CN118272415A - Recombinant corynebacterium glutamicum for producing L-lactic acid, construction method and application thereof - Google Patents
Recombinant corynebacterium glutamicum for producing L-lactic acid, construction method and application thereof Download PDFInfo
- Publication number
- CN118272415A CN118272415A CN202410429865.3A CN202410429865A CN118272415A CN 118272415 A CN118272415 A CN 118272415A CN 202410429865 A CN202410429865 A CN 202410429865A CN 118272415 A CN118272415 A CN 118272415A
- Authority
- CN
- China
- Prior art keywords
- corynebacterium glutamicum
- lactic acid
- gene
- acid
- producing
- 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.)
- Pending
Links
- 241000186226 Corynebacterium glutamicum Species 0.000 title claims abstract description 89
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 title claims abstract description 77
- 238000010276 construction Methods 0.000 title claims abstract description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 50
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims abstract description 34
- 238000004519 manufacturing process Methods 0.000 claims abstract description 18
- 239000001384 succinic acid Substances 0.000 claims abstract description 17
- 108700040099 Xylose isomerases Proteins 0.000 claims abstract description 14
- 239000002994 raw material Substances 0.000 claims abstract description 14
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims abstract description 11
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims abstract description 11
- 108091022915 xylulokinase Proteins 0.000 claims abstract description 11
- 238000000855 fermentation Methods 0.000 claims description 46
- 230000004151 fermentation Effects 0.000 claims description 44
- 108090000623 proteins and genes Proteins 0.000 claims description 34
- 238000000034 method Methods 0.000 claims description 21
- 230000014509 gene expression Effects 0.000 claims description 12
- 101100462488 Phlebiopsis gigantea p2ox gene Proteins 0.000 claims description 8
- 101150060030 poxB gene Proteins 0.000 claims description 8
- 101150110790 xylB gene Proteins 0.000 claims description 8
- 101100096533 Drosophila melanogaster SelD gene Proteins 0.000 claims description 7
- 101100157012 Thermoanaerobacterium saccharolyticum (strain DSM 8691 / JW/SL-YS485) xynB gene Proteins 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- 238000003306 harvesting Methods 0.000 claims description 3
- 229940116871 l-lactate Drugs 0.000 claims 1
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 abstract description 40
- 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 abstract description 22
- 239000008103 glucose Substances 0.000 abstract description 22
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 abstract description 20
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 abstract description 20
- 235000000346 sugar Nutrition 0.000 abstract description 15
- 229910052799 carbon Inorganic materials 0.000 abstract description 7
- 230000002503 metabolic effect Effects 0.000 abstract description 7
- 230000037353 metabolic pathway Effects 0.000 abstract description 7
- 239000010902 straw Substances 0.000 abstract description 6
- 230000037361 pathway Effects 0.000 abstract description 5
- 241000282414 Homo sapiens Species 0.000 abstract description 4
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 230000004127 xylose metabolism Effects 0.000 abstract description 3
- 230000000903 blocking effect Effects 0.000 abstract 1
- 230000001276 controlling effect Effects 0.000 abstract 1
- 239000006185 dispersion Substances 0.000 abstract 1
- 238000012269 metabolic engineering Methods 0.000 abstract 1
- 230000009466 transformation Effects 0.000 abstract 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 78
- 239000004310 lactic acid Substances 0.000 description 39
- 235000014655 lactic acid Nutrition 0.000 description 39
- 238000006243 chemical reaction Methods 0.000 description 28
- 239000013612 plasmid Substances 0.000 description 17
- 239000002253 acid Substances 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 239000007788 liquid Substances 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 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 8
- 229940088598 enzyme Drugs 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 101150023641 ppc gene Proteins 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 6
- 239000007621 bhi medium Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 6
- 229910001868 water Inorganic materials 0.000 description 6
- 101150052264 xylA gene Proteins 0.000 description 6
- 239000003513 alkali Substances 0.000 description 5
- 235000010633 broth Nutrition 0.000 description 5
- 239000000835 fiber Substances 0.000 description 5
- 239000000413 hydrolysate Substances 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- -1 agriculture Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000006227 byproduct Substances 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000600 sorbitol Substances 0.000 description 4
- 238000010025 steaming Methods 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 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 3
- 238000001816 cooling Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 3
- 239000001095 magnesium carbonate Substances 0.000 description 3
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- 235000005985 organic acids Nutrition 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 101150011516 xlnD gene Proteins 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 102100029089 Xylulose kinase Human genes 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004140 cleaning 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
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000004880 explosion Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 2
- 229960003495 thiamine Drugs 0.000 description 2
- 235000019157 thiamine Nutrition 0.000 description 2
- 239000011721 thiamine Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000203809 Actinomycetales Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- ZAQJHHRNXZUBTE-WUJLRWPWSA-N D-xylulose Chemical compound OC[C@@H](O)[C@H](O)C(=O)CO ZAQJHHRNXZUBTE-WUJLRWPWSA-N 0.000 description 1
- FNZLKVNUWIIPSJ-RFZPGFLSSA-N D-xylulose 5-phosphate Chemical compound OCC(=O)[C@@H](O)[C@H](O)COP(O)(O)=O FNZLKVNUWIIPSJ-RFZPGFLSSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 101001004672 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Probable L-lactate dehydrogenase Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229930027917 kanamycin Natural products 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
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.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 OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 1
- 229960002064 kanamycin sulfate Drugs 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000004108 pentose phosphate pathway Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- BHZRJJOHZFYXTO-UHFFFAOYSA-L potassium sulfite Chemical compound [K+].[K+].[O-]S([O-])=O BHZRJJOHZFYXTO-UHFFFAOYSA-L 0.000 description 1
- 235000019252 potassium sulphite Nutrition 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
Abstract
The invention discloses a recombinant corynebacterium glutamicum for producing L-lactic acid, a construction method and application thereof. The construction method comprises the steps of carrying out metabolic engineering transformation on corynebacterium glutamicum, and regulating and controlling by a push-pull resistance strategy: firstly, knocking out metabolic pathways of acetic acid and succinic acid pathways of the plant, and blocking the dispersion of metabolic flows; secondly, replacing the lactate dehydrogenase starting promoter with a strong promoter by promoter engineering to enhance its access to L-lactic acid; then, xylose isomerase gene and xylulokinase gene are introduced into corynebacterium glutamicum to endow xylose metabolism capacity, so that L-lactic acid is produced by fermenting straw sugar. By applying the technical scheme of the invention, a xylose utilization way is constructed, lignocellulose is used as a corynebacterium glutamicum production raw material, the cost is reduced, the low-carbon concept is met, and the limited lower occupation competition of industrial raw material glucose and human beings is reduced.
Description
Technical Field
The invention relates to the technical field of biology, in particular to recombinant corynebacterium glutamicum for producing L-lactic acid, and a construction method and application thereof.
Background
L-lactic acid has been widely used in various fields such as foods, medicines, agriculture, cosmetics, etc., and in particular, L-lactic acid having high optical purity, polylactic acid polymerized from L-lactic acid has been widely used for producing biodegradable plastics due to its biodegradability and biocompatibility. Microbial fermentation has become the most mature method for lactic acid production. The microbial fermentation has the advantages of high yield, high product purity, mild reaction conditions, no environmental pollution and the like. The microbial fermentation feedstock is typically glucose, corn starch, potato starch, and the like. The strains used for fermentation mainly comprise bacillus, lactobacillus, rhizopus, aspergillus and engineering strains.
Currently, the industrial raw materials used industrially for the fermentative production of corynebacterium glutamicum are mainly glucose obtained by purification of starch hydrolysates or molasses. However, this industrial application on the one hand limits the other applications of starch hydrolysates or purified glucose and on the other hand also creates a great competition for the limited cultivated land available to humans. Of all inexpensive carbon sources, lignocellulose is the most promising resource due to its abundance and availability. At present, lactic acid bacteria, bacillus and the like are commonly used for lactic acid fermentation in industry, but most of lactic acid bacteria have complex gene editing operation, and have high nutrition requirements during culture, and a large amount of nutrient elements are required to be added during fermentation. The gene editing operation of bacillus is more complex, and the fermentation usually needs 50 ℃ with high temperature, thus greatly increasing the energy consumption. The corynebacterium glutamicum has low nutrition requirement, and expensive organic nitrogen sources such as yeast powder and the like are not needed to be added in the fermentation process, so that the production cost is greatly reduced. Secondly, corynebacterium glutamicum has a fast fermentation rate, and can synthesize a large amount of lactic acid in a relatively short time. This results in a high production efficiency in industrial production.
Corynebacterium glutamicum (Corynebacterium glutamicum) belongs to the genus Corynebacterium of the order Actinomycetales, and is a gram-positive bacterium which is spore-free and whose cells are arranged in the shape of a short bar. The corynebacterium glutamicum has the advantages of easy culture, no spore production and the like, is an important industrial production strain as a food safety microorganism, is a novel microorganism capable of being used for L-lactic acid fermentation production, and has higher sugar acid conversion rate. In general, the synthesis of lactic acid by corynebacterium glutamicum has the advantages of high efficiency, rapidness, strong tolerance, sustainability, high product purity and the like, and becomes one of ideal choices for lactic acid production. However, although Corynebacterium glutamicum is relatively easy to genetically engineer, little research has been conducted on the fermentation of Corynebacterium glutamicum to produce L-lactic acid.
Disclosure of Invention
The invention aims to provide a recombinant corynebacterium glutamicum for producing L-lactic acid, and a construction method and application thereof, so as to solve the technical problem that no suitable microorganism can efficiently and stably utilize lignocellulose to synthesize lactic acid in the prior art.
