of described NADH kinases encoding gene pos5 expression cassette is specially the sequence 2 in sequence table;
The expression vector of the described gene phbCAB that contains synthetic gather-3-hydroxybutyrate ester is specially pBHR68.
The application in improving poly--3-hydroxybutyrate ester output of above-mentioned recombinant bacterium or above-mentioned recombinant vectors is also in the scope of protection of the invention.
In an embodiment of the present invention, described recombinant vectors is the carrier obtaining between the AvrII of the pos5 expression casette insertion expression vector pBHR68 shown in sequence 2 and NheI restriction enzyme site.
Of the present invention experimental results show that, the recombinant bacterium that the present invention builds is for all importing by the gene of NADH kinase gene and synthetic gather-3-hydroxybutyrate ester the recombinant bacterium that intestinal bacteria obtain, in this recombinant bacterium, NADH kinase gene can promote in cell that reduced coenzyme NADH is to the conversion of NADPH, thereby improved that NADPH relies on poly--3-hydroxybutyrate ester synthetic, this project bacterium was compared with former bacterial strain has higher PHB production efficiency.
Embodiment
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.
In following embodiment, relate to molecular biology and operate enzyme used, be all purchased from MBI Fermentas company, corresponding operation steps is carried out according to product description completely; Plasmid extraction, DNA fragmentation reclaim test kit used purchased from Beijing Bo Maide biotech company, and corresponding operation is carried out according to product description completely; The DNA relating in embodiment synthesizes and examining order is completed by Beijing Bo Maide biotech company.
The bacteria culture medium relating in following embodiment is as follows, and as indicated without special, the sterilising conditions of substratum is 121 ℃, 20 minutes:
LB-Amp liquid nutrient medium: every liter of substratum contains 5g/L yeast extract, 10g/L peptone, 10g/LNaCl and 100 μ g/mL penbritins, and all the other are water.
LB-Amp solid medium: every liter of substratum contains 15g/L agar, 5g/L yeast extract, 10g/L peptone, 10g/L NaCl and 100 μ g/mL penbritins, and all the other are water.
MSG-Amp substratum: every liter of substratum is containing yeast extract 5g/L, peptone 10g/L, glucose 20g/L, (NH
4)
2sO
42g/L, MgSO
40.4g/L, Na
2hPO
412H
2o9.65g/L, KH
2pO
41.5g/L, trace element solution I10mL/L, trace element solution II1mL/L and 100 μ g/mL penbritins, all the other are water;
Every liter of trace element solution I contains: Fe (III)-NH
4-Citrate5g/L and CaCl
22H
2o2g/L, all the other are 0.5M HCl;
Every liter of trace element solution II contains: ZnSO
47H
2o100mg/L, MnCl
24H
2o30mg/L, H
3bO
3300mg/L, CoCl
26H
2o200mg/L, CuSO
45H
2o10mg/L, NiCl
26H
2o20mg/L, NaMoO
42H
2o30mg/L, all the other are 0.5M HCl.
The structure of embodiment 1, recombinant bacterium E.coli JM109 (pBHR68pos5)
One, the structure of recombinant expression vector pBHR68pos5
1, the acquisition of expression vector p19pdc
One section of double-stranded DNA of synthetic, its nucleotides sequence is classified the sequence 1 in sequence table as, wherein in sequence table, sequence 1 is that pyruvic carboxylase promotor, sequence 1 are multiple clone site from 5 ' end 213-248 position Nucleotide from 5 ' end 28-208 position Nucleotide, and sequence 1 is transcription terminator from 5 ' end 249-334 position Nucleotide.
The method that double chain DNA fragment shown in the sequence of above-mentioned synthetic 1 is cloned by TA is connected to T carrier pMD19-T Simple(purchased from Japanese Takara company) in, expression vector p19pdc obtained.
2, the acquisition of NADH kinase gene pos5
Take Saccharomyces Cerevisiae in S accharomyces cerevisiae s288c(ATCC204508) genomic dna be template, take K1 and K2 as upstream and downstream primer amplification.
Primer is as follows:
K1：GAAAA
GGATCCAAGGAGATATACCATGAGTACGTTGGATTCACATTCCCTA（
BamHI）
K2：GAAAA
GGTACCTTAATCATTATCAGTCTGTCTCTTGGT（
KpnI）。
Obtain the PCR product of 1232bp size, through order-checking, this PCR product has sequence 2 in sequence table and, from the Nucleotide shown in 5 ' end 223-1416 position, is NADH kinase gene pos5, and the kinase whose aminoacid sequence of NADH of its coding is the sequence 3 in sequence table.
3, the acquisition of recombinant vectors p19pos5
1), with the expression vector p19pdc obtaining in BamHI and KpnI double digestion step 1, reclaim the carrier large fragment that size is about 3.0kb;
2) with the PCR product of the 1232bp size obtaining in BamHI and KpnI double digestion step 2, recovery is about the enzyme of 1220bp and cuts product;
3) the carrier large fragment and the step 2 that step 1) are obtained) enzyme of the 1220bp that obtains cuts product and is connected, obtaining connecting the method for product by chemical conversion imports in E.coli JM109, and coat LB-Amp solid medium, cultivate 16h, obtain transformant for 37 ℃.
Extract the plasmid of transformant, with BamHI and KpnI double digestion, enzyme is cut the positive plasmid of plasmid that product size is about 3.0kb and 1.2kb, and called after p19pos5 is recombinant expression vector.
Recombinant expression vector p19pos5 is sent to order-checking, and this carrier of result is for inserting from the gene pos5 shown in 5 ' end 223-1416 position Nucleotide the carrier that expression vector p19pdc obtains by sequence in sequence table 2.
4, build recombinant expression vector pBHR68pos5
1) with XbaI enzyme cutting plasmid pBHR68(Spiekermann P, Rehm BHA, Kalscheuer R, et al.A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds.Arch Microbiol, 1999,171:73-80; Public Ke Cong Beijing University of Chemical Technology obtains; Agarose gel electrophoresis reclaims and to obtain the carrier framework that size is about 8.1kb, wherein contains its nucleotides sequence of PHB synthetic gene phbCAB(and classifies sequence 4 as) and Amp resistant gene and the replicon in promoter sequence, pBluescript II SK (-) source.
2) with AvrII and NheI enzyme, cut the above-mentioned 3 recombinant vectors p19pos5 that obtain, agarose gel electrophoresis reclaims and obtains the DNA fragmentation that size is about 1.5kb.
Through order-checking, this size is about the nucleotides sequence of the DNA fragmentation of 1.5kb and classifies sequence 2 as, and shown in sequence 2 is pos5 expression casette, and pos5 expression casette comprises pyruvic carboxylase promotor, pos5 gene and the terminator that drives pos5 genetic expression;
The nucleotides sequence of pyruvic carboxylase promotor is classified in sequence table sequence 2 as from 5 ' end 16-196 position Nucleotide;
The nucleotides sequence of pos5 gene is classified in sequence table sequence 2 as from 5 ' end 223-1416 position Nucleotide;
The nucleotides sequence of terminator is classified in sequence table sequence 2 as from 5 ' end 1429-1514 position Nucleotide.
3) use T
4dNA ligase Connection Step 1) and step 2) in the fragment that reclaims, obtain connecting product.The method that connection product is transformed by electricity imports in E.coli JM109, and coats LB-Amp solid medium, cultivates 16h for 37 ℃.
Extract the plasmid of transformant, with EcoRI enzyme, cut, enzyme is cut the positive plasmid of plasmid that product size is about 3.4kb and 6.3kb, and called after pBHR68pos5 is recombinant expression vector.
Recombinant expression vector pBHR68pos5 is sent to order-checking, and this carrier of result is the carrier obtaining between the AvrII of the pos5 expression casette insertion expression vector pBHR68 shown in sequence 2 and NheI restriction enzyme site.
Two, the structure of recombinant bacterium E.coli JM109 (pBHR68pos5)
The method that above-mentioned one the 4 recombinant plasmid pBHR68pos5 that obtain are transformed by electricity is transformed in e. coli jm109, obtains recombinant bacterium.
Extracting the plasmid of recombinant bacterium, send to order-checking, is recombinant plasmid pBHR68pos5, the recombinant bacterium called after recombinant bacterium E.coli JM109 (pBHR68pos5) that contains this recombinant plasmid.
The method that control plasmid pBHR68 is transformed by electricity is transformed into e. coli jm109, builds contrast recombinant bacterium E.coli JM109 (pBHR68).
Embodiment 2, recombinant bacterium E.coli JM109 (pBHR68pos5) application in preparation PHB
1, fermentation culture
The acquisition of seed liquor: the recombinant bacterium E.coli JM109 (pBHR68pos5) of two preparations by embodiment 1 is cultivated to 14h(37 ℃ of shaking table in LB-Amp liquid nutrient medium, 200rpm) as seed liquor;
Fermentation: 4% inoculum size is inoculated into seed liquor in MSG-Amp liquid nutrient medium by volume, 37 ℃ of shaking tables, 200rpm, fermentation culture 48h, collects tunning, obtains PHB.
To contrast recombinant bacterium E.coli JM109 (pBHR68) fermentation culture according to the method described above, collect control fermentation product.
2, detect PHB content
Vapor-phase chromatography (Gas chromatography, GC) is carried out detection by quantitative to bacterium, measures the biomass of cell and the content of the interior PHB of born of the same parents.
Concrete detection method is as follows:
HP6890 type gas chromatograph is used in gas chromatographic analysis, and chromatographic column is HP-5 capillary column, column length 30m, and internal diameter 320 μ m, stationary phase is the phenyl methyl polysiloxane that 25nm is thick; Detector is flame ionization detector (Flame ionization detector, FID); With high pure nitrogen, as carrier gas, hydrogen is as combustion gas, and air is combustion-supporting gas.
The condition of gas chromatographic analysis is as follows:
Column temperature:
80 ℃ of beginnings, stop 1.5min;
The speed of 30 ℃/min is warmed up to 140 ℃, stops 0min;
The speed of 40 ℃/min is warmed up to 220 ℃, stops 1min.
Total time is 6.5min.
Post is pressed:
10psi starts, and stops 1.5min;
The speed of 2.5psi/min boosts to 20psi, stops 0.5min.
(psi is pressure unit, pound/square inch, and 1psi=6.89476kPa)
Injection port: temperature is 200 ℃, is used shunt mode, and splitting ratio is 30.
Detector: temperature is 220 ℃, hydrogen flowing quantity 30mL/min, air flow quantity 400mL/min.
Detecting step is as follows:
1) by above-mentioned 1 tunning obtaining, centrifugal (10000g, 10min) collects thalline, then with recentrifuge after distilled water wash; By cell frost drying, measure dry cell weight (Cell dry weight, CDW);
2) get 50mg stem cell in esterification pipe, (by volume percentage composition is 3% the vitriol oil to be dissolved in methyl alcohol to add 2mL esterifying liquid, obtain sulphuric acid soln, by final concentration, be that 1g/L adds phenylformic acid in sulphuric acid soln as interior mark again, obtain esterifying liquid), 2mL chloroform, covered and enclosed, esterification 4h in 100 ℃ of baking ovens; Be cooled to after room temperature, add 1mL distilled water, fully vibration, stratification; After chloroform is completely separated with water, get chloroform phase, be PHB, chloroform is carried out to gas chromatographic analysis mutually;
3) use the microsyringe of Agilent company, sample size is 1 μ L, adopts marker method to carry out quantitative analysis to PHB, according to peak area quantification.
PHB content is defined as the ratio of PHB to dry cell weight, PHB output=PHB content * dry cell weight.
Adopt identical method that control fermentation product is detected.Content and the dry cell weight result of bacterium cylinder accumulation PHB are as shown in table 1, result shows, E.coli JM109 (pBHR68pos5) does not have good PHB throughput at above-mentioned culture condition than not containing the kinase whose control strain E.coli of NADH JM109 (pBHR68), improves the output of PHB.
