CN101906394B - 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
- Publication number
- CN101906394B CN101906394B CN 201010228730 CN201010228730A CN101906394B CN 101906394 B CN101906394 B CN 101906394B CN 201010228730 CN201010228730 CN 201010228730 CN 201010228730 A CN201010228730 A CN 201010228730A CN 101906394 B CN101906394 B CN 101906394B
- Authority
- CN
- China
- Prior art keywords
- hydroxybutyric acid
- gene
- engineering bacteria
- kgd
- bacillus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 44
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 title claims abstract description 12
- 108090000489 Carboxy-Lyases Proteins 0.000 title claims abstract description 12
- WHBMMWSBFZVSSR-UHFFFAOYSA-N R3HBA Natural products CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 claims abstract description 34
- SJZRECIVHVDYJC-UHFFFAOYSA-N 4-hydroxybutyric acid Chemical compound OCCCC(O)=O SJZRECIVHVDYJC-UHFFFAOYSA-N 0.000 claims abstract description 27
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 27
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 27
- 229920001634 Copolyester Polymers 0.000 claims abstract description 22
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 238000000855 fermentation Methods 0.000 claims abstract description 9
- 230000004151 fermentation Effects 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 229940006015 4-hydroxybutyric acid Drugs 0.000 claims description 25
- 241000588724 Escherichia coli Species 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 20
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 12
- 101710088194 Dehydrogenase Proteins 0.000 claims description 10
- 239000013604 expression vector Substances 0.000 claims description 10
- 241000481518 Ralstonia eutropha H16 Species 0.000 claims description 9
- 241000186570 Clostridium kluyveri Species 0.000 claims description 8
- 230000000968 intestinal effect Effects 0.000 claims description 8
- 241001528539 Cupriavidus necator Species 0.000 claims description 6
- 229930091371 Fructose Natural products 0.000 claims description 6
- 239000005715 Fructose Substances 0.000 claims description 6
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 230000007062 hydrolysis Effects 0.000 claims description 6
- 238000006460 hydrolysis reaction Methods 0.000 claims description 6
- 101000746457 Neisseria gonorrhoeae UPF0213 protein in glnA 3'region Proteins 0.000 claims description 5
- 239000005516 coenzyme A Substances 0.000 claims description 5
- 229940093530 coenzyme a Drugs 0.000 claims description 5
- 150000002373 hemiacetals Chemical class 0.000 claims description 5
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 claims description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 4
- 229930195725 Mannitol Natural products 0.000 claims description 4
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims description 4
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 claims description 4
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 claims description 4
- 239000000594 mannitol Substances 0.000 claims description 4
- 235000010355 mannitol Nutrition 0.000 claims description 4
- 108020002872 3-oxoacid CoA-transferase Proteins 0.000 claims description 3
- 230000003570 biosynthesizing effect Effects 0.000 claims description 3
- 108700005078 Synthetic Genes Proteins 0.000 claims description 2
- 150000004676 glycans Chemical class 0.000 claims description 2
- 229920001282 polysaccharide Polymers 0.000 claims description 2
- 239000005017 polysaccharide Substances 0.000 claims description 2
- 238000003259 recombinant expression Methods 0.000 claims description 2
- 230000008521 reorganization Effects 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 3
- WHBMMWSBFZVSSR-UHFFFAOYSA-M 3-hydroxybutyrate Chemical compound CC(O)CC([O-])=O WHBMMWSBFZVSSR-UHFFFAOYSA-M 0.000 abstract description 2
- 239000013612 plasmid Substances 0.000 description 41
- 239000002609 medium Substances 0.000 description 37
- 108020004414 DNA Proteins 0.000 description 21
- 239000007788 liquid Substances 0.000 description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- 239000012634 fragment Substances 0.000 description 19
- 235000015097 nutrients Nutrition 0.000 description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 19
- 239000007787 solid Substances 0.000 description 17
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 description 16
- 229920000903 polyhydroxyalkanoate Polymers 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 14
- 239000001888 Peptone Substances 0.000 description 13
- 108010080698 Peptones Proteins 0.000 description 13
- 229940041514 candida albicans extract Drugs 0.000 description 13
- 239000000178 monomer Substances 0.000 description 13
- 235000019319 peptone Nutrition 0.000 description 13
- 239000012138 yeast extract Substances 0.000 description 13
- 101150108412 kgd gene Proteins 0.000 description 12
- 238000013467 fragmentation Methods 0.000 description 11
- 238000006062 fragmentation reaction Methods 0.000 description 11
- 108091008146 restriction endonucleases Proteins 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- 230000005611 electricity Effects 0.000 description 9
- 239000003292 glue Substances 0.000 description 9
- 229930027917 kanamycin Natural products 0.000 description 9
- 235000011149 sulphuric acid Nutrition 0.000 description 9
- 239000001117 sulphuric acid Substances 0.000 description 9
- -1 sulphuric acid kanamycins Chemical class 0.000 description 9
- 239000000284 extract Substances 0.000 description 8
- 244000005700 microbiome Species 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 235000013619 trace mineral Nutrition 0.000 description 8
- 239000011573 trace mineral Substances 0.000 description 8