In order to achieve the above object, according to one aspect of the present invention, there is provided a construction method of recombinant Corynebacterium glutamicum producing L-lactic acid. The construction method comprises the steps of introducing xylose isomerase genes and xylulokinase genes into corynebacterium glutamicum.
Further, the xylose isomerase gene is xylA and the xylulokinase gene is xylB.
Further, the construction method also comprises the following modification of corynebacterium glutamicum: knocking out a gene encoding acetic acid of corynebacterium glutamicum, and/or knocking out a gene encoding succinic acid of corynebacterium glutamicum.
Further, the genes encoding acetic acid are poxB, pta and/or acka; the genes encoding succinic acid are ppc and/or pyc.
Further, a strong promoter was used to enhance the expression of L-lactate dehydrogenase.
Further, the strong promoter is one or more of Psod, ptuf or Ptac.
Further, the enhanced expression of L-lactic acid was performed by replacing the L-lactate dehydrogenase promoter Pldh with a strong promoter.
According to another aspect of the present invention, there is provided a recombinant corynebacterium glutamicum obtained by the above-described construction method of recombinant corynebacterium glutamicum producing L-lactic acid.
According to another aspect of the present invention, a method for producing L-lactic acid is provided. The method comprises the following steps: (1) fermenting and culturing the recombinant corynebacterium glutamicum; (2) isolating and harvesting L-lactic acid.
Further, the fermentation culture uses lignocellulose as a raw material for producing L-lactic acid by recombinant corynebacterium glutamicum.
By applying the technical scheme of the invention, a xylose utilization way is constructed, lignocellulose is used as a corynebacterium glutamicum production raw material, the cost is reduced, the low-carbon concept is met, and the limited lower occupation competition of industrial raw material glucose and human beings is reduced.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 shows the L-lactic acid production, sugar acid conversion, etc. of the strains 13032, D-AA, D-pyc, and D-ppc in example 2;
FIG. 2 shows the L-lactic acid production, sugar acid conversion, etc. of the strain after the promoter engineering reinforcement in example 2; and
FIGS. 3 and 4 show the L-lactic acid production and sugar acid conversion, respectively, of the strain constructed with the heterologous xylose isomerase and xylulokinase in example 2.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The application will be described in detail below with reference to the drawings in connection with embodiments.
The inventors found that, since xylose is a major component of lignocellulose (about 30% of total sugars), whereas wild Corynebacterium glutamicum lacks the ability to utilize xylose, one major technical hurdle in using lignocellulose as a feedstock for Corynebacterium glutamicum production is the efficient use of xylose.
In order to overcome the defects in the prior art, the strain capable of efficiently and stably utilizing fermentation raw materials (lignocellulose) to produce L-lactic acid is obtained by genetically engineering corynebacterium glutamicum, a strain library for producing L-lactic acid is widened, and a stable strain with high sugar acid conversion rate and difficult bacteria contamination is provided. Specifically, the application provides the following technical scheme.
According to an exemplary embodiment of the present invention, there is provided a construction method of recombinant Corynebacterium glutamicum producing L-lactic acid. The construction method comprises the steps of introducing xylose isomerase genes and xylulokinase genes into corynebacterium glutamicum.
The "introduction" may be in any suitable manner known in the art, for example in the form of a plasmid, or integrated into the genome, with Corynebacterium glutamicum. In one embodiment, the enzyme-encoding gene integrated into the genome is placed under the control of suitable regulatory elements.
The invention constructs and expresses xylose isomerase gene and xylulokinase gene in corynebacterium glutamicum, successfully utilizes the mixed solution of glucose and xylose (fermentation raw material containing lignocellulose), and realizes the efficient synthesis of lactic acid by using lignocellulose hydrolysate to metabolize glucose and xylose simultaneously. The lignocellulose hydrolysate is used as the production raw material of the corynebacterium glutamicum, so that the production cost is reduced, the low-carbon concept is met, and the limited lower competition of the industrial raw material glucose and human beings is reduced;
In a preferred embodiment, the xylose isomerase gene is xylA and the xylulokinase gene is xylB.
According to an exemplary embodiment of the invention, the construction method further comprises the following modifications to Corynebacterium glutamicum: knocking out a gene encoding acetic acid of corynebacterium glutamicum, and/or knocking out a gene encoding succinic acid of corynebacterium glutamicum. Knocking out byproducts of lactic acid metabolic pathways, and improving the conversion efficiency and yield of lactic acid.
It will be appreciated by those skilled in the art that gene knockouts may be made in a manner known in the art such that the activity of the enzyme is reduced or inactivated. The knockout operation is directed to the initiation of an endogenous enzyme gene of C.glutamicum, such that the aforementioned endogenous enzyme activity of C.glutamicum is reduced or inactivated.
The coding sequence of another gene can be used for replacing the coding sequence of acetic acid and/or succinic acid in the original corynebacterium glutamicum by a homologous recombination and other genetic engineering modes, so that the activity of the endogenous enzyme of the corynebacterium glutamicum is reduced or inactivated. The gene replacing these endogenous enzymes may be a gene to be expressed or inserted in an enhanced manner.
In a preferred embodiment, the gene encoding acetic acid is poxB, pta and/or acka; the genes encoding succinic acid are ppc and/or pyc.
Since it is difficult to achieve overexpression (i.e., double copy, triple copy, etc.) of a gene in Corynebacterium glutamicum, it is generally selected to replace the promoter upstream of the gene with a promoter having higher expression strength. In a preferred embodiment, the lactate upper limit valve is increased by over-expressing lactate dehydrogenase by promoter engineering, preferably using a strong promoter to enhance expression of L-lactate dehydrogenase; more preferably, the strong promoter is one or more of Psod, ptuf or Ptac. The selected strong promoter Psod, ptuf or Ptac is derived from corynebacterium glutamicum, belongs to an endogenous promoter, and does not need to carry out codon optimization and compatibility consideration; furthermore, the strong promoters selected are all promoters which have been shown to enhance the expression of endogenous genes of C.glutamicum. In the present application, the inventors found that the Psod promoter is particularly suitable as a promoter for enhancing expression of lactate dehydrogenase in Corynebacterium glutamicum. The fermentation verification data is shown in FIG. 2. According to an exemplary embodiment of the present application, the enhanced expression of L-lactic acid is performed by replacing the L-lactate dehydrogenase promoter Pldh with a strong promoter.
In a preferred embodiment of the present application, there is provided a construction method of a genetically engineered strain producing lactic acid, the construction method comprising:
(1) Firstly, knocking out poxB, pta, acka genes of corynebacterium glutamicum encoding acetic acid, and reducing byproduct metabolic flow;
(2) Knocking out ppc and pyc genes of corynebacterium glutamicum encoding succinic acid on the basis of (1), and reducing byproduct metabolic flow;
(3) The strong promoter Psod, ptuf, ptac is used for replacing the L-LDH promoter Pldh on the basis of the step (2) to carry out the enhanced expression of the L-lactic acid and strengthen the metabolic flow of the main channel;
(4) Constructing xylose isomerase xylA and xylB genes simultaneously on the basis of the step (3) to construct a xylose metabolism path, and constructing a xylose utilization path to produce lactic acid;
Glucose enters the corynebacterium glutamicum body, L-lactic acid is generated through a glycolysis path, and other bypass metabolic flow paths such as acetic acid, succinic acid and the like exist in the process of converting pyruvic acid into lactic acid, so that the accumulation of lactic acid is reduced. Therefore, the knockout of the acetic acid gene poxB, pta, acka and the succinic acid ppc gene can better introduce metabolic flow into a lactic acid pathway, and the yield of lactic acid is improved. Similarly, the expression of the strong promoter Psod, ptuf, ptac of the L-lactic acid dehydrogenase gene can greatly improve the upper limit of the metabolic flow valve and ensure higher rate of synthesizing L-lactic acid.
Further, since the carbon source accounts for 30% or more of the cost of lactic acid fermentation, it is desired that biosynthesis of lactic acid can be performed by a cheaper carbon source such as lignocellulose hydrolysate or the like. The xylose is present in the lignocellulose hydrolysate, so that the natural corynebacterium glutamicum has no capability of directly utilizing xylose, and therefore, a complete xylose metabolic pathway needs to be assembled in the strain to utilize xylose as a substrate. Among the xylose metabolic pathways, the XI pathway is the most commonly used in microorganisms, xylose is converted into xylulose by xylose isomerase, then into xylulose-5-phosphate by xylulokinase, and then into pentose phosphate pathway to produce the target product. In this example, xylose isomerase xylA and xylulokinase genes xylB were constructed to construct a xylose metabolic pathway, and a xylose utilization pathway was constructed to produce lactic acid.
According to an exemplary embodiment of the present invention, there is provided a recombinant corynebacterium glutamicum obtained by a construction method of a recombinant corynebacterium glutamicum producing L-lactic acid constructed by the above construction method.
According to an exemplary embodiment of the present invention, a method for producing L-lactic acid is provided. The method comprises the following steps: (1) Fermenting the recombinant corynebacterium glutamicum of claim 8; (2) isolating and harvesting L-lactic acid. Preferably, the fermentation culture is conducted using lignocellulose as a starting material for producing L-lactic acid from recombinant Corynebacterium glutamicum, for example, the fermentation starting material lignocellulose prepared with reference to CN 202010294852.1.
