CN1246453C - Recombinant hygrophilous aeromonad producing copolymer PHBHHx and its construction and application - Google Patents
Recombinant hygrophilous aeromonad producing copolymer PHBHHx and its construction and application Download PDFInfo
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- CN1246453C CN1246453C CN 03130839 CN03130839A CN1246453C CN 1246453 C CN1246453 C CN 1246453C CN 03130839 CN03130839 CN 03130839 CN 03130839 A CN03130839 A CN 03130839A CN 1246453 C CN1246453 C CN 1246453C
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Abstract
The present invention discloses a recombined aeromonas hydrophila of the copolymer PHBHHx of 3-hydroxy butyric acid (HB) and 3-hydrodecemycaproic acid (HHx) in one strain, a construction method and application thereof. The recombined aeromonas hydrophila contains beta-ketothiolase genes phbA and acetoacetyl coenzyme A reductase genes phbB. The construction method comprises: 1) constructing recombined plasmids which contain the phbA and the phbB; 2) converting the recombined plasmids into aeromonas hydrophila; 3) sieving out recombined aeromonas hydrophila which contains the recombined plasmids. The 3HHx content of the PHBHHx produced by the recombined aeromonas hydrophila is 3 to 16 wt%. The proportion of 3HHx is lowered, and the variation range is wide. Therefore, the crystallization time of the PHBHHx is shortened, and the strength of the PHBHHx is increased. The content of the PHBHHx is increased in cells. Simultaneously, the present invention proves that antibiotics and inducers IPTG are not requisite in the process of producing the new polyester PHBHHx with controllable 3HHx content, and therefore, the present invention can lower production cost.
Description
Technical field
The present invention relates to produce reorganization Aeromonas hydrophila and construction process and the application of 3-hydroxybutyric acid (HB) and 3-hydroxycaproic acid (HHx) copolymer p HBHHx, belong to using microbe and field of fermentation engineering.
Background technology
Polyhydroxyalkanoate (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.Because its some physical properties and traditional, seemingly by oil synthetic plastics such as polyethylene, polypropylene-base, but can be synthetic by the reproducible energy, therefore and can degrade fully enters natural ecological circulation, is considered to a kind of and may substitutes " biodegradable plastic " of nondegradable conventional plastic and cause the extensive attention of countries in the world scientific circles and industrial community.
Poly--the R-3-butyric ester (poly-R-3-hydroxybutyric acid PHB) is the simplest member in the PHA family, also be study at most, the most thorough member.PHB behind the purifying on some performance similar in appearance to thermoplastics, mechanical property is similar to polypropylene (PP), degree of crystallinity is 55-80%, property is crisp, the jackknifing elongation is very low, and will cracking when Heating temperature is higher than 10 ℃ of fusing points (180 ℃), these character have increased the difficulty of the aftertreatment processing of PHB, have limited its range of application greatly.Other monomeric physicalies of mixing PHA have bigger influence in the multipolymer.As monomeric the mixing of 3-hydroxypentanoic acid HV the crystalline texture of hydroxybutyric acid and hydroxyl pentanoate copolymer PHBV is obviously changed, bring hardness decline, strength degradation, fusing point to descend, but decomposition temperature does not descend; And along with the adding of HV, crystalline crystal regularity descends and presents different crystal habits, and this all has improved action to the PHA performance.
The copolymer p HBHHx of 3-hydroxybutyric acid (HB) and 3-hydroxycaproic acid (HHx) is the novel pha copolymer of discovered in recent years.The physicals of PHBHHx has very big change along with the variation of 3HHx content, when 3HHx is increased to 17mol% by 0mol%, its tensile strength is reduced to 20MPa by 43MPa, and corresponding elongation at break is increased to 850% by 5%, and its degree of crystallinity drops to 18% by 60%.This shows that the flexibility of PHBHHx and toughness all improve a lot than PHB.Wild-type Aeromonas hydrophila CGMCC 1816 can synthesize PHBHHx with lauric acid during as substratum, but its 3HHx ratio is higher, and its variation range is narrower, between 12～18mol% (15.31-22.54wt%).Because too high 3HHx content can cause the crystallization time of PHBHHx to prolong and make its strength degradation, and the PHBHHx that contains different ratios 3HHx has different using values.Therefore, it is significant to produce the PHBHHx that the 3HHx component ratio can regulate and control.
Many PHB synthesis bacteriums, as really supporting Luo Shi bacillus, Alcaligenes, bulkholderia cepasea, acinetobacter calcoaceticus, pseudomonas, genus bacillus, giving birth to the moving glue bacterium of branch etc., all have β-Tong Liuxiemei gene (phbA) and Acetoacetyl-CoA reductase gene (phbB), these two enzyme catalysis D-(-)-3-hydroxybutyric acid coenzyme A (3HB-CoA) is synthetic.The gene of these two enzymes at first obtains from really supporting to clone the Luo Shi bacillus, and identified function (the Slater S.C. of its product, Voige W.H., Dennis D.E.Cloning and expression in Escherichia coli of theAlcaligenes eutrophus H16 poly-β-hydroxybutyrate biosynthetic pathway.J.Bacteriol., 1988,170:4431-4436; Schubert P., Steinb ü chel A., Schlegel H.G.Cloning of the Alcaligenes eutrophus genes for synthesis ofpoly-β-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli.J.Bacteriol., 1988,170:5837-5847).
Summary of the invention
The purpose of this invention is to provide a kind of reorganization Aeromonas hydrophila, contain the gene of catalysis D-(-)-two enzymes of 3-hydroxybutyric acid coenzyme A (3HB-CoA) synthetic in this bacterium, can produce the different PHBHHx of 3HHx component ratio.
Reorganization Aeromonas hydrophila provided by the present invention is the Aeromonas hydrophila that has imported β-Tong Liuxiemei gene phbA and Acetoacetyl-CoA reductase gene phbB.
Second purpose of the present invention provides a kind of method that makes up the reorganization Aeromonas hydrophila.
A kind of method that makes up the reorganization Aeromonas hydrophila may further comprise the steps:
1) makes up the recombinant plasmid that contains phbA and phbB gene;
2) transform this recombinant plasmid in Aeromonas hydrophila;
3) filter out the reorganization Aeromonas hydrophila that contains recombinant plasmid.
Described phbA and phbB gene can come from really supports Luo Shi bacillus, Alcaligenes, bulkholderia cepasea, acinetobacter calcoaceticus, pseudomonas, genus bacillus, gives birth to the moving glue bacterium of branch and other can synthesize the bacterium of PHB.
Described conversion recombinant plasmid to the method in the Aeromonas hydrophila is, earlier the mode of recombinant plasmid with electroporation is transformed among the E.coli S17-1, and with the E.coli S17-1 that contains recombinant plasmid as the donor bacterium, carry out mixed culture with Aeromonas hydrophila as recipient bacterium.
Described Aeromonas hydrophila is an Aeromonas hydrophila.
The 3rd purpose of the present invention provides a kind of method of utilizing above-mentioned reorganization Aeromonas hydrophila to produce PHBHHx.
A kind of with the reorganization Aeromonas hydrophila produce the regulatable 3-hydroxybutyric acid of component ratio (3HB) and-method of the copolymer p HBHHx of 3-hydroxycaproic acid (3HHx), may further comprise the steps:
1) shake-flask culture reorganization Aeromonas hydrophila produces PHBHHx;
2) add auxiliary carbon source, produce the PHBHHx that contains different ratios 3HHx.
Described auxiliary carbon source is preferably Sunmorl N 60S.
Also add inductor IPTG and kantlex in the described substratum where necessary.
Product is after separation and purification, with the content of gas chromatographic detection PHBHHx and 3HHx.
Advantage of the present invention and positively effect are: the constructed reorganization Aeromonas hydrophila that obtains can be produced 3HHx content
Advantage of the present invention and positively effect are: the constructed reorganization Aeromonas hydrophila that obtains can be produced the new polyester PHBHHx that 3HHx content can be regulated and control, its 3HHx content is 3-16wt%, the ratio of 3HHx reduces, and the variation range broad, thereby make the crystallization time shortening of PHBHHx and its intensity is increased.The present invention produces PHBHHx with the constructed reorganization Aeromonas hydrophila that obtains, and can improve the content of PHBHHx in cell.The present invention has proved that also microbiotic and inductor IPTG are not necessary in the process of the new polyester PHBHHx that production 3HHx content can be regulated and control, therefore can reduce production costs.
Description of drawings
Fig. 1 is the structure collection of illustrative plates of recombinant plasmid pTG01.
Fig. 2 is the electrophoretogram of PCR.
Embodiment
The preparation of embodiment 1, reorganization Aeromonas hydrophila QYZ1
Donor bacterium: intestinal bacteria E.coli S17-1 (Chinese common micro-organisms culture presevation administrative center)
Recipient bacterium: Aeromonas hydrophila (Aeromonas hydrophila) CGMCC 1816 (Chinese common micro-organisms culture presevation administrative center)
Foreign gene: plasmid pUCAB
Carrier: pBBR1MCS2
LB substratum: peptone 10g/L
Yeast extract powder 5g/L
Sodium-chlor 10g/L
1) structure of plasmid pTG01:
As shown in Figure 1, plasmid pUCAB contains to come since really supporting Luo Shi bacillus (U.S. type culture collection center, phbA ATCC17699) and phbB gene (its accession number in GenBank is J04987).With KpnI and HindIII digested plasmid pUCAB, its small segment is inserted into the KpnI/HindIII site of carrier pBBR1MCS2, promptly obtain recombinant plasmid pTG01.
2) with the mode that engages plasmid pTG01 is transformed among the Aeromonas hydrophila CGMCC 1816:
Earlier plasmid pTG01 is transformed among the E.coli S17-1 in the mode of electroporation, again with the E.coliS17-1 that contains pTG01 as the donor bacterium, with Aeromonas hydrophila CGMCC 1816 as recipient bacterium, on the LB flat board, two kinds of bacterium are mixed in 30 ℃ of following incubations 24 hours, use the potassium phosphate buffer re-suspended cell of 100mmol/L then, to be coated on the LB flat board that contains 60mg/L penbritin and 50mg/L kantlex after the cell suspension dilution, select single bacterium colony, be reorganization Aeromonas hydrophila QYZ1.
3) with PCR method reorganization Aeromonas hydrophila QYZ1 is verified:
PhbA that comes out according to clone in different bacteriums and the conserved sequence of phbB design a pair of primers F-phbA:5 ' TACCACATGGG (C/T) ATCAC (C/G) GC3 ' and R-phbB:3 ' TTGAT (G/A) AG (G/C) CG (G/C) CGGTTCCG 5 ', increase as template with the plasmid that extracts in the reorganization Aeromonas hydrophila QYZ1, obtain the specific fragment of a treaty 1200bp, prove the reorganization Aeromonas hydrophila QYZ1 that contains plasmid pTG01, as shown in Figure 2.
Embodiment 2: utilize reorganization Aeromonas hydrophila QYZ1 to produce the PHBHHx that contains different 3HHx ratios
Bacterial classification: reorganization Aeromonas hydrophila QYZ1
Culture condition: cultivated 200 rev/mins of shaking speed 48 hours for 30 ℃
Inoculum size: 10%
Substratum: Na
2HPO
412H
2O 3.5g/L
KH
2PO
4 0.70g/L
(NH
4)
2SO
4 2.0g/L
KCl 0.5g/L
NaCl 1.0g/L
MgSO
4·7H
2O 0.8g/L
Yeast powder 1.0g/L
Lauric acid 4.0g/L
Trace element solution 1%
IPTG 0.2g/L
Kantlex 50mg/L
Consisting of of trace element solution: every liter of solution contains 5g Fe (III)-NH
4-Citrate, 2g CaCl
22H
2O, 10mgZnSO
47H
2O, 3mg MnCl
24H
2O, 30mg H
3BO
3, 20mg CoCl
26H
2O, 1mg CuSO
45H
2O, 2mg NiCl
26H
2O and 3mg NaMoO
42H
2O.
The Aeromonas hydrophila of will recombinating is inserted in the above-mentioned substratum, 30 ℃ of following shake-flask culture 48 hours, and 200 rev/mins of shaking speed.Respectively 12 hours and 24 hours at twice equivalent add Sunmorl N 60S as auxiliary carbon source.After cultivating end, centrifugal (10000g, 20min), collect thalline, use deionized water and washing with alcohol thalline respectively, frost drying (35 ℃) thalline is got stem cell esterification (He W N, Tian W D about 30 milligrams then, Zhang G, ChenG Q, Zhang Z M.Production of novel polyhydroxyalkanoates by Pseudomonasstutzeri 1317 from glucose and soybean oil.FEMS Microbiol.Lett., 1998,169:45-49), with the content of gas chromatographic detection PHBHHx and the content of 3HHx, the condition of gas-chromatography is: column temperature is from 80 ℃ of beginnings, temperature programming to 220 ℃.
Experimental result: when the Sunmorl N 60S that adds is respectively 0,4,8, during 12g/L, the dry cell weight of the reorganization Aeromonas hydrophila QYZ1 that obtains reaches 2.20,2.79,2.66 respectively, 3.09g/L, the content of PHBHHx is respectively 41.45%, 41.09%, 39.71%, 37.52% in the cell, and the content of 3HHx is respectively 15.61wt%, 9.94wt%, 7.45wt%, 3.68wt%.As seen, by changing the concentration of the auxiliary carbon source Sunmorl N 60S that adds, can obtain a series of PHBHHx that contain different 3HHx ratios.
Embodiment 3:IPTG is to the influence of 3HHx ratio among the PHBHHx of reorganization Aeromonas hydrophila QYZ1 production
Bacterial classification: reorganization Aeromonas hydrophila QYZ1
Culture condition: cultivated 200 rev/mins of shaking speed 48 hours for 30 ℃
Inoculum size: 10%
Substratum: Na
2HPO
412H
2O 3.5g/L
KH
2PO
4 0.70g/L
(NH
4)
2SO
4 2.0g/L
KCl 0.5g/L
NaCl 1.0g/L
MgSO
4·7H
2O 0.8g/L
Yeast powder 1.0g/L
Lauric acid 4.0g/L
Trace element solution 1%
IPTG 0g/L or 0.2g/L
Kantlex 50mg/L
Consisting of of trace element solution: every liter of solution contains 5g Fe (III)-NH
4-Citrate, 2g CaCl
22H
2O, 10mgZnSO
47H
2O, 3mg MnCl
24H
2O, 30mg H
3BO
3, 20mg CoCl
26H
2O, 1mg CuSO
45H
2O, 2mg NiCl
26H
2O and 3mg NaMoO
42H
2O.
The Aeromonas hydrophila of will recombinating is inserted in the above-mentioned substratum, 30 ℃ of following shake-flask culture 48 hours, and 200 rev/mins of shaking speed.Respectively 12 hours and 24 hours at twice equivalent add Sunmorl N 60S as auxiliary carbon source.After cultivating end, centrifugal (10000g, 20min), collect thalline, use deionized water and washing with alcohol thalline respectively, frost drying (35 ℃) thalline then, get the stem cell esterification (He WN etc., 1998) about 30 milligrams, with the content of gas chromatographic detection PHBHHx and the content of 3HHx, the condition of gas-chromatography is: column temperature is from 80 ℃ of beginnings, temperature programming to 220 ℃.
Experimental result: when the Sunmorl N 60S that adds in substratum is 0g/L, the dry cell weight that IPTG does not add group and the reorganization Aeromonas hydrophila QYZ1 that obtains of IPTG interpolation group reaches 1.92g/L and 2.20g/L respectively, the content of PHBHHx is respectively 46.75% and 41.45% in the cell, and the content of 3HHx is respectively 15.66wt% and 15.61wt%; When the Sunmorl N 60S that adds in substratum is 8g/L, the dry cell weight that IPTG does not add group and the reorganization Aeromonas hydrophila QYZ1 that obtains of IPTG interpolation group reaches 2.55g/L and 2.66g/L respectively, the content of PHBHHx is respectively 50.65% and 39.71% in the cell, and the content of 3HHx is respectively 7.73wt% and 7.45wt%.As seen, for produce the PHBHHx that contains different 3HHx ratios from the reorganization Aeromonas hydrophila, the interpolation of IPTG is not necessary; And, when not adding IPTG, can also improve the content of PHBHHx in the cell.This can save cost greatly in industrial production.
Embodiment 4: kantlex is to the influence of 3HHx ratio among the PHBHHx of reorganization Aeromonas hydrophila QYZ1 production
Bacterial classification: reorganization Aeromonas hydrophila QYZ1
Culture condition: cultivated 200 rev/mins of shaking speed 48 hours for 30 ℃
Inoculum size: 10%
Substratum: Na
2HPO
412H
2O 3.5g/L
KH
2PO
4 0.70g/L
(NH
4)
2SO
4 2.0g/L
KCl 0.5g/L
NaCl 1.0g/L
MgSO
4·7H
2O 0.8g/L
Yeast powder 1.0g/L
Lauric acid 4.0g/L
Trace element solution 1%
Kantlex is respectively 0mg/L, 25mg/L, 50mg/L
Consisting of of trace element solution: every liter of solution contains 5g Fe (III)-NH
4-Citrate, 2g CaCl
22H
2O, 10mgZnSO
47H
2O, 3mg MnCl
24H
2O, 30mg H
3BO
3, 20mg CoCl
26H
2O, 1mg CuSO
45H
2O, 2mg NiCl
26H
2O and 3mg NaMoO
42H
2O.
The Aeromonas hydrophila of will recombinating is inserted in the above-mentioned substratum, 30 ℃ of following shake-flask culture 48 hours, and 200 rev/mins of shaking speed.Respectively 12 hours and 24 hours at twice equivalent add the 8g/L Sunmorl N 60S as auxiliary carbon source.After cultivating end, centrifugal (10000g, 20min), collect thalline, use deionized water and washing with alcohol thalline respectively, frost drying (35 ℃) thalline then, get the stem cell esterification (He WN etc., 1998) about 30 milligrams, with the content of gas chromatographic detection PHBHHx and the content of 3HHx, the condition of gas-chromatography is: column temperature is from 80 ℃ of beginnings, temperature programming to 220 ℃.