- 102000012410 DNA Ligases Human genes 0.000 description 7
- 108010061982 DNA Ligases Proteins 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 230000008676 import Effects 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 101100489694 Clostridium kluyveri (strain ATCC 8527 / DSM 555 / NCIMB 10680) 4hbD gene Proteins 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 229920001013 poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Polymers 0.000 description 6
- 239000007789 gas Substances 0.000 description 5
- YEJRWHAVMIAJKC-UHFFFAOYSA-N 4-Butyrolactone Chemical compound O=C1CCCO1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 description 4
- SJZRECIVHVDYJC-UHFFFAOYSA-M 4-hydroxybutyrate Chemical compound OCCCC([O-])=O SJZRECIVHVDYJC-UHFFFAOYSA-M 0.000 description 3
- 101150056162 4hbD gene Proteins 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000006652 catabolic pathway Effects 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000007479 molecular analysis Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- 108010053763 Pyruvate Carboxylase Proteins 0.000 description 2
- 102100039895 Pyruvate carboxylase, mitochondrial Human genes 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000005886 esterification reaction Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 2
- 235000019421 lipase Nutrition 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000003822 preparative gas chromatography Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 108010021680 2-oxoglutarate decarboxylase Proteins 0.000 description 1
- IPLKGJHGWCVSOG-UHFFFAOYSA-N 4-chlorobutanoic acid Chemical compound OC(=O)CCCCl IPLKGJHGWCVSOG-UHFFFAOYSA-N 0.000 description 1
- 108010093796 4-hydroxybutyrate dehydrogenase Proteins 0.000 description 1
- 108010003902 Acetyl-CoA C-acyltransferase Proteins 0.000 description 1
- 241000588986 Alcaligenes Species 0.000 description 1
- 241000193033 Azohydromonas lata Species 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 241000589518 Comamonas testosteroni Species 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241001600125 Delftia acidovorans Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000216646 Hydrogenophaga pseudoflava Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241001049988 Mycobacterium tuberculosis H37Ra Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241001206171 Stomoxys omega Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 241000588902 Zymomonas mobilis Species 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 108091000039 acetoacetyl-CoA reductase Proteins 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- ORILYTVJVMAKLC-UHFFFAOYSA-N adamantane Chemical compound C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 description 1
- 229910001573 adamantine Inorganic materials 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000000567 combustion gas Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000599 controlled substance Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000013190 lipid storage Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 108700004029 pol Genes Proteins 0.000 description 1
- 101150088264 pol gene Proteins 0.000 description 1
- 229920000520 poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Polymers 0.000 description 1
- 229920000070 poly-3-hydroxybutyrate Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229920002725 thermoplastic elastomer Polymers 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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 high-molecular biologic polyester in the microbe, 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 content of 4HB monomer 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 a series of different materialogy character such as elastomerics from adamantine highly crystalline body to softness.(tensile strength of 64～100mol%), 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 4HB monomer mix the speed of having accelerated enzymolysis and hydrolysis, hydrolysis rate depends on the monomer whose composition, when 4HB content is more high, P3HB4HB is by more fast (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, the carbon source that is used for producing the 3HB monomer comprises conventional sucrose, glucose, fructose and butyric acid etc., and mixing of 4HB monomer need be to the carbon source of adding in the substratum with the 4HB similar, 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 is also bigger to cytotoxicity, usually need add in the mode of batch feeding during the fermentation, and also very complicated comparatively of the fermentation strategy of mixed carbon source.For example, 4 hydroxybutyric acid is considered to most effectively the carbon source of mixing the 4HB monomer in copolyesters, but it is a kind of controlled drug in China, is difficult to buy in 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;
Said three - hydroxybutyric acid and 4 - hydroxy acid copolyester biosynthesis genes, including the necessary support from Roche true bacteria Ralstonia eutropha poly-3 - hydroxybutyrate synthetic gene phaCAB (from GENBANK number AM260479 5 'terminal nucleotides No. 1557353-1561203); from g's Clostridium Clostridium kluyveri 4 - hydroxybutyrate acyl-coenzyme A: coenzyme A transferase gene orfZ (GENBANK number CP000673 from the 5' end of the first position 3066385-3067674 nucleotides); derived from Clostridium Clostridium kluyveri Klinefelter 4 - hydroxybutyrate dehydrogenase gene 4hbD (GENBANK number CP000673 from the 5 'end of the first 3061070-3062185 nucleotides), and derived from Mycobacterium Mycobacterium tuberculosis bacilli 2 - oxoglutarate decarboxylase gene kgd (GENBANK number CP000611 from the 5 'end nucleotide No. 1390667-1394311).
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, namely 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, corresponding operation steps 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.
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, 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.Positive 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
A synthetic double-stranded DNA, the sequence is as follows:5′-tgataagaattccctaggactagtttacgctcatgatcgcggcatgtcctgatatttttcctctaaaaaagataaaaagtcttttcgcttcggcagaagaggttcatcatgaacaaaaattcggcatttttaaaaatgcctatagctaaatccggaacgacactttagaggtttctgggtcatcctgattcagacatagtgttttgaatataggatccgagctcaagcttagatctggtacctctagacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactcttcctgt
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.Positive 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.Positive 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.Positive 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.Positive 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.Positive 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.Positive 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 pressure unit, namely the 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 the dry cell weight of PHA, 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 (7)
1. engineering bacteria that utilizes the carbohydrate carbon source to produce 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters, it is characterized in that containing the 2-oxoglutaric acid decarboxylase gene kgd, described engineering bacteria 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.coii JM109SG (pBHR68orfZ+pMCS4HB) is contained is as shown in Figure 2; And described 2-oxoglutaric acid decarboxylase gene kgd is the 2-oxoglutaric acid decarboxylase gene kgd of mycobacterium tuberculosis (Mycobacterium tuberculosis); Wherein said engineering bacteria is transferred to the reorganization bacterium that obtains behind the host bacterium for the recombinant expression vector with 3-hydroxybutyric acid and the required gene of 4 hydroxybutyric acid copolyesters biosynthesizing, described 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 and derive from the 4 hydroxybutyric acid dehydrogenase gene 4hbD of Ke Shi clostridium Clostridium kluyveri.
2. the described engineering bacteria of claim 1, wherein the carbohydrate carbon source is 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, wherein the host bacterium is that intestinal bacteria or Luo Shi really support the wild-type of bacillus, or be the succsinic acid hemiacetal dehydrogenase gene mutant strain of intestinal bacteria or the very foster bacillus of Luo Shi.
4. the described engineering bacteria of claim 3, wherein this project bacterium is the succsinic acid hemiacetal dehydrogenase gene mutant strain that intestinal bacteria or Luo Shi really support bacillus.
5. the described engineering bacteria of claim 3, wherein the carrier that sets out of this project bacterium recombinant expressed carrier in intestinal bacteria is pBluescript II SK (-) and pBBR1MCS-2 (NCBI GenBank number be U23751); The carrier that sets out of really supporting carrier recombinant expressed in the bacillus at Luo Shi is pBBR1MCS-2.
6. method of producing 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters: with the arbitrary described engineering bacteria of claim 1～5, under the situation of adding the carbohydrate carbon source, cultivate by fermentation and obtain 3-hydroxybutyric acid and 4 hydroxybutyric acid copolyesters.
7. the method for the described production of claim 6 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 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010228730 CN101906394B (en) | 2010-07-16 | 2010-07-16 | Engineering bacterium containing 2-ketoglutarate decarboxylase gene kgd and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010228730 CN101906394B (en) | 2010-07-16 | 2010-07-16 | Engineering bacterium containing 2-ketoglutarate decarboxylase gene kgd and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101906394A CN101906394A (en) | 2010-12-08 |
| CN101906394B true CN101906394B (en) | 2013-08-07 |
Family
ID=43261981
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010228730 Expired - Fee Related CN101906394B (en) | 2010-07-16 | 2010-07-16 | Engineering bacterium containing 2-ketoglutarate decarboxylase gene kgd and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101906394B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105586304B (en) * | 2014-11-18 | 2019-03-05 | 北京化工大学 | A kind of recombinant bacterium producing ethyl alcohol acid-based polymer and its application |
| CN109112151B (en) * | 2017-06-23 | 2021-10-29 | 北京蓝晶微生物科技有限公司 | A gene cassette for finely regulating the composition ratio of 4-hydroxybutyric acid in copolymers and its application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6759219B2 (en) * | 1997-03-03 | 2004-07-06 | Metabolix, Inc. | Methods for the biosynthesis of polyesters |
-
2010
- 2010-07-16 CN CN 201010228730 patent/CN101906394B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| Jin Seol Kim et al.Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha.《Biochemical Engineering Journal》.2005,第169-174页. * |
| Valentin HE et al.Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown on glucose.《J Biotechnol》.1997,第58卷第33-38页. * |
| Zhang,Y. et al.alpha-ketoglutarate decarboxylase [Mycobacterium tuberculosis H37Ra].《GenBank数据库》.2010, * |
| Zheng-Jun Li et al.Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli.《Metabolic Engineering》.2010,第352-359页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101906394A (en) | 2010-12-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Production of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli | |
| US10829793B2 (en) | Transformant that produces copolymerized PHA containing 3HH unit, and method for producing said PHA | |
| Budde et al. | Production of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains | |
| AU2007332240B2 (en) | Method for preparing butanol through butyryl-coa as an intermediate using bacteria | |
| CN101058799B (en) | A method for producing polyhydroxyalkanoate and its special engineering bacteria | |
| Jambunathan et al. | Engineered biosynthesis of biodegradable polymers | |
| CN111705029A (en) | A kind of method for efficiently producing 3-hydroxypropionic acid (3HP) and derivatives thereof based on halophilic bacteria | |
| CN109295085A (en) | A kind of genetic engineering bacteria for producing lactic acid and 3-hydroxybutyric acid copolyester and its construction method and application | |
| WO2024169578A1 (en) | Recombinant bacterium which produces poly(hydroxy fatty acid-co-lactic acid), and application thereof | |
| CN117165617A (en) | Strain for producing P34HB by utilizing xylose as well as construction method and application thereof | |
| CN101906394B (en) | Engineering bacterium containing 2-ketoglutarate decarboxylase gene kgd and application thereof | |
| EP2906707A1 (en) | Polyhydroxyalkanoate copolymer compositions and methods of making the same | |
| CN102382789B (en) | Engineering bacterium containing 2-oxoglutarate decarboxylase gene kgd and applications thereof | |
| CN101445813B (en) | Method for producing 3-hydracrylic acid | |
| Gaur et al. | Efficient bioproduction of poly (3-hydroxypropionate) homopolymer using engineered Escherichia coli strains | |
| Liu et al. | Optimization of poly (-3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis using sodium acetate as a carbon source by Rhodococcus sp. lz1 via response surface methodology | |
| He et al. | Engineering Halomonas bluephagenesis for pilot production of terpolymers containing 3-hydroxybutyrate, 4-hydroxybutyrate and 3-hydroxyvalerate from glucose | |
| CN103740630B (en) | A kind of engineering bacteria containing NADH kinase gene and uses thereof | |
| Mothes et al. | Synthesis of poly (3‐hydroxybutyrate‐co‐4‐hydrobutyrate) with a target mole fraction of 4‐Hydroxybutyric acid units by two‐stage continuous cultivation of Delftia acidovorans P4a | |
| CN117143793B (en) | Method for producing 5-carbon compound or polymer thereof | |
| US20180371509A1 (en) | Pha-producing microorganism having sucrose assimilability, and method for producing pha using said microorganism | |
| Miyake et al. | Phosphotransacetylase as a key factor in biological production of polyhydroxybutyrate | |
| JP6078839B2 (en) | Mutant polyhydroxyalkanoic acid binding protein and method for producing polyhydroxyalkanoic acid using the same | |
| Choi et al. | Production of poly (3-hydroxybutyrate)[P (3HB)] with high P (3HB) content by recombinant Escherichia coli harboring the Alcaligenes latus P (3HB) biosynthesis genes and the E. coli ftsZ gene | |
| Trotsenko et al. | Biosynthesis of poly (3-Hydroxybutyrate) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and its regulation in bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Engineering bacterium containing 2-ketoglutarate decarboxylase gene kgd and application thereof Effective date of registration: 20140507 Granted publication date: 20130807 Pledgee: Zhongxing United Investment Company Limited Pledgor: Tianjin GreenBio Materials Co., Ltd. Registration number: 2014990000333 |
|
| PLDC | Enforcement, change and cancellation of contracts on pledge of patent right or utility model | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20140708 Granted publication date: 20130807 Pledgee: Zhongxing United Investment Company Limited Pledgor: Tianjin GreenBio Materials Co., Ltd. Registration number: 2014990000333 |
|
| PLDC | Enforcement, change and cancellation of contracts on pledge of patent right or utility model | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Engineering bacterium containing 2-ketoglutarate decarboxylase gene kgd and application thereof Effective date of registration: 20140715 Granted publication date: 20130807 Pledgee: Beijing Zhonglian new material investment management center (limited partnership) Pledgor: Tianjin Green Biosciences Limited Company Registration number: 2014990000573 |
|
| PLDC | Enforcement, change and cancellation of contracts on pledge of patent right or utility model | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130807 Termination date: 20190716 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |