CN105586304B - A kind of recombinant bacterium producing ethyl alcohol acid-based polymer and its application - Google Patents
A kind of recombinant bacterium producing ethyl alcohol acid-based polymer and its application Download PDFInfo
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Abstract
The present invention provides a kind of recombinant bacterium for producing ethyl alcohol acid-based polymer and its applications.The recombinant bacterium is to obtain PHB biosynthesis operon phbCAB, polyhydroxyalkanoate particle binding-protein gene phaP, CoA transferase gene pct, isocitrate lyase gene aceA, isocitric dehydrogenase kinase gene aceK and glyoxylate reductase gene ghrA importing host e. coli；The Escherichia coli are E.coli JM109 or E.coli JM109ldhA.The present invention can synthesize two kinds of ethyl alcohol acid-based polymer --- glycolic, lactic acid and 3-hydroxybutyrate copolyesters, glycolic and 3-hydroxybutyrate copolyesters, and the yield of biomass and polymer of the recombinant bacterium in shaking flask culture can also reach higher level, have preferable industrial applications prospect.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of recombinant bacterium for producing ethyl alcohol acid-based polymer and its answers
With.
Background technique
Glycolic (glycolic acid, hydroxyacetic acid) alias hydroxyacetic acid or Alpha-hydroxy acetic acid, are most
Simple hydroxycarboxylic acid.As important chemical products and organic synthesis intermediate, glycolic is in chemical industry, medicine, weaving, metallurgy
Etc. multiple fields be widely used.For example, the mixed acid of 2% glycolic and 1% formic acid is a kind of cleaning agent of high efficiency, low cost,
It can be used for the cleaning of boiler, pipeline, condenser and heat exchanger etc.；Glyclolic acid molecules are small, can effectively permeate pore, solve skin
The problems such as aging, wrinkle, dark sore, it is commonly used for cosmetic additive agent；Glycolic is commonly used in textile industry as dyeing and finishing wool fiber
And the crosslinking couplant of cellulose fabric；In addition, glycolic also act as fungicide, bonding agent, petroleum demulsifier, welding compound with
And chemical assistant etc..2011, glycolic market was 93,300,000 dollars, it is contemplated that the coming years will be with the speed per year over 10%
Increase, glycolic market can exceed that 200,000,000 dollars within 2018.
Ethyl alcohol acid-based polymer (glycolate based polymers) is a kind of with good biocompatibility and life
The high molecular material of Biodegradable is polymerized by glycolic and other hydroxy carboxylic acid monomers.Ethyl alcohol acid-based polymer can be with
Traditional petroleum resources plastics are substituted as common packaging materials, can be used for the higher field of biomedicine of added value, than
Such as prepare operation suture thread, medicine controlled release carrier, fracture fixation material, tissue engineering bracket and suture supporting material.Cream
The copolyesters (poly lactate-co-glycolate) of acid and glycolic, abbreviation PLGA or PGLA are the more ethyl alcohol of research
Acid-based polymer is usually used in preparing the controlled release preparations such as micro-capsule, and the controlled release as a variety of drugs, albumen and other macromoleculars carries
Body.PLGA is used for medical instruments field by U.S. Food and Drug Administration (FDA) approval, has on multiple products at present
City, such as the first of the World Health Organization (WHO) approval disposably vaccinate microballoon, the first polypeptide control-release microsphere injection in the whole world
Agent etc., Japan, France and China etc. also successively ratify to use PLGA in field of medicaments.
Ethyl alcohol acid-based polymer is industrially mainly prepared by chemically synthesized method.For example, with purer glycolide
For precursor, polyglycolic acid can be obtained by ring-opening polymerisation；Using glycolide and lactide as precursor, can be obtained by ring-opening polymerisation
Obtain glycolic lactic acid copolyesters etc..Heavy metal ion is generally required as catalyst during chemical polymerization, is widely used at present
Tin saline catalyst there is cytotoxicity, the considerations of for human safety, the ethyl alcohol acidic group for limiting chemical method synthesis is poly-
Object is closed in the application in the fields such as medicine and food.2011, Matsumoto etc. was prominent using the reinforcing of escherichia coli fatty acid oxidation
Mutant LS5218 realizes the biosynthesis of the polymer containing glycolic acid monomers for the first time.This is also to utilize microorganism so far
Fermentation obtains the single report of ethyl alcohol acid-based polymer.It is overexpressed the PHA for deriving from pseudomonad Pseudomonas sp.61-3
Polymerase PhaC1STQK and CoA-transferase Pct from Erichsen Megasphaera Megasphaera elsdenii, with ten
Diacid and glycolic are substrate, the ethyl alcohol acid constituents for being 17% containing content of monomer in the polymer of bacterium synthesis, other hydroxyls
Fatty acid mono 3-hydroxybutyrate (3-HB), 3- hydroxycaproic acid (3-HHx), 3- Hydroxyoctanoic acid (3-HO), 3- hydroxydecanoic acid (3-
HD) and the content of 3- hydroxyl dodecanoic acid (3-HDD) is respectively 19%, 22%, 26%, 8% and 8%.Since Escherichia coli are to rouge
Fat acid and glycolic utilizing status are poor, and leading to dry cell weight is only 0.64g/L, and polymer content is also at reduced levels, only
Account for the 4.8% of dry cell weight.Since biomass and polymer content are all lower, cause the extraction of polymer extremely difficult, also without
Method studies its materialogy performance.
Summary of the invention
It is an object of the present invention to provide a kind of recombinant bacteriums.
Recombinant bacterium provided by the present invention is to close polyhydroxyalkanoate particle binding-protein gene phaP, PHB biology
At operon phbCAB, CoA transferase gene pct, isocitrate lyase gene aceA, isocitric dehydrogenase kinases base
Because aceK and glyoxylate reductase gene ghrA imports what host e. coli obtained.
In above-mentioned recombinant bacterium, the Escherichia coli are E.coli JM109 or E.coli JM109ldhA.
In above-mentioned recombinant bacterium, the E.coli JM109ldhA is the bacterium after E.coli JM109 missing ldhA gene.
In above-mentioned recombinant bacterium, the construction method of the E. coli JM109ldhA includes the following steps:
(1) DNA fragmentation as shown in SEQ ID No.3 is prepared；
(2) plasmid pKD46 is converted into E.coli JM109, obtains E.coli JM109 (pKD46)；
(3) the Red recombination system expression in E.coli JM109 (pKD46) bacterial strain is induced, then by the DNA fragmentation
It is transferred in E.coli JM109 (pKD46) bacterial strain, by recombination, obtains ldhA gene by the recombinant bacterium of Kan gene replacement,
It is denoted as E.coli JM109ldhA-K (pKD46)；
(4) the plasmid pKD46 in E.coli JM109ldhA-K (pKD46) bacterial strain is removed, obtained recombinant bacterium note
Make E.coli JM109ldhA-K；
(5) plasmid pCP20 is converted into E.coli JM109ldhA-K bacterial strain, removes Kan gene, obtained recombinant bacterium
It is denoted as E.coli JM109ldhA.
It is described to manipulate polyhydroxyalkanoate particle binding-protein gene phaP, PHB biosynthesis in above-mentioned recombinant bacterium
Sub- phbCAB, CoA transferase gene pct, isocitrate lyase gene aceA, isocitric dehydrogenase kinase gene aceK
It is specifically that expression cassette 1 and expression cassette 2 are imported into host with the glyoxylate reductase gene ghrA method for importing host e. coli
Escherichia coli.
In above-mentioned recombinant bacterium, the expression cassette 1 is promoter sequence, polyhydroxyalkanoate particle binding-protein gene
PhaP and PHB biosynthesis operon phbCAB is sequentially connected；
The expression cassette 2 is promoter sequence, CoA transferase gene pct, glyoxylate reductase gene ghrA, different lemon
What lemon lyase gene aceA and isocitric dehydrogenase kinase gene aceK was sequentially connected.
In above-mentioned recombinant bacterium, the expression cassette is to import host strain by expression vector, and the expression cassette 1 passes through recombination
Expression vector p19-PCAB imports host strain, and the recombinant expression carrier p19-PCAB is that the expression cassette 1 is connected into pMD19-T
The restriction enzyme site of Pst I and EcoR I obtain；The expression cassette 2 imports host by recombinant expression carrier pMCS-pctAAK
Bacterium, the recombinant expression carrier pMCS-pctAAK are EcoR I and the Xho I that the expression cassette 2 is connected into pBBR1MCS-2
Restriction enzyme site obtains.
In above-mentioned recombinant bacterium, the sequence of the expression cassette 1 is as shown in SEQ ID No.1；The sequence of the expression cassette 2 is such as
Shown in SEQ ID No.2；
72-650 in the sequence such as SEQ ID No.1 of the polyhydroxyalkanoate particle binding-protein gene phaP
Shown in nucleic acid molecule；
682-4395 nucleotide point in the sequence such as SEQ ID No.1 of the PHB biosynthesis operon phbCAB
Shown in son；
The sequence of the CoA transferase gene pct is as shown in 72-1625 nucleic acid molecules in SEQ ID No.2；
1655-2593 nucleic acid molecules in the sequence such as SEQ ID No.2 of the glyoxylate reductase gene ghrA
It is shown；
2623-3927 nucleotide point in the sequence such as SEQ ID No.2 of the isocitrate lyase gene aceA
Shown in son；
4110-5826 nucleosides in the sequence such as SEQ ID No.2 of the isocitric dehydrogenase kinase gene aceK
Shown in acid molecule；
The promoter sequence is as shown in 13-55 nucleic acid molecules in SEQ ID No.1 or in SEQ ID No.2
Shown in 13-55 nucleic acid molecules；
The sequence of the ldhA gene is as shown in SEQ ID No.4.
The application of recombinant bacterium described above in production ethyl alcohol acid-based polymer also belongs to protection scope of the present invention.
Another object of the present invention is to provide a kind of method for producing ethyl alcohol acid-based polymer.
The method of production ethyl alcohol acid-based polymer provided by the invention includes the following steps: utilizable with Escherichia coli
Monosaccharide is substrate, and fermented and cultured recombinant bacterium described above obtains ethyl alcohol acid-based polymer.
In the above method, when it is described go out bacterium germination be E.coli JM109 when, the ethyl alcohol acid-based polymer is by ethyl alcohol
The copolymer that acid, lactic acid and 3-hydroxybutyrate polymerize；When it is described go out bacterium germination be E.coli JM109ldhA when, the second
Alcohol acid-based polymer is the copolymer being polymerize by glycolic and 3-hydroxybutyrate.
In the above method, the utilizable monosaccharide of Escherichia coli is specially glucose and/or mannitol.
In the above method, the culture medium that the fermented and cultured uses is MSG-Amp-Kan fluid nutrient medium and/or LBG-
Amp-Kan fluid nutrient medium and/or MSM-Amp-Kan fluid nutrient medium and/or LBM-Amp-Kan fluid nutrient medium.
In the above method, the preparation method of the MSG-Amp-Kan fluid nutrient medium: every liter of culture medium yeast containing 5g is extracted
Object, 20g glucose, 2g (NH4)2SO4、0.4g MgSO4、9.65g Na2HPO4·12H2O、1.5g KH2PO4, the micro member of 10mL
Plain solution I, 1mL trace element solution II, 0.1g ampicillin and 0.05g kanamycins, remaining is water；
The preparation method of the LBG-Amp-Kan fluid nutrient medium: every liter of culture medium contains 5g yeast extract, 10g egg
White peptone, 10g NaCl, 20g glucose, 0.1g ampicillin and 0.05g kanamycins, remaining is water；
The preparation method of the MSM-Amp-Kan fluid nutrient medium: every liter of culture medium yeast extract containing 5g, 20g sweet dew
Alcohol, 2g (NH4)2SO4、0.4g MgSO4、9.65g Na2HPO4·12H2O、1.5g KH2PO4, 10mL trace element solution I, 1mL
Trace element solution II, 0.1g ampicillin and 0.05g kanamycins, remaining is water；
The preparation method of the LBM-Amp-Kan fluid nutrient medium: every liter of culture medium contains 5g yeast extract, 10g egg
White peptone, 10g NaCl, 20g mannitol, 0.1g ampicillin and 0.05g kanamycins, remaining is water；
The preparation method of every liter of trace element solution I: 5g Fe (III)-NH4- Citrate and 2g CaCl2·
2H2O, remaining is 0.5M HCl；
The preparation method of every liter of trace element solution II: 100mg ZnSO4·7H2O、30mg MnCl2·4H2O、
300mg H3BO3、200mg CoCl2·6H2O、10mg CuSO4·5H2O、20mg NiCl2·6H2O、30mgNaMoO4·
2H2O, remaining is 0.5M HCl.
In the above method, the time of the fermented and cultured is 48-96h；Specially 72h.
In the above method, the temperature of the fermented and cultured is 30-40 DEG C；Specially 37 DEG C.
In the above method, the step of the recombinant bacterium described above in fermented and cultured before also need recombinant bacterium described above
The step of being cultivated.
In the above method, by the method that recombinant bacterium described above is cultivated: recombinant bacterium described above is inoculated in culture
It is cultivated in base.
In the above method, the culture medium is LB-Amp-Kan fluid nutrient medium；The incubation time is 16h；The training
Supporting temperature is 37 DEG C.
It is a still further object of the present invention to provide a kind of glycolic, lactic acid and 3-hydroxybutyrate copolyesters.
Glycolic, lactic acid and 3-hydroxybutyrate copolyesters provided by the invention are prepared according to method described above
's.
The last one of the invention purpose is to provide a kind of glycolic and 3-hydroxybutyrate copolyesters.
Glycolic and 3-hydroxybutyrate copolyesters provided by the invention is prepared according to method described above.
The present invention provides a kind of recombinant bacterium for producing ethyl alcohol acid-based polymer and its applications.Pass through the table in Escherichia coli
Up to the relevant gene of eight metabolic pathways, and combine the knockout to Escherichia coli endogenous gene, obtain can with glucose or
The carbohydrate of the cheap and simples such as mannitol is single carbon source, and fermentation obtains the engineered strain of ethyl alcohol acid-based polymer.This hair
It is bright to synthesize two kinds of ethyl alcohol acid-based polymer --- glycolic, lactic acid and 3-hydroxybutyrate copolyesters, glycolic and
3-hydroxybutyrate copolyesters, and the yield of biomass and polymer of the recombinant bacterium in shaking flask culture can also reach higher water
It is flat, there is preferable industrial applications prospect.
Detailed description of the invention
Fig. 1 is p19-PCAB Vector map.
Fig. 2 is pMCS-pctAAK Vector map.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Enzyme used in molecular biology manipulations involved in following embodiments is purchased from NEB (New England Biolabs)
Company；The kit that plasmid extracts, DNA fragmentation recycling is used is purchased from Beijing Bo Maide biotech company；Involved in embodiment
DNA synthesis and examining order by Beijing Bo Maide biotech company complete.
Plasmid pKD13: the public can be from E.coli Genetic Resources at Yale CGSC, The Coli
Genetic Stock Center is obtained, number CGSC#7633.
Plasmid pKD46: the public can be from E.coli Genetic Resources at Yale CGSC, The Coli
Genetic Stock Center is obtained, number CGSC#7739.
Plasmid pCP20: the public can be from E.coli Genetic Resources at Yale CGSC, The Coli
Genetic Stock Center is obtained, number CGSC#7637.
Bacteria culture media involved in following embodiments (is such as indicated without special, the sterilising conditions of culture medium are 121 as follows
DEG C, 20 minutes):
LB-Amp fluid nutrient medium: every liter of culture medium contains 5g yeast extract, 10g peptone, 10g NaCl and 0.1g
Ampicillin, remaining is water；
LB-Amp solid medium: every liter of culture medium contains 15g agar, 5g yeast extract, 10g peptone, 10g
NaCl and 0.1g ampicillin, remaining is water；
LB-Kan fluid nutrient medium: every liter of culture medium contains 5g yeast extract, 10g peptone, 10gNaCl and 0.05g
Kanamycins, remaining is water；
LB-Kan solid medium: every liter of culture medium contains 15g agar, 5g yeast extract, 10g peptone, 10gNaCl
With 0.05g kanamycins, remaining is water；
LB-Amp-Kan fluid nutrient medium: every liter of culture medium contain 5g yeast extract, 10g peptone, 10g NaCl,
0.1g ampicillin and 0.05g kanamycins, remaining is water；
LB-Amp-Kan solid medium: every liter of culture medium contain 15g agar, 5g yeast extract, 10g peptone,
10gNaCl, 0.1g ampicillin and 0.05g kanamycins, remaining is water；
LBG-Amp-Kan solid medium: every liter of culture medium contain 15g agar, 5g yeast extract, 10g peptone,
10g NaCl, 20g glucose, 0.1g ampicillin and 0.05g kanamycins, remaining is water；
LBM-Amp-Kan solid medium: every liter of culture medium contain 15g agar, 5g yeast extract, 10g peptone,
10g NaCl, 20g mannitol, 0.1g ampicillin and 0.05g kanamycins, remaining is water；
MSG-Amp-Kan culture medium: every liter of culture medium yeast extract containing 5g, 20g glucose, 2g (NH4)2SO4、0.4g
MgSO4、9.65g Na2HPO4·12H2O、1.5g KH2PO4, 10mL trace element solution I, 1mL trace element solution II, 0.1g
Ampicillin and 0.05g kanamycins, remaining is water；
MSM-Amp-Kan culture medium: every liter of culture medium yeast extract containing 5g, 20g mannitol, 2g (NH4)2SO4、0.4g
MgSO4、9.65g Na2HPO4·12H2O、1.5g KH2PO4, 10mL trace element solution I, 1mL trace element solution II, 0.1g
Ampicillin and 0.05g kanamycins, remaining is water；
Every liter of trace element solution I:5g Fe (III)-NH4- Citrate and 2g CaCl2·2H2O, remaining is 0.5M
HCl；
Every liter of trace element solution II:100mg ZnSO4·7H2O、30mg MnCl2·4H2O、300mg H3BO3、200mg
CoCl2·6H2O、10mg CuSO4·5H2O、20mg NiCl2·6H2O、30mg NaMoO4·2H2O, remaining is 0.5M HCl.
The building of embodiment 1, recombinant expression carrier p19-PCAB
1, DNA shown in sequence 1 in artificial synthesized sequence table, wherein 13-55 nucleotide are promoter sequence, the
72-650 nucleotide are phaP gene order, and 682-4395 nucleotide are phbCAB operon sequence.
2, with the DNA sequence dna synthesized in Pst I and EcoR I double digestion step 1, the gene piece that size is about 4.4kb is recycled
Section.
3, with Pst I and EcoR I double digestion carrier pMD19-T (being purchased from Takara company, Japan), recycling size is about
The carrier segments of 2.6kb.
4, the genetic fragment that step 2 obtains is connect (the solution I company of Takara with the carrier segments that step 3 obtains
Connect kit), it obtains connection product and E.coli JM109 is imported by the method for chemical conversion (Pu Luomaige biotechnology has
Limit company, catalog number (Cat.No.) P9751) in, and it is coated on LB-Amp solid medium, 37 DEG C of culture 16h obtain transformant.
5, the plasmid of transformant is extracted, with Pst I and EcoR I double digestion, digestion products size is about 4.4kb and 2.6kb
Plasmid be positive plasmid, this recombinant expression carrier is named as p19-PCAB, as shown in Figure 1.
Obtained recombinant expression carrier p19-PCAB is subjected to sequence verification, the results showed that in the Pst of pMD19-T carrier
The nucleic acid molecule as shown in sequence 1 in sequence table is inserted between I and EcoR I restriction enzyme site, shows that carrier is correct.
The building of embodiment 2, recombinant expression carrier pMCS-pctAAK
1, DNA sequence dna shown in sequence 2 in artificial synthesized sequence table, wherein 13-55 nucleotide are promoter sequence；
72-1625 nucleotide are pct gene order；1655-2593 nucleotide are ghrA gene order；2623-3927
Position nucleotide is aceA gene order；4110-5826 nucleotide are aceK gene order；
2, with the DNA sequence dna synthesized in EcoR I and Xho I double digestion step 1, the gene piece that size is about 5.8kb is recycled
Section；
3, with EcoR I and Xho I double digestion carrier pBBR1MCS-2, (public can be according to NCBI GenBank
The sequence of U23751 oneself synthesis), the carrier segments that recycling size is about 5.1kb；
4, the genetic fragment that step 2 obtains is connect (the solution I company of Takara with the carrier segments that step 3 obtains
Connect kit), it obtains connection product and is imported into E.coli JM109 by the method for chemical conversion, and it is solid to be coated on LB-Kan
Body culture medium, 37 DEG C of culture 16h, obtains transformant.
5, the plasmid of transformant is extracted, with Eco I and Xho I double digestion, digestion products size is about 5.8kb and 5.1kb
Plasmid be positive plasmid, this recombinant expression carrier is named as pMCS-pctAAK, as shown in Figure 2.
Obtained recombinant expression carrier pMCS-pctAAK is subjected to sequence verification, the results showed that in pBBR1MCS-2 carrier
EcoR I and Xho I restriction enzyme site between insert the nucleic acid molecule as shown in sequence 2 in sequence table, show that carrier is correct.
The building of embodiment 3, e. coli jm109 ldhA
1, primer ldhAF:5 '-CTCCCCTGGAATGCAGGGGAGCGGCAAGATT used in ldhA gene knockout is synthesized
AAACCAGTTCGTTCGGGCAATTCCGGGGATCCGTCGACC-3 ' and ldhAR:5 '-TATTTTTAGTAGCTTAAATGTGA
TTCAACATCACTGGAGAAAGTCTTATGTGTAGGCTGGAGCTGCTTCG-3 ' is used using plasmid pKD13 as template
LdhAF and ldhAR primer PCR expands to obtain the DNA fragmentation of 1.5kb or so, the sequence of the segment as shown in SEQ ID No.3,
Agarose gel electrophoresis purifies obtained DNA fragmentation；
2, with the Escherichia coli containing pKD46 are cultivated in LB-Amp fluid nutrient medium under conditions of 30 DEG C, plasmid is extracted
PKD46 is converted into F-strain E.coli JM109 using the method for electrotransformation, and is coated on LB-Amp solid medium,
30 DEG C of cultures for 24 hours, obtain transformant, obtain E.coli JM109 (pKD46)；
3, E.coli JM109 (pKD46) is inoculated into LB-Amp fluid nutrient medium, 30 DEG C of culture 4h, is added Arabic
Sugar continues to cultivate 1.5h, the competent cell of E.coli JM109 (pKD46) is next prepared, by step to final concentration 20g/L
The DNA fragmentation obtained in 1 is transferred in the competent cell of E.coli JM109 (pKD46), and is coated on LB-Kan solid culture
Base, 37 DEG C of cultures for 24 hours, obtain transformant；
4, it using the method for bacterium colony PCR, using ldhAF and ldhAR as primer, will be sequenced after PCR product purifying, screening is just
True ldhA gene has been replaced by the clone of Kan resistant gene, obtains E.coli JM109ldhA-K (pKD46)；
5, E.coli JM109ldhA-K (pKD46) is inoculated in LB liquid medium, 42 DEG C of subcultures three times, remove
PKD46 plasmid is removed, E.coli JM109ldhA-K is obtained；
The E.coli JM109ldhA-K is ldhA gene by the E.coli JM109 of Kan gene replacement；
6, E.coli JM109ldhA-K is inoculated into LB-Kan fluid nutrient medium, 37 DEG C of cultures for 24 hours, are transferred to LB-
In Kan fluid nutrient medium, 37 DEG C of culture 2h prepare E.coli JM109ldhA-K competent cell；
7, the 30 DEG C of Escherichia coli of culture containing pCP20 in LB-Amp fluid nutrient medium extract plasmid pCP20, utilize electricity
PCP20 is transformed into E.coli JM109ldhA-K competent cell by the method for conversion, and is coated on LB-Amp solid culture
Base, 37 DEG C of cultures for 24 hours, obtain transformant；
8, it using the method for bacterium colony PCR, using ldhAF and ldhAR as primer, will be sequenced after PCR product purifying, screening
The clone of Kan gene delection, the final mutant E.coli JM109ldhA for obtaining ldhA gene delection.
Embodiment 4, recombinant bacterium E.coli JM109 (p19-PCAB+pMCS-pctAAK) and recombinant bacterium E.coli
The building of JM109ldhA (p19-PCAB+pMCS-pctAAK)
1, the recombinant plasmid p19-PCAB that embodiment 1 obtains and the recombinant plasmid pMCS-pctAAK that embodiment 2 obtains are led to
The method for crossing electrotransformation is transformed into E.coli JM109, and is coated on LB-Amp-Kan solid medium, and 37 DEG C are cultivated for 24 hours,
It obtains as recombinant bacterium E.coli JM109 (p19-PCAB+pMCS-pctAAK).
2, the recombinant plasmid p19-PCAB that embodiment 1 obtains and the recombinant plasmid pMCS-pctAAK that embodiment 2 obtains are led to
The method for crossing electrotransformation is transformed into E.coli JM109ldhA, and is coated on LB-Amp-Kan solid medium, 37 DEG C of cultures
For 24 hours, recombinant bacterium E.coli JM109ldhA (p19-PCAB+pMCS-pctAAK) is obtained.
Embodiment 5, recombinant bacterium E.coli JM109 (p19-PCAB+pMCS-pctAAK) are in production ethyl alcohol acid-based polymer
In application
1, fermented and cultured
(1) acquisition of seed liquor: by recombinant bacterium E.coli JM109 (p19-PCAB+pMCS-pctAAK) in LB-Amp-
16h (37 DEG C of shaking tables, 200rpm) is cultivated in Kan fluid nutrient medium is used as seed liquor；
(2) ferment: seed liquor is inoculated into MSG-Amp-Kan fluid nutrient medium, LBG- by 4% inoculum concentration by volume
Amp-Kan fluid nutrient medium, MSM-Amp-Kan fluid nutrient medium, in LBM-Amp-Kan fluid nutrient medium, 37 DEG C of shaking tables,
200rpm, fermented and cultured 72h collect tunning.
2, the polymer content of detection bacterium intracellular accumulation
Quantitative detection is carried out by gas chromatography (Gas chromatography, the GC) polymer intracellular to bacterium,
Measure the ratio of each monomer in biomass, intracellular polymer content and the polymer of cell.
The specific detection method is as follows: gas chromatographic analysis uses 6890 type gas chromatograph of HP, and chromatographic column is HP-5 capillary
Tubing string, column length 30m, 320 μm of internal diameter, stationary phase is the methyl-polysiloxane of 25nm thickness；Detector is flame ion inspection
It surveys device (Flame ionization detector, FID)；Use high pure nitrogen as carrier gas, for hydrogen as combustion gas, air is to help
Combustion gas.
The condition of gas chromatographic analysis is as follows:
(1) column temperature: 80 DEG C of beginnings stop 1.5min；The rate of 30 DEG C/min is warming up to 140 DEG C, stops 0min；40℃/
The rate of min is warming up to 220 DEG C, stops 1min.Total time is 6.5min.
(2) column pressure: 10psi starts, and stops 1.5min；The rate of 2.5psi/min boosts to 20psi, stops 0.5min.
(psi is pressure unit, i.e., pound/square inch, 1psi=6.89476kPa)
(3) injection port: temperature is 200 DEG C, uses shunt mode, split ratio 30.
(4) detector: temperature is 220 DEG C, hydrogen flowing quantity 30mL/min, air mass flow 400mL/min.
(5) detecting step is as follows:
1) it by tunning obtained in above-mentioned steps 1, is centrifuged (10000g, 10min) and collects thallus, then use distilled water
It is centrifuged again after washing twice；Cell is freezed dried, is measured dry cell weight (Cell dry weight, CDW)；
2) take 50mg stem cell in esterification pipe, it (is 3% the concentrated sulfuric acid is molten by volumn concentration that 2mL esterifying liquid, which is added,
In methanol, sulfuric acid-methanol solution is obtained, it is according still further to final concentration of 1g/L that benzoic acid is molten as internal standard addition sulfuric acid-methanol
In liquid, obtain esterifying liquid), 2mL chloroform, covered and enclosed reacts 4h in 100 DEG C of baking ovens；After being cooled to room temperature, be added 1mL go from
Sub- water sufficiently vibrates, stratification；After chloroform phase and water phase are kept completely separate, chloroform phase is taken, carries out gas chromatographic analysis；
3) microsyringe of agilent company is used, sample volume is 1 μ L, is quantitatively divided using internal standard method polymer
Analysis, according to peak area quantification.
Polymer content is defined as polymer to the ratio of dry cell weight, and polymer output=polymer content × cell is dry
Weight, each content of monomer are defined as the molar content of each monomer in polymer.
After testing, the result of the dry cell weight, polymer output and the polymer monomer content that obtain in each culture medium is such as
Shown in table 1:
The synthesis situation of ethyl alcohol acid-based polymer in table 1, E.coli JM109 (p19-PCAB+pMCS-pctAAK)
Note: GA monomer is glycolic acid monomers；LA monomer is lactic acid monomer；3HB monomer is 3-hydroxybutyrate monomer
As seen from Table 1, the recombinant bacterial strain that the present invention constructs can be in addition glucose or mannitol
For in the simple mineral salts medium of carbon source or complicated culture medium, fermentation, which obtains, contains glycolic, lactic acid and 3-hydroxybutyrate
The polymer (glycolic, lactic acid and 3-hydroxybutyrate copolyesters) of three kinds of monomers, wherein complicated culture medium is more advantageous to polymer
Synthesis, polymer can account for the 56.2%-60.8% of dry cell weight, and glycolic acid monomers content is 16%-17%, lactic acid monomer
Content is 7%-8%, and 3-hydroxybutyrate content of monomer is 75%-77%.
Embodiment 6, recombinant bacterium E.coli JM109ldhA (p19-PCAB+pMCS-pctAAK) are poly- in production ethyl alcohol acidic group
Close the application in object
1, fermented and cultured
(1) acquisition of seed liquor: by recombinant bacterium E.coli JM109ldhA (p19-PCAB+pMCS-pctAAK) in LB-
16h (37 DEG C of shaking tables, 200rpm) is cultivated in Amp-Kan fluid nutrient medium is used as seed liquor；
(2) ferment: seed liquor is inoculated into MSG-Amp-Kan fluid nutrient medium, LBG- by 4% inoculum concentration by volume
Amp-Kan fluid nutrient medium, MSM-Amp-Kan fluid nutrient medium, in LBM-Amp-Kan fluid nutrient medium, 37 DEG C of shaking tables,
200rpm, fermented and cultured 72h collect tunning.
2, the polymer content of detection bacterium intracellular accumulation
It is carried out according to the method for step 2 in embodiment 5.After testing, dry cell weight, the polymer obtained in each culture medium
The results are shown in Table 2 for yield and polymer monomer content:
The synthesis situation of ethyl alcohol acid-based polymer in table 2, E.coli JM109ldhA (p19-PCAB+pMCS-pctAAK)
Note: GA monomer is glycolic acid monomers；3HB monomer is 3-hydroxybutyrate monomer
As seen from Table 2, the recombinant bacterial strain that the present invention constructs can be in addition glucose or mannitol
For in the simple mineral salts medium of carbon source or complicated culture medium, fermentation, which obtains, contains two kinds of lists of glycolic and 3-hydroxybutyrate
The polymer (glycolic and 3-hydroxybutyrate copolyesters) of body, wherein complicated culture medium is more advantageous to the synthesis of polymer, polymerization
Object can account for the 30.6%-32.9% of dry cell weight, and glycolic acid monomers content is 11%-13%, 3-hydroxybutyrate content of monomer
For 87%-89%.
Claims (9)
1. a kind of recombinant bacterium is by polyhydroxyalkanoate particle binding-protein gene phaP, PHB biosynthesis operon
PhbCAB, CoA transferase gene pct, isocitrate lyase gene aceA, isocitric dehydrogenase kinase gene aceK and
Glyoxylate reductase gene ghrA imports what host e. coli obtained.
2. recombinant bacterium according to claim 1, which is characterized in that the Escherichia coli are E.coli JM109 or E.coli
JM109 ldhA；
The E.coli JM109 ldhA is the bacterium after E.coli JM109 missing ldhA gene.
3. recombinant bacterium according to claim 2, which is characterized in that the structure of the E. coli JM109 ldhA
Construction method includes the following steps:
(1) DNA fragmentation as shown in SEQ ID No.3 is prepared；
(2) plasmid pKD46 is converted into E.coli JM109, obtains E.coli JM109 (pKD46)；
(3) the Red recombination system expression in E.coli JM109 (pKD46) bacterial strain is induced, then the DNA fragmentation is transferred to
In E.coli JM109 (pKD46) bacterial strain, by recombination, ldhA gene is obtained by the recombinant bacterium of Kan gene replacement, is denoted as
E.coli JM109 ldhA-K(pKD46)；
(4) the plasmid pKD46 in E.coli JM109 ldhA-K (pKD46) bacterial strain is removed, obtained recombinant bacterium is denoted as
E.coli JM109 ldhA-K；
(5) plasmid pCP20 is converted into E.coli JM109 ldhA-K bacterial strain, removes Kan gene, obtained recombinant bacterium note
Make E.coli JM109 ldhA.
4. recombinant bacterium according to claim 1 to 3, which is characterized in that described to combine polyhydroxyalkanoate particle
Protein gene phaP, PHB biosynthesis operon phbCAB, CoA transferase gene pct, isocitrate lyase gene
AceA, isocitric dehydrogenase kinase gene aceK and glyoxylate reductase gene ghrA import the method tool of host e. coli
Body is to import host e. coli by expression cassette 1 and expression cassette 2；
The expression cassette 1 is promoter sequence, polyhydroxyalkanoate particle binding-protein gene phaP and PHB biosynthesis behaviour
Indulge what sub- phbCAB was sequentially connected；
The expression cassette 2 is promoter sequence, CoA transferase gene pct, glyoxylate reductase gene ghrA, isocitric acid
What lyase gene aceA and isocitric dehydrogenase kinase gene aceK was sequentially connected.
5. recombinant bacterium according to claim 4, which is characterized in that the sequence of the expression cassette 1 such as SEQ ID No.1 institute
Show；The sequence of the expression cassette 2 is as shown in SEQ ID No.2；
72-650 nucleosides in the sequence such as SEQ ID No.1 of the polyhydroxyalkanoate particle binding-protein gene phaP
Shown in acid molecule；
The sequence of the PHB biosynthesis operon phbCAB such as 682-4395 nucleic acid molecule institutes in SEQ ID No.1
Show；
The sequence of the CoA transferase gene pct is as shown in 72-1625 nucleic acid molecules in SEQ ID No.2；
The sequence of the glyoxylate reductase gene ghrA is as shown in 1655-2593 nucleic acid molecules in SEQ ID No.2；
The sequence of the isocitrate lyase gene aceA such as 2623-3927 nucleic acid molecule institutes in SEQ ID No.2
Show；
4110-5826 nucleotide point in the sequence such as SEQ ID No.2 of the isocitric dehydrogenase kinase gene aceK
Shown in son；
Promoter sequence 13- as shown in 13-55 nucleic acid molecules in SEQ ID No.1 or in SEQ ID No.2
Shown in 55 nucleic acid molecules；
The sequence of the ldhA gene is as shown in SEQ ID No.4.
6. application of any recombinant bacterium of claim 1-5 in production ethyl alcohol acid-based polymer.
7. a kind of method for producing ethyl alcohol acid-based polymer includes the following steps: using Escherichia coli utilizable monosaccharide the bottom of as
Object, any recombinant bacterium of fermented and cultured claim 1-5, obtains ethyl alcohol acid-based polymer.
8. according to the method described in claim 7, it is characterized by: when it is described go out bacterium germination be E.coli JM109 when, it is described
Ethyl alcohol acid-based polymer is the copolymer being polymerize by glycolic, lactic acid and 3-hydroxybutyrate；When the bacterium germination out is
When E.coli JM109 ldhA, the ethyl alcohol acid-based polymer is the copolymerization being polymerize by glycolic and 3-hydroxybutyrate
Object；
The utilizable monosaccharide of Escherichia coli is specially glucose and/or mannitol；
The culture medium that the fermented and cultured uses is MSG-Amp-Kan fluid nutrient medium and/or LBG-Amp-Kan fluid nutrient medium
And/or MSM-Amp-Kan fluid nutrient medium and/or LBM-Amp-Kan fluid nutrient medium；
The time of the fermented and cultured is 48-96h；The temperature of the fermented and cultured is 30-40 DEG C.
9. according to the method described in claim 8, it is characterized by: the time of the fermented and cultured is 72h；
The temperature of the fermented and cultured is 37 DEG C.
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