CN101906394A - Engineering bacterium containing 2-ketoglutarate decarboxylase gene kgd and application thereof - Google Patents
Engineering bacterium containing 2-ketoglutarate decarboxylase gene kgd and application thereof Download PDFInfo
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Images
Abstract
The invention belongs to the fields of a gene engineering technology and a fermentation technology, in particular to an engineering bacterium which contains 2-ketoglutarate decarboxylase gene kgd and is used for producing 3-hydroxybutyrate and 4-hydroxy acid copolyester by utilizing a carbohydrate carbon source. Single carbohydrate carbon source with relative low price can be used to produce P3HB4HB by utilizing the engineering bacterium to effectively decrease the production cost thereof and propel the mass industrialized production and the commercial application development thereof.
Description
Technical field
The invention belongs to genetic engineering technique and fermentation technical field, relate more specifically to a kind of engineering bacteria that contains the 2-oxoglutaric acid decarboxylase gene kgd that utilizes carbohydrate carbon source production 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters and uses thereof.
Background technology
Polyhydroxyalkanoate (polyhydroxyalkanoates, be called for short PHA) be that a class extensively is present in the intravital high-molecular biologic polyester of microorganism, in cell mainly as reserve substance (the Anderson AJ of carbon source and energy, Dawes EA.Occurrence, metabolism, metabolic role, and industrial use of bacterial polyhydroxyalkanoates.Microbiol Rev, 1990,54:450-472).3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters [poly (3-hydroxybutyrate-co-4-hydroxybutyrate), be called for short P3HB4HB] be a member (molecular formula I) in the PHA family, it equals discovery (Doi Y in the very foster bacillus Ralstonia eutropha of Luo Shi in 1988 by Doi Y at first, Kunioka M, Nakamura Y, et al.Nuclear magnetic-resonance studies on unusual bacterial copolyesters of3-hydroxybutyrate and 4-hydroxybutyrate.Macromolecules, 1988,21:2722-2727).When adding 4 hydroxybutyric acid or 4-chloro-butyric acid in substratum, R.eutropha can synthesize P3HB4HB, and the monomeric content of 4HB can be regulated between 0 to 49mol%.
I wherein x and y represents the polymerization degree
According to compositing monomer 3-hydroxybutyric acid (3-hydroxybutyrate in the P3HB4HB polymkeric substance, be called for short 3HB) and 4 hydroxybutyric acid (4-hydroxybutyrate, abbreviation 4HB) difference of ratio, P3HB4HB has from adamantine highly crystalline body to a series of different materialogy character such as softish elastomericss.(64～100mol%), the tensile strength of P3HB4HB can be brought up to 104Mpa by 17Mpa, shows the character of thermoplastic elastomer when 4HB content was higher.When the 4HB ratio is increased to 82mol% by 0mol%, the elongation at break of P3HB4HB can be increased to 1320% (Saito Y by 5%, Nakamura S, Hiramitsu M, et al.Microbial synthesis and properties of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) .Polym Int, 1996,39:169-174).The melt temperature of P3HB4HB is along with the increase of 4HB monomer ratio reduces earlier, raise gradually then, second-order transition temperature is then along with the increase of 4HB monomer ratio is linear reduction trend (Ishida K, Wang Y, Inoue Y.Comonomer unit composition and thermal properties of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) s biosynthesized by Ralstonia eutropha.Biomacromolecules, 2001,2:1285-1293).
The 4HB monomer does not contain side chain after being aggregated to copolyesters, makes P3HB4HB both can be degraded by the PHA degrading enzyme, again can be by lipase hydrolysis.The monomeric speed of having quickened enzymolysis and hydrolysis of mixing of 4HB, hydrolysis rate depends on the monomer whose composition, when 4HB content is high more, P3HB4HB is by fast more (the Saito Y of the speed of lipase hydrolysis, Nakamura S, Hiramitsu M, et al.Microbial synthesis and properties of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) .Polym Int, 1996,39:169-174; Doi Y, Kanesawa Y, Kunioka M, et al.Biodegradation of microbial copolyesters:poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and poly (3-hydroxybutyrate-co-4-hydroxybutyrate) .Macromolecules, 1990,23:26-31), therefore, P3HB4HB has the degradation property more more excellent than other PHA material, is considered to one of PHA material that has most application prospect.
Up to now, the wild-type bacterium that can synthesize P3HB4HB that has been found that comprises that Luo Shi really supports bacillus Ralstonia eutropha, extensive Alcaligenes Alcaligenes latus, Comamonas acidovorans Comamonas acidovorans, Comamonas testosteroni Comamonas testosteroni and class yellow are bitten hydrogen bacterium Hydrogenophaga pseudoflava.In general, 3HB in the P3HB4HB copolyesters and 4HB monomer are synthesized by dissimilar carbon sources respectively, be used for producing the monomeric carbon source of 3HB and comprise conventional sucrose, glucose, fructose and butyric acid etc., and the monomeric carbon source that need in substratum, add with the 4HB similar of mixing of 4HB, for example 4 hydroxybutyric acid, gamma-butyrolactone and 1,4-butyleneglycol etc.Selecting for use of carbon source is relevant with the kind of producing bacterium, and different microorganism hobbies are utilized different carbon sources.The price of 4HB similar carbon source is very expensive, and pair cell toxicity is also bigger, usually need add during the fermentation in the mode of batch feeding, and also very complicated comparatively of the fermentation strategy of mixed carbon source.For example, 4 hydroxybutyric acid is considered to mix most effectively the monomeric carbon source of 4HB in copolyesters, but it is a kind of controlled drug in China, is difficult to buy on market with the form of chemical.The interpolation of 4HB similar carbon source has become the biggest problem of restriction P3HB4HB large-scale industrial production and application.If can utilize the relatively cheap single carbohydrate carbon source of price to produce P3HB4HB, will reduce its production cost effectively, promote the exploitation of its large-scale industrial production and commercial applications.
Summary of the invention
The purpose of this invention is to provide a kind of engineering bacteria that contains the 2-oxoglutaric acid decarboxylase gene kgd that utilizes the carbohydrate carbon source to produce 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters, wherein the carbohydrate carbon source can be glucose, Sunmorl N 60S, fructose, the monose that N.F,USP MANNITOL etc. can be utilized by engineering bacteria is perhaps through containing the polysaccharide of above-mentioned product after the hydrolysis.
Above-mentioned engineering bacteria provided by the invention be R.eutropha H16 (pMCS4HBRE) or E.coli JM109SG (pBHR68orfZ+pMCS4HB) it-; Wherein the physical map of the contained expression vector of R.eutropha H16 (pMCS4HBRE) as shown in Figure 1, the physical map of the expression vector that E.coli JM109SG (pBHR68orfZ+pMCS4HB) is contained is as shown in Figure 2;
The present invention also provides and has obtained above-mentioned engineering bacteria, obtains the bacterium of recombinating after soon the recombinant expression vector of 3-hydroxybutyric acid and the required gene of 4 hydroxybutyric acid copolyesters biosynthesizing is transferred to the host bacterium;
Described 3-hydroxybutyrate and the required gene of 4 hydroxybutyric acid copolyesters biosynthesis comprise that deriving from Luo Shi really supports gathering-3-hydroxybutyrate ester synthetic gene phaCAB (being 5 of AM260479 ' end 1557353-1561203 position nucleotides from GENBANK number) of bacillus Ralstonia eutropha; Derive from the 4-maloyl group coacetylase of Ke Shi clostridium Clostridium kluyveri: CoA-transferase gene orfZ (being 5 of CP000673 ' end 3066385-3067674 position nucleotides from GENBANK number); Derive from the 4 hydroxybutyric acid dehydrogenase gene 4hbD (being 5 of CP000673 ' end 3061070-3062185 position nucleotides from GENBANK number) of Ke Shi clostridium Clostridium kluyveri and derive from the 2-oxoglutaric acid decarboxylase gene kgd (being 5 of CP000611 ' end 1390667-1394311 position nucleotides from GENBANK number) of Much's bacillus Mycobacterium tuberculosis.
The biosynthetic pathway of P3HB4HB as shown in Figure 3.
The wild-type that described host bacterium can be intestinal bacteria or Luo Shi really supports bacillus also can be the succsinic acid hemiacetal dehydrogenase gene mutant strain that intestinal bacteria or Luo Shi really support bacillus, preferred succsinic acid hemiacetal dehydrogenase gene mutant strain.
The carrier that sets out of described carrier recombinant expressed in intestinal bacteria is pBluescript II SK (-) (NCBI GenBank searching number is X52330) and pBBR1MCS-2 (NCBI GenBank number be U23751).The described carrier that sets out of really supporting carrier recombinant expressed in the bacillus at Luo Shi is pBBR1MCS-2 (NCBIGenBank number be U23751).
The method of production 3-hydroxybutyric acid provided by the present invention and 4 hydroxybutyric acid copolyesters is with above-mentioned engineering bacteria, under the situation of adding the carbohydrate carbon source, cultivates by fermentation and obtains 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters.
The leavening temperature of described colibacillus engineering is 28-38 ℃, is preferably 37 ℃.
The leavening temperature that described Luo Shi really supports the bacillus engineering bacteria is 30-33 ℃.
Described fermention medium is following three kinds any one, and following substratum is all prepared with deionized water:
1) every liter of substratum contains: yeast extract 4-6g/L, and peptone 8-12g/L, NaCl 8-12g/L, carbohydrate carbon source 18-22g/L, all the other are water.
2) every liter of substratum contains: yeast extract 4-6g/L, peptone 8-12g/L, carbohydrate carbon source 20-60g/L, (NH
4)
2SO
41.8-2.2g/L; MgSO
40.38-0.45g/L; Na
2HPO
412H
2O 9.6-9.7g/L; KH
2PO
41.2-1.8g/L, trace element solution I 8-12mL/L, trace element solution II 0.8-1.2mL/L, all the other are water; Every liter of trace element solution I contains: Fe (III)-NH
4-Citrate 4-6g/L, CaCl
22H
2O1.8-2.2g/L, all the other are 0.4-0.6M HCl; Every liter of trace element solution II contains: ZnSO
47H
2O 80-120mg/L, MnCl
24H
2O 25-35mg/L, H
3BO
3280-320mg/L, CoCl
26H
2O 180-220mg/L, CuSO
45H
2O 8-12mg/L, NiCl
26H
2O 18-22mg/L, NaMoO
42H
2O 28-32mg/L, all the other are 0.4-0.6M HCl.
3) every liter of substratum contains: carbohydrate carbon source 20-60g/L, (NH
4)
2SO
41.8-2.2g/L; MgSO
40.38-0.45g/L; Na
2HPO
412H
2O 9.6-9.7g/L; KH2PO4 1.2-1.8g/L, trace element solution I 8-12mL/L, trace element solution II 0.8-1.2mL/L, all the other are water; Every liter of trace element solution I contains: Fe (III)-NH4-Citrate 4-6g/L, CaCl
22H
2O 1.8-2.2g/L, all the other are 0.4-0.6M HCl; Every liter of trace element solution II contains: ZnSO
47H
2O 80-120mg/L, MnCl
24H
2O25-35mg/L, H
3BO
3280-320mg/L, CoCl
26H
2O 180-220mg/L, CuSO
45H
2O 8-12mg/L, NiCl
26H
2O 18-22mg/L, NaMoO
42H
2O 28-32mg/L, all the other are 0.4-0.6M HCl.
In actual culturing process, can in above-mentioned substratum, add certain density microbiotic again to keep the stability of plasmid, as 30-50 μ g/mL sulphuric acid kanamycin and/or 60-100 μ g/mL penbritin.
The present invention is by the means of molecular biology and metabolic engineering, structure can utilize the carbohydrate carbon source through fermentation to produce the engineering bacteria of 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters, need not carbon source (for example 4 hydroxybutyric acid, the gamma-butyrolactone and 1 of 4HB similar, 4-butyleneglycol etc.) interpolation, promptly utilize relatively inexpensive carbon source through fermentation, reduced the production cost of 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters.
Description of drawings
The physical map of Fig. 1: pMCS4HBRE (Kan-R, kalamycin resistance gene; OrfZ, 4-maloyl group coenzyme A: thiophorase gene; 4hbD, the 4 hydroxybutyric acid dehydrogenase gene; Kgd, the 2-oxoglutaric acid decarboxylase gene);
The physical map of Fig. 2: pBHR68orfZ and pMCS4HB, wherein, left side figure is the physical map of pBHR68orfZ, right figure is physical map (Amp-R, the ampicillin resistance gene of pMCS4HB; Kan-R, kalamycin resistance gene; OrfZ, 4-maloyl group coenzyme A: thiophorase gene; PhaC, the PHA pol gene; PhaA, the 3-ketothiolase gene; PhaB, the Acetoacetyl-CoA reductase gene that NADPH-relies on; 4hbD, the 4 hydroxybutyric acid dehydrogenase gene; Kgd, the 2-oxoglutaric acid decarboxylase gene);
The biosynthetic pathway of Fig. 3: P3HB4HB.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Relate to the used enzyme of molecular biology operation among the following embodiment, all available from MBI Fermentas company, respective phases of operation is carried out according to relevant product description fully; Plasmid extracts, dna fragmentation reclaims used test kit available from U.S. OMEGA Bio-Tek company, and corresponding operation is carried out according to its product description fully; The primer that relates in the experiment is synthetic to be finished by Beijing AudioCodes biotech company with dna sequencing work.
The bacteria culture medium that relates among the following embodiment is as follows, as does not have special indicating, and substratum is all prepared with deionized water; As do not have special indicating, the sterilising conditions of substratum is 115 ℃, 20 minutes:
The LB liquid nutrient medium: every liter of substratum contains 5g/L yeast extract, 10g/L peptone, 10g/LNaCl, and pH 7.0, and all the other are water.
The LB-Amp liquid nutrient medium: every liter of substratum contains 5g/L yeast extract, 10g/L peptone, 10g/L NaCl and 100 μ g/mL penbritins, and all the other are water.
The 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.
The LB-Km liquid nutrient medium: every liter of substratum contains the 5g/L yeast extract, the 10g/L peptone, and 10g/LNaCl and 50 μ g/mL sulphuric acid kanamycins, all the other are water.
The LB-Km solid medium: every liter of substratum contains 15g/L agar, the 5g/L yeast extract, and the 10g/L peptone, 10g/L NaCl and 50 μ g/mL sulphuric acid kanamycins, all the other are water.
The LB-Km-Amp liquid nutrient medium: every liter of substratum contains the 5g/L yeast extract, the 10g/L peptone, and 10g/L NaCl, 50 μ g/mL sulphuric acid kanamycins and 100 μ g/mL penbritins, all the other are water.
The LB-Km-Amp solid medium: every liter of substratum contains 15g/L agar, the 5g/L yeast extract, and the 10g/L peptone, 10g/L NaCl, 50 μ g/mL sulphuric acid kanamycins and 100 μ g/mL penbritins, all the other are water.
The LBG-Km-Amp liquid nutrient medium: every liter of substratum contains the 5g/L yeast extract, the 10g/L peptone, and 10g/L NaCl, 20g/L glucose, 50 μ g/mL sulphuric acid kanamycins and 100 μ g/mL penbritins, all the other are water.
The LBM-Km-Amp liquid nutrient medium: every liter of substratum contains the 5g/L yeast extract, the 10g/L peptone, and 10g/L NaCl, 20g/L N.F,USP MANNITOL, 50 μ g/mL sulphuric acid kanamycins and 100 μ g/mL penbritins, all the other are water.
The LBGN-Km liquid nutrient medium: every liter of substratum contains the 5g/L yeast extract, the 10g/L peptone, and 10g/L NaCl, 20g/L Sunmorl N 60S and 50 μ g/mL sulphuric acid kanamycins, all the other are water.
The LBF-Km liquid nutrient medium: every liter of substratum contains the 5g/L yeast extract, the 10g/L peptone, and 10g/L NaCl, 20g/L fructose and 50 μ g/mL sulphuric acid kanamycins, all the other are water.
Embodiment 1, structure plasmid pBHR68orfZ
With ClaI and EcoRI two cut plasmid pCK3 (
B, Gott schalk G.Mol ecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri.J Bacteriol, 1996,178:871-880), glue reclaims the fragment that obtains 1.8kb, contains orfZ gene and promoter sequence thereof.
2. with ClaI and two plasmid pBHR68 (the Spiekermann P that cut of EcoRI, 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), glue reclaims the fragment that obtains 8.1kb, contains phaCAB gene and promoter sequence thereof, the Amp resistant gene and the replicon in pBluescript II SK (-) source.
3. use the fragment that reclaims in T4DNA ligase enzyme Connection Step 1 and the step 2; After ligation is finished, obtain plasmid pBHR68orfZ.The method that transforms by electricity imports to the E.coli JM109 (buying from Chinese common micro-organisms culture presevation administrative center), and coats the LB-Amp solid medium, cultivates 16h for 37 ℃;
4. the picking mono-clonal is to the LB-Amp liquid nutrient medium from the LB-Amp solid medium, and (37 ℃ of shaking tables 200rpm), extract plasmid, and confirm by ClaI and EcoRI double digestion whether plasmid construction is successful to cultivate 16h.Male mono-clonal E.coli JM109 (pBHR68orfZ) is after above-mentioned steps is handled, and it is two segment DNA fragments that enzyme is cut product, and size is 8.1kb and 1.8kb.
1. contain the pyruvic carboxylase promotor and (be called for short P
Pdc) structure of expression vector pPpdc
Artificial synthetic one section double-stranded DNA, its sequence is as follows: 5 '-tgataagaattccctaggactagtttacgctcatgatcgcggcatgtcctgatatt tttcctctaaaaaagataaaaagtcttttcgcttcggcagaagaggttcatcatga acaaaaattcggcatttttaaaaatgcctatagctaaatccggaacgacactttag aggtttctgggtcatcctgattcagacatagtgttttgaatataggatccgagctc aagcttagatctggtacctctagacgcccgccataaactgccaggcatcaaattaa gcagaaggccatcctgacggatggcctttttgcgtttctacaaactcttcctgt
Wherein contain the pyruvic carboxylase promoter sequence that derives from mobile Zymomonas mobilis, multiple clone site, and transcription termination sequence.Its method by TA clone is connected among the T carrier pMD19-T Simple (available from Japanese Takara company), obtains expression vector pPpdc.
2.4hbD the clone of gene and expression vector pPpdc4hbD make up
1) under the condition of the polymerase chain reaction of standard, with plasmid pCK3 (
B, Gottschalk G.Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri.J Bacteriol, 1996,178:871-880) be template, with P1:GCT
GGATCCAAGGAGATATACCATGAAGTTATTAAAATTGGCACCTG (
BamH I -) and P2:GCT
GGTACCCTCTTAAGATGGGATATTTAATG (
KpnI) obtain the dna fragmentation of 1185bp for the upstream and downstream primer amplification, show through order-checking, this fragment contains the 4 hydroxybutyric acid dehydrogenase gene 4hbD (being 5 of CP000673 ' end 3061070-3062185 position nucleotide sequence from GENBANK number) of Ke Shi clostridium Clostridium kluyveri, after cutting processing with restriction enzyme BamHI/KpnI enzyme, reclaim the dna fragmentation that contains 4 hydroxybutyric acid dehydrogenase gene 4hbD.
2) cut the plasmid pPpdc that obtains in the treatment step 1 with restriction enzyme BamHI/KpnI enzyme, obtain the carrier segments of 3.0kb.
3) use T4 dna ligase Connection Step 1) and step 2) the middle fragment that reclaims; After ligation is finished, obtain plasmid pPpdc4hbD.The method that transforms by electricity imports to the E.coli JM109 (buying from Chinese common micro-organisms culture presevation administrative center), and coats the LB-Amp solid medium, cultivates 16h for 37 ℃;
4) the picking mono-clonal is to the LB-Amp liquid nutrient medium from the LB-Amp solid medium, and (37 ℃ of shaking tables 200rpm), extract plasmid, and confirm by BamHI and KpnI double digestion whether plasmid construction is successful to cultivate 16h.Male mono-clonal E.coli JM109 (pPpdc4hbD) is after above-mentioned steps is handled, and it is two segment DNA fragments that enzyme is cut product, and size is 3.0kb and 1.2kb.
3.kgd the clone of gene and expression vector pPpdckgd make up
1) under the condition of the polymerase chain reaction of standard, be template with the genome of mycobacterium tuberculosis Mycobacterium tuberculosis H37Ra, with P3:GCT
AAGCTTAAGGAGATATACCATGTACCGCAAGTTCCGCGACGAC (
HindIII) and P4:GCT
TCTAGACGTTCGGCTGAGCGAACTCAG (
XbaI) obtain the dna fragmentation of 3693bp for the upstream and downstream primer amplification, show through order-checking, this fragment contains the 2-oxoglutaric acid decarboxylase gene kgd (being 5 of CP000611 ' end 1390667-1394311 position Nucleotide from GENBANK number) of mycobacterium tuberculosis Mycobacterium tuberculosisH37Ra, after cutting processing with restriction enzyme HindIII/XbaI enzyme, reclaim the dna fragmentation that contains the 2-oxoglutaric acid decarboxylase gene kgd.
2) cut the plasmid pPpdc that obtains in the treatment step 1 with restriction enzyme HindIII/XbaI enzyme, obtain the carrier segments of 3.0kb.
3) use T4 dna ligase Connection Step 1) and step 2) the middle fragment that reclaims; After ligation is finished, obtain plasmid pPpdckgd.The method that transforms by electricity imports to the E.coli JM109 (buying from Chinese common micro-organisms culture presevation administrative center), and coats the LB-Amp solid medium, cultivates 16h for 37 ℃;
4) the picking mono-clonal is to the LB-Amp liquid nutrient medium from the LB-Amp solid medium, and (37 ℃ of shaking tables 200rpm), extract plasmid, and confirm by HindIII and XbaI double digestion whether plasmid construction is successful to cultivate 16h.Male mono-clonal E.coli JM109 (pPpdckgd) is after above-mentioned steps is handled, and it is two segment DNA fragments that enzyme is cut product, and size is 3.0kb and 3.7kb.
4. the structure of plasmid pMCS4HB
1) with the plasmid pPpdckgd that obtains in the restriction enzyme A vrII/XhoI treatment step 3, glue reclaims the dna fragmentation that obtains 4.0kb, contains P
PdcThe kgd gene of transcribing.
2) with the plasmid pPpdc4hbD that obtains in the restriction enzyme NheI/XhoI treatment step 2, glue reclaims the dna fragmentation that obtains 4.2kb, as carrier, wherein contains P
PdcThe 4hbD gene of transcribing.
3) use T4DNA ligase enzyme Connection Step 1) and step 2) the middle fragment that reclaims; After ligation is finished, obtain plasmid pPpdc4hbD-kgd.The method that transforms by electricity imports to the E.coli JM109 (buying from Chinese common micro-organisms culture presevation administrative center), and coats the LB-Amp solid medium, cultivates 16h for 37 ℃;
4) the picking mono-clonal is to the LB-Amp liquid nutrient medium from the LB-Amp solid medium, and (37 ℃ of shaking tables 200rpm), extract plasmid, and confirm by EcoRI and XhoI double digestion whether plasmid construction is successful to cultivate 16h.Male mono-clonal E.coli JM109 (pPpdc4hbD-kgd) is after above-mentioned steps is handled, and it is two segment DNA fragments that enzyme is cut product, and size is 5.5kb and 2.7kb.
5) with restriction enzyme EcoRI/XhoI treatment step 4) in the plasmid pPpdc4hbD-kgd that obtains, glue reclaims the dna fragmentation that obtains 5.5kb, wherein contains P
PdcThe 4hbD gene and the P that transcribe
PdcThe kgd gene of transcribing.
6) handle carrier pBBR1MCS-2 (NCBI GenBank number is U23751) with restriction enzyme EcoRI/XhoI, glue reclaims the dna fragmentation that obtains 5.1kb, as carrier.
7) use T4DNA ligase enzyme Connection Step 5) and step 6) in the fragment that reclaims; After ligation is finished, obtain plasmid pMCS4HB.The method that transforms by electricity imports to the E.coli JM109 (buying from Chinese common micro-organisms culture presevation administrative center), and coats the LB-Km solid medium, cultivates 16h for 37 ℃;
8) the picking mono-clonal is to the LB-Km liquid nutrient medium from the LB-Km solid medium, and (37 ℃ of shaking tables 200rpm), extract plasmid, and confirm by EcoRI and XhoI double digestion whether plasmid construction is successful to cultivate 16h.Male mono-clonal E.coli JM109 (pMCS4HB) is after above-mentioned steps is handled, and it is two segment DNA fragments that enzyme is cut product, and size is 5.5kb and 5.1kb.
Embodiment 3 makes up plasmid pMCS4HBRE
With ClaI and EcoRI two cut plasmid pCK3 (
B, Gottschalk G.Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri.J Bacteriol, 1996,178:871-880), glue reclaims the fragment that obtains 1.8kb, contains orfZ gene and promoter sequence thereof.
2. with ClaI and the two plasmid vector pBBR1MCS-2 (NCBI GenBank number is U23751) that cut of EcoRI, obtain the dna fragmentation of 5.1kb, as carrier.
3. use the fragment that reclaims in T4DNA ligase enzyme Connection Step 1 and the step 2; After ligation is finished, obtain plasmid pMCSorfZ.The method that transforms by electricity imports to the E.coli JM109 (buying from Chinese common micro-organisms culture presevation administrative center), and coats the LB-Km solid medium, cultivates 16h for 37 ℃;
4. the picking mono-clonal is to the LB-Km liquid nutrient medium from the LB-Km solid medium, and (37 ℃ of shaking tables 200rpm), extract plasmid, and confirm by ClaI and EcoRI double digestion whether plasmid construction is successful to cultivate 16h.Male mono-clonal E.coli JM109 (pMCSorfZ) is after above-mentioned steps is handled, and it is two segment DNA fragments that enzyme is cut product, and size is 5.1kb and 1.8kb.
5. with the plasmid pPpdc4hbD-kgd that obtains in the restriction enzyme EcoRI/XhoI Processing Example 2, glue reclaims the dna fragmentation that obtains 5.5kb, wherein contains P
PdcThe 4hbD gene and the P that transcribe
PdcThe kgd gene of transcribing; Next handle with the T4DNA polysaccharase, make the terminal flush endization of its DNA.
6. with the plasmid pMCSorfZ that obtains in the restriction enzyme EcoRI treatment step 4, glue reclaims the carrier segments that obtains 6.9kb, wherein contains Km resistant gene, replicon and orfZ gene, next handles with the T4DNA polysaccharase, makes the terminal flush endization of its DNA.
7. use the fragment that reclaims in T4DNA ligase enzyme Connection Step 5 and the step 6; After ligation is finished, obtain plasmid pMCS4HBRE.The method that transforms by electricity imports to the E.coli JM109 (buying from Chinese common micro-organisms culture presevation administrative center), and coats the LB-Km solid medium, cultivates 16h for 37 ℃;
8. the picking mono-clonal is to the LB-Km liquid nutrient medium from the LB-Km solid medium, and (37 ℃ of shaking tables 200rpm), extract plasmid, and whether cut the affirmation plasmid construction by the ClaI enzyme successful to cultivate 16h.Male mono-clonal E.coli JM109 (pMCS4HBRE) is after above-mentioned steps is handled, and enzyme is cut product and is the unique DNA fragment of 12.4kb for size.
The structure of embodiment 4 engineering bacteria E.coli JM109SG (pBHR68orfZ+pMCS4HB) and utilize glucose and mannose ferment is produced the shake flat experiment of P3HB4HB
1. the method cotransformation that the plasmid pMCS4HB that makes up among the plasmid pBHR68orfZ that makes up among the embodiment 1 and the embodiment 2 is transformed by electricity is to intestinal bacteria succsinic acid hemiacetal dehydrogenase mutant E.coli JM109SG (Li ZJ, Shi ZY, Jian J, et al.Production of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli.Metab Eng, 2010,12:352-359), make up engineering bacteria E.coli JM109SG (pBHR68orfZ+pMCS4HB).
2. (37 ℃ of shaking tables are 200rpm) as seed liquor E.coli JM109SG (pBHR68orfZ+pMCS4HB) to be cultivated 12h in the LB-Km-Amp liquid nutrient medium; 4% inoculum size is inoculated into seed liquor in LBG-Km-Amp or the LBM-Km-Amp liquid nutrient medium by volume, 37 ℃ of shaking tables, and 200rpm cultivates 48h.
3. (Gas chromatography, GC) to the tunning qualitative detection, the result shows that the PHA component that adopts this method production to obtain is 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters (P3HB4HB) to vapor-phase chromatography.
The vapor-phase chromatography of carrying out PHA content in the thalline according to following method detects:
HP 6890 type gas chromatographs are used in gas chromatographic analysis, and chromatographic column is the HP-5 capillary column, and column length 30m, internal diameter 320 sun, stationary phase are the thick phenyl methyl polysiloxane of 25nm; Detector be flame ionization detector (Flame ionization detector, FID); As carrier gas, hydrogen is as combustion gas with high pure nitrogen, 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 0.5min.
Total time is 6min.
Post is pressed:
10psi begins, and stops 1.5min;
2.5psi/min speed boost to 20psi, stop 0.5min.
(psi is a pressure unit, i.e. pound/square inch, 1psi=6.89476kPa)
Injection port: temperature is 200 ℃, uses shunt mode, and splitting ratio is 30～100.
Detector: temperature is 220 ℃, hydrogen flowing quantity 30mL/min, air flow quantity 400mL/min.
The detection step is as follows:
1) with E.coli JM109SG (pBHR68orfZ+pMCS4HB) the bacterium liquid of above-mentioned fermentation culture, centrifugal (10000rpm 10min) collects bacterium, then with recentrifuge behind the distilled water wash; With the cell frost drying, and the mensuration dry cell weight (Cell dry weight, CDW);
2) get 30-50mg left and right trunk cell in the esterification pipe, add 2mL esterifying liquid (vitriol oil of 3% volume is dissolved in the methyl alcohol, contains the 2g/L phenylformic acid as interior mark), 2mL chloroform, covered and enclosed, esterification 4h in 100 ℃ of baking ovens; After being cooled to room temperature, add 1mL distilled water, fully vibration, standing demix; After treating chloroform and water separating fully, get chloroform and carry out gas chromatographic analysis mutually;
3) difference of concentration per sample, sample size is 0.4-1 μ L, uses the microsyringe of Agilent company.Adopt marker method that PHA is carried out quantitative analysis, according to peak area quantification.PHA content is defined as the ratio of PHA pair cell dry weight, PHA output=PHA content * dry cell weight.
Content and the dry cell weight result of accumulation P3HB4HB are as shown in table 1 in the thalline, the result shows, E.coliJM109 SG (pBHR68orfZ+pMCS4HB) can synthesize P3HB4HB under above-mentioned culture condition, and the content of P3HB4HB can reach 50 left and right sides % of dry cell weight.
Table 1 bacterial strain E.coli JM109 SG (pBHR68orfZ+pMCS4HB) cultivates accumulation P3HB4HB and shakes a bottle result
The structure of embodiment 5 engineering bacteria R.eutropha H16 (pMCS4HBRE) and utilize Sunmorl N 60S and the shake flat experiment of fructose fermentative production P3HB4HB
1. the plasmid pMCS4HBRE that makes up among the embodiment 3 is transformed among the R.eutropha H16 (ATCC 17699) (having contained phaCAB) by the method that electricity transforms, makes up engineering bacteria R.eutropha H16 (pMCS4HBRE).
2. (30 ℃ of shaking tables are 200rpm) as seed liquor R.eutropha H16 (pMCS4HBRE) to be cultivated 12h in the LB-Km liquid nutrient medium; 4% inoculum size is inoculated into seed liquor in LBGN-Km or the LBF-Km liquid nutrient medium by volume, 30 ℃ of shaking tables, and 200rpm cultivates 48h.
3. the P3HB4HB that detects in the engineering bacteria according to the method among the embodiment 4 accumulates, found that, R.eutropha H16 (pMCS4HBRE) can produce P3HB4HB under above-mentioned culture condition, in the LBGN-Km substratum, dry cell weight reaches 6.02g/L, P3HB4HB content reaches 51% of dry cell weight, and wherein the 4HB monomer content is 1.5mol%; In the LBF-Km substratum, dry cell weight reaches 6.71g/L, and P3HB4HB content reaches 59% of dry cell weight, and wherein the 4HB monomer content is 2.1mol%.
Claims (10)
1. an engineering bacteria that utilizes the carbohydrate carbon source to produce 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters is characterized in that containing the 2-oxoglutaric acid decarboxylase gene kgd.
2. the described engineering bacteria of claim 1, wherein the carbohydrate carbon source can be for one of glucose, Sunmorl N 60S, fructose, N.F,USP MANNITOL or its combination or through containing the polysaccharide of one of glucose, Sunmorl N 60S, fructose, N.F,USP MANNITOL after the hydrolysis.
3. the described engineering bacteria of claim 1 is one of R.eutropha H16 (pMCS4HBRE) or E.coli JM109SG (pBHR68orfZ+pMCS4HB); Wherein the physical map of the contained expression vector of R.eutropha H16 (pMCS4HBRE) as shown in Figure 1, the physical map of the expression vector that E.coli JM109SG (pBHR68orfZ+pMCS4HB) is contained is as shown in Figure 2.
4. the arbitrary described engineering bacteria of claim 1～3 is for the recombinant expression vector with 3-hydroxybutyric acid and the required gene of 4 hydroxybutyric acid copolyesters biosynthesizing obtains the bacterium of recombinating after being transferred to the host bacterium.
5. the described engineering bacteria of claim 4, wherein said 3-hydroxybutyric acid and the required gene of 4 hydroxybutyric acid copolyesters biosynthesizing comprise that deriving from Luo Shi really supports gathering-3-butyric ester synthetic gene phaCAB of bacillus Ralstonia eutropha, derive from the 4-maloyl group coenzyme A of Ke Shi clostridium Clostridium kluyveri: thiophorase gene orfZ derives from the 4 hydroxybutyric acid dehydrogenase gene 4hbD of Ke Shi clostridium Clostridium kluyveri and derives from the 2-oxoglutaric acid decarboxylase gene kgd of mycobacterium tuberculosis Mycobacteriumtuberculosis.
6. the described engineering bacteria of claim 4, wherein the wild-type that the host bacterium can be intestinal bacteria or Luo Shi really supports bacillus also can be the succsinic acid hemiacetal dehydrogenase gene mutant strain that intestinal bacteria or Luo Shi really support bacillus.
7. the described engineering bacteria of claim 6 is the succsinic acid hemiacetal dehydrogenase gene mutant strain that intestinal bacteria or Luo Shi really support bacillus.
8. the carrier that sets out of the described engineering bacteria of claim 6 recombinant expressed carrier in intestinal bacteria is pBluescript II SK (-) and pBBR1MCS-2 (NCBI GenBank number is U23751); The carrier that sets out of really supporting carrier recombinant expressed in the bacillus at Luo Shi is pBBR1MCS-2.
9. method of producing 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters:, under the situation of adding the carbohydrate carbon source, cultivate by fermentation and obtain 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters with the arbitrary described engineering bacteria of claim 1～8.
10. the method for the described production of claim 9 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters, wherein the leavening temperature of colibacillus engineering is 28-38 ℃; The leavening temperature that described Luo Shi really supports the bacillus engineering bacteria is 30-33 ℃.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102382789A (en) * | 2010-09-02 | 2012-03-21 | 天津国韵生物材料有限公司 | Engineering bacterium containing 2-oxoglutarate decarboxylase gene kgd and applications thereof |
| CN105586304A (en) * | 2014-11-18 | 2016-05-18 | 北京化工大学 | Recombinant strain for producing glycolate based polymers and application of recombinant strain |
| WO2018233703A1 (en) * | 2017-06-23 | 2018-12-27 | 北京蓝晶微生物科技有限公司 | Gene cassette for fine control of composition ratio of 4-hydroxybutanoic acid in copolymer and application thereof |
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| US20030113884A1 (en) * | 1997-03-03 | 2003-06-19 | Silke Hein | Methods for the biosynthesis of polyesters |
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| US20030113884A1 (en) * | 1997-03-03 | 2003-06-19 | Silke Hein | Methods for the biosynthesis of polyesters |
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| 《Biochemical Engineering Journal》 20051231 Jin Seol Kim et al Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha 第169-174页 , 2 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102382789A (en) * | 2010-09-02 | 2012-03-21 | 天津国韵生物材料有限公司 | Engineering bacterium containing 2-oxoglutarate decarboxylase gene kgd and applications thereof |
| CN102382789B (en) * | 2010-09-02 | 2014-06-04 | 天津国韵生物材料有限公司 | Engineering bacterium containing 2-oxoglutarate decarboxylase gene kgd and applications thereof |
| CN105586304A (en) * | 2014-11-18 | 2016-05-18 | 北京化工大学 | Recombinant strain for producing glycolate based polymers and application of recombinant strain |
| WO2018233703A1 (en) * | 2017-06-23 | 2018-12-27 | 北京蓝晶微生物科技有限公司 | Gene cassette for fine control of composition ratio of 4-hydroxybutanoic acid in copolymer and application thereof |
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