For example: cutting wheat straw into small sections of 2-3cm, cleaning to remove impurities such as sediment and the like, and performing steam explosion in a steam explosion reactor. The absolute dry weight of the straw is as follows: deionized water 1:4 ratio, absolute dry weight of straw: the proportion of potassium hydroxide is 10: 1. potassium sulfite: the potassium hydroxide ratio is 1:3, pouring the mixture into a rotary stirrer for presoaking for 48 hours, steaming at 155 ℃ for 3 hours in a continuous steaming machine after presoaking is finished, collecting steaming liquid after steaming, cleaning the steamed straw with a 500-mesh screen until the cleaned water is clear and free of impurities, collecting the cleaned alkali-treated fiber sample, and measuring the water content of the alkali-treated fiber sample. Straw raw materials are mixed according to a solid-liquid ratio of 1:40 in deionized water, and adjusting the initial pH to 2.5 with 1% sulfuric acid, soaking for 24 hours, completely removing alkali liquor to make the pH stable at 5, squeezing out water with a 500 mesh screen to obtain a fiber sample subjected to alkali removal treatment, and preparing for hydrolysis. The hydrolysis steps are as follows: taking 100g (absolute dry weight) of each group of fiber samples subjected to alkali removal treatment, respectively adding 3ml of clostridium cellulosum with the concentration of 5 multiplied by 109CFU, carrying out prehydrolysis for 48 hours, adding each group of fiber samples into a hydrolysis tank after the prehydrolysis is finished, regulating the pH of a hydrolysis system to be 5.2, adding 500FPU (filter paper enzyme activity unit) of cellulase, carrying out hydrolysis for 72 hours at 55 ℃, adding 1g of activated carbon powder to adsorb pigment and impurities after the hydrolysis is finished, carrying out centrifugal filtration to obtain an original sugar solution, and determining the content of reducing sugar in the hydrolysate (the method is adopted to prepare lignocellulose in the subsequent embodiment of the application). According to a typical embodiment of the application, the corynebacterium glutamicum has a growth optimum temperature of 30 to 32 ℃, preferably 30 ℃, and an optimum pH of 7.0 to 8.0, preferably 7.0. Therefore, the fermentation mode is selected to be at 30-32 ℃, preferably 30 ℃,10 g/L of calcium chloride is added to the culture medium at the initial stage of fermentation to adjust the pH to 7.0-8.0, preferably 7.0, and 10g/L of sodium bicarbonate and 40g/L of basic magnesium carbonate are added after 24 hours of fermentation to maintain the pH. The optimal fermentation time is 96 hours, and is shown in figure 4. The advantageous effects of the present application will be further described below with reference to examples.
The initial strain used in the experiment is Corynebacterium glutamicum ATCC 13032, the specific gene sequence is obtained by inquiring and confirming the NCBI of genome comparison and is extracted and synthesized from the genome, and the used plasmid is also the existing plasmid.
Experiment medium:
The common chemical reagent is purchased from Shanghai national drug reagent group; yeast powder, peptone was purchased from Oxiod company; kanamycin sulfate was purchased from Shanghai; the high fidelity enzymes, taq enzymes were all purchased from the biological sciences company of Nanjinouzan; gibson assembled enzyme was purchased from Beijing full gold Biotechnology Co., ltd; DNA purification kits, plasmid miniprep kit from Thermo FISHER SCIENTIFIC; primer synthesis and gene sequencing are undertaken by the company Jin Weizhi, su.
LB medium: 5 g.L -1 yeast powder, 10 g.L -1 tryptone, 10 g.L -1 sodium chloride, pH 7.0, and adding 1.8% agar powder to obtain LB solid culture medium.
BHI medium: 38.5 g.L -1 brain-heart leaching solution, and adding 1.8% agar powder to obtain LB solid culture medium.
Secondary medium: the BHI medium was supplemented with 20 g.L -1 of glucose.
BHIS: 91 g.L -1 sorbitol (sorbitol) was added to BHI medium.
BHIS +2% glycine: BHI medium was supplemented with 91 g.L -1 sorbitol (sorbitol) and 20 g.L -1 glycine.
10% (V/V) tween 80: and (5) filtering and sterilizing.
10% Glycerol: and (5) sterilizing at high temperature.
TG buffer:10% glycerol+1 mM Tris, and sterilized at high temperature.
CGXII medium, see Table 1:
TABLE 1
Supplementing microelements:
volume: 100mL.
The components are as follows: 2.5g urea ,0.125g MgSO4·7H2O,5mg FeSO4·7H2O,5mg MnSO4H2O,0.05mg ZnSO4·7H2O,0.1mg CuSO4,0.01mg NiCl2·6H2O,100μg biotin, 5mg CaCl 2 and 0.25mg thiamine (thiamine).
Seed BHI medium: the BHI medium was supplemented with 4 g.L -1 of glucose.
Fermentation medium: the CGXII medium was supplemented with 100 g.L -1 of glucose.
The fermentation mode of corynebacterium glutamicum:
Corynebacterium glutamicum strain was used as a control for anaerobic fermentation.
1) Preparation of first-level seed liquid
The correct monoclonal was picked and incubated in BHI tubes (5 mL), 30℃at 200rpm for 16h, pH 7.0.
2) Preparation of secondary seed liquid
4ML of the primary seed solution was inoculated into 100mL of the secondary seed medium, and the culture was carried out at 30℃and 200rpm for 24-28 hours at pH7.0.
3) Lactic acid fermentation
And culturing the secondary seed liquid to OD 600 -14-16, and collecting bacteria. The whole secondary seed liquid is poured into a centrifugal bottle, the centrifugal bottle is centrifuged at 6000rpm for 5min, the supernatant is discarded, and 3-4mL of fermentation CG (CG) culture medium is added for resuspension. After OD 600 value is measured, the fermentation medium is inoculated according to the initial OD 600 which is more than or equal to 50. 50g/L glucose, 10g/L sodium bicarbonate and 40g/L basic magnesium carbonate powder were fed at 24h of fermentation. During fermentation, samples are taken every 24 hours, and the fermentation period is 120 hours and the pH value is 7.0.
4) HPLC detection
The fermentation broths of 24 hours, 48 hours, 72 hours, 96 hours and 120 hours are detected by HPLC, specifically, 300 mu L of the fermentation broth is taken, centrifuged for 2min at 10000 rpm.min -1, the supernatant is collected, and the impurities are removed by filtration through a water-based filter membrane of 0.22 mu m. 2.5mM H 2SO4 was used as mobile phase, the flow rate was varied to 0.6ml min -1, the column temperature was 60℃and the amount of sample introduced was 10. Mu.l, the differential detector temperature was 50 ℃. Glucose and organic acids (e.g., lactic acid, acetic acid, succinic acid) were analyzed using a differential detector.
The experimental apparatus used is shown in table 2:
TABLE 2 Main instrumentation
The PCR reaction system and conditions in the following examples are shown in tables 3 to 6:2×taq DNA polymerase:
Table 32 ×Taq PCR amplification reaction system
TABLE 42 XTaq PCR amplification reaction conditions
Phanta EVO HS Super-Fidelity high-Fidelity DNA polymerase:
TABLE 5Phanta EVO HS Super-FIDELITY PCR amplification reaction system
TABLE 6Phanta EVO HS Super-FIDELITY PCR amplification reaction conditions
1. The corynebacterium glutamicum electrotransformation step:
a. cell activation: a5 mL BHI tube was inoculated with the monoclonal or bacterial solution at 30℃overnight.
B. Competent cell culture: the activated broth was inoculated with 25mL to BHIS +2% glycine medium containing 0.1% tween 80, with an initial OD 600 of about 0.2.
C. Competent cell preparation.
After 3-4 hours of incubation at 30℃OD 600 was about 0.9.
And ii.4500rpm,4 ℃, centrifuging for 5min, and discarding the supernatant.
Cells were resuspended using 25mL pre-chilled TG buffer.
Iv.4500rpm,4 ℃, centrifuge for 5min, discard supernatant.
V. resuspended cells using 25mL of pre-chilled 10% glycerol.
Vi.4500rpm,4 ℃, centrifuge for 5min, discard supernatant.
On ice, cells are resuspended with undipped liquid to a final volume of about 500 μl (e.g. insufficient supplementation with pre-chilled 10% glycerol).
D. Electric conversion:
mu.L of cells were added to 10. Mu.L of plasmid, which was added to a pre-chilled cuvette (2 mM diameter), 2.5kV, 25. Mu.F.
Immediately adding 4mL BHIS culture medium (preheated at 46 ℃) and thermally shocking at 46 ℃ for 6min.
Cooling to normal temperature, and incubating at 30deg.C for 60min.
Centrifugation at 4500rpm for 5min was performed on BHI plates with corresponding resistance (Kan-25, i.e.25 mg/L kanamycin resistance).
E. Plate culture at 30℃for 2d.
2. Recombinant screening step:
a. The growing monoclonal in 1e was streaked onto the corresponding resistant BHI and incubated overnight.
B. The bacteria of step 2a are dissolved in 100 μl of sterile water, streaked on a bhi+10% sucrose plate, and each monoclonal is streaked with a region and grown for 48-72 h.
C. And 5-10 single clones are selected from a streaking area with sparse growth, streaking is carried out on a flat plate of BHI+K and BHI+10% sucrose respectively, and colony PCR is carried out on clones with BHI+10% sucrose growth and BHI+K non-growth.
Example 1
A recombinant corynebacterium glutamicum constructed by the following method:
Step1, designing 500-1000bp homologous arms on the upper and lower sides of a gene poxB, and constructing PK18msB-poxB-kan knockout gene recombinant plasmid; among them, PK18msB (Plasmid # 177839) is a common Plasmid trade name.
And 2, transferring the gene recombinant plasmid constructed in the step 1 into corynebacterium glutamicum ATCC 13032 through an electrotransformation method to obtain recombinant corynebacterium glutamicum 13032-D-poxB, and preserving the strain of the recombinant corynebacterium glutamicum for later use.
Step 3: designing homologous arms of 500-1000bp up and down of genes ptah and acka, and constructing PK18msB-pta-acka-kan knockout gene recombinant plasmid;
And 4, transferring the gene recombinant plasmid constructed in the step 1 into corynebacterium glutamicum 13032-D-poxB by an electrotransformation method to obtain recombinant corynebacterium glutamicum 13032-D-AA, and preserving the strain of the recombinant corynebacterium glutamicum for later use.
And 5, respectively constructing PK18msB-pyc-kan and PK18msB-ppc-kan, and respectively transferring into corynebacterium glutamicum 13032-D-AA by an electrotransformation method to obtain recombinant corynebacterium glutamicum 13032-D-pyc and 13032-D-ppc.
And 6, respectively constructing PK18msB-Pldh:: ptac-ka, PK18msB-Pldh:: psod-kan and PK18msB-Pldh:: ptuf-kan, and respectively transferring into corynebacterium glutamicum 13032-D-ppc by an electrotransformation method to obtain the recombinant corynebacterium glutamicum 13032-Ptac,13032-Psod and 13032-Ptuf with the promoter engineering reinforced.
And 7, constructing pEKI-Ptac-xylA-xylB gene recombinant plasmid, transferring the constructed gene recombinant plasmid into corynebacterium glutamicum 13032-Psod by an electrotransformation method to obtain recombinant corynebacterium glutamicum 13032-xylAB, and preserving the recombinant corynebacterium glutamicum strain for later use.
PK18msB-poxB-kan,PK18msB-pta-acka-kan,PK18msB-ppc-kan,PK18msB-pyc-kan,PK18msB-Pldh::Ptac-kan,PK18msB-Pldh::Psod-kan,PK18msB-Pldh::Ptuf-kan,pEKI-Ptac-xylA-xylB The gene sequences of (2) are shown in SEQ ID NO.1-8, and the primer sequences of the recombinant genes are shown in Table 7.
The gene recombinant plasmid was transferred into Corynebacterium glutamicum by the following method:
Step A1, constructing competent cells of corynebacterium glutamicum;
Step A2, transferring competent cells obtained in the step A1 into plasmids with correct sequencing by electrochemical method; and step A3, obtaining the corynebacterium glutamicum containing the gene recombinant plasmid through resistance screening and PCR verification.
TABLE 7 primer list of recombinant genes
The PK18msB and pEKI plasmids were constructed by the following method:
Corresponding primers derived from Corynebacterium glutamicum poxB, pta-acka, pyc, ppc, pldh and Ptac, ptuf, psod, xylA, xylB homology arms derived from NCBI sequences were designed, the PCR-derived products were purified, the concentrations were measured and ligated using GIBSON, transformed into E.coli competence by chemical means, kan plates were applied and clone verified, and forward primer sequencing was used to verify that pEKI-Ptac-LRpck was obtained.
Specifically, gibson was assembled to obtain purified linearized vectors and fragments with homologous end sequences (15-20 bp) by PCR or restriction using the full gold companySeamless Cloning and Assembly the kit is subjected to rapid recombination, and the reaction system is shown in Table 8.
Taking a single segment recombination method as an example:
Cloning vector usage amount: [0.02 Xcloning vector base pair ] ng (0.03 pmol).
Amount of insert used: [0.04 Xcloning vector base pair ] ng (0.06 pmol).
Table 8gibson assembled reaction System
Reaction conditions: single fragment recombination reaction at 50 ℃ for 15min; cooling to 4 ℃ or immediately cooling on ice.
Example 2
Fermentation experiments were performed using the strains constructed in example 1:
corynebacterium glutamicum strains are collected for anaerobic fermentation after aerobic enrichment:
Firstly, picking a monoclonal in a BHI test tube (5 mL) with pH of 7.0, culturing at 30 ℃ and 200rpm for 16 hours to activate the strain, and taking the strain as a first-stage seed solution;
then 4mL of the primary seed liquid is inoculated into a 500mL shaking bottle containing 100mL of secondary seed culture medium BHI plus 20g.L -1 glucose, and the culture is carried out for 24-28 hours at the pH of 7.0 and the temperature of 30 ℃ and the rpm of 200 to enrich the seed liquid with high density content;
And culturing the secondary seed liquid to OD 600 -14-16, and collecting bacteria. The whole secondary seed liquid is poured into a centrifugal bottle, the centrifugal bottle is centrifuged at 6000rpm for 5min, the supernatant is discarded, and 3-4mL of fermentation CG (CG) culture medium is added for resuspension. After OD 600 value is measured, the fermentation medium is inoculated according to the initial OD 600 which is more than or equal to 50, and the pH value is 7.0. 50g/L glucose, 10g/L sodium bicarbonate and 40g/L basic magnesium carbonate powder were fed at 24h of fermentation. In the fermentation process, sampling is carried out every 24 hours, and the fermentation period is 120 hours.
HPLC detection, analysis of glucose and organic acids (e.g. lactic acid, acetic acid, succinic acid) using a differential detector:
The fermentation broths of 24 hours, 48 hours, 72 hours, 96 hours and 120 hours are detected by HPLC, specifically, 300 mu L of the fermentation broth is taken, centrifuged for 2min at 10000 rpm.min -1, the supernatant is collected, and the impurities are removed by filtration through a water-based filter membrane of 0.22 mu m. 2.5mM H 2SO4 was used as mobile phase, the flow rate was varied to 0.6ml min -1, the column temperature was 60℃and the amount of sample introduced was 10. Mu.l, the differential detector temperature was 50 ℃. Glucose and organic acids (e.g., lactic acid, acetic acid, succinic acid) were analyzed using a differential detector.
Results: FIG. 1 shows that the strains with knocked out ppc do not produce succinic acid SA and the strains with knocked out pyc still have a succinic acid pathway as seen from the data for 96h fermentation alignments of 13032, D-AA, D-pyc and D-ppc shake flasks. The subsequent operations are thus carried out on the basis of 13032-D-ppc. The lactic acid yield of the original strain 13032 reaches 42g/L, the sugar acid conversion rate is about 0.6, compared with the knocked-out strain D-AA lactic acid yield reaching 52g/L, the sugar acid conversion rate is about 0.78, the D-ppc lactic acid yield reaches 72g/L, the sugar acid conversion rate is 0.87, the D-ppc lactic acid yield is 70% higher than that of the original strain 13032, and the conversion rate is 45%.
FIG. 2 shows a 96h shake flask fermentation comparison of 13032 and promoter-enhanced strains, and it is known from the data that the lactic acid yield of the starting strain 13032 reaches 42g/L, and the sugar acid conversion rate is approximately 0.6; the D-ppc lactic acid yield reaches 72g/L, and the sugar acid conversion rate is about 0.87; in contrast, the yield of Psod lactic acid of the promoter engineering strain reaches 100g/L, the sugar-acid conversion rate is 0.95, and the yield of the promoter engineering strain is 39% higher than that of the chassis D-ppc lactic acid in the previous step, and the sugar-acid conversion rate is 10% higher. The subsequent operations are therefore carried out on the basis of 13032-Psod.
FIG. 3 and FIG. 4 show that the comparison of the 13032 and 13032-Psod and 13032-xylAB strains by shake flask fermentation for 96 hours, and according to data, the construction of a xylose metabolism path of coding xylose isomerase xylA and xylB gene xylB can better utilize the mixed solution of glucose and xylose, improve the yield of lactic acid of the strains, and the yield of the 13032-xylAB lactic acid of the strains can reach 140g/L, the conversion rate reaches 0.97, and the yield of lactic acid of the strains is improved by 40 percent compared with that of the 13032-Psod lactic acid.
