CN112852795A - Psicose 3-epimerase mutant, engineering bacterium for expressing mutant and application - Google Patents
Psicose 3-epimerase mutant, engineering bacterium for expressing mutant and application Download PDFInfo
- Publication number
- CN112852795A CN112852795A CN202010496928.9A CN202010496928A CN112852795A CN 112852795 A CN112852795 A CN 112852795A CN 202010496928 A CN202010496928 A CN 202010496928A CN 112852795 A CN112852795 A CN 112852795A
- Authority
- CN
- China
- Prior art keywords
- mutant
- psicose
- epimerase
- mutation
- amino acid
- 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.)
- Granted
Links
- LKDRXBCSQODPBY-JDJSBBGDSA-N D-allulose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@H]1O LKDRXBCSQODPBY-JDJSBBGDSA-N 0.000 title claims abstract description 64
- 241000894006 Bacteria Species 0.000 title claims abstract description 40
- BJHIKXHVCXFQLS-PUFIMZNGSA-N D-psicose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PUFIMZNGSA-N 0.000 claims abstract description 32
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 29
- 229930091371 Fructose Natural products 0.000 claims abstract description 25
- 239000005715 Fructose Substances 0.000 claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 21
- 238000010353 genetic engineering Methods 0.000 claims abstract description 14
- 102220350767 c.470T>A Human genes 0.000 claims description 72
- 102220034241 rs483352780 Human genes 0.000 claims description 58
- 230000000694 effects Effects 0.000 claims description 47
- 230000035772 mutation Effects 0.000 claims description 46
- 238000006243 chemical reaction Methods 0.000 claims description 35
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 30
- 244000063299 Bacillus subtilis Species 0.000 claims description 24
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 24
- 239000013598 vector Substances 0.000 claims description 22
- 150000007523 nucleic acids Chemical class 0.000 claims description 17
- 241000588724 Escherichia coli Species 0.000 claims description 16
- 102000039446 nucleic acids Human genes 0.000 claims description 16
- 108020004707 nucleic acids Proteins 0.000 claims description 16
- 102220480322 Copper-transporting ATPase 2_Y44A_mutation Human genes 0.000 claims description 12
- 125000000539 amino acid group Chemical group 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 12
- 238000002835 absorbance Methods 0.000 claims description 10
- 102200016128 rs34631763 Human genes 0.000 claims description 10
- 102220486824 Gap junction beta-2 protein_D117H_mutation Human genes 0.000 claims description 9
- 238000006555 catalytic reaction Methods 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000006467 substitution reaction Methods 0.000 claims description 8
- 230000003197 catalytic effect Effects 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 241000276408 Bacillus subtilis subsp. subtilis str. 168 Species 0.000 claims description 5
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 claims description 5
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 5
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 5
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 3
- 102100026974 Sorbitol dehydrogenase Human genes 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 239000005515 coenzyme Substances 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 108010020957 ribitol 2-dehydrogenase Proteins 0.000 claims description 3
- 101100083746 Caenorhabditis elegans pmk-3 gene Proteins 0.000 claims description 2
- 101100184605 Escherichia coli mobA gene Proteins 0.000 claims description 2
- 101100391699 Pseudomonas viridiflava gacA gene Proteins 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 238000006345 epimerization reaction Methods 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 101150055347 repA2 gene Proteins 0.000 claims description 2
- 101150107738 repB gene Proteins 0.000 claims description 2
- 101150057627 trxB gene Proteins 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 54
- 102000004190 Enzymes Human genes 0.000 abstract description 54
- 238000000855 fermentation Methods 0.000 abstract description 18
- 230000004151 fermentation Effects 0.000 abstract description 18
- 238000013537 high throughput screening Methods 0.000 abstract description 7
- QYMFNZIUDRQRSA-UHFFFAOYSA-N dimethyl butanedioate;dimethyl hexanedioate;dimethyl pentanedioate Chemical compound COC(=O)CCC(=O)OC.COC(=O)CCCC(=O)OC.COC(=O)CCCCC(=O)OC QYMFNZIUDRQRSA-UHFFFAOYSA-N 0.000 description 55
- 108090000623 proteins and genes Proteins 0.000 description 18
- 102000004169 proteins and genes Human genes 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 11
- 210000003000 inclusion body Anatomy 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 8
- 230000003248 secreting effect Effects 0.000 description 7
- 230000028327 secretion Effects 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 230000006872 improvement Effects 0.000 description 5
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- BJHIKXHVCXFQLS-PQLUHFTBSA-N keto-D-tagatose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-PQLUHFTBSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000002741 site-directed mutagenesis Methods 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- 102000008300 Mutant Proteins Human genes 0.000 description 3
- 108010021466 Mutant Proteins Proteins 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 108090001066 Racemases and epimerases Proteins 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
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 239000008057 potassium phosphate buffer Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 229960004793 sucrose Drugs 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 238000002742 combinatorial mutagenesis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 235000021472 generally recognized as safe Nutrition 0.000 description 2
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 210000004767 rumen Anatomy 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000003765 sweetening agent Substances 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- COEXAQSTZUWMRI-STQMWFEESA-N (2s)-1-[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound C([C@H](N)C(=O)NCC(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=C(O)C=C1 COEXAQSTZUWMRI-STQMWFEESA-N 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- VNYMOTCMNHJGTG-JBDRJPRFSA-N Ala-Ile-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O VNYMOTCMNHJGTG-JBDRJPRFSA-N 0.000 description 1
- YCRAFFCYWOUEOF-DLOVCJGASA-N Ala-Phe-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 YCRAFFCYWOUEOF-DLOVCJGASA-N 0.000 description 1
- YEBZNKPPOHFZJM-BPNCWPANSA-N Ala-Tyr-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O YEBZNKPPOHFZJM-BPNCWPANSA-N 0.000 description 1
- JSHVMZANPXCDTL-GMOBBJLQSA-N Arg-Asp-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JSHVMZANPXCDTL-GMOBBJLQSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- MSBDSTRUMZFSEU-PEFMBERDSA-N Asn-Glu-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O MSBDSTRUMZFSEU-PEFMBERDSA-N 0.000 description 1
- SEMWSADZTMJELF-BYULHYEWSA-N Asp-Ile-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O SEMWSADZTMJELF-BYULHYEWSA-N 0.000 description 1
- HJCGDIGVVWETRO-ZPFDUUQYSA-N Asp-Lys-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC(O)=O)C(O)=O HJCGDIGVVWETRO-ZPFDUUQYSA-N 0.000 description 1
- BJDHEININLSZOT-KKUMJFAQSA-N Asp-Tyr-Lys Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(O)=O BJDHEININLSZOT-KKUMJFAQSA-N 0.000 description 1
- ALMIMUZAWTUNIO-BZSNNMDCSA-N Asp-Tyr-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ALMIMUZAWTUNIO-BZSNNMDCSA-N 0.000 description 1
- LKHMGNHQULEPFY-ACZMJKKPSA-N Cys-Ser-Glu Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O LKHMGNHQULEPFY-ACZMJKKPSA-N 0.000 description 1
- BJHIKXHVCXFQLS-UYFOZJQFSA-N D-fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- 108030002106 D-psicose 3-epimerases Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102100025101 GATA-type zinc finger protein 1 Human genes 0.000 description 1
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 description 1
- UWKPRVKWEKEMSY-DCAQKATOSA-N Gln-Lys-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O UWKPRVKWEKEMSY-DCAQKATOSA-N 0.000 description 1
- KUBFPYIMAGXGBT-ACZMJKKPSA-N Gln-Ser-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O KUBFPYIMAGXGBT-ACZMJKKPSA-N 0.000 description 1
- ITYRYNUZHPNCIK-GUBZILKMSA-N Glu-Ala-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O ITYRYNUZHPNCIK-GUBZILKMSA-N 0.000 description 1
- RDDSZZJOKDVPAE-ACZMJKKPSA-N Glu-Asn-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RDDSZZJOKDVPAE-ACZMJKKPSA-N 0.000 description 1
- QJCKNLPMTPXXEM-AUTRQRHGSA-N Glu-Glu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O QJCKNLPMTPXXEM-AUTRQRHGSA-N 0.000 description 1
- VGBSZQSKQRMLHD-MNXVOIDGSA-N Glu-Leu-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VGBSZQSKQRMLHD-MNXVOIDGSA-N 0.000 description 1
- IDEODOAVGCMUQV-GUBZILKMSA-N Glu-Ser-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O IDEODOAVGCMUQV-GUBZILKMSA-N 0.000 description 1
- YQAQQKPWFOBSMU-WDCWCFNPSA-N Glu-Thr-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O YQAQQKPWFOBSMU-WDCWCFNPSA-N 0.000 description 1
- RZMXBFUSQNLEQF-QEJZJMRPSA-N Glu-Trp-Asn Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N RZMXBFUSQNLEQF-QEJZJMRPSA-N 0.000 description 1
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 description 1
- 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 description 1
- DWUKOTKSTDWGAE-BQBZGAKWSA-N Gly-Asn-Arg Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DWUKOTKSTDWGAE-BQBZGAKWSA-N 0.000 description 1
- CQZDZKRHFWJXDF-WDSKDSINSA-N Gly-Gln-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CN CQZDZKRHFWJXDF-WDSKDSINSA-N 0.000 description 1
- XVYKMNXXJXQKME-XEGUGMAKSA-N Gly-Ile-Tyr Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 XVYKMNXXJXQKME-XEGUGMAKSA-N 0.000 description 1
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 1
- GAFKBWKVXNERFA-QWRGUYRKSA-N Gly-Phe-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 GAFKBWKVXNERFA-QWRGUYRKSA-N 0.000 description 1
- CUVBTVWFVIIDOC-YEPSODPASA-N Gly-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)CN CUVBTVWFVIIDOC-YEPSODPASA-N 0.000 description 1
- AFMOTCMSEBITOE-YEPSODPASA-N Gly-Val-Thr Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O AFMOTCMSEBITOE-YEPSODPASA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- BIAKMWKJMQLZOJ-ZKWXMUAHSA-N His-Ala-Ala Chemical compound C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)Cc1cnc[nH]1)C(O)=O BIAKMWKJMQLZOJ-ZKWXMUAHSA-N 0.000 description 1
- ZJSMFRTVYSLKQU-DJFWLOJKSA-N His-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N ZJSMFRTVYSLKQU-DJFWLOJKSA-N 0.000 description 1
- KYFGGRHWLFZXPU-KKUMJFAQSA-N His-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N KYFGGRHWLFZXPU-KKUMJFAQSA-N 0.000 description 1
- VUUFXXGKMPLKNH-BZSNNMDCSA-N His-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CC3=CN=CN3)N VUUFXXGKMPLKNH-BZSNNMDCSA-N 0.000 description 1
- LEDRIAHEWDJRMF-CFMVVWHZSA-N Ile-Asn-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 LEDRIAHEWDJRMF-CFMVVWHZSA-N 0.000 description 1
- SLQVFYWBGNNOTK-BYULHYEWSA-N Ile-Gly-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)N)C(=O)O)N SLQVFYWBGNNOTK-BYULHYEWSA-N 0.000 description 1
- LPFBXFILACZHIB-LAEOZQHASA-N Ile-Gly-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)O)C(=O)O)N LPFBXFILACZHIB-LAEOZQHASA-N 0.000 description 1
- KOPIAUWNLKKELG-SIGLWIIPSA-N Ile-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N KOPIAUWNLKKELG-SIGLWIIPSA-N 0.000 description 1
- OVDKXUDMKXAZIV-ZPFDUUQYSA-N Ile-Lys-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N)C(=O)O)N OVDKXUDMKXAZIV-ZPFDUUQYSA-N 0.000 description 1
- XMYURPUVJSKTMC-KBIXCLLPSA-N Ile-Ser-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N XMYURPUVJSKTMC-KBIXCLLPSA-N 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- SUPVSFFZWVOEOI-UHFFFAOYSA-N Leu-Ala-Tyr Natural products CC(C)CC(N)C(=O)NC(C)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 SUPVSFFZWVOEOI-UHFFFAOYSA-N 0.000 description 1
- REPPKAMYTOJTFC-DCAQKATOSA-N Leu-Arg-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O REPPKAMYTOJTFC-DCAQKATOSA-N 0.000 description 1
- IGUOAYLTQJLPPD-DCAQKATOSA-N Leu-Asn-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IGUOAYLTQJLPPD-DCAQKATOSA-N 0.000 description 1
- HPBCTWSUJOGJSH-MNXVOIDGSA-N Leu-Glu-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HPBCTWSUJOGJSH-MNXVOIDGSA-N 0.000 description 1
- XBCWOTOCBXXJDG-BZSNNMDCSA-N Leu-His-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CN=CN1 XBCWOTOCBXXJDG-BZSNNMDCSA-N 0.000 description 1
- WXZOHBVPVKABQN-DCAQKATOSA-N Leu-Met-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)O)C(=O)O)N WXZOHBVPVKABQN-DCAQKATOSA-N 0.000 description 1
- JLYUZRKPDKHUTC-WDSOQIARSA-N Leu-Pro-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O JLYUZRKPDKHUTC-WDSOQIARSA-N 0.000 description 1
- WFCKERTZVCQXKH-KBPBESRZSA-N Leu-Tyr-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O WFCKERTZVCQXKH-KBPBESRZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- GQUDMNDPQTXZRV-DCAQKATOSA-N Lys-Arg-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O GQUDMNDPQTXZRV-DCAQKATOSA-N 0.000 description 1
- IMAKMJCBYCSMHM-AVGNSLFASA-N Lys-Glu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN IMAKMJCBYCSMHM-AVGNSLFASA-N 0.000 description 1
- GFWLIJDQILOEPP-HSCHXYMDSA-N Lys-Ile-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CCCCN)N GFWLIJDQILOEPP-HSCHXYMDSA-N 0.000 description 1
- RBEATVHTWHTHTJ-KKUMJFAQSA-N Lys-Leu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O RBEATVHTWHTHTJ-KKUMJFAQSA-N 0.000 description 1
- SUZVLFWOCKHWET-CQDKDKBSSA-N Lys-Tyr-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O SUZVLFWOCKHWET-CQDKDKBSSA-N 0.000 description 1
- OZVXDDFYCQOPFD-XQQFMLRXSA-N Lys-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCCN)N OZVXDDFYCQOPFD-XQQFMLRXSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- QXEVZBXTDTVPCP-GMOBBJLQSA-N Met-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCSC)N QXEVZBXTDTVPCP-GMOBBJLQSA-N 0.000 description 1
- UZVWDRPUTHXQAM-FXQIFTODSA-N Met-Asp-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O UZVWDRPUTHXQAM-FXQIFTODSA-N 0.000 description 1
- UOENBSHXYCHSAU-YUMQZZPRSA-N Met-Gln-Gly Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O UOENBSHXYCHSAU-YUMQZZPRSA-N 0.000 description 1
- OOSPRDCGTLQLBP-NHCYSSNCSA-N Met-Glu-Val Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OOSPRDCGTLQLBP-NHCYSSNCSA-N 0.000 description 1
- QZPXMHVKPHJNTR-DCAQKATOSA-N Met-Leu-Asn Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O QZPXMHVKPHJNTR-DCAQKATOSA-N 0.000 description 1
- UROWNMBTQGGTHB-DCAQKATOSA-N Met-Leu-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O UROWNMBTQGGTHB-DCAQKATOSA-N 0.000 description 1
- HOZNVKDCKZPRER-XUXIUFHCSA-N Met-Lys-Ile Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HOZNVKDCKZPRER-XUXIUFHCSA-N 0.000 description 1
- AOFZWWDTTJLHOU-ULQDDVLXSA-N Met-Lys-Tyr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AOFZWWDTTJLHOU-ULQDDVLXSA-N 0.000 description 1
- JOYFULUKJRJCSX-IUCAKERBSA-N Met-Met-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)NCC(O)=O JOYFULUKJRJCSX-IUCAKERBSA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- WIVCOAKLPICYGY-KKUMJFAQSA-N Phe-Asp-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N WIVCOAKLPICYGY-KKUMJFAQSA-N 0.000 description 1
- FIRWJEJVFFGXSH-RYUDHWBXSA-N Phe-Glu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 FIRWJEJVFFGXSH-RYUDHWBXSA-N 0.000 description 1
- LWPMGKSZPKFKJD-DZKIICNBSA-N Phe-Glu-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O LWPMGKSZPKFKJD-DZKIICNBSA-N 0.000 description 1
- YRHRGNUAXGUPTO-PMVMPFDFSA-N Phe-Trp-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)N[C@@H](CCCCN)C(=O)O)N YRHRGNUAXGUPTO-PMVMPFDFSA-N 0.000 description 1
- SWXSLPHTJVAWDF-VEVYYDQMSA-N Pro-Asn-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SWXSLPHTJVAWDF-VEVYYDQMSA-N 0.000 description 1
- XYAFCOJKICBRDU-JYJNAYRXSA-N Pro-Phe-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O XYAFCOJKICBRDU-JYJNAYRXSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 241001508466 Pseudomonas cichorii Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000192031 Ruminococcus Species 0.000 description 1
- 241000134861 Ruminococcus sp. Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- RNFKSBPHLTZHLU-WHFBIAKZSA-N Ser-Cys-Gly Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)O)N)O RNFKSBPHLTZHLU-WHFBIAKZSA-N 0.000 description 1
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 0.000 description 1
- PTWIYDNFWPXQSD-GARJFASQSA-N Ser-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CO)N)C(=O)O PTWIYDNFWPXQSD-GARJFASQSA-N 0.000 description 1
- WLJPJRGQRNCIQS-ZLUOBGJFSA-N Ser-Ser-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O WLJPJRGQRNCIQS-ZLUOBGJFSA-N 0.000 description 1
- XQJCEKXQUJQNNK-ZLUOBGJFSA-N Ser-Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O XQJCEKXQUJQNNK-ZLUOBGJFSA-N 0.000 description 1
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 1
- KZUJCMPVNXOBAF-LKXGYXEUSA-N Thr-Cys-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(O)=O KZUJCMPVNXOBAF-LKXGYXEUSA-N 0.000 description 1
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 1
- ZSPQUTWLWGWTPS-HJGDQZAQSA-N Thr-Lys-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O ZSPQUTWLWGWTPS-HJGDQZAQSA-N 0.000 description 1
- GYUUYCIXELGTJS-MEYUZBJRSA-N Thr-Phe-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O GYUUYCIXELGTJS-MEYUZBJRSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- OBAMASZCXDIXSS-SZMVWBNQSA-N Trp-Glu-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N OBAMASZCXDIXSS-SZMVWBNQSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- IELISNUVHBKYBX-XDTLVQLUSA-N Tyr-Ala-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 IELISNUVHBKYBX-XDTLVQLUSA-N 0.000 description 1
- KSVMDJJCYKIXTK-IGNZVWTISA-N Tyr-Ala-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 KSVMDJJCYKIXTK-IGNZVWTISA-N 0.000 description 1
- NLMXVDDEQFKQQU-CFMVVWHZSA-N Tyr-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NLMXVDDEQFKQQU-CFMVVWHZSA-N 0.000 description 1
- LYPKCSYAKLTBHJ-ILWGZMRPSA-N Tyr-Trp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CC4=CC=C(C=C4)O)N)C(=O)O LYPKCSYAKLTBHJ-ILWGZMRPSA-N 0.000 description 1
- KRXFXDCNKLANCP-CXTHYWKRSA-N Tyr-Tyr-Ile Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 KRXFXDCNKLANCP-CXTHYWKRSA-N 0.000 description 1
- COSLEEOIYRPTHD-YDHLFZDLSA-N Val-Asp-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 COSLEEOIYRPTHD-YDHLFZDLSA-N 0.000 description 1
- PIFJAFRUVWZRKR-QMMMGPOBSA-N Val-Gly-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O PIFJAFRUVWZRKR-QMMMGPOBSA-N 0.000 description 1
- XXROXFHCMVXETG-UWVGGRQHSA-N Val-Gly-Val Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXROXFHCMVXETG-UWVGGRQHSA-N 0.000 description 1
- MLADEWAIYAPAAU-IHRRRGAJSA-N Val-Lys-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N MLADEWAIYAPAAU-IHRRRGAJSA-N 0.000 description 1
- VENKIVFKIPGEJN-NHCYSSNCSA-N Val-Met-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N VENKIVFKIPGEJN-NHCYSSNCSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 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 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 238000000113 differential scanning calorimetry Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 108010076718 lysyl-glutamyl-tryptophan Proteins 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000000291 postprandial effect Effects 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000009962 secretion pathway Effects 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000021092 sugar substitutes Nutrition 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 108700004896 tripeptide FEG Proteins 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000001515 vagal effect Effects 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a psicose 3-epimerase mutant and a genetic engineering bacterium for expressing the mutant. The invention screens the psicose 3-epimerase mutant which can be efficiently expressed in the fermentation process by a high-throughput screening method, can catalyze fructose to be efficiently converted into D-psicose, provides an efficient production path of key enzymes required in the production process of the D-psicose, obviously reduces the production cost of the D-psicose, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of industrial biology, and particularly relates to a psicose 3-epimerase mutant, a construction method of a genetic engineering strain secreting and expressing psicose 3-epimerase, and application of the genetic engineering strain in fermentation production of a psicose 3-epimerase preparation.
Background
D-psicose (D-allulose) is an epimer at the carbon-3 position of D-fructose, is a 'healthy sugar' with the functions of improving lipid metabolism, reducing postprandial blood sugar and zero calorie, has 70 percent of cane sugar sweetness, and has the taste and the characteristics close to those of cane sugar, thereby being an ideal substitute of cane sugar. Related studies have shown that D-psicose can decrease the body's absorption of fructose and glucose in the diet by competitively inhibiting the ingress and egress of transporters, with a zero-glycemic response in rats. Most of D-psicose is absorbed by small intestine, enters blood, participates in circulation, and is finally discharged out of body in the form of urine through kidney, and the blood sugar content has no obvious fluctuation in the process. Recent reports indicate that D-psicose can induce the release of GLP-1, thereby activating vagal afferent signals to limit feeding and hyperglycemia. Therefore, the D-psicose can be used as sugar substitute for obesity and diabetes, and can also be used as an ideal sweetener for healthy people. In addition, the D-psicose is added in the food processing process, so that the gel property of the food can be effectively improved, the oxidation degree of the Maillard reaction in the food processing process is reduced, and the flavor of the food is improved. The U.S. Food and Drug Administration (FDA) has certified D-psicose as GRAS (generally recognized as safe) as a component of food additives. The D-psicose has wide application prospect in the fields of food, health care, medicine and the like.
Psicose 3-epimerase (or tagatose 3-epimerase) is capable of epimerizing D-fructose to D-psicose, and is a key enzyme in the biological production of psicose. At present, a large number of psicose 3-epimerases of microbial origin have been discovered and reported. With the progress of the research, the protein structures of some important psicose 3-epimerases, such as psicose 3-epimerases derived from Agrobacterium tumefaciens, tagatose 3-epimerases derived from Pseudomonas cichorii, and the like, were resolved. Based on the important information provided by the protein structures, researchers have also directed evolution on some key enzymes to improve the activity and stability of the enzymes, so that the enzymes are more suitable for industrial production.
The high-efficiency enzyme production process is another important factor for determining whether the enzyme can be really applied to industrial production. Researchers use host bacteria such as escherichia coli, bacillus subtilis, corynebacterium glutamicum and the like as starting strains to construct engineering strains for heterologous expression of the psicose 3-epimerase. The bacillus subtilis has high-efficiency protein expression and secretion functions, and is an ideal host bacterium for producing the key enzyme by fermentation. Chinese patent CN105602925B demonstrates that D-psicose 3-epimerase (RDPE) derived from rumen bacteria (Ruminococcus sp) is efficiently secreted extracellularly in bacillus subtilis through a non-classical secretion pathway. The secretion characteristic of RDPE can greatly reduce the cost of enzyme production by fermentation, thereby reducing the production cost of the final product allulose. Chinese patent CN105602879B constructs a Bacillus subtilis engineering strain capable of efficiently secreting RDPE. There is still a need to develop a mutant psicose 3-epimerase that has high catalytic activity, strong stability, and is easier to produce by fermentation.
Disclosure of Invention
The invention provides a psicose 3-epimerase mutant, and also relates to a construction method of a genetic engineering strain secreting and expressing the psicose 3-epimerase, and application of the genetic engineering strain in fermentation production of a psicose 3-epimerase preparation.
The invention adopts the following technical scheme:
the present invention provides a psicose 3-epimerase mutant having an activity of catalyzing epimerization of D-fructose into D-psicose, the amino acid sequence of which comprises a mutation of an amino acid residue at least one position among S36, Y44, D117, F157, C165, I196, Q251, I265 corresponding to SEQ ID No. 1.
According to an embodiment of the invention, the amino acid sequence of the mutant comprises a mutation of an amino acid residue at any two of positions S36, Y44, D117, F157, C165, I196, Q251, I265. In one embodiment, the amino acid sequence of the mutant comprises mutations at the above two positions, and at least one of the mutation positions is any one of F157, C165, Q251, I265; preferably, one of the mutation sites of the amino acid sequence of the mutant is F157 site, and the other mutation site is any one of C165, Q251 and I265. In yet another embodiment, the amino acid sequence of the mutant comprises mutations at two positions: Y44/F157, Y44/I196, D117/I196, F157/C165A, F157/Q251, F157/I265, or I196/Q251.
According to an embodiment of the invention, the amino acid sequence of the mutant comprises a mutation of an amino acid residue at any three of positions S36, Y44, D117, F157, C165, I196, Q251, I265. In one embodiment, the amino acid sequence of the mutant comprises a mutation at F157/C165 site, and a mutation at any one of S36, Y44, D117, I196, Q251, I265; preferably, the amino acid sequence of the mutant comprises a mutation at the F157/C165/I196 site, or the mutant comprises a mutation at the F157/C165/Q251 site.
According to an embodiment of the invention, the mutation of the amino acid residue in at least one of the positions S36, Y44, D117, F157, C165, I196, Q251, I265 is selected from the group consisting of substitution of the residue in at least one of the positions S36N, Y44A, D117H, F157Y, C165A, I196F, Q251T, I265L. In one embodiment, the amino acid sequence of the mutant includes the F157Y mutation; in one embodiment, the amino acid sequence of the mutant comprises any one mutation site combination of Y44A/F157Y, Y44A/I196F, D117H/I196F, F157Y/C165A, F157Y/Q251T, F157Y/I265L and I196F/Q251T, preferably F157Y/C165A. In one embodiment, the amino acid sequence of the mutant comprises any one mutation site combination of F157Y/C165A/S36N, F157Y/C165A/Y44A, F157Y/C165A/D117H, F157Y/C165A/I196F, F157Y/C165A/Q251T, F157Y/C165A/I265L, preferably F157Y/C539165 165A/I196F or F157Y/C165A/Q251T.
According to an embodiment of the invention, the mutant has more than 70% homology, such as more than 80% homology, further such as more than 90%, more than 95%, more than 98% homology with the amino acid sequence shown in SEQ ID NO. 1.
The present invention also provides a nucleic acid encoding the above-described psicose 3-epimerase mutant.
The invention also provides a genetically engineered bacterium expressing the psicose 3-epimerase mutant, which comprises a nucleic acid encoding the psicose 3-epimerase mutant.
According to the embodiment of the invention, the genetically engineered bacterium is a recombinant strain obtained by connecting the nucleic acid with a vector to obtain a recombinant vector and then introducing the recombinant vector into a host bacterium.
According to an embodiment of the present invention, the host bacterium is any one of escherichia coli, bacillus subtilis, corynebacterium glutamicum, lactic acid bacteria, or yeast species, for example, bacillus subtilis such as b.subtilis 168, WB600, WB800N, 1a751, FZB24, SCK 6; or E.coli such as E.coli BL21(DE3), BL21(DE3) pLysS, Rosetta (DE3), Endotoxin-Free BL21(DE3), BL21 trxB (DE3) and JM 109.
According to an embodiment of the present invention, the vector may be selected from vectors carrying a repB Bacillus subtilis replicon, such as any one of pHP13-43, pHT43, pHT304, pMK3, pMK4, pHCMC04, pHCMC05, pMA5, pHY300PLK, pYH-P43. Preferably, the vector is selected from the group consisting of the bacillus subtilis expression vector pNWP43N containing the constitutive expression promoter P43.
According to an embodiment of the invention, the nucleic acid is ligated to the vector by ligase or PCR recombination to form a recombinant vector.
According to an embodiment of the present invention, the genetically engineered bacterium expresses a psicose 3-epimerase mutant.
The invention also provides a construction method of the genetic engineering bacteria, which comprises the steps of connecting the nucleic acid with a vector to obtain a recombinant vector, and then introducing the recombinant vector into host bacteria to obtain a recombinant strain.
The invention provides application of the genetic engineering bacteria in preparation of a psicose 3-epimerase mutant.
The invention further provides a preparation method of the psicose 3-epimerase mutant, which comprises the step of culturing the genetic engineering bacteria to express the nucleic acid for coding the psicose 3-epimerase mutant.
According to an embodiment of the invention, the temperature of the cultivation is 35-40 ℃, preferably 37 ℃; the culture time is 24-72h, preferably 48 h.
According to an embodiment of the present invention, the culturing is performed under stirring or shaking conditions, for example, at a stirring speed of 100-.
According to an embodiment of the present invention, the preparation method further comprises the step of isolating the psicose 3-epimerase mutant from the culture.
The invention also provides application of the psicose 3-epimerase mutant in preparation of D-psicose, wherein the mutant takes fructose as a substrate to perform catalytic reaction.
The invention further provides a production method of D-psicose, which comprises the step of contacting the psicose 3-epimerase mutant with fructose to perform catalytic reaction.
According to an embodiment of the invention, the temperature of the catalytic reaction is between 40 and 80 ℃; preferably, the reaction system has a fructose mass volume concentration of 20-80%, for example 50%.
In one embodiment, the method for producing D-psicose comprises the steps of: fructose is added into a culture medium, the genetic engineering bacteria are cultured, and D-psicose is separated from the culture.
In still another preferred embodiment, the method for producing D-psicose comprises the steps of: culturing the genetically engineered bacterium of the invention to express a nucleic acid encoding the psicose 3-epimerase mutant; isolating the psicose 3-epimerase mutant from the culture; adding the separated psicose 3-epimerase mutant into fructose for catalytic reaction to obtain D-psicose.
The invention also provides a screening method of the high-activity mutant of the psicose 3-epimerase, which comprises the following steps:
(1) establishing a mutant library, and constructing, culturing and separating strains;
(2) adding fructose for reaction;
(3) adding ribitol dehydrogenase (KRDH) and coenzyme NADH to perform a second reaction to obtain a reaction solution;
(4) detecting the absorbance change of the reaction solution at 340nm, and selecting the mutant with high catalytic activity.
In the step (4), the more the absorbance decrease trend is obvious, the more D-psicose is generated, which indicates that the catalytic activity of the mutant is higher.
The screening method further comprises the step of carrying out rescreening by detecting the content of the D-psicose in the reaction solution through high performance liquid chromatography.
Advantageous effects
According to the invention, a high-throughput screening method of the mutant is constructed according to the catalytic property of the psicose 3-epimerase, and a batch of mutants of the psicose 3-epimerase are screened out, the specific activity, the thermal stability and the heterologous expression water average of the mutant are obviously improved compared with those of wild type, and the mutant can be efficiently expressed in escherichia coli and bacillus subtilis. Therefore, the invention also provides the genetic engineering bacteria capable of secreting and expressing the psicose, the psicose 3-epimerase activity in the fermentation liquor of the genetic engineering bacteria can reach 1436U/mL, the high-efficiency production of key enzymes required in the production process of the D-psicose is realized, the production cost of the psicose is obviously reduced, and the genetic engineering bacteria have wide application prospects.
Drawings
FIG. 1 is a schematic diagram of a high throughput screening strategy for mutants;
FIG. 2 shows the results of determination of denaturation temperature (Tm) of RDPE mutants from wild type;
FIG. 3 shows the results of comparing the amount of inclusion bodies produced by heterologous expression of the RDPE mutant and the wild type in E.coli;
FIG. 4 shows the comparison of the relative enzyme activities of the RDPE mutant F157Y/C165A/I196F and the wild type at different temperatures (30, 40, 50, 60, 70, 80 ℃);
FIG. 5 shows the expression of the RDPE mutant in the engineered Bacillus subtilis strain B-3-1 (M: protein molecular weight standard, 1: 18h fermentation supernatant, 2: 24h fermentation supernatant, 3: 30h fermentation supernatant, 4: 42h fermentation supernatant).
Detailed Description
Amino acids in the present invention are represented by a single or three letter code and have the following meanings: a: ala (alanine); r: arg (arginine); n: asn (asparagine); d: asp (aspartic acid); c: cys (cysteine); q: gln (glutamine); e: glu (glutamic acid); g: gly (glycine); h: his (histidine); i: ile (isoleucine); l: leu (leucine); k: lys (lysine); m: met (methionine); f: phe (phenylalanine); p: pro (proline); s: ser (serine); t: thr (threonine); w: trp (tryptophan); y: tyr (tyrosine); v: val (valine).
In the present invention, "homology" has the conventional meaning in the art and refers to "identity" between two nucleic acid or amino acid sequences, the percentage of which represents the statistically significant percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment (best alignment), the differences between the two sequences being randomly distributed over their entire length.
Within the context of the present invention, the variants are described in terms of their mutation at a specific residue, the position of which is determined by alignment with the wild-type enzyme sequence SEQ ID NO.1 or by reference to the enzyme sequence SEQ ID NO. 1. In the context of the present invention, it also relates to any variant carrying these same mutations at functionally equivalent residues.
In the present invention, the terms "primer" and "primer strand" are used interchangeably and refer to an initial nucleic acid fragment, typically an RNA oligonucleotide, DNA oligonucleotide or chimeric sequence that is complementary to a primer binding site formed by all or part of a target nucleic acid molecule. The primer strand may comprise natural, synthetic or modified nucleotides. The lower limit of the primer length is the minimum length required for a stable duplex to form under the conditions of the nucleic acid amplification reaction.
In the present invention, the terms "mutant" and "variant" are used interchangeably, and "modification" or "mutation" refers to an amino acid sequence of a wild-type protein, such as the wild-type sequence SEQ ID NO:1, the psicose 3-epimerase from ruminobacteria, or a sequence derived from such an enzyme, comprising alterations, i.e. substitutions, insertions and/or deletions, at one or more positions, and still retaining its activity. Mutants can be obtained by various techniques known in the art. Exemplary techniques for modifying a DNA sequence encoding a wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis, and construction of synthetic oligonucleotides, whereby the modified DNA sequence is expressed in a host bacterium to yield mutants having amino acid sequence substitutions, insertions, and/or deletions. In the present invention, the expression "the mutant includes a mutation at position … …" or "the amino acid sequence of the mutant includes a mutation at position … …" has the same meaning, and indicates that a substitution, insertion and/or deletion occurs at a specific site in the amino acid sequence of the mutein. The term "substitution" with respect to an amino acid position or residue means that the amino acid at the specified position has been replaced with another amino acid. Substitutions may be conservative or non-conservative.
The term "corresponding to" as used herein has the meaning commonly understood by a person of ordinary skill in the art. Specifically, "corresponding to" means the position of one sequence corresponding to a specified position in the other sequence after alignment of the two sequences by homology or sequence identity. Thus, for example, in the case of "amino acid residue corresponding to position 40 of the amino acid sequence shown in SEQ ID NO: 1", if a 6 XHis tag is added to one end of any of the amino acid sequences shown in SEQ ID NO:1, position 40 of the resulting mutant corresponding to the amino acid sequence shown in SEQ ID NO:1 may be position 46 of the mutant. In one embodiment, the mutation site is determined according to homology alignment software, e.g., where the sequence is substituted or deleted at a site other than the X site of the particular mutation site as compared to SEQ ID NO:1, but where the homology alignment software corresponds the mutated amino acid residue to the X site of SEQ ID NO:1, then the sequence still belongs to the sequence corresponding to SEQ ID NO:1, where the X site is mutated.
In a specific embodiment, the homology or sequence identity may be 90% or more, preferably 95% or more, more preferably 98% homology. The mutation site and its substitution are expressed herein by the position number of the mutation site and the amino acid type of the site, for example, S36N indicates that the serine at the position corresponding to the 36 th position of SEQ ID NO:1 is substituted with asparagine in alignment with SEQ ID NO: 1. In the present invention, "/" is used to indicate a combination of mutation sites, for example, "Y44/F157" indicates that both tyrosine at position 44 and phenylalanine at position 157 are mutated, and comprises two mutation sites, and the mutation sites are double mutants. By analogy, "F157/C165/I196" indicates that the corresponding three sites are mutated simultaneously, and is a triple mutant.
As used herein, "psicose" and "D-psicose", "fructose" and "D-fructose" all have the same meaning and are used interchangeably.
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Example 1 establishment of a high throughput screening method for mutants
Firstly, the Escherichia coli expressing RDPE is intracellular enzyme, the thallus is required to be crushed, the crushed supernatant is taken as crude enzyme liquid, and a substrate is added for reaction. In order to improve the efficiency, the catalytic reaction can be carried out by means of a whole-cell reaction. Meanwhile, the inventor finds that the RDPE gene has a certain background expression in E.coli BL21 host cells, and the protein expression amount without IPTG meets the enzyme activity determination requirement, so that the step of adding IPTG for induction can be omitted.
Secondly, the reaction catalyzed by the RDPE is a reversible conversion reaction between fructose and psicose, enzyme activity can be calculated only in a high-efficiency liquid phase mode, a large amount of detection time is consumed, and the method is not suitable for high-throughput screening. Therefore, ribitol dehydrogenase (KRDH) derived from Klebsiella oxide and coenzyme NADH were added to the whole-cell transformation reaction solution to carry out the second reaction. Because KRDH has reducing capacity to allulose but not to fructose, and NADH is converted into NAD + in the reaction process, the generation amount of fructose converted into allulose under the catalysis of RDPE in the first round of reaction can be determined by detecting the absorbance change of the reaction solution at 340 nm. The more pronounced the tendency to decrease in absorbance, the higher the allulose formed.
Based on the two aspects, the invention establishes a strategy for efficiently screening the RDPE mutant library by the enzyme-linked reaction, optimizes conditions such as reaction mode, determination mode, reaction temperature and the like, and finally realizes high-throughput screening of the mutant of the psicose 3-epimerase or the tagatose 3-epimerase, wherein the process schematic diagram is shown in figure 1. The specific method comprises the following steps:
(1) selecting transformant inoculation bacteria from a mutant library, placing the transformant inoculation bacteria in a 96-hole deep-hole plate, culturing for 16h at 37 ℃ and 600rpm, wherein a culture medium is an LB culture medium containing 100 mu g/ml ampicillin;
(2) centrifuging to remove supernatant, adding 100 μ l of 100mM potassium phosphate buffer solution with pH7.5 to resuspend thallus, adding 1% fructose solution, and reacting at 50 deg.C and 300rpm for 1 h;
(3) centrifuging, sucking 100 μ l of the supernatant into a 96-well plate, adding 100 μ l of 50mM pH7.5 potassium phosphate buffer containing 10U KRDH and 2mM NADH, and mixing;
(4) and (3) placing the 96-well plate in a microplate reader, setting the temperature to be 30 ℃, performing kinetic determination at 340nm, wherein the more violent the decrease of absorbance, the higher the content of D-psicose in the solution is, and the higher the enzyme activity of the RDPE is indirectly reacted.
Example 2 improvement of enzyme Activity of RDPE by site-directed saturation mutagenesis and combinatorial mutagenesis
Carrying out homology comparison analysis on the amino acid sequences of psicose epimerase (RDPE) from rumen bacteria and psicose epimerase and tagatose epimerase reported in a Genbank database; and simultaneously, carrying out structure prediction on the RDPE, carrying out homologous modeling on the wild type protein of the enzyme by using software such as Swiss-Model, Phyre2, Discovery Studio and the like, carrying out molecular docking by using fructose molecules as substrates, thereby predicting the catalytic sites and the substrate binding sites of the wild type enzyme, and analyzing the action of amino acid residues near the sites, thereby designing the amino acid sequence of the RDPE mutant. Through the analysis, S36, G38, Y44, L110, D117, F122, F157, C165, D194, I196, L207, G215, V244, Q251 and I265 corresponding to SEQ ID No.1 are selected as mutation sites to carry out saturation mutation, and a single-site saturation mutation library is established.
Then, the high-throughput screening strategy in example 1 is used to screen transformants in the mutant library, 8 mutants with 1.2 times higher enzyme activity are screened from more than 3000 transformants and sequenced. After sequence analysis, corresponding mutant expression strains are cultured, 0.1mM IPTG is added for induction, then a Ni column is used for purifying mutant protein, and high performance liquid chromatography is used for re-screening mutants with improved enzyme activity. The specific method comprises the following steps:
using 8% fructose (potassium phosphate buffer, pH8.0, containing 1mM Mn2+) as a substrate, RDPE wild type and mutant pure enzyme were added at appropriate concentrations, and the reaction was carried out at 60 ℃ for 20 min. After the reaction is finished, the reaction product is treated in a boiling water bath for 5min, and the content of the product D-psicose is measured by HPLC, so that the specific enzyme activity of the RDPE wild type and the mutant is calculated, and the result is shown in Table 1.
TABLE 1 specific enzyme Activity of RDPE wild type and Single mutants
| RDPE wild type or mutant | Specific activity (U/mg) | Relative enzyme activity (%) |
| WT | 406.63 | 100.00 |
| S36N | 513.26 | 126.22 |
| Y44A | 533.23 | 131.13 |
| D117H | 554.58 | 136.38 |
| F157Y | 556.52 | 136.86 |
| C165A | 551.09 | 135.53 |
| I196F | 561.40 | 138.06 |
| Q251T | 508.37 | 125.02 |
| I265L | 516.61 | 127.05 |
And combining the 8 sites in pairs to construct 28 double-mutant expression strains. The mutant protein is purified and activity determination is carried out by HPLC, the result shows that the enzyme activity of 21 double mutants is obviously reduced compared with that of the first round of single mutant, the enzyme activity of F157Y/C165A in the rest seven mutants is obviously improved, the relative enzyme activity is 1.53 times of that of a wild type, and the specific result is shown in Table 2.
TABLE 2 specific enzyme Activity of RDPE wild type and Single mutants
Double mutants F157Y/C165A with the most obvious improvement of enzyme activity are selected to be combined with S36N, Y44A, D117H, I196F, Q251T and I265L respectively to construct 6 triple mutants, mutant proteins are purified, activity determination is carried out by HPLC, and the enzyme activity is shown in Table 3. Thus, the enzyme activity of the triple mutant F157Y/C165A/S36N is obviously reduced, the enzyme activities of F157Y/C165A/I196F and F157Y/C165A/Q251T are obviously improved compared with the wild type, and the enzyme activity of the triple mutant F157Y/C165A is obviously improved compared with that of the double mutant F157Y/C165.
TABLE 3 specific enzyme Activity of RDPE wild type and triple mutants
| RDPE wild type or mutant | Specific activity (U/mg) | Relative enzyme activity (%) |
| WT | 406.63 | 100.00 |
| F157Y/C165A/S36N | 277.72 | 68.30 |
| F157Y/C165A/Y44A | 497.18 | 122.27 |
| F157Y/C165A/D117H | 439.06 | 107.98 |
| F157Y/C165A/I196F | 699.71 | 172.08 |
| F157Y/C165A/Q251T | 677.13 | 166.73 |
| F157Y/C165A/I265L | 480.90 | 118.26 |
Example 3 improvement of RDPE stability by site-directed saturation mutagenesis and combinatorial mutagenesis
Selecting mutants (I196F, F157Y/C165A, F157Y/I265L, F157Y/C165A/I196F, F157Y/C165A/Q251T) with relative enzyme activity of more than 150% from the RDPE single mutant, the double mutant and the triple mutant, and carrying out thermal stability determination. The specific determination method comprises the following steps: heat-treating the RDPE mutant at 60 ℃ for 30 min, 60 min, 120 min and 240min, then determining enzyme activity by taking fructose as a substrate, defining the enzyme activity of each mutant which is not subjected to heat treatment as 100%, and calculating the residual enzyme activity after heat treatment. The results are shown in Table 4, and it can be seen that the thermal stability of the selected five mutants is improved to a certain extent compared with that of the wild type, wherein the thermal stability of the triple mutant F157Y/C165A/I196F is improved most remarkably.
TABLE 4 comparison of the thermal stabilities of the RDPE wild type and the mutants
The protein denaturation temperature (Tm) of the wild type RDPE and the triple mutant F157Y/C165A/I196F with the most significant improvement of enzyme activity and stability are further determined by differential scanning calorimetry, and the results are shown in FIG. 2. The results showed that the Tm of the wild type RDPE was 69.3 ℃ and the protein denaturation temperature of the triple mutant F157Y/C165A/I196F was 79.9 ℃. The significant increase in Tm indicates that the thermostability of the mutant RDPE is significantly enhanced compared to the wild type.
Example 4 site-directed mutagenesis to increase the soluble expression level of RDPE in E.coli
In the above examples, the expression of the RDPE mutant was performed in E.coli BL21, and since the previous experiments found that the expression of the RDPE wild type in E.coli forms inclusion bodies, the induction expression was performed at low temperature and low rotation speed (20 ℃, 100 rpm). In experiments, the generation amount of the inclusion body of the protein is obviously influenced by the mutation of different sites, wherein the generation amount of the inclusion body of the S36N, F157Y, Y44A/F157Y, F157Y/C165A, F157Y/I265L, F157Y/C165A/I196F and F157Y/C165A/Q251T mutants is reduced compared with the wild type, and the generation amount of other mutants is improved compared with the wild type. In order to quantify the generation amount of the inclusion bodies, the suspension after ultrasonic crushing is taken and diluted with water properly, then the absorbance of the suspension is measured at 600nm, the absorbance of the RDPE wild type at 600nm is taken as 1, the relative value of the absorbance of each mutant is calculated, and the turbidity is taken as a quantitative index for measuring the generation amount of the inclusion bodies. FIG. 3 shows the absorbance relative values of each mutant with reduced inclusion body production, and it can be seen that when the triple mutant F157Y/C165A/I196F is expressed in E.coli, the inclusion body production is significantly reduced, and the soluble protein level is increased. Further attempts were made to increase the temperature for induced expression of the mutant to 25 ℃ and the amount of inclusion bodies produced was still small. The reduction of the inclusion body is beneficial to expressing more proteins into an active soluble form, improves the efficiency and the yield of producing the RDPE enzyme by fermenting escherichia coli, and has stronger application prospect.
Example 5 site-directed mutagenesis to increase the secretory expression level of RDPE in Bacillus subtilis
As RDPE can be secreted and expressed in the Bacillus subtilis, the secretory expression levels of mutants F157Y/C165A, F157Y/I265L, F157Y/C165A/I196F and F157Y/C165A/Q251T in the Bacillus subtilis are further analyzed. Constructing expression engineering strains B-0, B-1, B-2, B-3 and B-4 of RDPE wild type and mutant by using pMA5 as a vector and B.subtilis 168 as a host bacterium, culturing for 48h by using an SR culture medium, and determining the enzyme activity of RDPE secreted into a fermentation broth. The results are shown in table 5, and it can be seen that the secretion expression amount of the selected RDPE mutant in bacillus subtilis is significantly improved compared with that of the wild type.
TABLE 5 secretory expression levels of RDPE mutants and wild type in Bacillus subtilis
Example 6 site-directed mutagenesis to increase the optimum reaction temperature and fructose conversion of RDPE
The enzyme activities of the RDPE wild type and the mutant F157Y/C165A/I196F at different temperatures (30, 40, 50, 60, 70 and 80 ℃) are measured, the relative enzyme activity is calculated by taking the highest enzyme activity as 100%, and the influence of the temperature on the enzyme activity is researched, as shown in figure 4. The optimum temperature of the mutant F157Y/C165A/I196F is increased from 60 ℃ to 70 ℃, and the mutant still has relative enzyme activity of more than 65% at 80 ℃, while the wild type has relative enzyme activity of only about 30% at 80 ℃.
Using fructose of 50% concentration as a substrate, 20U of RDPE wild type and mutants F157Y/C165A, F157Y/I265L, F157Y/C165A/I196F and F157Y/C165A/Q251T were added per g of fructose to carry out conversion reactions at 60, 70 and 80 ℃ respectively, and the results of equilibrium conversion measurements were shown in Table 6. The result shows that the thermal stability of the wild type RDPE is poor, the conversion rate is reduced along with the temperature increase, the thermal stability of the 4 mutants is obviously improved compared with that of the wild type RDPE, the conversion rate is gradually increased along with the temperature increase and reaches about 33 percent at most, and the method has a very strong industrial application prospect.
TABLE 6 catalytic efficiency for fructose at different temperatures for RDPE mutants and wild type
| RDPE wild type and mutant | Conversion at 60 (%) | Conversion at 70 degree C (%) | Conversion at 80 ℃ (%) |
| WT | 26.3 | 25.6 | 24.8 |
| F157Y/C165A | 28.6 | 30.1 | 32.5 |
| F157Y/I265L | 28.2 | 29.8 | 31.8 |
| F157Y/C165A/I196F | 30.4 | 31.9 | 33.5 |
| F157Y/C165A/Q251T | 27.9 | 29.5 | 31.2 |
Example 7 construction of Bacillus subtilis secretion engineered Strain
In order to further improve the secretion expression amount of the RDPE mutant F157Y/C165A/I196F in Bacillus subtilis, a Bacillus subtilis expression vector pNWP43N containing a constitutive expression promoter P43 is used, and Bacillus subtilis 168 is used as a host bacterium to construct an expression engineering strain B-3-1 of the RDPE mutant F157Y/C165A/I196F, wherein the specific method is as follows: the gene fragment of the RDPE mutant F157Y/C165A/I196F is amplified by PCR and is connected to the vector in an enzyme digestion mode. Adding the ligation product into B.subtilis 168 competence, mixing uniformly, recovering at 37 ℃ and 200rpm for 1.5h, taking all mixed solution, coating LB plate containing chloramphenicol (25 mug/mL), and verifying colony PCR to obtain the bacillus subtilis secretion engineering strain B-3-1.
Example 8 production of enzyme by fermentation of Bacillus subtilis
Carrying out 5L scale fermentation tank fermentation by using the engineering strain B-3-1, optimizing the fermentation condition, culturing for 48h by using an SR culture medium under the conditions of 37 ℃ and 300rpm, and determining that the RDPE activity in the fermentation liquid reaches 1436U/mL. The results show that the stability of the mutant plasmid of the invention is greatly improved, thereby improving the secretion expression amount of the heterologous protein RDPE. Meanwhile, the RDPE mutant secreted into the culture medium was detected by SDS-PAGE (FIG. 5), and the results showed that the target protein was secreted and expressed in a large amount into the fermentation medium and the host impurity protein was less.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> psicose 3-epimerase mutant, engineering bacterium expressing mutant and application
<130> CPCN20110339
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 291
<212> PRT
<213> Ruminococcus sp.
<400> 1
Met Lys Tyr Gly Ile Tyr Tyr Ala Tyr Trp Glu Lys Glu Trp Asn Gly
1 5 10 15
Asp Tyr Lys Tyr Tyr Ile Asp Lys Ile Ser Lys Leu Gly Phe Asp Ile
20 25 30
Leu Glu Ile Ser Cys Gly Ala Phe Ser Asp Tyr Tyr Thr Lys Asp Gln
35 40 45
Glu Leu Ile Asp Ile Gly Lys Tyr Ala Lys Glu Lys Gly Val Thr Leu
50 55 60
Thr Ala Gly Tyr Gly Pro His Phe Asn Glu Ser Leu Ser Ser Ser Glu
65 70 75 80
Pro Asn Thr Gln Lys Gln Ala Ile Ser Phe Trp Lys Glu Thr Leu Arg
85 90 95
Lys Leu Lys Leu Met Asp Ile His Ile Val Gly Gly Ala Leu Tyr Gly
100 105 110
Tyr Trp Pro Val Asp Tyr Ser Lys Pro Phe Asp Lys Lys Arg Asp Leu
115 120 125
Glu Asn Ser Ile Lys Asn Met Lys Ile Ile Ser Gln Tyr Ala Glu Glu
130 135 140
Tyr Asp Ile Met Met Gly Met Glu Val Leu Asn Arg Phe Glu Gly Tyr
145 150 155 160
Met Leu Asn Thr Cys Asp Glu Ala Leu Ala Tyr Val Glu Glu Val Gly
165 170 175
Ser Ser Asn Val Gly Val Met Leu Asp Thr Phe His Met Asn Ile Glu
180 185 190
Glu Asp Asn Ile Ala Ala Ala Ile Arg Lys Ala Gly Asp Arg Leu Tyr
195 200 205
His Phe His Ile Gly Glu Gly Asn Arg Lys Val Pro Gly Lys Gly Met
210 215 220
Leu Pro Trp Asn Glu Ile Gly Gln Ala Leu Arg Asp Ile Asn Tyr Gln
225 230 235 240
His Ala Ala Val Met Glu Pro Phe Val Met Gln Gly Gly Thr Val Gly
245 250 255
His Asp Ile Lys Ile Trp Arg Asp Ile Ile Gly Asn Cys Ser Glu Val
260 265 270
Thr Leu Asp Met Asp Ala Gln Ser Ala Leu His Phe Val Lys His Val
275 280 285
Phe Glu Val
290
Claims (10)
1. A psicose 3-epimerase mutant having an activity of catalyzing epimerization of D-fructose to D-psicose, the amino acid sequence of which comprises a mutation of an amino acid residue at least one position corresponding to S36, Y44, D117, F157, C165, I196, Q251, I265 of SEQ ID No. 1;
preferably, the mutant has more than 70% homology, such as more than 80% homology, and further such as more than 90%, more than 95%, more than 98% homology with the amino acid sequence shown in SEQ ID NO. 1.
2. The mutant according to claim 1, wherein the amino acid sequence comprises mutation of amino acid residues at any two of positions S36, Y44, D117, F157, C165, I196, Q251, I265;
preferably, at least one mutation site of the mutant is any one of F157, C165, Q251 and I265;
preferably, one of the mutation sites of the amino acid sequence of the mutant is F157 site, and the other mutation site is any one of C165, Q251 and I265;
preferably, the mutant comprises mutations at two positions: Y44/F157, Y44/I196, D117/I196, F157/C165A, F157/Q251, F157/I265, or I196/Q251;
preferably, the amino acid sequence thereof comprises mutation of amino acid residues at any three of positions S36, Y44, D117, F157, C165, I196, Q251, I265;
preferably, the mutant comprises a mutation at F157/C165 site, and a mutation at any one of S36, Y44, D117, I196, Q251, I265;
preferably, the mutant comprises a mutation at the F157/C165/I196 site, or the mutant comprises a mutation at the F157/C165/Q251 site.
3. The mutant according to any one of claims 1-2, wherein the mutation of the amino acid residue at least one of S36, Y44, D117, F157, C165, I196, Q251, I265 is selected from the group consisting of a substitution of the residue at least one of S36N, Y44A, D117H, F157Y, C165A, I196F, Q251T, I265L;
preferably, the amino acid sequence of the mutant comprises any one mutation site combination of Y44A/F157Y, Y44A/I196F, D117H/I196F, F157Y/C165A, F157Y/Q251T, F157Y/I265L and I196F/Q251T;
preferably, the amino acid sequence of the mutant comprises any one mutation site combination of F157Y/C165A/S36N, F157Y/C165A/Y44A, F157Y/C165A/D117H, F157Y/C165A/I196F, F157Y/C165A/Q251T and F157Y/C165A/I265L.
4. Nucleic acid encoding a psicose 3-epimerase mutant according to any one of claims 1 to 3.
5. A genetically engineered bacterium expressing a psicose 3-epimerase mutant comprising a nucleic acid encoding the psicose 3-epimerase mutant of any one of claims 1 to 3;
preferably, the genetic engineering bacteria are recombinant strains obtained by connecting the nucleic acid with a vector to obtain a recombinant vector and then introducing the recombinant vector into host bacteria;
preferably, the host bacterium is any one of escherichia coli, bacillus subtilis, corynebacterium glutamicum, lactic acid bacteria, or yeast, for example, any one selected from b.subtilis 168, WB600, WB800N, 1a751, FZB24, or SCK6 bacillus subtilis; or any one selected from E.coli BL21(DE3), BL21(DE3) pLysS, Rosetta (DE3), Endotoxin-Free BL21(DE3), BL21 trxB (DE3), JM109 E.coli;
preferably, the vector is selected from the group consisting of repB Bacillus subtilis replicon-carrying vectors, such as any one of pHP13-43, pHT43, pHT304, pMK3, pMK4, pHCMC04, pHCMC05, pMA5, pHY300PLK, pYH-P43;
preferably, the vector is selected from the group consisting of bacillus subtilis expression vector pNWP43N containing constitutive expression promoter P43;
preferably, the genetically engineered bacterium expresses a psicose 3-epimerase mutant.
6. The method for constructing a genetically engineered bacterium according to claim 5, comprising the step of ligating the nucleic acid to a vector to obtain a recombinant vector, and introducing the recombinant vector into a host bacterium to obtain a recombinant strain.
7. Use of the genetically engineered bacterium of claim 5 in the preparation of a psicose 3-epimerase mutant.
8. A method for producing a mutant psicose 3-epimerase, comprising the step of culturing the genetically engineered bacterium of claim 7 to express a nucleic acid encoding the mutant psicose 3-epimerase;
preferably, the temperature of the culture is 35-40 ℃; the culture time is 24-72;
preferably, the culturing is performed under stirring or shaking conditions, such as a stirring rotation speed of 100-1000 rpm;
preferably, the preparation method further comprises the step of isolating the psicose 3-epimerase mutant from the culture.
9. A method for producing D-psicose, comprising contacting the psicose 3-epimerase mutant of any one of claims 1 to 3 with fructose to perform a catalytic reaction;
preferably, the temperature of the catalytic reaction is 40-80 ℃;
preferably, in the reaction system, the mass volume concentration of the fructose is 20-80%;
preferably, the method for producing D-psicose comprises the steps of: adding fructose into a culture medium, culturing a genetic engineering bacterium expressing the psicose 3-epimerase mutant, and separating D-psicose from the culture;
preferably, the method for producing D-psicose comprises the steps of: culturing a genetically engineered bacterium to express a nucleic acid encoding the psicose 3-epimerase mutant; isolating the psicose 3-epimerase mutant from the culture; adding the separated psicose 3-epimerase mutant into fructose for catalytic reaction to obtain D-psicose.
10. A screening method of a high-activity mutant of psicose 3-epimerase comprises the following steps:
(1) establishing a mutant library, and constructing, culturing and separating strains;
(2) adding fructose for reaction;
(3) adding ribitol dehydrogenase (KRDH) and coenzyme NADH to perform a second reaction to obtain a reaction solution;
(4) detecting the absorbance change of the reaction solution at 340nm, and selecting a mutant with high catalytic activity;
preferably, the screening method further comprises the step of rescreening by detecting the content of the D-psicose in the reaction solution by high performance liquid chromatography.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010496928.9A CN112852795B (en) | 2020-06-03 | 2020-06-03 | Psicose 3-epimerase mutant, engineering bacterium for expressing mutant and application |
| JP2022506812A JP7348457B2 (en) | 2020-06-03 | 2020-12-15 | Psicose 3-epimerase mutant, genetically engineered bacteria for expressing it, immobilized enzyme and immobilization method |
| EP20938719.0A EP4026903A4 (en) | 2020-06-03 | 2020-12-15 | Allulose 3-epimerase mutant, engineered bacterium expressing same, and immobilized enzyme and immobilization method thereof |
| PCT/CN2020/136574 WO2021244005A1 (en) | 2020-06-03 | 2020-12-15 | Allulose 3-epimerase mutant, engineered bacterium expressing same, and immobilized enzyme and immobilization method thereof |
| US17/753,739 US20220340944A1 (en) | 2020-06-03 | 2020-12-15 | Allulose 3-epimerase mutant, engineered bacterium expressing same, and immobilized enzyme and immobilization method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010496928.9A CN112852795B (en) | 2020-06-03 | 2020-06-03 | Psicose 3-epimerase mutant, engineering bacterium for expressing mutant and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112852795A true CN112852795A (en) | 2021-05-28 |
| CN112852795B CN112852795B (en) | 2022-04-08 |
Family
ID=75996116
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010496928.9A Active CN112852795B (en) | 2020-06-03 | 2020-06-03 | Psicose 3-epimerase mutant, engineering bacterium for expressing mutant and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112852795B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480359A (en) * | 2022-03-22 | 2022-05-13 | 河南中大恒源生物科技股份有限公司 | Method for producing psicose 3-epimerase by high-density fermentation |
| CN114561417A (en) * | 2022-04-27 | 2022-05-31 | 中国科学院天津工业生物技术研究所 | Corynebacterium glutamicum engineering strain for preparing psicose and application thereof |
| CN116004596A (en) * | 2022-10-20 | 2023-04-25 | 天津科技大学 | Acid-resistant epimerase mutant and application thereof |
| CN116676300A (en) * | 2023-04-27 | 2023-09-01 | 江南大学 | High-temperature-stability D-psicose 3-epimerase mutant enzyme and application thereof |
| CN116875626A (en) * | 2023-06-07 | 2023-10-13 | 山东福洋生物科技股份有限公司 | Construction method of genetically engineered bacterium for producing D-psicose by one-step method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952358A (en) * | 2014-05-06 | 2014-07-30 | 中国科学院天津工业生物技术研究所 | Escherichia coli engineered strain for producing allitol by virtue of biotransformation as well as construction method and application thereof |
| CN108239633A (en) * | 2016-12-26 | 2018-07-03 | 上海立足生物科技有限公司 | A kind of mutant for the D-Psicose -3- epimerases that catalytic activity is improved and its application |
-
2020
- 2020-06-03 CN CN202010496928.9A patent/CN112852795B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952358A (en) * | 2014-05-06 | 2014-07-30 | 中国科学院天津工业生物技术研究所 | Escherichia coli engineered strain for producing allitol by virtue of biotransformation as well as construction method and application thereof |
| CN108239633A (en) * | 2016-12-26 | 2018-07-03 | 上海立足生物科技有限公司 | A kind of mutant for the D-Psicose -3- epimerases that catalytic activity is improved and its application |
Non-Patent Citations (2)
| Title |
|---|
| NOBUYUKI URANO ET AL.: "Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 * |
| 宋连明: "生物催化剂的高通量筛选方法", 《河北化工》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480359A (en) * | 2022-03-22 | 2022-05-13 | 河南中大恒源生物科技股份有限公司 | Method for producing psicose 3-epimerase by high-density fermentation |
| CN114561417A (en) * | 2022-04-27 | 2022-05-31 | 中国科学院天津工业生物技术研究所 | Corynebacterium glutamicum engineering strain for preparing psicose and application thereof |
| CN114561417B (en) * | 2022-04-27 | 2022-08-09 | 中国科学院天津工业生物技术研究所 | Corynebacterium glutamicum engineering strain for preparing psicose and application thereof |
| CN116004596A (en) * | 2022-10-20 | 2023-04-25 | 天津科技大学 | Acid-resistant epimerase mutant and application thereof |
| CN116676300A (en) * | 2023-04-27 | 2023-09-01 | 江南大学 | High-temperature-stability D-psicose 3-epimerase mutant enzyme and application thereof |
| CN116676300B (en) * | 2023-04-27 | 2024-03-08 | 江南大学 | High-temperature-stability D-psicose 3-epimerase mutant enzyme and application thereof |
| CN116875626A (en) * | 2023-06-07 | 2023-10-13 | 山东福洋生物科技股份有限公司 | Construction method of genetically engineered bacterium for producing D-psicose by one-step method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112852795B (en) | 2022-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112852795B (en) | Psicose 3-epimerase mutant, engineering bacterium for expressing mutant and application | |
| EP4026903A1 (en) | Allulose 3-epimerase mutant, engineered bacterium expressing same, and immobilized enzyme and immobilization method thereof | |
| CN108018278B (en) | D-psicose 3-epimerase mutant with improved catalytic efficiency | |
| EP3438256A1 (en) | 3-epimerase and polynucleotide encoding same | |
| CN113462678B (en) | Glutamic acid decarboxylase mutant | |
| CN110904088B (en) | High-temperature-resistant D-psicose3-epimerase, mutant and application thereof | |
| CN111394410A (en) | High-catalytic-activity neuraminic acid synthase and application thereof | |
| CN114934035B (en) | Thermophilic acidic III type pullulanase mutant with improved starch degradation capacity as well as preparation method and application thereof | |
| US20030166172A1 (en) | Thermotoga neapolitana xylose isomerase polypeptides and nucleic acids encoding same | |
| CN115806955A (en) | Method for reducing GI value of rice starch and application thereof | |
| CN106119235B (en) | A kind of DPE and its application from bulkholderia cepasea | |
| CN113061593B (en) | L-malate dehydrogenase mutant and application thereof | |
| CN113373135B (en) | Mutant of D-psicose 3-epimerase and application thereof | |
| CN115896081A (en) | Aspartase mutant and application thereof | |
| CN110358745B (en) | 4-xylitol dehydrogenase mutant and application thereof | |
| CN110846288B (en) | Glutathione bifunctional enzyme mutant and application thereof | |
| CN115011622A (en) | Screening method and application of D-psicose 3-epimerase mutant | |
| JP7445947B2 (en) | Arabinose isomerase mutant | |
| CN110452899B (en) | Glucose isomerase, mutant and application of mutant in preparation of D-fructose | |
| CN111057698B (en) | L-arabinose isomerase, mutant and application thereof | |
| CN110904087B (en) | L-arabinose epimerase mutant and application thereof | |
| KR102312807B1 (en) | Amylases having resistance to starch decomposition activity in Bifidobacterium and uses thereof | |
| CN114752581B (en) | Alpha-galactosidase mutant and application thereof | |
| CN113583991B (en) | Amylosucrase SaAS and encoding gene and application thereof | |
| KR20180026820A (en) | A fucose isomerase and a use of the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230810 Address after: Building 1-201-A218, Building 8, Huiying Industrial Park, No. 86 Zhonghuan West Road, Tianjin Binhai New Area Pilot Free Trade Zone (Airport Economic Zone), 300000 Patentee after: Tiangong Biotechnology (Tianjin) Co.,Ltd. Address before: No.32, Xiqi Road, Airport Economic Zone, Binhai New Area, Tianjin 300308 Patentee before: TIANJIN INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY, CHINESE ACADEMY OF SCIENCES |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230902 Address after: Building 1-201-4, Building 8, Huiying Industrial Park, No. 86 Zhonghuan West Road, Tianjin Binhai New Area Pilot Free Trade Zone (Airport Economic Zone), 300000 Patentee after: Tianjin Yihe Biotechnology Co.,Ltd. Address before: Building 1-201-A218, Building 8, Huiying Industrial Park, No. 86 Zhonghuan West Road, Tianjin Binhai New Area Pilot Free Trade Zone (Airport Economic Zone), 300000 Patentee before: Tiangong Biotechnology (Tianjin) Co.,Ltd. |