In the intestinal bacteria that table 1 contains different expression vectors, PHB's is synthetic
Claims (10)
1. build a method for recombinant bacterium, for the gene phbCAB of NADH kinases encoding gene pos5 and synthetic gather-3-hydroxybutyrate ester is imported in object bacterium, obtain recombinant bacterium.
2. method according to claim 1, is characterized in that: the gene phbCAB of described NADH kinases encoding gene pos5 and synthetic gather-3-hydroxybutyrate ester imports in object bacterium by recombinant vectors;
Described recombinant vectors is that NADH kinases encoding gene pos5 expression cassette is inserted in the expression vector of the gene phbCAB that contains synthetic gather-3-hydroxybutyrate ester, the recombinant vectors obtaining.
3. method according to claim 2, is characterized in that: described NADH kinases encoding gene pos5 expression cassette comprises pyruvic carboxylase promotor, NADH kinases encoding gene pos5 and the terminator that driving N ADH kinases encoding gene pos5 expresses.
4. according to arbitrary described method in claim 1-3, it is characterized in that:
Described NADH kinases encoding gene pos5 derives from Saccharomyces Cerevisiae in S accharomyces cerevisiae s288c;
The kinase whose aminoacid sequence of described NADH is the sequence 3 in sequence table.
5. according to arbitrary described method in claim 1-4, it is characterized in that:
The expression vector of the described gene phbCAB that contains synthetic gather-3-hydroxybutyrate ester is pBHR68;
The nucleotides sequence of described NADH kinases encoding gene pos5 is classified sequence 2 in sequence table as from 5 ' end 223-1416 position Nucleotide;
The nucleotides sequence of the gene phbCAB of described synthetic gather-3-hydroxybutyrate ester is classified the sequence 4 in sequence table as;
The nucleotides sequence of described NADH kinases encoding gene pos5 expression cassette is classified the sequence 2 in sequence table as;
The described bacterium that sets out is intestinal bacteria, and described intestinal bacteria are specially E.coli JM109.
6. the recombinant bacterium that arbitrary described method builds in claim 1-5.
7. produce a method for poly--3-hydroxybutyrate ester, the recombinant bacterium claimed in claim 6 that comprises the steps: to ferment, collects tunning, gathered-3-hydroxybutyrate ester.
8. method according to claim 7, is characterized in that:
Described leavening temperature is 30 ℃-40 ℃; Described fermentation time is 40-50h;
Described leavening temperature is specially 37 ℃; Described fermentation time is specially 48h.
9. a recombinant vectors of preparing poly--3-hydroxybutyrate ester, for NADH kinases encoding gene pos5 expression cassette being inserted in the expression vector of the gene phbCAB that contains synthetic gather-3-hydroxybutyrate ester, the recombinant vectors obtaining;
Described NADH kinases encoding gene pos5 expression cassette specifically comprises pyruvic carboxylase promotor, NADH kinases encoding gene pos5 and the terminator that driving N ADH kinases encoding gene pos5 expresses;
The nucleotide sequence of described NADH kinases encoding gene pos5 expression cassette is specially the sequence 2 in sequence table;
The expression vector of the described gene phbCAB that contains synthetic gather-3-hydroxybutyrate ester is specially pBHR68.
10. recombinant bacterium claimed in claim 6 or recombinant vectors claimed in claim 9 application in improving poly--3-hydroxybutyrate ester output.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755210A (en) * | 2016-12-12 | 2017-05-31 | 安徽翠鸟生物技术有限公司 | Coenzyme mixed solution and preparation method thereof |
| CN113755356A (en) * | 2021-10-19 | 2021-12-07 | 浙江大学 | Gene engineering bacterium for extracellularly secreting tocotrienol and application thereof |
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2013
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755210A (en) * | 2016-12-12 | 2017-05-31 | 安徽翠鸟生物技术有限公司 | Coenzyme mixed solution and preparation method thereof |
| CN113755356A (en) * | 2021-10-19 | 2021-12-07 | 浙江大学 | Gene engineering bacterium for extracellularly secreting tocotrienol and application thereof |
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