Experimental result: when the kantlex that adds in substratum is respectively 0mg/L, 25mg/L, during 50mg/L, the dry cell weight of resulting reorganization Aeromonas hydrophila QYZ1 reaches 2.52g/L respectively, 2.43g/L and 2.43g/L, the content of PHBHHx is respectively 47.13%, 48.90% and 52.12% in the cell, the content of 3HHx is respectively 9.03wt%, 9.44wt% and 9.28wt%.As seen, for produce the PHBHHx that contains different 3HHx ratios from the reorganization Aeromonas hydrophila, antibiotic interpolation is not necessary.This can save cost greatly in industrial production.
Claims (10)
1, a kind of reorganization Aeromonas hydrophila is the Aeromonas hydrophila that has imported β-Tong Liuxiemei gene phbA and Acetoacetyl-CoA reductase gene phbB.
2, a kind of method that makes up the described reorganization of claim 1 Aeromonas hydrophila may further comprise the steps:
1) makes up the recombinant plasmid that contains phbA and phbB gene;
2) transform this recombinant plasmid in Aeromonas hydrophila;
3) filter out the reorganization Aeromonas hydrophila that contains recombinant plasmid.
3, according to the described method of claim 2, it is characterized in that: described phbA and phbB gene come from the bacterium that can synthesize PHB.
4, according to the described method of claim 3, it is characterized in that: the described bacterium that can synthesize PHB is very foster Luo Shi bacillus, Alcaligenes, bulkholderia cepasea, acinetobacter calcoaceticus, pseudomonas, genus bacillus or the moving glue bacterium of living branch.
5, according to the described method of claim 2, it is characterized in that: described conversion recombinant plasmid to the method in the Aeromonas hydrophila is, earlier the mode of recombinant plasmid with electroporation is transformed among the E.coli S17-1, and with the E.coli S17-1 that contains recombinant plasmid as the donor bacterium, mix cultivation with Aeromonas hydrophila as recipient bacterium.
6, according to the described method of claim 2, it is characterized in that: described Aeromonas hydrophila is Aeromonas hydrophila CGMCC 1816.
7, a kind of with the described reorganization of claim 1 Aeromonas hydrophila produce the regulatable 3-hydroxybutyric acid of component ratio (3HB) and-method of the copolymer p HBHHx of 3-hydroxycaproic acid (3HHx), may further comprise the steps:
1) shake-flask culture reorganization Aeromonas hydrophila produces PHBHHx;
2) add auxiliary carbon source, produce the PHBHHx that contains different ratios 3HHx.
8, method according to claim 7 is characterized in that: described auxiliary carbon source is a Sunmorl N 60S.
9, method according to claim 7 is characterized in that: be used in and add or do not add the ratio that inductor IPTG controls 3HHx among the PHBHHx in the substratum.
10, method according to claim 7 is characterized in that: be used in and add or do not add the ratio that antibiosis is usually controlled 3HHx among the PHBHHx in the substratum; Described microbiotic is kantlex preferably.
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| CN1807649B (en) * | 2006-01-26 | 2012-07-04 | 深圳大学 | Exogenous gene expression system of Chlamydomonasreinhardtii and method for constructing and producing PHB transgenic algae |
| EP2129785B2 (en) * | 2007-03-28 | 2021-11-17 | Genomatica, Inc. | Enhanced production of fatty acid derivatives |
| CN101696431B (en) * | 2009-10-29 | 2011-11-09 | 辽宁大学 | Method for producing copolymer PHBHHx by metabolism control of aeromonas hydrophila |
| CN102120973B (en) * | 2010-12-08 | 2012-10-10 | 清华大学 | Halomonas strain and application thereof |
| CN103232955A (en) * | 2013-03-27 | 2013-08-07 | 南开大学 | Burkholderia sp. and method for fermentation synthesis of PHA by adopting the same |
| CN108660166A (en) * | 2018-05-31 | 2018-10-16 | 保定百恩杰生物科技有限公司 | The culture medium and its fermentation process of primary gram of Hall bacterium fermenting and producing PHA of sensitive plant |
| CN108641996B (en) * | 2018-06-19 | 2020-12-01 | 广东容大生物股份有限公司 | Fermentation medium of bacillus licheniformis and production method thereof |
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