The specific sequences involved are:
SEQ ID NO.1：
GCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCGAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATGGGTACCGAGCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCCGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTGCTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGAGGGGACAGGCGAGAGACGATGCCAAAGAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCctaattgtttctgccgcgggcaactaccttccaggtacggaaagtgccatcggcttcccgaacgtagacctcatccaccaaattaatcacgttgcaggcatggttgggcacgatgttgagctgctccccgattactggaagtagcactttatctggccagaaaatggttgcgtgatgctccgacaaagcagagattcgggcttcaggattccccagaacaaaaccattgccatcaatccatgctggtttatcagtgctgaggattttggatcccgcatccaaaatgatccgacgatctgacacatttcggctgaccacagtagacagcaccgtcattgccacctgcttctcagtgcatgctcccgaggtgatctgctgggaatcgttaaacacatacacgcctggtcgcatctcatcgattgcgtctgtaaactgcgcagacggcgaggaaccgccagaagtcaggccgccagcaagtcgctggacgctgttgtttagagcctgaagctcatcagctgctgcctgctcaccatttcccgggccataagaatgcccaggaaaagtaaacactcctgcatacctgctgcccagcgcctcgcggatctgactcaattctgaagcagtcgccgtgactccacttctacgatgtcccgaatccacttcaatcagagccttgatatcttcccgcaaacccgccgtcgcctgtgccatctctaccgaatccacgccaatggaaatttctccggggatcgcgttcaggcgttgcactgcatgatcggttagatacagcggatatgcaataaagatgtccgtaaaacctgcgccggcaaaaatttccgcctcgccaatggttgcgcaggtgatccctcgggcaccggcgtcgacctgcatctgcgcaatttcaatgattttgtgcgttttcacatgcggacgcagggcaatctcatgggcaccggcgtgagctgccatcctggaaatgttggcagttaagcgctcgcggtcaatgagaacagcaggtgtatcaatcatcatctgaactcctcaacgttatggctattgtgttgcctaacttggtgcgacttcaattgtgctcttaaattccagttgttgccacgcttcgcccgcctgccagggagtttcaggcccgggtgggtactcggtaccaaacagggatgcccacttttgggatttccgtcgttgctgggcagtctggtttggtttcagcccgtcgaaaccaaacacagatgcccaccgatcgcctatttaaacatcgtgggcacgcctgtctggtcgcctcgaaaactggccaaatttagaccccgaccccacaaaaagtgacctcacagaatcgcctctaagcgcctcaaagaacccgacccacgctgacagtcccctacgatttccgacgtcttaaatcgccacacagcgccgtggtctaaaacaccaacaaaagagttgtaactgtaccgaccattcgttacagttacgatccatgacttcagaaaccttacaggcgcaagcgcctacgaaaacccaacgttgggctttcctcgccgttatcagcggtggtctctttctgatcggtgtagacaactcgattctctacaccgcactccctctgctgcgtgaacagctcgcagccaccgaaacccaagcgttgtggatcatcaacgcatatcccctgctcatggcgggccttcttttgggtaccggcactttgggtgacaaaatcggccaccgccggatgttcctcatgggcttgagcattttcggaatcgcttcacttggtgctgcgtttgctccaactgcgtgggctcttgttgctgcgagagctttccttggcatcggtgcggcaacgatgatgcctgcaaccttggctctgatccgcattacgtttgaggatgagcgtgagcgcaacactgcaattggtatttggggttccgtggcaattcttggcgctgcggcaggcccgatcattggtggtgcgctgttggaattcttctggtggggttcAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCTAGAGGATCGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTTCTGAATTTGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAG
SEQ ID NO.2：
TCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCGAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATGGGTACCGAGCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCCGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTGCTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGAGGGGACAGGCGAGAGACGATGCCAAAGAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCcttcgccaaaatacgagcaacgtccaacgccacgttaccgacaccaacaaccgctaccttctcagcagaaagatcccagttgcgttcaaagttcgggttgccatcatagaaaccaacgaactcgccagcgccccacgaaccttccagatcagaacctggaacccgaagatcctggtcgccagtagcgccagtggagaacacgatcgcgtcataaaactcacgcaactcctcaacagtgatgtccttgccgacctcaatgttgcccaagaaacgcagctgctccttgtccatcacattgtgcagggacttcacgatgcccttgatgcgagggtgatcaggcgcaacaccataacggatcaaaccgaaaggcgctggcatacgttcaaaaagatcaatctgcacgtccgtgtcggatttcatcaacaaatcagacgcgtagattcctgctggacctgcaccgacaacggcaacacgcaaagggcgagacatataaagttcgattccttaaaggggttctaaaaaatgtggagtatgtgagcgggggttccacttgtagattcgactcctatcggggtgcgactgctaatggtgccctgctatcaaccctccatgatacgtggtaagtgcagactaataaaggccagtcggggagtattgggggctttgctggggtcagatttgtcacgctgcgcgctttcatagaccccattaatggggggtgaagagctgtaaagtaccgctaaaaactttgcaaagggtgcttcgcaacttgtaaccgctccgtattgttttctacggcaataagcatttgtgctgctcaaagcgtggaattgagatcggtttgaaaattacaaaataaaactttgcaaaccgggctgtacgcaaggcggacgaacgctaaactatgtaagaaatcacaacctcccctcattagtgccaggaggcacaagcctgaagtgtcatcaatgagaaggttcaggctgaaattagaaaggcgatgttctcctggttaggatccaccacaaatcgctctgatcagcggttttgtggtggatttttgcgtttttaaggggtgaaactgcacggatccaccacagatcccagttttcctttggaacgtggtggatccttgccctggagcttcacaggaatcgcttgttggcccctagacctcttggggttgcgaattttcgtccccaccgaacattaaaaggccggttttggtcgaaaatttgctctaacaccttgctattatgcgaatattcgttccatttcatcgaattccagcaacccgtaacgagaagttgaacaggaaacctgcagtaaccccgcagaaatcacatcagccccaattgtcccaaaagtaactcccccagaatcgcttctaagggcctaactcgcccaaagtcaaactaggggacatcgcactcctaaaggcccttaaatcgccacctaccaaatagccccaagtcaaaacagctagaaccaactcagtggccgcacggcattcgccatatccacaagtgcgtaacggtggtgcgggaacggtgcagaacgtgcctgaatgcggagtgcctcggagatgccggtgcgcaggcctttttgggagaacgggtattcaaacaaagggttcgcggaggcggaagctttgagttttcgctctcgaagccagctgaggcctgccgacatgatggcaattttgatttggttgaagcggggttcgtttgtggggatttcggtgagtcggcgggcagcccgtcggatgcgggattcactcaaattggagctgaccagcaacaagatggtggtgagggtggccattcggtagtgggtggatgattgggggagtttgtctagggcttggactgcgagttcgatttggttttcggccatgagttggcgggcgagcccgaacgcggaggacacggtggtggggttggttgcccagacaagtgcgtagaggcgcagtgaatggaagcggactacgtgtgggtcgctggagatAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCTAGAGGATCGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTTCTGAATTTGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAGGCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGA.
SEQ ID NO.3：
GCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCGAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATGGGTACCGAGCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCCGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTGCTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGAGGGGACAGGCGAGAGACGATGCCAAAGAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCagctgatcctaccgatcgctgtggcagtgaccaaccgtctgacagttgctgatctggctgataccttcgcggtgtacccatcattgtcaggttcgattactgaagcagcacgtcagctggttcaacatgatgatctaggctaatttttctgagtcttagattttgagaaaacccaggattgctttgtgcactcctgggttttcactttgttaagcagttttggggaaaagtgcaaagtttgcaaagtttagaaatattttaagaggtaagatgtctgcaggtggaagcgtttaaatgcgttaaacttggccaaatgtggcaacctttgcaaggtgaaaaactggggcggggttagatcctggggggtttatttcattcactttggcttgaagtcgtgcaggtcaggggagtgttgcccgaaaacattgagaggaaaacaaaaaccgatgtttgattgggggaatcgggggttacgatactaggacgcagtgactgctatcacccttggcggtctcttgttgaaaggaataattactctaacctttctgtaaaaagccccgcgtcttcctcatggaggaggcggggctttttgggccaagatgggagatgggtgagttggatttggtctgattcgacacttttaagggcagagatttgaagatggagaccaaggctcaaagggaatccatgccgtcttggtttaatactgcacccgtctaatgaaaatcattactattaggtgtcatgatggaccatgcacacgattcctgctcaccaactctgcgccgtgatttggaggtcactggccagctccaacctgagaaagctgtcgatttagcagcgccgcacgaagggaaggttgccaatataacgaaggtgacctcctcaaatatggagcacaccatcacgcaggcctcaaaagctaaggaggtggtggtgctcattggtcactccctgctgcccacatttcaggatttggaaaaagacattctgcactttcaggcaggtaataaagggcgattttctgtagcgattgttgatcctgatcgcagtgcagatgtggttgccagatttaggccaaaacagattccggtggcatacgtggtgaaagatggcgccagcattgcggagttcaactcgctcaacaaggagccgAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCTAGAGGATCGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTTCTGAATTTGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAG.
SEQ ID NO.4：
GCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCGAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATGGGTACCGAGCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCCGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTGCTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGAGGGGACAGGCGAGAGACGATGCCAAAGAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCctggaaatccgtgaagctggcacccacgttgagtacgtcgtcgagcagacccgtaagtcccttgctggcctggatgctgctgagctggccaacaccgttatcgcgtatgagccagtgtgggctatcggcaccggtaaggttgcttccgcagctgacgctcaggaagtgtgcaaggctatccgcggtctgatcgtggagcttgcaggcgacgaggtcgctgagggcctgcgtattctttacggtggttctgttaaggcagaaaccgtcgctgagatcgtcggtcagcctgacgtcgacggcggacttgtcggtggcgcttccctcgacggtgaagcattcgccaagctggctgccaacgctgcgagcgttgcttaaagtacagagctttaaagcacagccttaaagcacagccttaaagcacaagcactgtagaagtgcggttttgatgagcccatgaaagccatcgaaatcaatcgcccagctaaacacctgttttgctgggtgattttttatctcatgcacgccaacaccctcaatgtgaaagagtgtttaaagtagttatccagccggctgggtagtactcgtgtatactgtctaaagttattcgaaatcaggtgggcataaggttcacctgggttctcaaacggcaaaggaacattttccacatggcattgacgcttcaaatcatcctcgtcgtcgccagcctgctcatgacggttttcgtcttgctgcacaagggcaaaggcggcggactctccagcctcttcggtggcggtgtgcagtccaatctttcgggctccactgttgttgaaaagaacctggatcgcgtcaccattttggttgccgttatctggattgtgtgcattgtcgcactcaacctcatccagacttattcataagacacgagcttaaaaagagcggttcccttttcataggggagccgcttttttgggttttgtcgacctgttgtctccccactgttcctcggtgtgcactttcgacaccaagatttcggcaaagtggtggtcaaaattggaaaatcttggtgcctaattcacatacattccaattttccctaaggacatctttaaaggggaactgtttcccgacggaacgtggagtctataaaaccgcaggttaaaacgctgccaAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCTAGAGGATCGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTTCTGAATTTGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAG.
SEQ ID NO.5：
GCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCGAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATGGGTACCGAGCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCCGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTGCTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGAGGGGACAGGCGAGAGACGATGCCAAAGAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCcggtgcccgtggaattgttgatcacgatgtggtcgcggcgcagggctcccgtgcggcgcatttcatgaatgacaagatccacctgatcgcttaaggatcgtcccggaaccataccgataaaaatacgaatcggttcatgtgtttccgtgtcagaaagcctggttaccgtgatgatgtcatctttgcgtgggcctcccgacaacactgcacggccctgcgaacccaccgcaccccatttttcatacgaccacgggctacccgaacgctccggctcccacggctgctcagttcctgggaacaccatgcgattaaggtgcgctgcttgaattgcagaattatgcaagatgcgccgcaacaaaacgcgatcggccaaggtcaaagtggtcaatgtaatgaccgaaaccgctgcgatgaaacttatccacggcggtaaaaacctctcaattaggagcttgacctcattaatactgtgctgggttaattcgccggtgatcagcagcgcgccgtaccccaaggtgccgacactaatgcccgcgatcgtctccttcggtccaaaattcttctgcccaatcagccggatttgggtgcgatgcctgatcaatcccacaaccgtggtggtcaacgtgatggcaccagttgcgatgtgggtggcgttgtaaattttcctggatacccgccggttggttctggggaggatcgagtggattcccgtcgctgccgcatgccccaccgcttgtaaaacagccaggttagcagccgtaacccaccacggtttcggcaacaatgacggcgagagagcccaccacattgcgatttccgctccgataaagccagcgcccatatttgcagggaggattcgcctgcggtttggcgacattcggatccccggaactagctctgcaatgacctgcgcgccgagggaggcgaggtgggtggcaggttttagtgcgggtttaagcgttgccaggcgagtggtgagcagagacgctagtctggggagcgaaaccatGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAAAAAGGAGGCCCTTCAGatgaaagaaaccgtcggtaacaagattgtcctcattggcgcaggagatgttggagttgcatacgcatacgcactgatcaaccagggcatggcagatcaccttgcgatcatcgacatcgatgaaaagaaactcgaaggcaacgtcatggacttaaaccatggtgttgtgtgggccgattcccgcacccgcgtcaccaagggcacctacgctgactgcgaagacgcagccatggttgtcatttgtgccggcgcagcccaaaagccaggcgagacccgcctccagctggtggacaaaaacgtcaagattatgaaatccatcgtcggcgatgtcatggacagcggattcgacggcatcttcctcgtggcgtccaacccagtggatatcctgacctacgcagtgtggaaattctccggcttggaatggaaccgcgtgatcggctccggaactgtcctggactccgctcgattccgctacatgctgggcgaactctacgaagtggcaccaagctccgtccacgcctacatcatcggcgaacacggcgacactgaacttccagtcctgtcctccgcgaccatcgcaggcgtatcgcttagccgaatgctggacaaagacccagagcttgagggccgtctagagaaaattttcgaagacacccgcgacgctgcctatcacattatcgacgccaagggctccacttcctacggcatcggcatgggtcttgctcgcatcacccgcgcaatcctgcagaaccaagacgttgcagtcccagtctctgcactgctccacggtgaatacggtgaggaagacatctacatcggcaccccagctgtggtgaaccgccgaggcatccgccgcgttgtcgaactagaaatcaccgaccacgagatggaacgcttcaagcattccgcaaataccctgcgcgaaattcagaagcagttcttctaaatctttggcgcctagttggcgacgcaagtgtAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCTAGAGGATCGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTTCTGAATTTGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAG.
SEQ ID NO.6：
GCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCGAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATGGGTACCGAGCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCCGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTGCTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGAGGGGACAGGCGAGAGACGATGCCAAAGAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCcggtgcccgtggaattgttgatcacgatgtggtcgcggcgcagggctcccgtgcggcgcatttcatgaatgacaagatccacctgatcgcttaaggatcgtcccggaaccataccgataaaaatacgaatcggttcatgtgtttccgtgtcagaaagcctggttaccgtgatgatgtcatctttgcgtgggcctcccgacaacactgcacggccctgcgaacccaccgcaccccatttttcatacgaccacgggctacccgaacgctccggctcccacggctgctcagttcctgggaacaccatgcgattaaggtgcgctgcttgaattgcagaattatgcaagatgcgccgcaacaaaacgcgatcggccaaggtcaaagtggtcaatgtaatgaccgaaaccgctgcgatgaaacttatccacggcggtaaaaacctctcaattaggagcttgacctcattaatactgtgctgggttaattcgccggtgatcagcagcgcgccgtaccccaaggtgccgacactaatgcccgcgatcgtctccttcggtccaaaattcttctgcccaatcagccggatttgggtgcgatgcctgatcaatcccacaaccgtggtggtcaacgtgatggcaccagttgcgatgtgggtggcgttgtaaattttcctggatacccgccggttggttctggggaggatcgagtggattcccgtcgctgccgcatgccccaccgcttgtaaaacagccaggttagcagccgtaacccaccacggtttcggcaacaatgacggcgagagagcccaccacattgcgatttccgctccgataaagccagcgcccatatttgcagggaggattcgcctgcggtttggcgacattcggatccccggaactagctctgcaatgacctgcgcgccgagggaggcgaggtgggtggcaggttttagtgcgggtttaagcgttgccaggcgagtggtgagcagagacgctagtctggggagcgaaaccattagctgccaattattccgggcttgtgacccgctacccgataaataggtcggctgaaaaatttcgttgcaatatcaacaaaaaggcctatcattgggaggtgtcgcaccaagtacttttgcgaagcgccatctgacggattttcaaaagatgtatatgctcggtgcggaaacctacgaaaggattttttacccatgaaagaaaccgtcggtaacaagattgtcctcattggcgcaggagatgttggagttgcatacgcatacgcactgatcaaccagggcatggcagatcaccttgcgatcatcgacatcgatgaaaagaaactcgaaggcaacgtcatggacttaaaccatggtgttgtgtgggccgattcccgcacccgcgtcaccaagggcacctacgctgactgcgaagacgcagccatggttgtcatttgtgccggcgcagcccaaaagccaggcgagacccgcctccagctggtggacaaaaacgtcaagattatgaaatccatcgtcggcgatgtcatggacagcggattcgacggcatcttcctcgtggcgtccaacccagtggatatcctgacctacgcagtgtggaaattctccggcttggaatggaaccgcgtgatcggctccggaactgtcctggactccgctcgattccgctacatgctgggcgaactctacgaagtggcaccaagctccgtccacgcctacatcatcggcgaacacggcgacactgaacttccagtcctgtcctccgcgaccatcgcaggcgtatcgcttagccgaatgctggacaaagacccagagcttgagggccgtctagagaaaattttcgaagacacccgcgacgctgcctatcacattatcgacgccaagggctccacttcctacggcatcggcatgggtcttgctcgcatcacccgcgcaatcctgcagaaccaagacgttgcagtcccagtctctgcactgctccacggtgaatacggtgaggaagacatctacatcggcaccccagctgtggtgaaccgccgaggcatccgccgcgttgtcgaactagaaatcaccgaccacgagatggaacgcttcaagcaAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCTAGAGGATCGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTTCTGAATTTGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAG.
SEQ ID NO.7：
GCAACTACAGCTCAGGCGACAACCATACGCTGAGAGATCCTCACTACGTAGAAGATAAAGGCCACAAATACTTAGTATTTGAAGCAAACACTGGAACTGAAGATGGCTACCAAGGCGAAGAATCTTTATTTAACAAAGCATACTATGGCAAAAGCACATCATTCTTCCGTCAAGAAAGTCAAAAACTTCTGCAAAGCGATAAAAAACGCACGGCTGAGTTAGCAAACGGCGCTCTCGGTATGATTGAGCTAAACGATGATTACACACTGAAAAAAGTGATGAAACCGCTGATTGCATCTAACACAGTAACAGATGAAATTGAACGCGCGAACGTCTTTAAAATGAACGGCAAATGGTACCTGTTCACTGACTCCCGCGGATCAAAAATGACGATTGACGGCATTACGTCTAACGATATTTACATGCTTGGTTATGTTTCTAATTCTTTAACTGGCCCATACAAGCCGCTGAACAAAACTGGCCTTGTGTTAAAAATGGATCTTGATCCTAACGATGTAACCTTTACTTACTCACACTTCGCTGTACCTCAAGCGAAAGGAAACAATGTCGTGATTACAAGCTATATGACAAACAGAGGATTCTACGCAGACAAACAATCAACGTTTGCGCCGAGCTTCCTGCTGAACATCAAAGGCAAGAAAACATCTGTTGTCAAAGACAGCATCCTTGAACAAGGACAATTAACAGTTAACAAATAAAAACGCAAAAGAAAATGCCGATGGGTACCGAGCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCCGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTGCTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGAGGGGACAGGCGAGAGACGATGCCAAAGAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCcggtgcccgtggaattgttgatcacgatgtggtcgcggcgcagggctcccgtgcggcgcatttcatgaatgacaagatccacctgatcgcttaaggatcgtcccggaaccataccgataaaaatacgaatcggttcatgtgtttccgtgtcagaaagcctggttaccgtgatgatgtcatctttgcgtgggcctcccgacaacactgcacggccctgcgaacccaccgcaccccatttttcatacgaccacgggctacccgaacgctccggctcccacggctgctcagttcctgggaacaccatgcgattaaggtgcgctgcttgaattgcagaattatgcaagatgcgccgcaacaaaacgcgatcggccaaggtcaaagtggtcaatgtaatgaccgaaaccgctgcgatgaaacttatccacggcggtaaaaacctctcaattaggagcttgacctcattaatactgtgctgggttaattcgccggtgatcagcagcgcgccgtaccccaaggtgccgacactaatgcccgcgatcgtctccttcggtccaaaattcttctgcccaatcagccggatttgggtgcgatgcctgatcaatcccacaaccgtggtggtcaacgtgatggcaccagttgcgatgtgggtggcgttgtaaattttcctggatacccgccggttggttctggggaggatcgagtggattcccgtcgctgccgcatgccccaccgcttgtaaaacagccaggttagcagccgtaacccaccacggtttcggcaacaatgacggcgagagagcccaccacattgcgatttccgctccgataaagccagcgcccatatttgcagggaggattcgcctgcggtttggcgacattcggatccccggaactagctctgcaatgacctgcgcgccgagggaggcgaggtgggtggcaggttttagtgcgggtttaagcgttgccaggcgagtggtgagcagagacgctagtctggggagcgaaaccatCTCGAGCAGTAGGCGCGTAGGGTAAGTGGGGTAGCGGCTTGTTAGATATCTTGAAATCGGCTTTCAACAGCATTGATTTCGATGTATTTAGCTGGCCGTTACCCTGCGAATGTCCACAGGGTAGCTGGTAGTTTGAAAATCAACGCCGTTGCCCTTAGGATTCAGTAACTGGCACATTTTGTAATGCGCTAGATCTGTGTGCTCAGTCTTCCAGGCTGCTTATCACAGTGAAAGCAAAACCAATTCGTGGCTGCGAAAGTCGTAGCCACCTCGAGGAAGGAGATATACATatgaaagaaaccgtcggtaacaagattgtcctcattggcgcaggagatgttggagttgcatacgcatacgcactgatcaaccagggcatggcagatcaccttgcgatcatcgacatcgatgaaaagaaactcgaaggcaacgtcatggacttaaaccatggtgttgtgtgggccgattcccgcacccgcgtcaccaagggcacctacgctgactgcgaagacgcagccatggttgtcatttgtgccggcgcagcccaaaagccaggcgagacccgcctccagctggtggacaaaaacgtcaagattatgaaatccatcgtcggcgatgtcatggacagcggattcgacggcatcttcctcgtggcgtccaacccagtggatatcctgacctacgcagtgtggaaattctccggcttggaatggaaccgcgtgatcggctccggaactgtcctggactccgctcgattccgctacatgctgggcgaactctacgaagtggcaccaagctccgtccacgcctacatcatcggcgaacacggcgacactgaacttccagtcctgtcctccgcgaccatcgcaggcgtatcgcttagccgaatgctggacaaagacccagagcttgagggccgtctagagaaaattttcgaagacacccgcgacgctgcctatcacattatcgacgccaagggctccacttcctacggcatcggcatgggtcttgctcgcatcacccgcgcaatcctgcagaaccaagacgttgcagtcccagtctctgcactgctccacggtgaatacggtgaggaagacatctacatcggcaccccagctgtggtgaaccgccgaggcatccgccgcgttgtcgaactagaaatcaccgaccacgagatggaacgcttcaagcattccgcaaataccctgcgcgaaattcagaagcagttcttctaaatctttggcgcctagttggcgacgcaagtgtAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCTAGAGGATCGATCCTTTTTAACCCATCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATCAAGTTTCTGAATTTGATTCGTCCACAATTAAAAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACGCTGACGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGACAGCTGGAAAAACGCTGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGATTCTATCCTAAAAGACCAAACACAAGAATGGTCAGGTTCAGCCACATTTACATCTGACGGAAAAATCCGTTTATTCTACACTGATTTCTCCGGTAAACATTACGGCAAACAAACACTGACAACTGCACAAGTTAACGTATCAGCATCAGACAGCTCTTTGAACATCAACGGTGTAGAGGATTATAAATCAATCTTTGACGGTGACGGAAAAACGTATCAAAATGTACAGCAGTTCATCGATGAAG.
SEQ ID NO.8：
cagcggttaccgcactttttttccaggtgatttcaccctgaccagcgaagcggcactttagtgcatgaggtgtgcccctggtttcccctctttggagggttcaacccaaaaaagcacacaagcaaaaatgaaaatcatcatgagcaagttggtgcgaagcagcaacgcgctagctccaaaaaggtctccaggatctcgaggagatttttgagggggagggagtcgaggaagagccagagcagaaggcggggaaccgttctctgccgacagcgtgagccccccttaaaaatcaggccggggaggaaccggggagggatcagagctaggagcgagacaccctaaagggggggaaccgttttctgctgacggtgtttcgtttattagttttcagcccgtggatagcggagggtgagggcaagtgagagccagagcaaggacgggacccctaaaggggggaaccgttttctgctgacggtgtttcgtttattagttttcagcccgtggacggccgcgtttagcttccattccaagtgcctttctgacttgttggatgcgcctttcactgacacctagttcgcctgcaagctcacgagtcgagggatcagcaaccgattgagaacgggcatccaggatcgcagttttgacgcgaagttcgagcaactcgcctgtcatttctcggcgtttgtttgcttccgctaatcgctgtcgcgtctcctgcgcatacttactttctgggtcagcccatctgcgtgcattcgatgtagctgcgccccgtcgccccatcgtcgctagagctttccgccctcggctgctctgcgtttccacccgacgagcagggacgactggctggcctttagccacgtagccgcgcacacgacgcgccatcgtcaggcgatcacgcatggcgggaagatccggctcccggccgtctgcaccgaccgcctgggcaacgttgtacgccacttcatacgcgtcgatgatcttggcatcttttaggcgctcaccagcagctttgagctggtatcccacggtcaacgcgtggcgaaacgcggtctcgtcgcgcgctcgctctggatttgtccagagcactcgcacgccgtcgatcaggtcgccggacgcgtccagggcgctcggcaggctcgcgtccaaaatcgctagcgccttggcttctgcggtggcgcgttgtgccgcttcaatgcgggcgcgtccgctggaaaagtcctgctcaatgtactttttcggcttctgtgatccggtcatcgttcgagcaatctccattaggtcggccagccgatccacacgatcatgctggcagtgccatttataggctgtcggatcgtctgagacgtgcagcggccaccggctcagcctatgcgaaaaagcctggtcagcgccgaaaacacgagtcatttcttccgtcgttgcagccagcaggcgcatatttgggctggttttacctgctgcggcatacaccgggtcaatgagccagatgagctggcatttcccgctcagcggattcacgccgatccaagccggcgctttttctaggcgtgcccatttctctaaaatcgcgtagacctgcgggtttacgtgctcaatcttcccgccggcctggtggctgggcacatcgatgtcaagcacgatcaccgcggcatgttgcgcgtgcgtcagcgcaacgtactggcaccgcgtcagcgcttttgagccagcccggtagagctttggttgggtttcgccggtatccgggtttttaatccaggcgctcgcgaaatctcttgtcttgctgccctggaagctttcgcgtcccaggtgagcgagcagttcgcggcgatcttctgccgtccagccgcgtgagccgcagcgcatagcttcggggtgggtgtcgaacagatcggcggacaatttccacgcgctagctgtgactgtgtcctgcggatcggctagagtcatgtcttgagtgctttctcccagctgatgactgggggttagccgacgccctgtgagttcccgctcacggggcgttcaactttttcaggtatttgtgcagcttatcgtgttttcttcgtaaatgaacgcttaactaccttgttaaacgtggcaaataggcaggattgatggggatctagcttcacgctgccgcaagcactcagggcgcaagggctgctaaaggaagcggaacacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactccaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcggatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgcggaatcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagcagatcaattcgcgcgcgaaggcgaagcggcATGCATTTACGTTGACACCACGCAACGCAATTAATGTGAGTTAGCGCGAATTGATCTGGTTTGACAGCTTATCATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAAAAAGGAGGCCCTTCAGatgtatatcgggatagatcttggcacctcgggcgtaaaagttattttgctcaacgagcagggtgaggtggttgctgcgcaaacggaaaagctgaccgtttcgcgcccgcatccactctggtcggaacaagacccggaacagtggtggcaggcaactgatcgcgcaatgaaagctctgggcgatcagcattctctgcaggacgttaaagcattgggtattgccggccagatgcacggagcaaccttgctggatgctcagcaacgggtgttacgccctgccattttgtggaacgacgggcgctgtgcgcaagagtgcactttgctggaagcgcgagttccgcaatcgcgggtgattaccggcaacctgatgatgcccggatttactgcgcctaaattgctatgggttcagcggcatgagccggagatattccgtcaaatcgacaaagtattattaccgaaagattacttgcgtctgcgtatgacgggggagtttgccagcgatatgtctgacgcagctggcaccatgtggctggatgtcgcaaagcgtgactggagtgacgtcatgctgcaggcttgcgacttatctcgtgaccagatgcccgcattatacgaaggcagcgaaattactggtgctttgttacctgaagttgcgaaagcgtggggtatggcgacggtgccagttgtcgcaggcggtggcgacaatgcagctggtgcagttggtgtgggaatggttgatgctaatcaggcaatgttatcgctggggacgtcgggggtctattttgctgtcagcgaagggttcttaagcaagccagaaagcgccgtacatagcttttgccatgcgctaccgcaacgttggcatttaatgtctgtgatgctgagtgcagcgtcgtgtctggattgggccgcgaaattaaccggcctgagcaatgtcccagctttaatcgctgcagctcaacaggctgatgaaagtgccgagccagtttggtttctgccttatctttccggcgagcgtacgccacacaataatccccaggcgaagggggttttctttggtttgactcatcaacatggccccaatgaactggcgcgagcagtgctggaaggcgtgggttatgcgctggcagatggcatggatgtcgtgcatgcctgcggtattaaaccgcaaagtgttacgttgattgggggcggggcgcgtagtgagtactggcgtcagatgctggcggatatcagcggtcagcagctcgattaccgtacggggggggatgtggggccagcactgggcgcagcaaggctggcgcagatcgcggcgaatccagagaaatcgctcattgaattgttgccgcaactaccgttagaacagtcgcatctaccagatgcgcagcgttatgccgcttatcagccacgacgagaaacgttccgtcgcctctatcagcaacttctgccattaatggcgtaaTTAAGGCAGTTATTGGTGCCCATGCGAGAGTAGGGAACTGccaggcatcaaataaaacgaaaggcccagtctttcgactgagcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactcttttgtttatttttctaaatacattcaaatatgtatccgctcatgaattaattccgctagatgacgtgcggcttcgacctcctgggcgtggcgcttgttggttatttgtcgaacagataatggtttaccagattttccagttgttcctggcgaccactctgatgcaccggagacaaatgatgttcctgagcatatttggctaaatctgccagtgacatttggcctttcaggatttgctggcccaattcgctattccagccggaataacgctgcgcgatgcgtttatccagctcgccatcttcaatcatgcgcgctgcaattttcagcgccagtgccatcgtatccatcgcgccgatatgaccgtaaaacagatcatatttatcagtactttgacgacgtactttggcatcgaagttcagaccaccggtggtgaaaccgcctgctttgagaatttcatacatcaccagcgcattctcttccacactgttcgggaactggtcggtgtcccagcccagttgcgcatcgccacggttggcgtcgacagaaccgaacaggccaagcgcaatggcggtggctatttcatgatggaaagagtgacctgccagcgtcgcgtggttagcttcaatgttcagtttaatctctttttccagaccaaactgtttcaggaagccatagaccgtcgcggcatcgtaatcatattgatgtttggtcggttcttgcggtttcggttcgataagcaacgtgccctggaaaccgattttatgtttatgctcaaccaccatctgcataaagcggcccagttgttcacgctcctgacgcaagtcggtatttaacagcgtttcgtaaccttcacgaccgccccacaggacatagttttcaccgcccaatttatgggttgcttccatcgctgtaacaacttgcgttgccgcccagctgaagacttcaggatctgggttcgtcgccgcacccgcgccgtagcgagggtttgtaaagcagttggccgttccccacagcagcttcacgccgctctcttcttgcttgcctgccaggacatcaaccatttgcgcaaaattattgatgtactcttttaacgacgcgccctcaggggaaacatccacatcgtggaagcaataaaatggcacatgtaacttgtggaaaaactcaaatgcgacatctgctttacgcttcgccaacgccagtgcctcaccaggctgctgccacggacgattaaacgcccccacaccaaacatatccgccccgttccagcagaaggtgtgccagtagcaggcggcaaaacgcaagtgctcttccatacgcttacccaacaccagttcgtcgggattgtagtgacggaatgctaacgggtttgaggattttgagccttcataacgaacgcgatcgagctggtcaaaataggcttgcatCTGAAGGGCCTCCTTTTTGTTATCCGCTCACAATTCCACACATTATACGAGCCGATGATTAATTGTCAAcgcgctcgcggctggctgcggcacgacacgcgtctgagcagtattttgcgcgccgtcctcgtgggtcaggccggggtgggatcaggccaccgcagtaggcgcagctgatgcgatcctccactactgcgcgtcctcctggcgctgccgagcacgcagctcgtcggccagctcttcaaggtcggccacaagcgtttctaggtcgctcgcggcacttgcccagtcgcgtgatgctggcgcgtctgtcgtatcgagggcgcggaaaaatccgatcaccgtttttaaatcgacggcggcatcgagtgcgtcggactccagcgcgacatcggagagatccaccgctgatgcttcaggccagttttggtacttcgtcgtgaaggtcatgacaccattataacgaacgttcgttaaaaattctagccccaattctgataatttcttccggcactcctgcgaaaacctgcgagacttcttgcccagaaaaaacgccaagcg.
In the preferred embodiment of the application, corynebacterium glutamicum is taken as an initial strain, the metabolic pathways of the acetic acid poxB, pta-acka and succinic acid ppc are knocked out, a Psod promoter is screened, and the expression of the L-lactate dehydrogenase is enhanced through promoter engineering; finally, the xylose isomerase xylA and xylB genes are expressed and constructed on the basis of knocking out the strain, the mixed solution of glucose and xylose is successfully utilized, and the corynebacterium glutamicum is initiated to utilize lignocellulose hydrolysate to simultaneously metabolize glucose and xylose to efficiently synthesize lactic acid.
From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:
1) The byproducts of the lactic acid metabolic pathway are knocked out, so that the conversion efficiency and the yield of lactic acid are improved;
2) Through promoter engineering, the lactate dehydrogenase is overexpressed, so that the lactate upper limit valve is improved;
3) The use of PK18msB and PEKI genetic manipulation tools for C.glutamicum engineering is provided;
4) The xylose utilization way is constructed, lignocellulose is used as a corynebacterium glutamicum production raw material, the cost is reduced, the low-carbon concept is met, and the limited lower occupation competition of industrial raw material glucose and human beings is reduced;
5) The sugar acid conversion efficiency is improved.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A construction method of recombinant corynebacterium glutamicum for producing L-lactic acid, which is characterized by comprising the step of introducing xylose isomerase gene and xylulokinase gene into corynebacterium glutamicum.
2. The method according to claim 1, wherein the xylose isomerase gene is xylA and the xylulokinase gene is xylB.
3. The construction method according to claim 1, further comprising the following modifications to the corynebacterium glutamicum: knocking out a gene encoding acetic acid of corynebacterium glutamicum, and/or knocking out a gene encoding succinic acid of corynebacterium glutamicum.
4. A method of construction according to claim 3, wherein the gene encoding acetic acid is poxB, pta and/or acka; the gene encoding succinic acid is ppc and/or pyc.
5. The method according to any one of claims 1 to 4, wherein the expression of L-lactate dehydrogenase is enhanced using a strong promoter.
6. The method of claim 5, wherein the strong promoter is one or more of Psod, ptuf or Ptac.
7. The method according to claim 6, wherein the strong promoter is used in place of the L-lactate dehydrogenase promoter Pldh for enhanced expression of L-lactate.
8. A recombinant corynebacterium glutamicum obtained by the method of constructing a recombinant corynebacterium glutamicum producing L-lactic acid according to any one of claims 1 to 7.
9. A method for producing L-lactic acid, comprising: (1) Fermenting and culturing the recombinant corynebacterium glutamicum of claim 8; (2) isolating and harvesting L-lactic acid.
10. The method according to claim 9, wherein the fermentation culture takes lignocellulose as a raw material for producing L-lactic acid by the recombinant corynebacterium glutamicum.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118272415A true CN118272415A (en) | 2024-07-02 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0737742A2 (en) | Pentose fermentation by recombinant zymomonas | |
| CN114480240B (en) | Genetic engineering bacterium for producing fucosyllactose and production method thereof | |
| JP5714904B2 (en) | Production of amino acids from sucrose in Corynebacterium luglutamicum | |
| JP4124270B1 (en) | Glucose / Mannose / Xylose Parallel Fermentative Bacteria and Method for Producing Bioethanol Using the Same | |
| CN109207373B (en) | Microbial strain for high yield of citric acid and method for producing citric acid by fermenting starch sugar through microbial strain | |
| CN112063572B (en) | Recombinant escherichia coli capable of highly producing O-acetyl-L-homoserine and application thereof | |
| JP5496356B2 (en) | Xylitol producing strain introduced with arabinose metabolic pathway and xylitol producing method using the same | |
| Wang et al. | Propionic acid fermentation | |
| CN113564193A (en) | Microorganism gene expression fate community and construction method and application thereof | |
| CN116064345A (en) | Non-antibiotic genetic engineering bacteria for efficiently producing fucosyllactose and application thereof | |
| Mhatre et al. | Corynebacterium glutamicum as an efficient omnivorous microbial host for the bioconversion of lignocellulosic biomass | |
| CN113249238B (en) | Acid-resistant saccharomyces cerevisiae and application thereof in preparation of organic acid | |
| CN111411066B (en) | Double-way composite neuraminic acid-producing bacillus subtilis and construction method thereof | |
| CN112430560B (en) | 2-keto-L-gulonic acid production strain and construction method thereof | |
| CN111662832A (en) | Construction method and application of heat-resistant yeast engineering strain for producing xylitol under high-temperature aerobic condition | |
| CN111394410A (en) | High-catalytic-activity neuraminic acid synthase and application thereof | |
| CN113430127B (en) | Recombinant bacterium for producing L-lactic acid and application thereof | |
| CN118272415A (en) | Recombinant corynebacterium glutamicum for producing L-lactic acid, construction method and application thereof | |
| CN116676243A (en) | Construction method and application of recombinant escherichia coli producing 2' -fucosyllactose | |
| WO2019231393A1 (en) | D-psicose production using probiotic microorganisms | |
| CN116926089A (en) | Method for producing shikimic acid by whole cell catalysis | |
| CN113832092A (en) | Genetic engineering bacterium for improving yield of lactoyl-N-fucopentaose and production method thereof | |
| CN114456964B (en) | Recombinant yarrowia lipolytica for high yield of stigmasterol, construction method thereof, fermentation medium for producing stigmasterol and application | |
| CN113897405B (en) | Method for synthesizing 3' -sialyllactose by three-strain coupling fermentation at low cost | |
| CN118272397A (en) | Gene combination, genetically engineered bacterium containing same and application of genetically engineered bacterium in L-lysine production |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication |