CN116262915A - 3-异丙基苹果酸脱水酶突变体及其应用 - Google Patents
3-异丙基苹果酸脱水酶突变体及其应用 Download PDFInfo
- Publication number
- CN116262915A CN116262915A CN202111530562.3A CN202111530562A CN116262915A CN 116262915 A CN116262915 A CN 116262915A CN 202111530562 A CN202111530562 A CN 202111530562A CN 116262915 A CN116262915 A CN 116262915A
- Authority
- CN
- China
- Prior art keywords
- valine
- amino acid
- leu
- val
- isopropyl malate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010028984 3-isopropylmalate dehydratase Proteins 0.000 title claims abstract description 32
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims abstract description 61
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims abstract description 28
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000004474 valine Substances 0.000 claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 23
- 230000004151 fermentation Effects 0.000 claims abstract description 23
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims abstract description 22
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 150000001413 amino acids Chemical class 0.000 claims abstract description 15
- 150000005693 branched-chain amino acids Chemical class 0.000 claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 claims abstract description 11
- 230000035772 mutation Effects 0.000 claims abstract description 9
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 13
- 102220533321 Vesicle-associated membrane protein 2_A84I_mutation Human genes 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 9
- 241000186216 Corynebacterium Species 0.000 claims description 8
- 238000010276 construction Methods 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 150000007523 nucleic acids Chemical class 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 239000012620 biological material Substances 0.000 claims description 5
- 241000186146 Brevibacterium Species 0.000 claims description 4
- 241000319304 [Brevibacterium] flavum Species 0.000 claims description 4
- 238000010353 genetic engineering Methods 0.000 claims description 4
- 241000572303 Corynebacterium pekinense Species 0.000 claims description 3
- 108020004511 Recombinant DNA Proteins 0.000 claims description 2
- 239000013600 plasmid vector Substances 0.000 claims description 2
- 241000894007 species Species 0.000 claims description 2
- 239000013603 viral vector Substances 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims 1
- 101150019665 leuD gene Proteins 0.000 abstract description 18
- 229940024606 amino acid Drugs 0.000 abstract description 11
- 235000001014 amino acid Nutrition 0.000 abstract description 11
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 abstract description 9
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 abstract description 9
- 229960000310 isoleucine Drugs 0.000 abstract description 9
- 239000006227 byproduct Substances 0.000 abstract description 7
- 244000005700 microbiome Species 0.000 abstract description 6
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 abstract description 5
- 235000004279 alanine Nutrition 0.000 abstract description 5
- 230000004048 modification Effects 0.000 abstract description 4
- 238000012986 modification Methods 0.000 abstract description 4
- -1 leucine (L) Chemical class 0.000 abstract description 3
- 229960004295 valine Drugs 0.000 description 35
- 229960003136 leucine Drugs 0.000 description 21
- 102200072698 rs772008300 Human genes 0.000 description 13
- 101100123415 Methanosarcina acetivorans (strain ATCC 35395 / DSM 2834 / JCM 12185 / C2A) hacB1 gene Proteins 0.000 description 12
- 101100288829 Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) leuD1 gene Proteins 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- LMFXXZPPZDCPTA-ZKWXMUAHSA-N Ala-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N LMFXXZPPZDCPTA-ZKWXMUAHSA-N 0.000 description 4
- CWEAKSWWKHGTRJ-BQBZGAKWSA-N Ala-Gly-Met Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O CWEAKSWWKHGTRJ-BQBZGAKWSA-N 0.000 description 4
- LDLSENBXQNDTPB-DCAQKATOSA-N Ala-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LDLSENBXQNDTPB-DCAQKATOSA-N 0.000 description 4
- PNIGSVZJNVUVJA-BQBZGAKWSA-N Arg-Gly-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O PNIGSVZJNVUVJA-BQBZGAKWSA-N 0.000 description 4
- CZUHPNLXLWMYMG-UBHSHLNASA-N Arg-Phe-Ala Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 CZUHPNLXLWMYMG-UBHSHLNASA-N 0.000 description 4
- QUBKBPZGMZWOKQ-SZMVWBNQSA-N Arg-Trp-Arg Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCN=C(N)N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 QUBKBPZGMZWOKQ-SZMVWBNQSA-N 0.000 description 4
- ZDOQDYFZNGASEY-BIIVOSGPSA-N Asn-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N)C(=O)O ZDOQDYFZNGASEY-BIIVOSGPSA-N 0.000 description 4
- UYCPJVYQYARFGB-YDHLFZDLSA-N Asn-Phe-Val Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O UYCPJVYQYARFGB-YDHLFZDLSA-N 0.000 description 4
- FLJVGAFLZVBBNG-BPUTZDHNSA-N Asn-Trp-Arg Chemical compound N[C@@H](CC(=O)N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(=N)N)C(=O)O FLJVGAFLZVBBNG-BPUTZDHNSA-N 0.000 description 4
- HPNDBHLITCHRSO-WHFBIAKZSA-N Asp-Ala-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)NCC(O)=O HPNDBHLITCHRSO-WHFBIAKZSA-N 0.000 description 4
- PYXXJFRXIYAESU-PCBIJLKTSA-N Asp-Ile-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PYXXJFRXIYAESU-PCBIJLKTSA-N 0.000 description 4
- GGRSYTUJHAZTFN-IHRRRGAJSA-N Asp-Pro-Tyr Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)O)N)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O GGRSYTUJHAZTFN-IHRRRGAJSA-N 0.000 description 4
- IWLZBRTUIVXZJD-OLHMAJIHSA-N Asp-Thr-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O IWLZBRTUIVXZJD-OLHMAJIHSA-N 0.000 description 4
- JDDYEZGPYBBPBN-JRQIVUDYSA-N Asp-Thr-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JDDYEZGPYBBPBN-JRQIVUDYSA-N 0.000 description 4
- KNDCWFXCFKSEBM-AVGNSLFASA-N Asp-Tyr-Glu 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](CCC(O)=O)C(O)=O KNDCWFXCFKSEBM-AVGNSLFASA-N 0.000 description 4
- 108010090461 DFG peptide Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- RGAOLBZBLOJUTP-GRLWGSQLSA-N Gln-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H](CCC(=O)N)N RGAOLBZBLOJUTP-GRLWGSQLSA-N 0.000 description 4
- DSPQRJXOIXHOHK-WDSKDSINSA-N Glu-Asp-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O DSPQRJXOIXHOHK-WDSKDSINSA-N 0.000 description 4
- VFZIDQZAEBORGY-GLLZPBPUSA-N Glu-Gln-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VFZIDQZAEBORGY-GLLZPBPUSA-N 0.000 description 4
- VGUYMZGLJUJRBV-YVNDNENWSA-N Glu-Ile-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O VGUYMZGLJUJRBV-YVNDNENWSA-N 0.000 description 4
- MWMJCGBSIORNCD-AVGNSLFASA-N Glu-Leu-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O MWMJCGBSIORNCD-AVGNSLFASA-N 0.000 description 4
- NJCALAAIGREHDR-WDCWCFNPSA-N Glu-Leu-Thr 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)O)C(O)=O NJCALAAIGREHDR-WDCWCFNPSA-N 0.000 description 4
- UJMNFCAHLYKWOZ-DCAQKATOSA-N Glu-Lys-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O UJMNFCAHLYKWOZ-DCAQKATOSA-N 0.000 description 4
- YTSVAIMKVLZUDU-YUMQZZPRSA-N Gly-Leu-Asp Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O YTSVAIMKVLZUDU-YUMQZZPRSA-N 0.000 description 4
- JQFILXICXLDTRR-FBCQKBJTSA-N Gly-Thr-Gly Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)NCC(O)=O JQFILXICXLDTRR-FBCQKBJTSA-N 0.000 description 4
- MDOBWSFNSNPENN-PMVVWTBXSA-N His-Thr-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O MDOBWSFNSNPENN-PMVVWTBXSA-N 0.000 description 4
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 4
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 4
- FIJMQLGQLBLBOL-HJGDQZAQSA-N Leu-Asn-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O FIJMQLGQLBLBOL-HJGDQZAQSA-N 0.000 description 4
- BJWKOATWNQJPSK-SRVKXCTJSA-N Leu-Met-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N BJWKOATWNQJPSK-SRVKXCTJSA-N 0.000 description 4
- PTRKPHUGYULXPU-KKUMJFAQSA-N Leu-Phe-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O PTRKPHUGYULXPU-KKUMJFAQSA-N 0.000 description 4
- QWWPYKKLXWOITQ-VOAKCMCISA-N Leu-Thr-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QWWPYKKLXWOITQ-VOAKCMCISA-N 0.000 description 4
- HQVDJTYKCMIWJP-YUMQZZPRSA-N Lys-Asn-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O HQVDJTYKCMIWJP-YUMQZZPRSA-N 0.000 description 4
- MUXNCRWTWBMNHX-SRVKXCTJSA-N Lys-Leu-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O MUXNCRWTWBMNHX-SRVKXCTJSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- DJDFBVNNDAUPRW-GUBZILKMSA-N Met-Glu-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O DJDFBVNNDAUPRW-GUBZILKMSA-N 0.000 description 4
- VZBXCMCHIHEPBL-SRVKXCTJSA-N Met-Glu-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN VZBXCMCHIHEPBL-SRVKXCTJSA-N 0.000 description 4
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 4
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 4
- 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 4
- YFXXRYFWJFQAFW-JHYOHUSXSA-N Phe-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O YFXXRYFWJFQAFW-JHYOHUSXSA-N 0.000 description 4
- CQZNGNCAIXMAIQ-UBHSHLNASA-N Pro-Ala-Phe Chemical compound C[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O CQZNGNCAIXMAIQ-UBHSHLNASA-N 0.000 description 4
- OOLOTUZJUBOMAX-GUBZILKMSA-N Pro-Ala-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O OOLOTUZJUBOMAX-GUBZILKMSA-N 0.000 description 4
- ZSKJPKFTPQCPIH-RCWTZXSCSA-N Pro-Arg-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZSKJPKFTPQCPIH-RCWTZXSCSA-N 0.000 description 4
- HXOLCSYHGRNXJJ-IHRRRGAJSA-N Pro-Asp-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HXOLCSYHGRNXJJ-IHRRRGAJSA-N 0.000 description 4
- FKLSMYYLJHYPHH-UWVGGRQHSA-N Pro-Gly-Leu Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O FKLSMYYLJHYPHH-UWVGGRQHSA-N 0.000 description 4
- TYYBJUYSTWJHGO-ZKWXMUAHSA-N Ser-Asn-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O TYYBJUYSTWJHGO-ZKWXMUAHSA-N 0.000 description 4
- QPFJSHSJFIYDJZ-GHCJXIJMSA-N Ser-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)CO QPFJSHSJFIYDJZ-GHCJXIJMSA-N 0.000 description 4
- WSTIOCFMWXNOCX-YUMQZZPRSA-N Ser-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CO)N WSTIOCFMWXNOCX-YUMQZZPRSA-N 0.000 description 4
- KQNDIKOYWZTZIX-FXQIFTODSA-N Ser-Ser-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KQNDIKOYWZTZIX-FXQIFTODSA-N 0.000 description 4
- ZTPXSEUVYNNZRB-CDMKHQONSA-N Thr-Gly-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O ZTPXSEUVYNNZRB-CDMKHQONSA-N 0.000 description 4
- NWQCKAPDGQMZQN-IHPCNDPISA-N Trp-Lys-Leu Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O NWQCKAPDGQMZQN-IHPCNDPISA-N 0.000 description 4
- PRONOHBTMLNXCZ-BZSNNMDCSA-N Tyr-Leu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 PRONOHBTMLNXCZ-BZSNNMDCSA-N 0.000 description 4
- ASQFIHTXXMFENG-XPUUQOCRSA-N Val-Ala-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O ASQFIHTXXMFENG-XPUUQOCRSA-N 0.000 description 4
- LNYOXPDEIZJDEI-NHCYSSNCSA-N Val-Asn-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](C(C)C)N LNYOXPDEIZJDEI-NHCYSSNCSA-N 0.000 description 4
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 4
- NHXZRXLFOBFMDM-AVGNSLFASA-N Val-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)C(C)C NHXZRXLFOBFMDM-AVGNSLFASA-N 0.000 description 4
- CEKSLIVSNNGOKH-KZVJFYERSA-N Val-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](C(C)C)N)O CEKSLIVSNNGOKH-KZVJFYERSA-N 0.000 description 4
- MNSSBIHFEUUXNW-RCWTZXSCSA-N Val-Thr-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N MNSSBIHFEUUXNW-RCWTZXSCSA-N 0.000 description 4
- 108010039538 alanyl-glycyl-aspartyl-valine Proteins 0.000 description 4
- 108010041407 alanylaspartic acid Proteins 0.000 description 4
- 108010013835 arginine glutamate Proteins 0.000 description 4
- 108010008355 arginyl-glutamine Proteins 0.000 description 4
- 108010094001 arginyl-tryptophyl-arginine Proteins 0.000 description 4
- 108010060035 arginylproline Proteins 0.000 description 4
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 4
- 108010038633 aspartylglutamate Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 4
- 108010049041 glutamylalanine Proteins 0.000 description 4
- 108010037850 glycylvaline Proteins 0.000 description 4
- 108010038320 lysylphenylalanine Proteins 0.000 description 4
- 108010012581 phenylalanylglutamate Proteins 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 108010051110 tyrosyl-lysine Proteins 0.000 description 4
- 108010009962 valyltyrosine Proteins 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- RNQHMTFBUSSBJQ-WHFBIAKZSA-N 3-isopropylmalic acid Chemical compound CC(C)[C@H](C(O)=O)[C@H](O)C(O)=O RNQHMTFBUSSBJQ-WHFBIAKZSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 108090001042 Hydro-Lyases Proteins 0.000 description 3
- 102000004867 Hydro-Lyases Human genes 0.000 description 3
- 235000019454 L-leucine Nutrition 0.000 description 3
- 239000004395 L-leucine Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- BITYXLXUCSKTJS-ZETCQYMHSA-N (2S)-2-isopropylmalic acid Chemical compound CC(C)[C@](O)(C(O)=O)CC(O)=O BITYXLXUCSKTJS-ZETCQYMHSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241000186031 Corynebacteriaceae Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 235000019764 Soybean Meal Nutrition 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 238000006317 isomerization reaction Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 229940049920 malate Drugs 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- BITYXLXUCSKTJS-UHFFFAOYSA-N 2-isopropylmalic acid Chemical compound CC(C)C(O)(C(O)=O)CC(O)=O BITYXLXUCSKTJS-UHFFFAOYSA-N 0.000 description 1
- 101710175998 3-isopropylmalate dehydratase small subunit Proteins 0.000 description 1
- OPZJWMJPCNNZNT-DCAQKATOSA-N Ala-Leu-Met Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)O)N OPZJWMJPCNNZNT-DCAQKATOSA-N 0.000 description 1
- SSQHYGLFYWZWDV-UVBJJODRSA-N Ala-Val-Trp Chemical compound CC(C)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O SSQHYGLFYWZWDV-UVBJJODRSA-N 0.000 description 1
- AWPWHMVCSISSQK-QWRGUYRKSA-N Asp-Tyr-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O AWPWHMVCSISSQK-QWRGUYRKSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010049565 Muscle fatigue Diseases 0.000 description 1
- DPUOLKQSMYLRDR-UBHSHLNASA-N Phe-Arg-Ala Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 DPUOLKQSMYLRDR-UBHSHLNASA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- KISFXYYRKKNLOP-IHRRRGAJSA-N Val-Phe-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)O)N KISFXYYRKKNLOP-IHRRRGAJSA-N 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 241000186254 coryneform bacterium Species 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 235000020510 functional beverage Nutrition 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- ZPEZUAAEBBHXBT-RZVRUWJTSA-N l-valine l-valine Chemical compound CC(C)[C@H](N)C(O)=O.CC(C)[C@H](N)C(O)=O ZPEZUAAEBBHXBT-RZVRUWJTSA-N 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 101150081723 leuC gene Proteins 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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
- C12N9/88—Lyases (4.)
-
- 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
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01033—3-Isopropylmalate dehydratase (4.2.1.33)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提供一种3‑异丙基苹果酸脱水酶突变体及其应用。本发明通过对leuD基因修饰，将3‑异丙基苹果酸脱水酶小亚基的第84位氨基酸由丙氨酸(A)突变为其他氨基酸，如亮氨酸(L)、缬氨酸(V)或异亮氨酸(I)，实现了对3‑异丙基苹果酸脱水酶的突变，使得微生物产副产物亮氨酸的能力下降，产缬氨酸的能力与未修饰的菌株相比增强，最终提高了缬氨酸的产量。本发明还提供一种利用微生物生产缬氨酸的方法以及能够高效生产缬氨酸的基因工程菌，该工程菌可用于发酵生产缬氨酸、异亮氨酸、亮氨酸等支链氨基酸或其衍生物。
Description
技术领域
本发明属于基因工程和微生物发酵技术领域，具体地说，涉及一种3-异丙基苹果酸脱水酶突变体及其应用。
背景技术
L-缬氨酸(L-valine)，化学名称为L-α-氨基异戊酸，分子式为C5H11NO2，相对分子质量为117.15。L-缬氨酸呈白色结晶或结晶性粉末，无臭，味苦，在水中溶解度：25℃为88.5g/L，50℃为96.2g/L，不溶于冷乙醇、乙醚、丙酮。L-缬氨酸等电点为5.96，熔点315℃。
L-缬氨酸是人体八种必需氨基酸之一，又是三种支链氨基酸(包括缬氨酸、亮氨酸、异亮氨酸)之一，因其特殊的结构和功能，在人类生命代谢中具有特别重要的地位。L-缬氨酸可以广泛应用于医药工业、食品工业和饲料工业等。医药工业中，L-缬氨酸可作氨基酸输液、综合氨基酸制剂的主要成分，可治疗肝功能衰竭、中枢神经系统功能紊乱。食品工业中，L-缬氨酸可用作食品添加剂、营养增补液及风味剂等。L-缬氨酸也可用作氨基酸功能饮料与运动员饮料，有形成肌肉、强化肝功能、减轻肌肉疲劳等作用。饲料工业中，对动物的乳腺组织分泌乳汁有重要的促进作用。
目前L-缬氨酸的生产方法有三种：提取法、化学合成法、微生物发酵法。提取法和化学合成法由于原料来源受限制、生产成本高、污染环境，难以实现工业化生产。微生物发酵法生产L-缬氨酸具有原料成本低、反应条件温和、容易实现大规模生产等优点，是目前生产L-缬氨酸最主要的方法。但目前L-缬氨酸的菌种的发酵性能仍较差，且副产物亮氨酸较高，导致转化率仍较低，不能满足大规模工业化生产的需求。
发明内容
本发明的目的是提供一种3-异丙基苹果酸脱水酶突变体及其应用。
本发明的另一目的是提供产支链氨基酸的基因工程菌及其构建方法与应用。
发明人意外地发现，经过修饰谷氨酸棒杆菌的3-异丙基苹果酸脱水酶，使得微生物能够高效地生成缬氨酸，并且降低了副产物亮氨酸的含量，从而成功构建出能够高效生产缬氨酸的新微生物，从而完成本发明。
为了实现本发明目的，第一方面，本发明提供一种3-异丙基苹果酸脱水酶突变体，所述突变体包含3-异丙基苹果酸脱水酶第84位氨基酸由A到除A之外的其他氨基酸的突变，优选由A到L、V或I的突变。
本发明中，3-异丙基苹果酸脱水酶在NCBI上的参考序列编号为WP_003862260.1。
3-异丙基苹果酸脱水酶是亮氨酸合成末端途径第二个酶，催化α-异丙基苹果酸异构化生成β-异丙基苹果酸。3-异丙基苹果酸脱水酶由大小2个亚基组成，大亚基由leuC基因编码，小亚基由leuD基因编码，催化α-异丙基苹果酸异构化生成β-异丙基苹果酸。本发明通过对leuD基因修饰，将3-异丙基苹果酸脱水酶小亚基的第84位氨基酸由丙氨酸(A)突变为亮氨酸(L)、缬氨酸(V)或异亮氨酸(I)，实现了对3-异丙基苹果酸脱水酶的突变，使得微生物产副产物亮氨酸的能力下降，产缬氨酸的能力与未修饰的菌株相比增强，最终提高了缬氨酸的产量。
第二方面，本发明提供编码所述3-异丙基苹果酸脱水酶突变体的核酸分子。
第三方面，本发明提供含有所述核酸分子的生物材料，所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、病毒载体或工程菌。
第四方面，本发明提供所述核酸分子或含有所述核酸分子的生物材料的以下任一应用：
(1)用于支链氨基酸的发酵生产；
(2)用于提高缬氨酸，同时降低亮氨酸的发酵产量；
(3)用于构建产支链氨基酸的基因工程菌。
第五方面，本发明提供产支链氨基酸的基因工程菌的构建方法，利用基因工程手段，在具有支链氨基酸生产能力的细菌基因组中引入突变，使其编码的3-异丙基苹果酸脱水酶包含A84L、A84V或A84I突变位点。
所述细菌可以是棒杆菌属(Corynebacterium)或短杆菌属(Breviabacterium)等菌种。
优选地，所述细菌为谷氨酸棒杆菌(Corynebacterium glutamicum)、北京棒杆菌(Corynebacterium pekinense)或黄色短杆菌(Breviabacterium flavum)等，更优选谷氨酸棒杆菌MHZ-1012-2(参见CN106520655A，菌株MHZ-1012-2于2016年11月30日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏中心地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏编号为CGMCC No.13406)。
菌株MHZ-1012-2中leuD基因及其编码的3-异丙基苹果酸脱水酶小亚基的核苷酸序列和氨基酸序列分别如SEQ ID NO:1和5所示。
第六方面，本发明提供按照所述方法构建得到的基因工程菌。
第七方面，本发明提供所述基因工程菌的以下任一应用：
1)用于支链氨基酸的发酵生产；
2)用于提高缬氨酸，同时降低亮氨酸的发酵产量。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明首次发现当3-异丙基苹果酸脱水酶第84位氨基酸由丙氨酸(A)突变为亮氨酸(L)、缬氨酸(V)或异亮氨酸(I)后，缬氨酸产量均有提升，亮氨酸产量显著下降，尤其以第84位氨基酸由丙氨酸(A)突变为缬氨酸(V)效果最好。
上述突变位点可应用于谷氨酸棒杆菌，但不限于谷氨酸棒杆菌，如北京棒杆菌或黄色短杆菌等，用于产缬氨酸、异亮氨酸、亮氨酸等支链氨基酸或其衍生物。
本发明构建的3-异丙基苹果酸脱水酶突变菌株2-LeuDA84L、2-LeuDA84V、2-LeuDA84I的L-缬氨酸产量和转化率较出发菌株(谷氨酸棒杆菌MHZ-1012-2)显著提高，且L-亮氨酸产量大幅下降，同时仍能够保持较好的生长性能。其中，以突变菌株2-LeuDA84V表现最为突出，缬氨酸产量为7.9g/L，比出发菌株产量提高1.8g/L，提高了29.5％；副产物亮氨酸产量为0.5g/L，较出发菌株亮氨酸产量下降75.0％。
具体实施方式
本发明提供一种棒杆菌，其细胞内由leuD基因编码的3-异丙基苹果酸脱水酶小亚基的第84位氨基酸由丙氨酸(A)突变为亮氨酸(L)、缬氨酸(V)或异亮氨酸(I)，其对应的碱基由GCA分别突变为CTA、GTA或ATA。改造后的棒杆菌可用于产缬氨酸、异亮氨酸、亮氨酸等支链氨基酸或其衍生物。
本发明中，所述棒杆菌可以是谷氨酸棒杆菌(Corynebacterium glutamicum)、北京棒杆菌(Corynebacterium pekinense)或黄色短杆菌(Breviabacterium flavum)等。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
以下实施例中涉及的引物信息如表1所示。
表1引物信息
实施例1 3-异丙基苹果酸脱水酶突变菌株2-LeuDA84L、2-LeuDA84V、2-LeuDA84I的构建
以谷氨酸棒杆菌MHZ-1012-2为出发菌株，将MHZ-1012-2中leuD基因突变为编码SEQ ID NO:2、3、4所示的3-异丙基苹果酸脱水酶突变体的基因(编码的突变体的氨基酸序列分别如SEQ ID NO:6、7、8所示)，依次构建3-异丙基苹果酸脱水酶突变菌株2-LeuDA84L、2-LeuDA84V、2-LeuDA84I。具体构建方法如下：
1、质粒pK18mobsacB-leuDA84L的构建
利用Phusion超保真聚合酶(New England BioLabs)，以出发菌株MHZ-1012-2的基因组为模板，以leuDA84L-UP-1F/leuDA84L-UP-1R为引物，制备重组片段UP-1，以leuDA84L-DN-2F/leuDA84L-DN-2R为引物，制备重组片段DN-1；以质粒pk18-mob-sacB为模板，以leuDA84L-pk18-3F/leuDA84L-pk18-3R为引物获得片段pk18-1，经琼脂糖凝胶回收试剂盒(天根)纯化，随后按照吉普森组装试剂盒配置体系进行反应，反应体系如表2所示。
表2吉普森组装反应体系
| 组分 | UP-1 | DN-1 | pk18-1 | CE Buffer | CE Exnase | 无菌水 |
| 体积/μL | 1 | 1 | 2 | 4 | 2 | 10 |
将配制的反应体系于37℃反应30min，吸取10μL转化Trans1T1感受态细胞(TransGen Biotech)，挑取单克隆，通过菌落PCR鉴定插入的片段正确，进一步酶切鉴定得到片段插入pK18mobsacB的阳性克隆，最后将质粒送至金唯智生物科技有限公司进行测序，将测序正确的质粒命名为pK18mobsacB-leuDA84L。
2、质粒pK18mobsacB-leuDA84V、pK18mobsacB-leuDA84I的构建
按上述1相同的方法，将引物leuDA84L-UP-1R分别替换为leuDA84V-UP-1R、leuDA84I-UP-1R，将引物leuDA84L-DN-2F分别替换为leuDA84V-DN-2F、leuDA84I-DN-2F，所构建的质粒分别命名为pK18mobsacB-leuDA84V、pK18mobsacB-leuDA84I。
3、3-异丙基苹果酸脱水酶突变菌株2-LeuDA84L、2-LeuDA84V、2-LeuDA84I的构建
将上述1、2所述方法构建获得的重组质粒pK18mobsacB-leuDA84L、pK18mobsacB-leuDA84V、pK18mobsacB-leuDA84I分别转入出发菌株MHZ-1012-2中，在含有15mg/L的卡那霉素的选择培养基上选择交换重组子。培养的温度为30℃，倒置培养。将筛选获得的转化子过夜培养于液体脑心浸液培养基中，培养温度为30℃，回转摇床220rpm振荡培养。培养过程中，转化子发生第二次重组，通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4)，稀释液涂布在含有10％蔗糖的固体脑心浸液培养基上，33℃静置培养48h。对在此培养基上长出的转化子进行鉴定。通过PCR扩增目的序列，核苷酸测序分析，获得目的突变菌株，分别命名为2-LeuDA84L、2-LeuDA84V、2-LeuDA84I。
实施例2 3-异丙基苹果酸脱水酶突变菌株发酵生产L-缬氨酸
对实施例1构建的突变菌株进行发酵验证，具体方法如下：
1、培养基
种子培养基：豆粕抽提物15g/L，葡萄糖20g/L，硫酸铵7g/L，硫酸镁0.5g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，尿素2g/L，余量为水，pH 7.2。
发酵培养基：豆粕抽提物15g/L，葡萄糖60g/L，硫酸铵20g/L，硫酸镁0.5g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，尿素2g/L，碳酸钙40g/L，VB3 15mg/L，VH50μg/L，VB1·HCl 100μg/L，余量为水，pH 7.2。
2、摇瓶发酵生产L-缬氨酸
(1)种子培养：挑取斜面种子1环接至装有50mL种子培养基的500mL三角瓶中，30℃、220r/min振荡培养10-12h；
(2)发酵培养：将5mL种子液接种至装有50mL发酵培养基的500mL三角瓶中，30℃、220r/min振荡培养48h；
(3)取1mL发酵液离心(12000rpm，2min)，收集上清液，用HPLC检测酵液中的L-缬氨酸、L-亮氨酸。同时采用分光光度法检测发酵液在562nm下的OD值，结果如表3所示。
表3菌株生长和发酵产物含量检测结果
注：*表示与出发菌株相比具有显著差异，P<0.05。
实验结果显示，出发菌株MHZ-1012-2的L-缬氨酸产量仅为6.1g/L，3-异丙基苹果酸脱水酶突变菌株2-LeuDA84L、2-LeuDA84V、2-LeuDA84I的L-缬氨酸产量和转化率较出发菌株显著提高，且L-亮氨酸产量大幅下降，同时仍能够保持较好的生长性能。其中，以α-异丙基苹果酸合酶突变菌株2-LeuDA84V表现最为突出，缬氨酸产量为7.9g/L，比出发菌株产量提高1.8g/L，提高了29.5％；副产物亮氨酸产量为0.5g/L，较出发菌株亮氨酸产量下降75.0％。
由此可见，本发明提供的3-异丙基苹果酸脱水酶突变体及3-异丙基苹果酸脱水酶突变菌株对目标产物缬氨酸的产量提升具有显著的促进作用，对副产物亮氨酸产量有显著的降低作用。该3-异丙基苹果酸脱水酶突变体及其重组微生物为产缬氨酸、异亮氨酸及以其为前体的衍生物的生产菌株的构建提供借鉴。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 廊坊梅花生物技术开发有限公司
<120> 3-异丙基苹果酸脱水酶突变体及其应用
<130> KHP211124099.9
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 594
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggaaaaat ttaccaccca caccggcgtt ggcgttccac tgcagcgatc caacgtggac 60
accgaccaga tcatccccgc cgtctacctc aagcgcgtca cccgcacagg cttcgaagac 120
ggactgtttt ccaactggcg ccaaaacgac cccaactttg tcctcaacac cgacacctac 180
aagaacggct ccgttctcgt agcaggccct gactttggca ccggctcctc ccgcgagcac 240
gccgtctggg cactcatgga ctacggcttc cgcgctgtct tctcctcacg attcgccgac 300
atcttccgcg gcaactccgg aaaagctggc atgctcgccg gcatcatgga acagtccgac 360
atcgaacttc tgtggaagct catggaacaa accccgggcc tcgaactgac cgtgaacctg 420
gaaaagcaga tcgtcactgc aggcgacgta gtgatcagct tcgaagttga cccttacatt 480
cgctggcgtt tgatggaagg cctcgacgac gctggcctga ccctgcgcaa gctcgatgaa 540
attgaagact acgaggctaa gcgccctgcg tttaagccac gcactaacgc ttaa 594
<210> 2
<211> 594
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggaaaaat ttaccaccca caccggcgtt ggcgttccac tgcagcgatc caacgtggac 60
accgaccaga tcatccccgc cgtctacctc aagcgcgtca cccgcacagg cttcgaagac 120
ggactgtttt ccaactggcg ccaaaacgac cccaactttg tcctcaacac cgacacctac 180
aagaacggct ccgttctcgt agcaggccct gactttggca ccggctcctc ccgcgagcac 240
gccgtctggc tactcatgga ctacggcttc cgcgctgtct tctcctcacg attcgccgac 300
atcttccgcg gcaactccgg aaaagctggc atgctcgccg gcatcatgga acagtccgac 360
atcgaacttc tgtggaagct catggaacaa accccgggcc tcgaactgac cgtgaacctg 420
gaaaagcaga tcgtcactgc aggcgacgta gtgatcagct tcgaagttga cccttacatt 480
cgctggcgtt tgatggaagg cctcgacgac gctggcctga ccctgcgcaa gctcgatgaa 540
attgaagact acgaggctaa gcgccctgcg tttaagccac gcactaacgc ttaa 594
<210> 3
<211> 594
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggaaaaat ttaccaccca caccggcgtt ggcgttccac tgcagcgatc caacgtggac 60
accgaccaga tcatccccgc cgtctacctc aagcgcgtca cccgcacagg cttcgaagac 120
ggactgtttt ccaactggcg ccaaaacgac cccaactttg tcctcaacac cgacacctac 180
aagaacggct ccgttctcgt agcaggccct gactttggca ccggctcctc ccgcgagcac 240
gccgtctggg tactcatgga ctacggcttc cgcgctgtct tctcctcacg attcgccgac 300
atcttccgcg gcaactccgg aaaagctggc atgctcgccg gcatcatgga acagtccgac 360
atcgaacttc tgtggaagct catggaacaa accccgggcc tcgaactgac cgtgaacctg 420
gaaaagcaga tcgtcactgc aggcgacgta gtgatcagct tcgaagttga cccttacatt 480
cgctggcgtt tgatggaagg cctcgacgac gctggcctga ccctgcgcaa gctcgatgaa 540
attgaagact acgaggctaa gcgccctgcg tttaagccac gcactaacgc ttaa 594
<210> 4
<211> 594
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggaaaaat ttaccaccca caccggcgtt ggcgttccac tgcagcgatc caacgtggac 60
accgaccaga tcatccccgc cgtctacctc aagcgcgtca cccgcacagg cttcgaagac 120
ggactgtttt ccaactggcg ccaaaacgac cccaactttg tcctcaacac cgacacctac 180
aagaacggct ccgttctcgt agcaggccct gactttggca ccggctcctc ccgcgagcac 240
gccgtctgga tactcatgga ctacggcttc cgcgctgtct tctcctcacg attcgccgac 300
atcttccgcg gcaactccgg aaaagctggc atgctcgccg gcatcatgga acagtccgac 360
atcgaacttc tgtggaagct catggaacaa accccgggcc tcgaactgac cgtgaacctg 420
gaaaagcaga tcgtcactgc aggcgacgta gtgatcagct tcgaagttga cccttacatt 480
cgctggcgtt tgatggaagg cctcgacgac gctggcctga ccctgcgcaa gctcgatgaa 540
attgaagact acgaggctaa gcgccctgcg tttaagccac gcactaacgc ttaa 594
<210> 5
<211> 197
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Glu Lys Phe Thr Thr His Thr Gly Val Gly Val Pro Leu Gln Arg
1 5 10 15
Ser Asn Val Asp Thr Asp Gln Ile Ile Pro Ala Val Tyr Leu Lys Arg
20 25 30
Val Thr Arg Thr Gly Phe Glu Asp Gly Leu Phe Ser Asn Trp Arg Gln
35 40 45
Asn Asp Pro Asn Phe Val Leu Asn Thr Asp Thr Tyr Lys Asn Gly Ser
50 55 60
Val Leu Val Ala Gly Pro Asp Phe Gly Thr Gly Ser Ser Arg Glu His
65 70 75 80
Ala Val Trp Ala Leu Met Asp Tyr Gly Phe Arg Ala Val Phe Ser Ser
85 90 95
Arg Phe Ala Asp Ile Phe Arg Gly Asn Ser Gly Lys Ala Gly Met Leu
100 105 110
Ala Gly Ile Met Glu Gln Ser Asp Ile Glu Leu Leu Trp Lys Leu Met
115 120 125
Glu Gln Thr Pro Gly Leu Glu Leu Thr Val Asn Leu Glu Lys Gln Ile
130 135 140
Val Thr Ala Gly Asp Val Val Ile Ser Phe Glu Val Asp Pro Tyr Ile
145 150 155 160
Arg Trp Arg Leu Met Glu Gly Leu Asp Asp Ala Gly Leu Thr Leu Arg
165 170 175
Lys Leu Asp Glu Ile Glu Asp Tyr Glu Ala Lys Arg Pro Ala Phe Lys
180 185 190
Pro Arg Thr Asn Ala
195
<210> 6
<211> 197
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Glu Lys Phe Thr Thr His Thr Gly Val Gly Val Pro Leu Gln Arg
1 5 10 15
Ser Asn Val Asp Thr Asp Gln Ile Ile Pro Ala Val Tyr Leu Lys Arg
20 25 30
Val Thr Arg Thr Gly Phe Glu Asp Gly Leu Phe Ser Asn Trp Arg Gln
35 40 45
Asn Asp Pro Asn Phe Val Leu Asn Thr Asp Thr Tyr Lys Asn Gly Ser
50 55 60
Val Leu Val Ala Gly Pro Asp Phe Gly Thr Gly Ser Ser Arg Glu His
65 70 75 80
Ala Val Trp Leu Leu Met Asp Tyr Gly Phe Arg Ala Val Phe Ser Ser
85 90 95
Arg Phe Ala Asp Ile Phe Arg Gly Asn Ser Gly Lys Ala Gly Met Leu
100 105 110
Ala Gly Ile Met Glu Gln Ser Asp Ile Glu Leu Leu Trp Lys Leu Met
115 120 125
Glu Gln Thr Pro Gly Leu Glu Leu Thr Val Asn Leu Glu Lys Gln Ile
130 135 140
Val Thr Ala Gly Asp Val Val Ile Ser Phe Glu Val Asp Pro Tyr Ile
145 150 155 160
Arg Trp Arg Leu Met Glu Gly Leu Asp Asp Ala Gly Leu Thr Leu Arg
165 170 175
Lys Leu Asp Glu Ile Glu Asp Tyr Glu Ala Lys Arg Pro Ala Phe Lys
180 185 190
Pro Arg Thr Asn Ala
195
<210> 7
<211> 197
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Glu Lys Phe Thr Thr His Thr Gly Val Gly Val Pro Leu Gln Arg
1 5 10 15
Ser Asn Val Asp Thr Asp Gln Ile Ile Pro Ala Val Tyr Leu Lys Arg
20 25 30
Val Thr Arg Thr Gly Phe Glu Asp Gly Leu Phe Ser Asn Trp Arg Gln
35 40 45
Asn Asp Pro Asn Phe Val Leu Asn Thr Asp Thr Tyr Lys Asn Gly Ser
50 55 60
Val Leu Val Ala Gly Pro Asp Phe Gly Thr Gly Ser Ser Arg Glu His
65 70 75 80
Ala Val Trp Val Leu Met Asp Tyr Gly Phe Arg Ala Val Phe Ser Ser
85 90 95
Arg Phe Ala Asp Ile Phe Arg Gly Asn Ser Gly Lys Ala Gly Met Leu
100 105 110
Ala Gly Ile Met Glu Gln Ser Asp Ile Glu Leu Leu Trp Lys Leu Met
115 120 125
Glu Gln Thr Pro Gly Leu Glu Leu Thr Val Asn Leu Glu Lys Gln Ile
130 135 140
Val Thr Ala Gly Asp Val Val Ile Ser Phe Glu Val Asp Pro Tyr Ile
145 150 155 160
Arg Trp Arg Leu Met Glu Gly Leu Asp Asp Ala Gly Leu Thr Leu Arg
165 170 175
Lys Leu Asp Glu Ile Glu Asp Tyr Glu Ala Lys Arg Pro Ala Phe Lys
180 185 190
Pro Arg Thr Asn Ala
195
<210> 8
<211> 197
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Glu Lys Phe Thr Thr His Thr Gly Val Gly Val Pro Leu Gln Arg
1 5 10 15
Ser Asn Val Asp Thr Asp Gln Ile Ile Pro Ala Val Tyr Leu Lys Arg
20 25 30
Val Thr Arg Thr Gly Phe Glu Asp Gly Leu Phe Ser Asn Trp Arg Gln
35 40 45
Asn Asp Pro Asn Phe Val Leu Asn Thr Asp Thr Tyr Lys Asn Gly Ser
50 55 60
Val Leu Val Ala Gly Pro Asp Phe Gly Thr Gly Ser Ser Arg Glu His
65 70 75 80
Ala Val Trp Ile Leu Met Asp Tyr Gly Phe Arg Ala Val Phe Ser Ser
85 90 95
Arg Phe Ala Asp Ile Phe Arg Gly Asn Ser Gly Lys Ala Gly Met Leu
100 105 110
Ala Gly Ile Met Glu Gln Ser Asp Ile Glu Leu Leu Trp Lys Leu Met
115 120 125
Glu Gln Thr Pro Gly Leu Glu Leu Thr Val Asn Leu Glu Lys Gln Ile
130 135 140
Val Thr Ala Gly Asp Val Val Ile Ser Phe Glu Val Asp Pro Tyr Ile
145 150 155 160
Arg Trp Arg Leu Met Glu Gly Leu Asp Asp Ala Gly Leu Thr Leu Arg
165 170 175
Lys Leu Asp Glu Ile Glu Asp Tyr Glu Ala Lys Arg Pro Ala Phe Lys
180 185 190
Pro Arg Thr Asn Ala
195
Claims (10)
1.3-异丙基苹果酸脱水酶突变体，其特征在于，所述突变体包含3-异丙基苹果酸脱水酶第84位氨基酸由A到除A之外的其他氨基酸的突变，优选由A到L、V或I的突变；
其中，3-异丙基苹果酸脱水酶在NCBI上的参考序列编号为WP_003862260.1。
2.编码权利要求1所述突变体的核酸分子。
3.含有权利要求2所述核酸分子的生物材料，所述生物材料为重组DNA、表达盒、转座子、质粒载体、病毒载体或工程菌。
4.权利要求2所述核酸分子或权利要求3所述生物材料的以下任一应用：
(1)用于支链氨基酸的发酵生产；
(2)用于提高缬氨酸，同时降低亮氨酸的发酵产量；
(3)用于构建产支链氨基酸的基因工程菌。
5.产支链氨基酸的基因工程菌的构建方法，其特征在于，利用基因工程手段，在具有支链氨基酸生产能力的细菌基因组中引入突变，使其编码的3-异丙基苹果酸脱水酶包含A84L、A84V或A84I突变位点；其中，3-异丙基苹果酸脱水酶在NCBI上的参考序列编号为WP_003862260.1。
6.根据权利要求5所述的方法，其特征在于，所述细菌为棒杆菌属(Corynebacterium)或短杆菌属(Breviabacterium)菌种。
7.根据权利要求6所述的方法，其特征在于，所述细菌为谷氨酸棒杆菌(Corynebacterium glutamicum)、北京棒杆菌(Corynebacterium pekinense)或黄色短杆菌(Breviabacterium flavum)。
8.根据权利要求7所述的方法，其特征在于，所述细菌为谷氨酸棒杆菌MHZ-1012-2。
9.根据权利要求5-8任一项所述方法构建得到的基因工程菌。
10.权利要求9所述基因工程菌的以下任一应用：
1)用于支链氨基酸的发酵生产；
2)用于提高缬氨酸，同时降低亮氨酸的发酵产量。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111530562.3A CN116262915A (zh) | 2021-12-14 | 2021-12-14 | 3-异丙基苹果酸脱水酶突变体及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111530562.3A CN116262915A (zh) | 2021-12-14 | 2021-12-14 | 3-异丙基苹果酸脱水酶突变体及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116262915A true CN116262915A (zh) | 2023-06-16 |
Family
ID=86722332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111530562.3A Pending CN116262915A (zh) | 2021-12-14 | 2021-12-14 | 3-异丙基苹果酸脱水酶突变体及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116262915A (zh) |
-
2021
- 2021-12-14 CN CN202111530562.3A patent/CN116262915A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110468092B (zh) | 一株高产l-缬氨酸的基因工程菌及其构建方法与应用 | |
| CN110607313A (zh) | 一种高产l-赖氨酸的重组菌株及其构建方法与应用 | |
| CN104480058A (zh) | 一株高产l-亮氨酸工程菌及其应用 | |
| CN111411092B (zh) | 高产l-赖氨酸的谷氨酸棒状杆菌及其应用 | |
| WO2022143763A1 (zh) | 具有增强的l-谷氨酸生产力的菌株及其构建方法与应用 | |
| CN113201524B (zh) | 肌醇-3-磷酸合酶突变体及其在构建高产谷氨酰胺的谷氨酸棒杆菌中的应用 | |
| CN113278571A (zh) | 一种棒状杆菌工程菌构建方法及应用 | |
| CN112646766A (zh) | 一种改造基因bbd29_04920的产l-谷氨酸的重组菌株及其构建方法与应用 | |
| CN114181288B (zh) | 制备l-缬氨酸的方法及其所用的基因与该基因编码的蛋白质 | |
| CN114957414B (zh) | 一种RosR突变体及其重组微生物与应用 | |
| CN114717237A (zh) | 一种ep6启动子与其相关生物材料及应用 | |
| CN110862940B (zh) | 一种谷氨酸棒杆菌工程菌及其在制备l-色氨酸中的应用 | |
| CN116262915A (zh) | 3-异丙基苹果酸脱水酶突变体及其应用 | |
| CN113308426A (zh) | 一种改造tk基因5′端序列的重组棒状杆菌及其应用 | |
| TW202202621A (zh) | 新穎啟動子及使用其生產麩胱甘肽的方法 | |
| CN117165545A (zh) | α-异丙基苹果酸合酶突变体、重组微生物及其应用 | |
| CN112538491B (zh) | 一种基于yh66_08550基因的产l-异亮氨酸的重组菌株及其构建方法与应用 | |
| CN116262914A (zh) | 一种3-异丙基苹果酸脱水酶突变体及其应用 | |
| CN117247914A (zh) | 乙酰羟酸合酶突变体及其应用 | |
| CN115490761B (zh) | 基于赖氨酸外排蛋白构建的重组微生物及生产赖氨酸的方法 | |
| CN115678820A (zh) | 产缬氨酸的重组微生物及其构建方法和应用 | |
| CN112266891B (zh) | 一种产l-氨基酸的重组菌株及其构建方法与应用 | |
| CN113278572B (zh) | 一种改造hts基因5′端序列的重组棒状杆菌及其应用 | |
| CN117586977A (zh) | 一种支链氨基酸氨基转移酶突变体及其重组微生物与应用 | |
| CN117586998A (zh) | 一种二羟酸脱水酶突变体及其重组微生物与应用 |
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 |