CN1807595B - Recombinant bacteria producing polyhydroxy fatty acid ester and its uses - Google Patents
Recombinant bacteria producing polyhydroxy fatty acid ester and its uses Download PDFInfo
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Abstract
The invention discloses a recombinant bacteria producing polyhydroxy fatty acid ester and its uses, and also discloses the process for obtaining the bacterial strain and its application, compared with the natural Delftia sp.GBSX267, the culture and fermentation of the P(3HB-co4HB) obtained from the recombinant R-Delftia sp.GBR008 can increase the yield of P(3HB-co-4HB) under oxygen insufficiency condition.
Description
Technical field
The present invention relates to the production field of polyhydroxyalkanoate, more specifically the invention discloses a kind of bacteria producing polyhydroxy fatty acid ester and application of reorganization.
Background technology
Polyhydroxyalkanoate especially poly butyric ester (polyhydroxyalkanoates, PHA) be a class by microorganism synthetic macromolecule polyester, its molecular weight generally by several ten thousand to millions of, extensively is present in the natural multiple microbe.Its some physical properties and traditional by oil synthetic plastics such as polyethylene, polypropylene-base seemingly, can be synthetic by the reproducible energy, and can degrade fully, enter natural ecological circulation, be considered to a kind of " biodegradable plastic " that may substitute nondegradable conventional plastic, caused the extensive concern of countries in the world scientific circles and industrial community.
The common feature of biological decomposable plastics is not only at nature and can be decomposed fully by microorganism and alleviate the pollution of land and ocean and the CO that is discharged when the burning disposal of discarded back
2Amount all is starkly lower than synthetic plastics, thereby can play active effect to the atmospheric environment of preserving our planet, minimizing Greenhouse effect.
And be the poly 3-hydroxy butyrate P (3HB) that monomer is formed with the 3-hydroxybutyric acid, because of the more crisp grade of its physical properties is not suitable for industrial application.P (3HB-co-4HB), the P (3HB-co-3HV) or the P (3HB-co-3HHx) that form with 4 hydroxybutyric acid, 3-hydroxypentanoic acid or the copolymerization of 3-hydroxycaproic acid respectively by the 3-hydroxybutyric acid, because they have good physical and mechanical property and (can regulate by the copolymerization ratio by the difference requirement, acquisition has different rigidity, crystallinity, fusing point and second-order transition temperature and is suitable for different purposes 7684 polymer materialss), particularly have biocompatibility and absorbability, can study one of the most popular PHA and become the world today as biomedical material.
1997, director researcher's clay fertilizer instrument of Japan RIKEN is controlled and wait to be found to eat sour feathering sporangium (Comomanas acidovorans IFO 13582, synonym Dai Erfu acidophilic bacteria Delftia acidovorans) benefit is gone into different lipid acid or alcohol and is controlled different fermentation conditions in the fermenting process, can obtain different types of PHA, thereby particularly obtaining to have good physical has the 3-hydroxybutyric acid of extensive use and the copolymer p (3HB-co-4HB) of 4 hydroxybutyric acid (to see JP08-089264 for details, JP09-098793).
In recent years, genetically modified microorganism is produced PHA, and to introduce the achievement of attracting attention most be that the succinyl semialdehyde pathways metabolism gene of the PHA biosynthetic pathway gene of alcaligenes eutrophus and Ke Shi clostridium (Clostridium kluyveri) is all recombinated in the e. coli host cell, expresses under suitable culture condition.Because the succinyl semialdehyde pathways metabolism is that initial substance produces 4-maloyl group coenzyme A intermediate with 6 carbon sugar or 5 carbon sugar, it means with combining of PHA biosynthetic pathway can not rely on precursor 4 hydroxybutyric acid or 1, and 4-butyleneglycol or gamma-butyrolactone produce the monomeric pha copolymer of 4HB.With glucose is that carbon source is cultivated this reorganization bacterium, can obtain molecular weight 1.8x10
6Da, amount of savings accounts for (P3HB-co-4HB) of dry cell weight 46% in the 4HB content 1.5mol%, cell.In addition, control etc. as far back as clay fertilizer instrument in 1988 and just to find that alcaligenes eutrophus can be by 4 hydroxybutyric acid, gamma-butyrolactone, 1,4-butyleneglycol and 1,6-hexylene glycol synthesize P (3HB-co-4HB) multipolymer.By regulating the composition of carbon, nitrogenous source, the ratio of 4HB can change in 0～100% scope in this multipolymer.The biological degradability of P (3HB-co-4HB) and material property and P (3HB) and P (3HB-co-3HV) are different, thereby have important use value.
With the gamma-butyrolactone is carbon source, with alcaligenes eutrophus 30 hours fermentation, obtains molecular weight 240,000～425 in nitrogen-free agar, 000, account for the copolymer p (3HB-co-4HB) of dry cell weight 29%.The gamma-butyrolactone consumption is increased to 25% by 10% in fermention medium, and the content of 4HB can be increased to 21% from 9% in the gained multipolymer.Add fructose in the above-mentioned substratum, can improve productive rate and the molecular weight of P (3HB-co-4HB), multipolymer content reaches 48% of dry cell weight in the cell, and the content of 4HB is reduced to 17% in the multipolymer.It is to produce bacterium with Comamonas acidovorans also that the clay fertilizer instrument is controlled etc., and with butyric acid or 1, the 4-butyleneglycol is a carbon source, and the product of acquisition is respectively P (3HB) and P (3HB-co-4HB) (wherein 4HB content is 94mol%).As with butyric acid or 1, the 4-butyleneglycol is a mixed carbon source, and then the content of 4HB in P (3HB-co-4HB) is with 1, and the ratio of 4-butyleneglycol in mixed carbon source increases and increase.The gained multipolymer is not a homogeneous, but contains the mixture (Appl.Envir.Microbiol.1998 64:3437-3443.) of 4HB different content multipolymer.
Kim etc. adopt methyl Bacillaceae (Methylobacterium sp.KCTC 0048) fermentation using bacteria, are carbon source with methyl alcohol and gamma-butyrolactone, also can obtain P (3HB-co-4HB).The polymkeric substance amount of savings is 2.0%～13.1% of a dry cell weight, and wherein the content of 4HB is 1.9%～5.5% (Biotechnol.Lett., 15,1017-1020 (1993) 36).
Thereby, thereby constantly utilize modern biotechnology that traditional bacterial strain is transformed and obtain new reorganization bacterium P (3HB-co-4HB) conveniently is easy to get is the problem that those skilled in the art put forth effort to solve always although at present by utilizing various microorganism, regulate forming and feed supplement and control the method that different fermentation conditions improves P (3HB-co-4HB) output during the fermentation of charcoal source, ammonia source in the substrate.
Summary of the invention
The invention discloses:
1. new reorganization bacteria producing polyhydroxy fatty acid ester GBR008;
2. the expression vector pGB_ZV of above-mentioned 1 reorganization bacterium;
3. method that obtains above-mentioned 1 described reorganization bacterium comprises:
(1) gene of the PHA depolymerizing enzyme of clone Dai Erfu bacterium obtains plasmid pGEMT_phaZ;
(2) clone's Vitreoscilla hemoglobin gene obtains plasmid pGEMT_VHb;
(3) obtain expression vector pGEMT_ZV;
(4) the bacteria producing polyhydroxy fatty acid ester GBR008 that obtains recombinating.
4. method of utilizing above-mentioned 1 described reorganization bacterium to produce P (3HB-co-4HB) comprises:
(1) the reorganization bacterium is cultivated;
(2) the reorganization bacterium is fermented;
(3) separate fermentation liquid extracts object P (3HB-co-4HB).
5. above-mentioned 5 described methods, the cultivation stage culture condition of the bacterium of wherein recombinating is 37 ℃, 220rpm cultivated 5 hours.
6. above-mentioned 5 described methods, the fermentation stage fermentation condition of the bacterium of wherein recombinating are 30 ℃ of fermentations of fed-batch fermentation 40 hours.
7. above-mentioned 5 described methods, the solvent that wherein extracts object is chloroform/dehydrated alcohol.
8. above-mentioned 8 method, wherein the volume ratio of solvent chloroform/dehydrated alcohol is 1: 4.
To the encode gene and the Vitreoscilla hemoglobin gene of intracellular PHA depolymerase of Dai Erfu bacterium of the present invention is cloned in the plasmid, obtained expression vector pGEMT_ZV, further it is transformed among the Delftiasp., thereby the screening transformant obtains the Delftia sp. of recombinant type: a kind of new reorganization bacteria producing polyhydroxy fatty acid ester GBR008.Utilize this reorganization bacterium to obtain P (3HB-co-4HB) through cultivating, fermenting, its content accounts for 30.29% of dry cell weight, wherein the molar content of 4HB is 17.2%, with the P that utilizes traditional Delftiaacidovorans to obtain (3HB-co-4HB) even compare under cultivation at oxygen lack, the fermentation condition and still can increase the output of P (3HB-co-4HB), thereby help industrialized scale operation.
Description of drawings
Accompanying drawing 1: the physical map of plasmid pGEMT_ZV;
Embodiment
Following examples only are further detailed the present invention, should not be construed as limitation of the present invention.
Used molecule clone technology among the present invention, the separation and purification, DNA that comprises DNA is synthetic, polymerase chain reaction (PCR), the digestion with restriction enzyme of DNA, the connection of DNA, intestinal bacteria transformation and selection all are to be familiar with very much in this area, at the MolecularCloning of works such as Sambrook, have in a laboratory manual one book fully and describe.
The model of gas chromatograph and manufacturer: Agilent company 6890 types
PGEM-T easy carrier, T4 ligase enzyme, AatII, SalI, EcoRI, NcoI restriction enzyme are all available from Promega company, E.coli DH5 α is available from Beijing ancient cooking vessel state biotech company, natural Dai Erfu bacterium (Delftia sp.) is separated from active sludge by this laboratory, the culture presevation GBSX267 of company.
If not otherwise specified, all reagent are commercially available among the present invention.
Among the present invention if not otherwise specified, born of the same parents' intensive amount per-cent (accumulation per-cent in the born of the same parents) is the weight percent that P (3HB-co-4HB) dry weight accounts for the cell gross dry weight, and the 4HB percentage composition is the molar percentage that the 4HB mole number accounts for 3HB and 4HB mole number sum among the P (3HB-co-4HB).
Embodiment 1: the clone of hemoglobin gene and plasmid construction
1.1 the gene clone of the PHA depolymerizing enzyme of Dai Erfu bacterium (Delftia sp.)
For the gene clone of the intracellular PHA depolymerase of the Dai Erfu bacterium of will encoding is adopted in plasmid
Primer 2,5 '-ATTAGCGTCGACCGGACTGGCCCGTGAGGTC-3 ' (SEQ NO.2)
By the method for pcr amplification, clone the PHA depolymerizing enzyme as template from natural Dai Erfu bacterium (Delftia sp.GBSX267), to separate the gene that obtains.The preparation of these primers is from the nucleotide sequence of the gene of the intracellular PHA depolymerase of Dai Erfu acidophilic bacteria (referring to Kasuya, K., GenBankSequence Database, AB003186,1998).Carry out PCR by following condition: at first 94 ℃ of sex change 5 minutes, 94 ℃ of sex change of 35 circulations are 50 seconds then, and 50 ℃ of annealing were extended after 3 minutes in 1 minute and 72 ℃, added that at last 72 ℃ were extended 10 minutes.The DNA that obtains through PCR separates the dna fragmentation of about 2kb down with agarose gel electrophoresis behind AatII and the SalI digestion with restriction enzyme, thereby this fragment is connected to subsequently and has digested the good pGEM-T easy through transforming with AatII and SalI restriction enzyme and connect generation recombinant plasmid pGB/phaZ on the empty carrier certainly.Give E.coli DH5 α (yeast powder 5g/l on the LB agar plate that contains penbritin (50ug/l) then by electroporation technology with recombinant plasmid transformed; Tryptones 10g/l; NaCl 10g/l; Agar powder 15g/l) thus the screening transformant obtains the E.coli DH5 α/pGB_phaZ of reorganization.
1.2 Vitreoscilla hemoglobin gene clone
Vitreoscilla hemoglobin gene is cloned in the plasmid in order to encode
Adopt primer 1; 5 '-GGACGCTGGGGTTAAAAGTAT-3 ' (SEQ NO.3)
Primer 2; 5 '-GTCCCAAGTTTTGGCAACAGC-3 ' (SEQ NO.4)
To separate the gene obtain from Vitreoscilla is template, and the method by pcr amplification obtains hemoglobin gene.The preparation of these primers is from the nucleotide sequence (referring to Khosla, C. and Bailey etc., GenBank Sequence Database, M30794,1993) of the hemoglobin gene of Vitreoscilla.Carry out PCR according to following condition: at first 94 ℃ of sex change 5 minutes, 94 ℃ of sex change of 35 round-robin are 50 seconds then, and 50 ℃ of annealing were extended after 1 minute in 1 minute and 72 ℃, added that at last 72 ℃ were extended 10 minutes.The DNA that process PCR obtains separates the dna fragmentation of about 650bp under agarose gel electrophoresis, thereby this fragment is connected to generation recombinant plasmid pGB_VHb on the pGEM-T easy T carrier subsequently.Containing penbritin (50ug/L), (yeast powder 5g/L on the LB agar plate of IPTG and X-Gal then for E.coli DH5 α recombinant plasmid transformed by electroporation technology; Tryptones 10g/L; NaCl 10g/L; Agar powder 15g/L) thus the screening transformant obtains the E.coli DH5 α/pGB_VHb of reorganization.
1.3 the structure of target practice plasmid
Respectively with EcoRI and NcoI digested plasmid pGB_VHb with EcoRI and NcoI digested plasmid pGB_phaZ, separate the VHb dna fragmentation of 650bp and the pGB_phaZ linear DNA fragment of about 5kb with agarose gel electrophoresis then, connect two linear fragments with the T4 ligase enzyme then and form plasmid pGB_ZV.Give E.coli DH5 α (yeast powder 5g/l on the LB agar plate that contains penbritin (50ug/l) then by electroporation technology with recombinant plasmid transformed; Tryptones 10g/l; NaCl10g/l; Agar powder 15g/l) thus select that mono-clonal is cultivated the extracting plasmid and enzyme is cut E.coli DH5 α/pGB_ZV that the evaluation and screening transformant obtains reorganization.
Embodiment 2: the acquisition of reorganization Dai Erfu bacterial strain.
Give natural Dai Erfu bacterium (Delftia sp.) then on the LB agar plate that contain penbritin (50ug/l) (yeast powder 5g/l with electroporation technology with recombinant plasmid transformed with the pGB_ZV plasmid; Tryptones 10g/l; NaCl 10g/l; Agar powder 15g/l, thus pH7.0) the screening transformant obtains the Delftia sp./pGB_ZV of recombinant type.And number rename to producing polyhydroxyalkanoate reorganization Dai Erfu bacterium R-Delftia sp.GBR008 according to the clone.
Embodiment 3: utilize natural Dai Erfu bacterium (Delftia sp.GBSX267) to produce P (3HB-co-4HB)
Thalli growth substratum (LB): yeast powder 5g/l; Tryptones 10g/l; NaCl 10g/l, pH7.0.
Fermention medium (g/L): 2g/L NH
4Cl, 0.2g/L MgSO
4.7H
2O, 9g/L Na
2HPO
4.12H
2O, 1.5g/L KH
2PO
4, 50ul/L penbritin and 1ml/L trace element are (among the 1mol/L HCl (g/L): 9.7g FeCl
3, 7.8g CaCl
2, 0.218g CoCl
2.6H
2O, 0.156g CuSO
4.5H
2O, 0.118g NiCl
3.6H
2O, 0.105g CrCl
3.6H
2O, 0.05g MnCl
2.4H
2O, 0.06g ZnSO
4.7H
2O), pH6.8.Glucose 10g/L.1,4-butyleneglycol concentration is 1% (V/V).
The natural Dai Erfu bacterium (Delftia sp.) that will contain-80 ℃ of preservations of 15% glycerine with each 100ul inoculum size is inoculated in 100ml (250ml triangular flask) the thalli growth substratum (LB).Cover with eight layers of gauze.37 ℃, 220rpm cultivated 5 hours.
Will be in LB cultured thalline 10ml bacterium liquid is inserted among the above-mentioned fermention medium 100ml that is contained in the 250ml triangular flask according to 1: 10 ratio, cover with 16 layers of sterile gauze.30 ℃, 220rpm cultivated 40 hours.Centrifugal collection thalline extracts P (3HB-co-4HB) multipolymer drying with the chloroform/ethanol precipitator method (volume ratio 1: 4) and weighs.
Carry out eight batches of experiments altogether, the results are shown in Table 1.
The natural Dai Erfu bacterium of table 1 (Delftia sp.GBSX267) shake flask fermentation experimental result
| Batch | Dry cell weight (CDW) (g/L) | P(34HB)/CDW(％) | 4HB content (%) |
| 1 | 2.89 | 27.55 | ?15.3 |
| 2 | 2.91 | 27.34 | ?15.7 |
| 3 | 3.05 | 28.37 | ?18.3 |
| 4 | 3.24 | 29.12 | ?16.9 |
| 5 | 2.69 | 26.69 | ?16.5 |
| 6 | 2.98 | 27.33 | ?14.4 |
| 7 | 3.07 | 30.05 | ?16.1 |
| 8 | 3.01 | 28.49 | ?17.3 |
| Mean value | 2.98 | 28.12 | ?16.3 |
Wherein: dry cell weight is the fermentation dry cell weight;
P (34HB)/CDW (%) accounts for the per-cent of dry cell weight for P (3HB-co-4HB) weight;
4HB content (%) accounts for the molar percentage of 3HB and 4HB mole number sum for 4HB mole number among the P (3HB-co-4HB).
Resulting fermentation dry cell weight average out to 2.98g/L, product is verified as 3-hydroxybutyric acid and the 4 hydroxybutyric acid multipolymer that contains 4HB16.3mol% by analysis.Detection method: take by weighing the 8-10mg stem cell, add the 2ml chloroform, add 1.7ml methyl alcohol then, add the 0.3ml vitriol oil again, 100 ℃ of esterifications 4 hours, liquid after the esterification adds 1ml water, takes off layer organic phase after the vibration and carries out gas chromatographic detection (Braunegg, G.et al.Eur.J.Microbiol.Biotechnol.1978,6,29-37), calculate P (3HB-co-4HB) content and account for 28.12% of dry cell weight, wherein the molar content of 4HB is 16.3%.
Embodiment 4: utilize reorganization Dai Erfu bacterium (R-Delftia sp.GBR008) to produce P (3HB-co-4HB)
Change the loading amount of fermention medium in the 250ml triangular flask and be increased to 150ml, and the gauze number of plies that covers is increased to 24 layers, supply with oxygen in the restriction culturing process, and changing fermented bacterium into reorganization bacterium (R-Delftia sp.GBR008) that embodiment 2 obtains, other experimental procedure is with embodiment 3.Eight batches of repeated experiments the results are shown in Table 2.
Table 2 reorganization Dai Erfu (R-Delftia sp.GBR008) shake flask fermentation experimental result
| Batch | Dry cell weight (CDW) (g/L) | P(34HB)/CDW(％) | 4HB content (%) |
| 1 | 3.56 | 32.56 | 18.5 |
| 2 | 3.26 | 30.61 | 16.4 |
| 3 | 3.19 | 29.53 | 17.3 |
| 4 | 3.24 | 29.28 | 16.9 |
| 5 | 3.29 | 28.97 | 15.3 |
| 6 | 3.18 | 29.33 | 17.8 |
| 7 | 3.43 | 31.29 | 17.3 |
| 8 | 3.02 | 30.73 | 18.1 |
| Mean value | 3.27 | 30.29 | 17.2 |
Wherein: dry cell weight, P (34HB)/CDW (%), 4HB content (%) are with table 1.
The average dry weight of resulting fermentation cell is 3.27g/L.Product verifies that by analysis (with embodiment 3) calculating mean value obtains 3-hydroxybutyric acid and the 4 hydroxybutyric acid multipolymer for containing 4HB 17.2%, and P (3HB-co-4HB) content accounts for 30.29% of dry cell weight, and wherein the molar content of 4HB is 17.2%.
By above-mentioned shake flask fermentation experimental result as can be known, utilize reorganization disclosed by the invention Dai Erfu bacterium (R-Delftiasp.GBR008) to compare with the P (3HB-co-4HB) that utilizes natural Dai Erfu bacterium Delftia sp.GBSX267 to obtain under the condition of oxygen lack to cultivate, fermenting to increase the output of P (3HB-co-4HB), helps industrialized scale operation.
SEQUENCE?LISTING
SEQ?NO.1
＜110〉Tianjin Green Biosciences Limited Company
＜120〉a kind of bacteria producing polyhydroxy fatty acid ester of reorganization and application
<160>4
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＜213〉artificial sequence
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Claims (5)
1. the bacteria producing polyhydroxy fatty acid ester GBR008 of a reorganization, its production method comprises:
(1) gene and the structure of the PHA depolymerizing enzyme of clone Dai Erfu bacterium obtain plasmid pGEMT_phaZ;
(2) clone's Vitreoscilla hemoglobin gene obtains plasmid pGEMT_VHb;
(3) obtain expression vector pGEMT_ZV, its physical map as shown in Figure 1;
(4) the expression vector transfection is obtained reorganization to natural Dai Erfu bacterium and produce poly-hydroxy fatty acid bacterium GBR008.
2. utilize the described reorganization of claim 1 to produce the method that poly-hydroxy fatty acid bacterium GBR008 produces 3-hydroxybutyric acid-4 hydroxybutyric acid multipolymer, comprising:
(1) poly-hydroxy fatty acid bacterium GBR008 being produced in described reorganization cultivates;
(2) poly-hydroxy fatty acid bacterium GBR008 being produced in described reorganization ferments;
(3) separate fermentation liquid extracts object 3-hydroxybutyric acid-4 hydroxybutyric acid multipolymer.
3. the described method of claim 2, wherein to produce the cultivation stage culture condition of poly-hydroxy fatty acid bacterium GBR008 be 37 ℃ in reorganization, 220rpm cultivated 5 hours.
4. the described method of claim 3, wherein the reorganization fermentation stage fermentation condition that produces poly-hydroxy fatty acid bacterium GBR008 fermented 40 hours for 30 ℃ for shaking bottle batch fermentation.
5. the described method of claim 2, the solvent that wherein extracts object is chloroform/dehydrated alcohol, wherein the volume ratio of chloroform/dehydrated alcohol is 1: 4.
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| Title |
|---|
| 于慧敏 等.透明颤菌血红蛋白基因在产PHB重组大肠杆菌中的引入.《微生物学报》.2001,第41卷(第5期),548-552. |
| 于慧敏等.透明颤菌血红蛋白基因在产PHB重组大肠杆菌中的引入.《微生物学报》.2001,第41卷(第5期),548-552. * |
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